Home

3 - Agilent Technologies

1. 1e 074 9000000 8000000 TIC 1901026 DNdata ms 7000000 TIC RELOCKCHECKA D data ms 6000000 5000000 4000000 3000000 2000000 1000000 1 0 SS SSS SS A 9 60 9 80 10 00 10 20 1040 1060 1080 11 00 11 20 11 40 Time Abundance TIC 1901026 D data ms TIC RELOCKCHECKA D data ms le 07 4 9000000 8000000 5 7000000 6000000 5000000 4000000 5 3000000 5 2000000 Lt 7 A J UU L JULY E 1000000 eem oS T c cp ue dm a a a a TT 7 14 00 16 00 18 00 Time Figure 4 Overlay of injections before and after column maintenance and relocking including a zoomed area of the four peaks from phenanthrene to pyrene Conclusions For More Information This application demonstrates the ability of the For more information on our products and services 7890 GC system to perform RTL at low pressures visit our Web site at www agilent com chem sub 5 psi such as those experienced when using a 320 um column in a GC MS system The average retention time variation before and after column maintenance for a 16 PAH mixture is less than 0 5 sec providing high confidence in peak assignments even with critical separations www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this
2. B CFT tee I S 4 Standard 30 m column 84 S y a a 7 J PFETPD RDPPTTTTTTTTFTTTTTTTTTTFTTTTEFTTTTTFTTTTTTTTTITTFTTTTTTTTFTTTTTTTTTITTTTTTITTTTTFTTTTTTTTTTTTTT 5 00 5 05 5 10 515 5 20 5 5 5 30 535 5 40 545 5 50 5 55 560 5 65 5 70 575 5 80 5 85 Time Figure 6 Reconstructed total ion chromatogram RTIC of three replicate SIM acquisitions of octafluoronaphtha lene using pulsed splitless injection with CFT tee left profiles and with a standard 30 m continuous column configuration right profiles Ix Chromatographic Character Beyond preserving signal the CFT device should exhibit reasonable chromatographic performance One indication of chromatographic integrity is the peak shape profiles of the fatty acid methyl esters FAMESs The result for GC MS analysis of a FAMEs standard acquired using a metabolomics method is shown in Figure 7 and suggests very little degradation of chromatography using this PCT This can be expected as the path is deacti vated and the path length in the channels in the PCT relative to the linear velocity suggests a relatively rapid transit through the device Another common chromatographic test used in organochlorine pesticide analysis as in USEPA method 8081 examines degradation of 4 4 DDT and Endrin This degradation test was developed to indicate the degree of activity of the injection port by examining the amounts of DD
3. Lu 219 90 5 69 80 4 70 4 A Relative Iso 60 Mass Abundance abundance lso mass abundance Iso ratio 69 00 382336 100 00 70 00 4302 1 13 7 219 00 461504 120 71 220 00 19976 4 33 50 4 502 00 51720 13 53 503 00 5073 9 81 40 30 4 20 4 10 4 i Ed de Ea Esp de dura tc d e d ui E 50 100 150 200 250 300 350 400 450 500 550 600 650 700 Figure 1 Autotune results for an ion source temperature of 230 C 100 4 69 90 219 80 4 70 4 J Relative Iso 60 _ Mass Abundance abundance lso mass abundance Iso ratio 69 00 425024 100 00 70 00 4657 1 10 7 219 00 395392 93 03 220 00 17000 4 30 50 4 502 00 24688 5 81 503 00 2563 10 38 40 4 30 4 20 4 10 Ll liL Je 50 100 150 200 250 300 350 400 450 500 550 600 650 700 Figure 2 Autotune results for an ion source temperature of 300 C Implications for Analytical Applications Although the tuning compound showed a spectral change that favored more fragmentation and all compounds could be expected to be influenced simi larly there are some advantages that can occur for less fragile compounds especially those that have higher boiling points and are late eluting in GC Analysis of the class of compounds known as per sistent organic pollutants POPs is likely to benefit from higher source temperatures To illustrate the aspects that need to be examined consider the six polychlorinated biphenyls PCBs acquired in full scan and presented in Figure 3 The Relative ab
4. UV Vis 230 nm 8 12112 500000 MS EIC m z 358 300000 100000 L 0 25 5 6 0 NE NEC Time min Figure 17 Extract from tuna 0 2 ppm 1 pl injected UV Vis 230 nm 10815 500000 MS EIC m z 358 300000 100000 4 0 25 5 75 10 DS B ma A Time min Figure 18 Extract from sardine 20 ppm 1 pl injected 25 Pesticides The following compound classes of pesticides have been analyzed triazines phenylurea herbicides methabenzthiaz uron diquat paraquat and mercaptobenzothiazol Carbamates and glyphosate also have been analyzed but with different equipment In most countries growing concern about the residues of pesticides in food products is evident Therefore regulations limiting the concentration of pesticides in foodstuffs have been introduced to protect consumers from contaminated food products Several methods are used to control these limits HPLC is recom mended for the analysis of low volatile compounds and for compounds that are unstable when heated Sample preparation Sample preparation and enrichment depend strongly on the matrix Drinking water samples for example must be extracted using solid phase extraction whereas vegetables are extracted with liquid liquid extraction after homo genization followed by additional cleaning and sample enrichment Control and data evaluation Quaternary pump Auto Column Diode es vacuum 7 sampler compa
5. In brief The following table reviews the detection techniques discussed in this chapter your decision ideally should reflect a balance between desired results and financial resources Detector Sensitivity Advantages Applications UV variable t Low cost Organic acids fatty wavelength universal acids after derivatization inorganic anions UV DAD Peak purity Antioxidants confirmation preservatives flavors colorants antiparasitic drugs mycotoxins pesticides vitamins amines after derivatization Fluorescence High sensitivity Artificial sweeteners mycotoxins vitamins carbamates glyphosate Electro High sensitivity Vitamins inorganic chemical anions Mass spectro Identity Carbamates lipids meter scan structure Mass spectro High selectivity Pesticides proteins meter SIM RI Universal Carbohydrates nonaromatic acids 105 106 Chapter 9 Derivatization chemistries Addition of UV visible chromophores When analyte concentrations are particularly low sample handling equipment for chemical derivatization can enhance the sensitivity and selectivity of results As discussed in chapter 6 such equipment is available both pre and postcolumn In this chapter we detail the chemistries that can be applied to food compounds and list the detection techniques for which they are best suited Labeling compounds with reagents that enable UV absorp tion is one
6. Agilent 190915 433 325 C 30 m x 250 um x 0 25 um In Front 55 Inlet He Aux Pressure 1 WZ r Contral Made M on f Flow 125 mL min Initial 0 min Pressure 2 7419 psi Ru miee Average Velocity 15 m 250 pm x 0 25 pm 57 48 cm sec jo 43493 min Holdup Time Rate Aux Pressure 2 M2 Hold Time min Run Time min Value mL min per mimin Aux Pressure 3 Nz min PCM A 1 Nz PCM A 2 MZ k Initial El Final value will be extended by GC run time Post Run fi 25 mL min e Figure 5B lower panel Cancel Typical pressure pulsed splitless injection parameters for constant flow Second Help column section configured as a 15 m column Results and Discussion Figure 6 shows the results for pressure pulsed splitless injections of octafluoronaphthalene OFN at 1 pg uL acquired in selected ion monitoring SIM with the two 15 m column and CFT tee con figuration and the standard 30 m continuous column configuration Both peak height and area remain the same indicating that there is no loss in signal This is as expected since no signal dilution is taking place There is a slight degradation in S N for the CFT tee results as the background noise is raised by about 35 due to the additional flow con troller The important point is that the signal is preserved at trace levels
7. Borosilicate 18740 60840 4mm with cup YES column packing Borosilicate 18740 80190 4mm with cup Borosilicate less liner 18740 80220 Splitless liner 5181 8818 18740 80200 1 5mm 140 L Direct liner Borosilicate General recommendation See notes at left regarding use Can be used without the glass wool in some applications but liners with glass wool are gen erally recommended for best reproducibility in fast injections Recommended only for small lt 0 5 uL vol umes depending on solvent and conditions The glass wool packing as supplied may not pro vide adequate mixing for good precision in split injections Liners can be packed with silanized glass wool positioned as in the straight 4 mm capillary liner part number 19251 60540 Taper liners are recommended for best perfor mance in this application particularly with labile samples and wide boilingrange mixtures Not recommended for use with EPC Quality discounts available please inquire Silanized glass wool 10 gm pesticide grade Agilent p n 5181 3317 www agilent com chem For More Information For more information on our products and services visit our Web site at www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with t
8. Figure 10 Offset overlay of RTLocked data file and the RTCorrected datafile show peaks overlapped and corrected for column maintainence fy Enhanced Data Analysis RTLPESTB M 7 RTLOCK3 D MS Data Not Quantitated File Method Chromatogram Spectrum Calibrate Quantitate Tools MERE H Feu Abund Edt Gtent Result Edition tene Peaks Parametric Retrieval Align GC Review Peak Purity 6000000 5000000 Results Screener 4000000 RTLock Setup 3000000 2000000 Enter RT Locking setup mode Figure 11 To define a new compound for RTLocking select RTLock Setup from the View menu in data analysis After selecting the compound to RTLock from the RTLock menu item select Calculate New Curve from Selected Peaks This will generate a new RT vs P cali bration curve The new RT vs P calibration curve equation will be displayed on the screen along with the correlation coefficient Select Yes to create a new or update an existing RTLock cali bration See Figure 12 From the RTLock menu you can also View current setpoints Calculate the RT vs P curve Restore the original chromatograms Report the RTLock calibration Unlock the method Relock the method View Help View Current Method Setpoints Calculate New Curve from Selected Peaks Restore Nominal Chromatogram Report RTLock Calibration Print Relock Method Unlock Method e 10 00 Figure 12
9. Polarity solubility in water Figure 70 Suitability of MS interfaces Should the electrode surface of the flow cell become severely contaminated as is likely for food matrixes the cell must be disassembled and the electrode removed and cleaned in a strong acid or other suitable cleaning agent Modern detectors are designed for ease of access and disas sembly Part one of this primer contains several applica tions of electrochemical detection 101 HPLC MS High pressure High vacuum liquid phase required Separation Produces large Typical MS quantities of volatilized solvent 100 1000 ml min gas No mass range limitation Can use inorganic buffers vacuum systems designed for low ml min gas load Depends on masss charge and mass range of analyzer Prefers volatile buffers About 1000 fold increase going from liquid to gas phase with typical LC solvent API interfaces Mass spectrometers nevertheless are more easily interfaced with GC equipment than with LC equipment The table at left lists the different operational conditions of LC and MS Early efforts to interface LC with MS used direct liquid injection and moving belt interfaces but these methods proved ineffective and unreliable In the 1980s thermospray and particle beam interfaces improved both the range of applicability and the reliability of LC MS However low sen sitivity the narrow mass and polarity range of analytes and frequ
10. y TIC 1802024 D data ms 1000000 3 f TIC 1801023 DNdata ms TIC 1803025 D data ms 600000 200000 4s VUE ew pum m 1 r 34 4 00 6 00 8 00 14 00 16 00 18 00 Time Figure 2 Overlay of three replicate injections of 2 ppm standard prior to column maintenance and relocking including a zoomed area of the four peaks from phenanthrene to pyrene A 45 cm length of column was removed from the column to simulate typical maintenance that may be performed on a column during routine use The method was then relocked and the sample re run to check the efficacy of relocking at low pressure The relocked method had a resulting pressure of 3 168 psig The change in locking pressure can over time provide guidance as to the extent at which column trimming can be undertaken with out the need for full re locking of the method Figure 3 shows an overlay of the before and after column trimming and the extent of the retention time change Figure 4 presents an overlay of chro matograms one of the originals and one after column trimming and relocking The last two columns of Table 2 compare the relocked retention times of target compounds to the originals Abundance 1e 07 9000000 4 8000000 TIC RELOCK1 D data ms 7000000 TIC 1902027 D data ms 6000000 5000000 4000000 30000004 2000000 I 1000000 4 M Dore a ripper EUER ERATES 9 40 9 60 9 80 10 00 10 20 10 40 10
11. AU Citric acid m Glucose Sample spectrum Citric acid Fructose overlaid with library spectrum match 994 0 190 Wavelength nm 276 Ethanol 0 5 10 15 20 Time min Figure 3 Analysis of citric acid in vodka ELI 4 Official M ethods of Analysis Food Compositions Additives Natural Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 Official M ethod AOAC 986 13 quinic malic citric acid in cranberry juice cocktail and apple juice Antioxidants The following compounds are used as antioxidants in food products Natural antioxidants vitamin C vitamin E Synthetic antioxidants e BHT butylated hydroxytoluene e BHA butylated hydroxyanisole TBHQ mono tert butylhydroquinone e THBP 2 4 5 trihydroxybutyrophenone e PG propyl gallate e OG octyl gallate e DG dodecyl gallate e Ionox 100 4 hydroxymethyl 2 6 di tert butyl phenol e NDGA nordihydroguaiaretic acid e TDPA 3 3 thiodipropionic acid e ACP ascorbyl palmitate Antioxidants may be naturally present in food or they may be formed by processes such as smoking Examples of natural antioxidants include tocopherols vitamin E and acsorbic acid vitamin C A second category of antioxidants comprises the wholly synthetic antioxidants When these antioxidants are added to foodstuffs they retard the onset of rancidity by preventing the oxidative degradation of lipids In most countries where antioxidants are permitted either singl
12. HPLC method performance Limit of detection Repeatability of RT over 10 runs areas over 10 runs 200 pg injected amount S N 2 0 1 96 5 96 mAU 1400 4 1000 4 Standard 6004 Dietary fat 200 Standard 15 20 25 30 Time min Figure 28 Analysis of a dietary fat triglyceride pattern Overlay of one sample and two standard chromatograms Norm 40 30 C10 9 9ng 20 C16 6 7 ng g 8 XE Z S i m 10 og 8 m 0 20 22 dr 26 28 30 3p Time min Figure 29 Trace analysis of triglycerides with a diode array and a variable wavelength detector in series 39 Carbohydrates The following carbohydrates have been analyzed glucose galactose raffinose fructose mannitol sorbitol lactose maltose cellobiose and sucrose Food carbohydrates are characterized by a wide range of chemical reactivity and molecular size Because carbohydrates do not possess chromophores or fluorophores they cannot be detected with UV visible or fluorescence techniques Nowadays however refractive index detection can be used to detect concentrations in the low parts per million ppm range and above whereas electrochemical detection is used in the analysis of sugars in the low parts per billion ppb range Sample preparation Degassed drinks can be injected directly after filtration More complex samples require more extensive treatment such as fat extraction and deproteination Sample cleanup to r
13. 9 56 es UENIRE 9 58 9 60 9 62 9 64 9 66 9 68 Figure 6 Overlaid extracted ion current chromatograms of one ion m z 300 for coronene acquired in full scan at source temperatures of 230 C and 300 C Source Bakeout There may be considerable temptation to use the higher source temperature for source cleaning by baking In other words when the user notices a higher background in the source or a reduction in response the ill conceived approach of baking the source clean may come to mind The result will be that garbage coating the source will be volatized further into the analyzer the other lenses will get dirtier as will the multiplier etc Baking is not a substitute for mechanical cleaning of the source However baking a source after a cleaning is a good approach and a macro that provides this option is given in Table 1 After a source has been cleaned and the MS system pumped down and checked to be leak free this macro can be implemented either Table 1 name Bake manually or in a sequence Note that the tempera ture limits in the tune file need to be altered to 300 and 200 for source and quadrupole respectively Manually the bakeout is called from the command line in TOP by macro bake mac enter bake 2 enter The 2 calls for a 2 hour bakeout and which can be set to anytime the user requires Copy the lines in Table 1 into Notepad and save the file as
14. Enhances diagnostics and troubleshooting func tionality with searchable complete user infor mation capability Keeps your systems in top condition and helps with routine troubleshooting Maximizes column and consumables utilization by optimizing replacement schedules Helps you meet regulatory requirements by keeping all maintenance event and run logs in a single easily accessible location Provides a link to the optional Web enabled Remote Advisor to back up your internal ser vice and support resources The following two case studies demonstrate some of the features and benefits of the Agilent LMD software E Agilent Technologies Configuration for Case Studies One PC with LMD monitors two Agilent 7890A GCs Agilent LMD software A 01 03 Advanced Firmware A 01 06 ChemStation B 03 02 The first case study demonstrates the feature ben efit of right advice alarm by tracking resource counters before results go bad Problem Oxygen is an enemy to most capillary GC columns especially polar columns In this study a polar column is used for analyzing trace oxygenates The baseline is getting worse after 400 runs because of septum leakage which may impact quantitative analysis for trace level analysis see Figure 1 upper chromatogram Tracking Resource Counters and Giving Alarm Before Results Go Bad Because LMD is configured to track the GC s inlet septum it reminds the user to change
15. Ill GC Edit Parameters Oven Temperature 186 3 C 9 d O M Oven Temp On column 2 flow is raised to 4 mL min the Holdup Time for back flow through column 1 will be less than this actually around 0 26 min But estimat ing that every 0 4 minute the front 15 m column section would be flushed at least once is very con servative and an adequate approximation Five to 10 column volumes will flush this front 15 m sec tion in less than 2 to 4 minutes which is relatively rapid Choose a time in this range for example 3 minutes and test the effectiveness of the backflush method by injecting a sample and follow this with a solvent blank injected under the non backflush GC MSD method There should be no sign of carryover Extend this Post Run time if there is carryover or further raise the Post Run temperature or both This is a very conservative approach Column or Inlet Servicing and Maintenance To change the liner septum cutback the column or replace the front 15 m column simply cool the inlet s and increase the flow on the back column column 2 to 4 mL min and set the front injec tion port pressure to OFF It is worth saving this method such as SERVICE Front M When the head of the column is removed from the injection port one can confirm that the carrier is flowing back up the column by immersing the tip in liquid Hold Time Run Time cimin min min Equilibration Time gt faz min Post R
16. formance and reliability of instruments in the lab LMD monitors multiple instruments in the lab con tinuously in real time and detects maintenance needs and instrument problems before a problem occurs These case studies demonstrated the bene fits of Agilent LMD software for customers Right advice before results go bad Real time instrument monitoring tracks the number of runs or the life of GC resources such as inlet septa liners and o rings Then when the user specified limit has been reached LMD generates an Alert Action for example it sends an alert email to specified user s Intuitive help in case of a problem Diagnostics and tests will help you solve problems and verify proper performance www agilent com chem Easy to use system tests including an inlet leak test and inlet decay test will help you do quick diagnostics A detector checkout test will provide con venient guidance to help you verify proper performance Extensive user document search capability LMD offers easy to find and easy to use instruc tions on how to change the consumables including the inlet septum and liner O ring and detailed consumable information including part numbers to help you select the right parts For More Information For more information on our products and services visit our Web site at www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequent
17. products software services 7i Agilent Technologies Productivity Tools Applications gt Return to Table of Contents gt Search entire document Retention Time Locking with the MSD Productivity ChemStation e e 9 e eo o l l 00 Q e Technical Overview 050 e 9 e e e e e e Introduction A retention time is the fundamental qualitative measurement of chromatography Most peak identi fication is performed by comparing the retention time of the unknown peak to that of a standard It is much easier to identify peaks and validate meth ods if there is no variation in the retention time of each analyte However shifts in retention time occur frequently Routine maintenance procedures such as column trimming alter retention times In a multi instru ment laboratory running duplicate methods the retention times for each instrument will differ from each other even when run under nominally identi cal conditions These differences in retention times mean that each instrument must have a separate calibration and integration event table making it time consuming to transfer methods from one instrument to another Differences in retention time also complicate comparison of data between instruments over time What Is Retention Time Locking Retention time locking RTL allows a close match of retention times on one Agilent Technologies GC MSD or GC system to those on another like system with the same nominal colum
18. 94 x DADs are best suited for universal rather than sensitive analysis for which electrochemical or fluorescence detection is more appropriate Fluorescence detectors Xenon Emission flash lamp monochromator Lens Qu irror Lens b A Photomultiplier Excitation Photodiode monochromator Sample Figure 62 Schematics of a fluorescence detector Fluorescence is a specific type of luminescence that is created when certain molecules emit energy previously absorbed during a period of illumination Luminescence detectors have higher selectivity than for example UV detectors because not all molecules that absorb light also emit it Fluorescence detectors are more sensitive than absorbance detectors owing to lower background noise Most fluorescence detectors are configured such that fluorescent light is recorded at an angle often at a right angle to the incident light beam This geometry reduces the likelihood that stray incident light will interfere as a background signal and ensures maximum S N for sensitive detection levels The new optical design of the Agilent 1100 Series fluores cence detector is illustrated in figure 62 A Xenon flash lamp is used to offer the highest light intensities for exci tation in the UV range The flash lamp ignites only for microseconds to provide light energy Each flash causes fluorescence in the flow cell and generates an individual data point for the chromatogram Since the lamp is not
19. Aldehydes Ketones 0G DG Enzymes 3 G Sulfonamides 7 Glycols phenols Aflatoxins E x Fatty acids Antibiotics Nitriles BHT BH THBQ a antioxidants Flavonoids Organo Alcohol TN a phosphorous PAHs 7 EUER Natural food dyes TM pesticides Aromatic amines Fat soluble vitamins derivative sr PCB of sugars Nitrosamine Essential oils Polymer monomers Triglycerides Phospho lipids Epoxides Fatty acid Aromatic esters methylester lt fl Figure 1 Match of analyte characteristics to carrier medium C C hydrocarbons Its selective detectors together with its ability to connect a mass spectrometer MS for peak identification make gas chromatography GC the most popular chromatographic method HPLC separates and detects at ambient temperatures For this reason agencies such as the U S Food and Drug Administration FDA have adopted and recommended HPLC for the analysis of thermally labile nonvolatile highly polar compounds Capillary electrophoresis CE is a relatively new but rap idly growing separation technique It is not yet used in the routine analysis of food however Originally CE was applied primarily in the analysis of biological macromolecules but it also has been used to separate amino acids chiral drugs vitamins pesticides inorganic ions organic acids dyes and surfactants t 2 3 Part 1 is a catalog of analyses of compounds in foods Each section features individual chromatogr
20. PAHs was chosen for this example as it covers a wide range of physical properties and provides several challenging separations This requires the retention time precision to be as tight as possible to ensure correct identification of the Table 2 Retention Time Precision of Low Pressure RTL Average retention RSD time Naphthalene 5 884 0 055 Acenapthylene 7 153 0 045 Acenaphthene 7 968 0 044 Fluorene 8 562 0 048 Phenanthrene 9 702 0 089 Anthracene 9 752 0 044 Fluoranthene 11 119 0 031 Pyrene 11 388 0 090 Chrysene 12 808 0 094 Benz a anthracene 12 855 0 046 Benz b fluoranthene 14 285 0 045 Benz k fluoranthene 14 314 0 069 Benz a pyrene 14 815 0 035 Indeno 1 2 3 cd pyrene 17 088 0 051 Dibenz a h anthracene 17 098 0 066 Benzo ghi perylene 17 725 0 089 peaks of interest and relocking must be effective or peak identification will fail on the different column Samples were injected in triplicate to measure the retention time precision of the locked method at different concentrations from 0 01 to 5 ppm Table 2 shows the performance metrics for the retention time and also the correlation co efficient for the analysis The initial retention times in Table 2 were achieved at a head pressure of 3 761 psig This setpoint was determined from the retention time calibration and relocking process to be appropriate to achieve the target retention time of 11 112 min for the locking compound pyrene Table 3 shows the calibration data
21. The 7890 GC s fifth generation EPC provides excel lent low pressure control and with third decimal place control of the pressure providing the precision demanded for RTL at low pressures This application explores the suitability of the 7890 for RTL at low pressures with a 320 um column and uses a translation of the method described in Agi lent Technologies publication 5989 6569EN Reli able transfer of existing Agilent 6890 5973GC MSD methods to the new 7890 5975 GC MSD Agilent Technologies Experimental The method used for this example was translated using the Agilent Method Translator software to convert to a 250 um id column method to a 320 um method A series of standards was run from 10 ppb to 5 ppm to illustrate the performance of the method followed by trimming approximately 45 cm off the column and relocking the method Table 1 Method Conditions Column HP 5 25 m x 0 32 um x 0 52 um p n 19091J 112 Carrier gas Helium constant pressure mode nominal 3 761 psi RTL peak Pyrene at 11 112 min Split splitless inlet 300 C pulsed splitless 7 psi for 0 3 in 50 mL min purge at 0 75 min Oven 55 C 1 1 min to 320 C 6 5 min at 22 88 C min Sample 1 pL injection PAH 0 01 to 5 ppm concentration range MSD Scan 45 400 u Samples 2 Autotune EM 200V Source 230 C Quad 150 C Transfer line 280 C Results and Discussion The test sample with 16 polynuclear aromatic hydrocarbons
22. This guide is specific for the Agilent Technologies 6890 Series GC used with the Agilent Technologies 5973 MSD Supplies In order to improve the system performance some supplies should be on hand These may not give significant improvements by themselves but when installed together they will give the best results Many of the referenced supplies have changed over the past few years and will continue to be improved It is important to stay informed and purchase the most recently updated versions of the consumables Refer to www agilent com chem for the latest comsumables available The GC carrier gas should be at least 99 999 helium Lower grades of helium are available and may contain impurities that can damage the GC column for example oxygen and contribute to the chemical noise background Even with a high purity gas there may be trace water oxygen and hydrocarbons Putting a trap in the carrier line will eliminate these contaminants see Figure 1 The mass spectrometer MS gas purifier trap is recommended and shipped with all new mass selective detectors MSDs It must be installed diagonally or vertically for optimum performance Do not install it horizontally High purity helium also increases the effective lifetime of traps Precleaned refrigeration grade 1 8 inch copper tubing should be used with high quality carrier gas Other tubing can be cleaned by running sol vents methanol ethyl acetate hexane through it
23. Troubleshooting chromatographic problems How Does It Work The process of RTL is to determine what adjust ment in inlet pressure is necessary to achieve the T P Agilent Technologies desired match in retention times To lock a given method for the first time or for the reasons above you must first develop a Retention Time vs Pressure RT vs P calibration curve Using an established method multiple five injections of the standard are used to calculate the reten tion times at predefined inlet pressures The use of an auto matic sampler simplifies this process The RT vs P calibration data for each of the five injec tions are saved and used to cor rect locked methods The five defined pressures are Target pressure 20 Target pressure 10 Target pressure nominal method pressure Target pressure 10 Target pressure 20 Even when using columns with the same part number same id stationary phase type phase ratio and same nominal length separate and different locking calibration curves may be needed Other examples of when separate locking calibration curves are required include Systems with different column outlet pressures MSD vacuum FID atmos pheric Columns differing from the nominal length by more than 15 for example due to trimming Systems where the predicted locking pressure falls outside the range of the current cali bration Selecting the Standard
24. but this factor must be weighed against cost per analysis when deciding whether to use a detector routinely The ability to use UV spectra to confirm the presence of cer tain food analytes and their metabolites and derivatives makes UV absorbance the most popular detection tech nique However for analytical problems requiring high sen sitivity and selectivity fluorescence detection is the method of choice Although electrochemical detectors are also highly sensitive and selective they are rarely used in food analysis Conductivity detectors on the other hand are well suited for the sensitive and selective analysis of cations and anions and thermal energy detectors are used for high sensitivity determination of nitrosamines down to 10 parts per trillion ppt Refractive index RI detectors are appropriate only if the above mentioned detectors are not applicable or if the concentration of analytes is high or both 86 Analytical parameters Limit of detection and limit of quantification Selectivity The most important parameters for food analysis are limit of detection LOD and limit of quantification LOQ linearity selectivity qualitative information The LOD and LOQ of an analytical system depend on the noise and drift of the detection equipment Absolute detec tor LOD can be determined by injecting a sample directly into the detector It is often expressed as minimum detect able level which is so
25. the user selects the peak used for RTL In this example the second peak has been selected The spectrum of the selected compound is also displayed and is used to confirm the RTL compound E Enhanced RTL setup RTLPESTB M is LOCKED IG Tx RTLock View Help E Window 2 TG Tx Ens TIC RTLOCK1 D 1945 i T i T T f T T T T T T T T T T Time gt 10 00 20 00 30 00 Ce TIC RTLOCK2 D 1845 LI XCR 0 999681 i Time gt 10 00 20 00 30 00 ELE Nominal TIC RTLOCK3 D 1945 l T T T T T T T T T T T T Time gt 10 00 20 00 30 00 Euge TIC RTLOCK4 D 1945 i T A T T T T T T L T T T T T Time gt 10 00 20 00 30 00 Abundance TIC RTLOCKS D 5118296 Time 10 00 20 00 30 00 246 96 308 321 300 Figure 3 When the RT vs P calibration curve equation is calculated the correlation coefficient is determined for the RTLocked compound in each of the five calibration samples The resulting coefficient is displayed at each peak The nominal or no change to pressure calibration sample has a correlation of 1 Calculating the RTLock Curve and Saving a Method Once the RTLock compound has been selected the new RT vs P curve for each compound will be displayed To select a new com pound and calculate new RT vs P curves reset the nominal MSTIC from the RTLock menu item Select a new compound as the RTL compound and from the RTLock menu item select Calcu late New Curve from Selected Peaks This will generat
26. value reported in the method translation software Make sure that the new method parameters are consistent with the hardware capabilities of where the new method will be used 3 Perform the RTL calibration runs for the new method Alternatively the method translation software can be used to calculate the RTL calibration points for the new method using those from the origi nal method 4 Retention time lock the new method using the locking refer ence standard from the original method The new method should be locked to the original reference standard retention time divided by the scale factor 5 Export the retention time table as a text file using the EXPORT func tion in the RTL SEARCH menu of the RTL ChemStation software 6 Divide the retention times in the table by the scale factor in a spreadsheet program like Microsoft Excel 7 Re import the new scaled table 8 Runarepresentative test mixture to validate the scaled method Several examples of scaling the HP RTL Pesticide Library are presented below Experimental All data were collected on Agilent 6890 Series GC systems All systems were equipped with Electronic pneumatics control EPC e Split splitless inlet e Automatic liquid sampler The GC AED system also included an Agilent G2350A atomic emission detector with GC AED ChemStation software rev B 00 00 for Microsoft Windows NT The GC micro ECD system was con trolled by Agil
27. 10 The calculations are also very simple To calculate when the flow pressure in the front column column 1 is to be reduced simply subtract the Holdup Time Figure 9C from the last compound s eS eee 9 00 9 50 10 00 10 50 11 00 11 50 12 00 a b ee Cee rene ee T T T T oorr T T i T 9 00 9 50 10 00 10 50 11 00 11 50 12 00 oT T T T T T T T T 9 00 9 50 10 00 10 50 11 00 11 50 12 00 Figure 10 Example of backflushing with flow or pressure control Upper panel RTIC of original six component standard The third peak is considered the last analyte and the fourth peak the beginning of the late eluting interferences Middle panel RTIC of the same standard with backflushing beginning at 10 1 min a where the first 15 m column column 1 flow is dropped and at b where column 2 flow is increased to 4 mL min Note that the last analyte is retained but the late eluters never enter the MSD Lower panel solvent blank run without backflush after the backflush method which shows no carryover 12 elution time After this last compound has eluted go into Post Run and set the second 15 m column 2 flow to 4 mL min or the pumping system maximum with the front column 1 pressure remaining low and the oven at the final pro grammed temperature This can best be accom plished in ramped flow mode or in pressure programming Do this for two to three column vol ume
28. 10 min 813 Purity Spectra C Differences C Peak Signals O Ratios Purity factor 994 557 49 of 49 spectra are within the thresh a Threshold 830 000 Set by user Reference Peak start and end spectra integrated 6 682 4 DAD1 A Sig 205 15 Ref 450 80 NLICLEO TANUC MD TI IDAD1 B Sig 270 40 Ref 450 80 NUCLEOT NUC MDTI In light of the complexity of most food samples the ability to check peak purity can reduce quantification errors In the most popular form of peak purity analysis several spectra acquired during peak elution are compared Normalized and overlaid these spectra can be evaluated with the naked eye or the computer can produce a comparison Figure 60 shows a peak purity analysis of antibiotics If a spectral library has been established during method development it can be used to confirm peak identity Analyte spectra can be compared with those stored in the library either inter actively or automatically after each run 93 Figure 61 shows both the quantitative and qualitative results of the analysis Part one of this primer contains several applications of UV absorbance DAD detection 10 20 30 Match 950 1 Metronidazole 7 Chloramphenicol 2 i Meticlorpindol 8 N Acetylsulfapyridine 3 Sulfapyridine 9 Ethopabate 4 Furazolidone 10 Benzothiazuron 5 Pyrazon 11 Nicarbazin 6 pronidazole Figure 61 Quantitative and qualita
29. 3 cd pyrene 1000000 500000 Dibenz a h anthracene Benzo ghi perylene T 2000 T 3000 T 4000 5000 6000 Concentration ppb Figure 1 GCMS calibration curves for 16 PAHs using 320 ym id column with low head pressure Figure 2 presents overlayed chromatograms for the replicate sample injections showing the excellent precision of the replicate injections summarized in Table 2 Abundance 3000000 2600000 TIC 1802024 D data ms 7 TIC 1801023 D data ms 2200000 TIC 1803025 D data ms 1800000 1400000 1000000 6000004 J J Lae 20000044 LN a CLE 9 60 9 80 10 00 10 20 10 40 10 60 10 80 11 00 11 20 11 40 Time Abundance 3800000 3400000 3000000 2600000 2200000 p Hesk 1800000 1400000 y TIC 1802024 D data ms 1000000 3 TIC 1801023 D data ms TIC 1803025 D data ms 600000 200000 4s oe ew A pum m 1 r 34 4 00 6 00 8 00 14 00 16 00 18 00 Time Figure 2 Overlay of three replicate injections of 2 ppm standard prior to column maintenance and relocking including a zoomed area of the four peaks from phenanthrene to pyrene A 45 cm length of column was removed from the column to simulate typical maintenance that may be performed on a column during routine use The method was then relocked and the sample re run to check the efficacy of relocking at low pressure The relocked
30. 4 diaminobutane Gradient at5min 45 B 3 methylamine 10 1 butylamine 17 1 5 diaminopentane at 30 min 45 B 4 ethylamine 11 tryptamine 18 hexylamine at 50 min 60 B 5 morpholine 12 diethylamine 19 histamine at 55 min 80 B 6 i propylamine 13 phenethylamine 20 heptylamine at 60 min 80 B 7 propylamine 14 3 methylbutylamine internal standard Stop time 60 min Post time 4 min Figure 39 Flow rate 1 ml min Analysis of amine standard with UV detection after derivatization Column compartment 60 2C Detector UV VWD 250 nm HPLC method performance Recovery rate gt 85 Limit of detection 50 150 ug l M ethod repeatability for 5 red wine analyses lt 5 Linearity 500 ug l to 20 mg l aa 21 0 Busto et al Solid phase extraction applied to the determination of biogenic amines in wines by HPLC Chromatographia 1994 38 9 10 571 578 49 Amino acids Both primary and secondary amino acids were analyzed in one run The amino acid composition of proteins can be used to determine the origin of meat products and thus to detect adulteration of foodstuffs Detection of potentially toxic amino acids is also possible through such analysis Through the use of chiral stationary phases as column material D and L forms of amino acids can be separated and quanti fied HPLC in combination with automated online derivatization is now a well accepted method for detecting amino acids owing to its short analysis time and relatively simple sample
31. 60 10 80 11 00 11 20 11 40 TIC RELOCK1 D data ms Abundance TIC 1902027 DNdata ms le 07 4 9000000 8000000 4 7000000 6000000 5000000 4000000 3000000 5 2000000 1000000 1 Time Time Figure 3 D V 16 00 18 00 Overlay of injection before and after column maintenance showing the extent of retention time variation including a zoomed area of the four peaks from phenanthrene to pyrene Abundance 1e 074 9000000 80000005 TIC 1901026 D data ms 7000000 TIC RELOCKCHECKA D data ms 6000000 5000000 4000000 3000000 2000000 1000000 k 0 SS SSS SS SS SS Soot 9 60 9 80 10 00 10 20 10 40 10 60 10 80 11 00 11 20 11 40 Time Abundance TIC 1901026 D data ms TIC RELOCKCHECKA D data ms le 07 4 9000000 8000000 7000000 6000000 5000000 4000000 5 3000000 5 2000000 nj ren A J UU L JUL ALZA E 1000000 Lae gt To cbe rr E T wy i a i ae 14 00 16 00 18 00 Time Figure 4 Overlay of injections before and after column maintenance and relocking including a zoomed area of the four peaks from phenanthrene to pyrene Conclusions For More Informa
32. 625 mL min El Apply OK Cancel Help Figure 4B lower panel Typical pressure pulsed splitless injection parameters for constant flow back injection port not used for injection but for column control Figures 5A and 5B show the constant flow mode settings for the two columns The front column flow is the typical 1 20 mL min but the back GC Edit Parameters Oven Temperature 45 2 C Agilent 180913 431 325 C 15m x 250 um x 0 25 um In Back 55 Inlet He Out Vacuum Aux Pressure 1 M2 Aux Pressure 2 M2 Aux Pressure 3 Wz PCM A 1 NZ PCM 4 2 N2 ie e EN column flow is slightly higher at 1 25 mL min to prevent any backflow Essentially the additional flow is equivalent to an extra meter of column length r Control Mode Flow Pressure Average vel Holdup Time M on 2 mL min He 45 C Oven E 4667 psi Dut Vacuum locity 38 824 cm sec 30 m 250 pm x 0 25 1 2555 min i Value mL min per walt Rate min Hold Time Run Time min min b Initial Final value will be extended by GC run time Post Run fi 2 mL min Help Figure 5A upper panel Typical pressure pulsed splitless injection parameters for constant flow First column section configured as a 30 m column Ix Ix GC Edit Parameters Oven Temperature 45 0 C Description
33. Aux Pressure 1 NZ Value Hald Time Run Time Aux Pressure 2 N2 mL min min min mL min per min Rate Aux Pressure 3 NZ PCM A 1 N2 PCM A 2 N2 b Initial Final value will be extended by GC run time Past Run fa mL min i Apply Cancel Help Figure 9C lower panel Adding backflushing in Post Run back column column 2 parameters 11 This backflow also prevents fines from the column cutting from entering the column Make the neces sary service and reattach and reload the analytical method If a completely new 15 m column 1 is installed it can be conditioned in situ by setting up the backflow condition with the oven at the condition ing column temperature Advanced Techniques Concurrent Backflushing If the fastest possible total analytical time is the highest priority one will realize that backflush can begin earlier than the elution of the last compo nent In other words backflushing can occur during the analytical acquisition thereby increas ing productivity After the last compound of inter est has passed the CFT tee and entered the back 15 m column the pressure or flow through the ear lier 15 m column can be dropped and compounds will cease moving forward and actually begin to retreat When the last compound elutes then the flow in the back column can be raised to complete backflushing This is demonstrated in Figure
34. Compound for RTLocking A specific compound usually one found in the normal method calibration standard must be chosen and then used for both developing the locking calibra tion and locking all future sys tems The compound or target peak should be easily identifi able symmetrical and should elute in the most critical part of the chromatogram Compounds that are very polar or subject to degradation should be avoided Once the target compound has been chosen and all other chro matographic parameters of the method have been determined the five calibration runs are per formed The resulting RT vs P calibration curve data are saved The software is then used to select and integrate the peak used for locking Creating an RTL Method The following is an overview of the actual steps taken to acquire the RT vs P calibration curve data selecting the compound peak to use and locking the method Acquiring the RTLock Data From Instrument Control select the Instrument menu and ZI GC MS Instrument 1 DEFAULT M Acquire RTLock Calibration Data Agilent Technologies GCs are the only ones supported See Figure 1 If you are using an automatic sampler the five sample injec tions will be made automatically This example illustrates how RTL works on a GC MS system Upon completion of the five sample analysis Data Analysis will begin and display the nomi nal MS total ion chromatogram TIC If the system
35. Figure 2 shows the locked chromatograms from a three component mixture run on GC AED and GC MS systems As can be seen the retention times are well matched between the two methods Table 1 Calculated Points for GC MS GC AED RTL Calibration The RTL Pesticide Library contains the retention times of the 567 pesti cides measured with GC FID The values measured with the FID would be the same observed with any detec tor that is operated at or near atmos pheric pressure Because retention time matching is critical in this appli cation the retention times for all the compounds in the table were also measured on the GC MS system after scaling as described here Figure 3 is a plot of the difference between the retention times measured on the GC FID and the GC MS systems The plot shows the retention times match well within 0 1 minute out to 30 minutes A few compounds at the end deviate outside this window with one compound 0 2 minute different The RTL Calibration Points from Original GC AED Method and GC MS RTL Calibration Calculated Calculated Pressure Ret Time Pressure Ret Time psi min psi min 33 1 15 346 24 27 15 346 30 4 15 919 21 18 15 919 27 6 16 578 17 934 16 578 24 8 17 338 14 654 17 338 22 1 18 242 11 449 18 242 Table 2 Comparison of Locking Pressures Calculated Using Measured and Predicted GC MS RTL Calibration Data GC MS Locking Runs Measured GC MS RTL Cal Points Using Measure
36. Quertitate Took View Help a amp iesango e muzssuNmi2p272 BERRA s il vv BOA S HEXADECANOLIDE Mille 254 EAE NISTO2 Match 98 400000 Figure 2 PCI and El spectra for Hexadeconolide A Enhanced Data Analysis FLAY_RTLM PERFMIX_E1 COMBO D MS Data Not Quantitated Die Method Chromatogram Spectrum Calibrate Quantitate Took wiew Help AOoSBes PROG BST u t BmiIa A m IRE os vfmiiiiiest uaX mbatT Hexadecanalidc El Spectrum using Cl source VM 138 492 a ag IM Sui LA Ji dk alu ll E Liu 120 140 NISTO2 spectrum 5 xj Figure 3 Acquired El spectrum compared to the NIST02 library reference spectrum The EI data in Figure 3 shows much more fragmen tation useful for compound identification The response for 255 is relatively small Using the NIST02 library the EI reference spectra for Hexadecanolide Oxacyclohelptadecan 2 one was retrieved with a 98 quality match Summary This data demonstrates the Agilent 5973 inert GC MSD s ability to acquire high quality EI spectra using the CI source The EI spectra can be searched against standard libraries for identifica tion while the CI spectra provide molecular weight information The ability to acquire both types of data without changing sources results in increased productivity For More Information For more information on our products and services visit our Web site at www agilent com chem www ag
37. RTLock view and menu choices www agilent com chem Summary RTLocking provides an easy and flexible tool that can be used to reduce the time and complexity often associated with routine chromatographic maintenance It allows methods to be trans ferred between like GC MS sys tems without time intensive edits to the quant database and reacquisition of standards It also simplifies the process for executing routine chromato graphic maintenance RTLocking can minimize mistakes and pro vide a productivity improvement for most applications by reduc ing the time and setpoint changes required to update a method For More Information For more information on our products and services visit our Web site at www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Printed in the USA May 5 2008 5989 8574EN a Agilent Technologies Low Pressure Retention Time Locking with the 7890A GC e e 8 e e e ee a o0 e Application 050 e 9 e e 9 e HPI e e Authors Courtney Milner and Russell Kinghorn BST International 41 Greenaway Street Bulleen VIC 3105 Australia Matthew S Klee Agilent Technologies Inc 2850 Centervil
38. Seal Injections 160 200 s Ay chiude Attributes Front Inlet Gold Seal age Time 54 05 10 55 90 00 0000 NN 60 Real time indicators Tests Front inlet O Ring injections Inedons o so Jm and alerts of preventive ME Front Inlet O Ring aye Time 54 10 56 21 180 00 00 00 HM 90 maintenance needs iS Rep JAA Front Inlet Split Vent Trap Injections 160 6 100 Logs amp Resuks Back Inlet Septum Injections 14 100 14 BH Tools Ii Back Inlet Liner Injections 14 500 3 Te i Back Inlet Liner age 2 00 00 00 59 t9 Maintenance Indicators Current Selection add New Maintenance Indicator Logs and Resuks Module 7090 CN10724125 Tasks z Name Instrument run counter 49 Tc monitor a resource and set a limit Description Stop Monitoring Four Alert Actions Tc config J i Setup value of Set Not Ready To view ae Warning Limit Warning Meere Set Service Due j To t wm Alert Actions C Set Not Ready Set Service Due Email e P mb Email Text Message Figure 2 LMD real time indicators and alerts of preventive maintenance needs arning limit on 7890 CN10734125 146 208 219 31 for Front Inlet Septum has been reached Message P From LMD agilent com Sent Thu 1 31 2008 12 51 PM To WANG CHUN XIAO A China ex2 ZOU YUN A China ex2 Cc Subject Warning limit on 7890 CN10734125 146 208 219 31 for Front Inlet Septum has been reached Warning limit on
39. Three dimensions of data chromatic light from the deuterium and tungsten lamps into the flow cell The light then disperses on the surface of a dif fraction grating and falls on the photodiode array The range varies from instrument to instrument The detector shown here is used to measure wavelengths from 190 to 950 nm using the twin lamp design In our example the array consists of 1024 diodes each of which measures a different narrow band spectrum Measur ing the variation in light intensity over the entire wavelength range yields an absorption spectrum The bandwidth of light detected by a diode depends on the width of the entrance slit In our example this width can be pro grammed to selected values from 1 to 16 nm If very high sensitivity is required the slit is opened to 16 nm for maxi mum light throughput If maximum spectral resolution is needed the slit is narrowed to 1 nm At this setting the fine structure of benzene can be detected even at 0 7 mAU full scale mAUFS see figure 57 Because the relative posi tions of the sample and the diffraction grating are reversed compared with a conventional instrument this configura tion is often referred to as reversed optics The most signifi cant differences between a conventional UV absorbance detector and a DAD are listed at left DADs connected to appropriate data evaluation units help optimize wavelengths for different compounds over the course of the run Maxima can be
40. acetonitrile 0 min 33 B 8 min 60 B 9 min 33 B 250 ul min 5 yl 40 C API ES positive or APCI negative 30 psig 350 C 6 l min 4000 volts 100 volts m z 120 820 Tt oun R 200000 8 8 m R Imne 100000 Ale g 0 CO SE I ei dull contr cu DI TET E te Mo CC 200 300 400 500 600 70 mz n 200000 WS T FB3 g 1091 a Bio oe 5 R Ag R Eg TETTE JE 1 i La ic Doni 1E dal ina 200 300 400 500 600 700 m z u 250000 FB2 8 150000 M n E Bun o n R g R 50000 8 3 B Ger GRUSS RSS E b eeke aa TE we ell dete come CLE sh levee is lI I 200 300 400 500 600 700 m z Figure 13 Mass spectra of Fumonisins B 1 2 3 when the fragmentor is ramped from 230 to 100V 100000 B FB MS EIC m z 723 m 60000 4 20000 250000 R i FB MS EIC m z 335 m 150000 50000 FB3 BF 300000 1 200000 MS EIC m z 707 r N 100000 4 A MS EIC m z 337 100000 m z a e ri um TEN r A E 1 2 3 4 2 6 7 8 Time min Figure 14 Identification of different Fumonisin species in corn extract by retention time with further confirmation through fragment ion 20 Mycotoxins The following mycotoxins have been analyzed aflatoxins G Gp B B M and M ochratoxin A zearalenone and patuline Mycotoxins are highly toxic compounds produced by fungi They can contaminate food products when storage conditions are favorable to fungal growth These toxins are of rel
41. application For trace level analysis the single tapered deactivated liner is recommended The Viton O ring 4 holding the liner in place should be replaced periodically to reduce the chance of leaking The seal and the washer in the bottom of the injection port 10 11 should be replaced whenever the reducing nut 12 is removed The recommended seal is gold plated to reduce metal catalyzed thermal degradation of analytes Septa 2 should be replaced quite fre quently for example every 100 injections The low bleed precored red septa should be used Keeping a beaker of septa in an oven at 250 C at all times will eliminate the need to condition the septum once it is in place A Merlin Microseal is highly recommended over a conventional septum nut and septum 1 2 The Microseal eliminates the need for septa and lasts for tens of thousands of injec tions without leaking It is most appropriate with the Automatic Liquid Sampler ALS injection tower and only works with untapered blunt tip syringe needles The Microseal has been improved this past year Older seals have a maximum pres sure rating of 30 psi The new seals are rated to 100 psi The new seal and nut are recommended The gold nuts stamped 303C are not compatible with the new seals A gray nut stamped 221B should be installed 1 Septum retainer nut e 2 Septum 3 Insert assembly 4 O ring 6 Split vent line 5 L
42. aromatics in river water A different online solid phase extraction system has been used to extract and analyze iso a acids in beer 40 Uses small amounts of organic solvents Differing batch to batch efficiencies can can run several samples at once and can reduce reproducibility Risk of irreversible be automated adsorption Degradation by surface catalysis can occur Also known as size exclusion chromatography gel perme ation chromatography GPC has become a standard tech nique for isolating compounds of low molecular weight from samples that contain compounds of high molecular weight such as oil or fats The separation is based on differ ences in size with higher molecular weight compounds retained less than smaller ones GPC has been used success fully to separate vitamins A D and E from glycerides in infant formula and clean up of pesticides in spices see chapter 2 page 22 ff Highly reproducible good automation Large amounts of solvents needed possibilities separation efficiency may differ from batch to batch 66 In brief Guard columns A guard column is connected in front of the analytical column to prevent contamination of the analytical column by the matrix Either the guard column can be included in analytical column design or both columns can be interconnected by a valve that when switched transfers fractions from the precolumn to the analytical column The latter technique is more flexibl
43. be used with multiple injections of more than 5 uL In addition to preventing solvent from flowing into the column glass wool or other packing provides a surface to retain a film of solvent which in turn helps retain early eluting com pounds Figure 2A shows loss of ana lytes eluting before C18 Figure 2B with glass wool packing in the liner shows complete recovery down to C14 Area Count 12 000 10 000 Response Factor RSD 8 000 023 2 16 C22 2 6 6 000 4 000 A Pd 0 Packed Liner 150 200 250 Total Injection Volume uL Figure 1 Linearity of multiple injections pA Inlet 40 C at injection 2500 A Open baffle liner 50 mL min purge for 2 5 min 30 m x 0 32 mm x 0 25 um HP 5 2000 C18 1500 C16 Septum extract 1000 C14 vial pierced 40 times 500 7 C12 C10 EERI 04 T T T T T T T T 0 2 5 5 75 10 12 5 15 17 5 20 22 5 Minutes pA B C14 6000 C16 i C18 Glass wool packed liner 5000 10 x 25 uL injections 4000 3000 2000 C12 1000 5 C10 9 i T T T T T T T T 0 2 5 5 75 10 12 5 15 17 5 20 22 5 Minutes Figure 2 Comparison of packed and open baffle liners using PTV sample C10 C44 in Hexane PTV Figure 3 is a chromatogram of an LVI of pesticides using a PTV inlet By injecting 25 uL divided into five injec tions good response is obtained from a 0 01 ppm mixture COC SVE is usually the preferred technique for samples with low bo
44. calibration data is entered into the RTL calibration dialog box the target time for methyl chlorpyri fos is entered as 5 532 minutes which is 16 596 minutes divided by 3 it GC Method Translation Table 4 compares the locking pres sures determined with measured and with calculated RTL calibration points As in the above GC MS exam ple the range of the locking pressures from the calculated data is only 0 11 psi 87 88 to 87 99 which corresponds to 0 003 minute Figure 5 compares the chro matograms of the RTL locking mix ture from both the original and the 3x scaled methods Note that while the chromatographic resolution is reduced the speed is increased by a factor of 3 Figure 6 shows a plot of the differ ence between the RTL Pesticide Library retention times divided by 3 and those of the 3x method The data were taken with a 36 component subset of the library The plot shows the retention times match well within 0 05 minute for all compounds even Criterion Translate Only Best Efficiency Fast Analysis None Speed qain 3 00000 Original Method Translated Method 30 Internal Diameter um 250 250 Film Unlock Thickness um 25 25 Phase Ratio 250 0 250 0 p Carrier Gas Helium lint Enter one Setpoint Ambient Pressure absolute psi Oven Temperature 3 ramp Program H
45. calorie sweeteners are widely used in foods and soft drinks Investigations of the toxicity of these compounds have raised questions as to whether they are safe to consume As a result their concentration in foods and beverages is regulated through legislation in order to prevent excessive intake Sample preparation Sample preparation depends strongly on the matrix to be analyzed For sample low in fat liquid extraction at low pH with ultrasonic bath stimulation can be used For samples with more complex matrices solid phase extraction liquid liquid extraction or steam distillation may be necessary Fluores cence detector Control and I data evaluation Quaternary i pump Auto Column Diode AT vacuum 77 sampler compart P degasser ment etector ps4 Water Methanol Derivatization agent Column Mobile phase Gradient Flow rate Post time Column compartment Injection volume o phthalaldehyde OPA mercapto propionic acid M PA 100 x 2 1 mm Hypersil ODS 5 um A 20 01 mM sodium acetate B 2 methanol start with 5 B at5 min 25 B at 10 min 35 B at 13 min 55 B at 18 min 80 B at 20 min 95 B 0 35 ml min 5 min 40 C Tul Injector program for online derivatization 1 Draw 5 0 ul from vial 3 borate buffer 2 Draw 0 0 ul from vial 0 water 3 Draw 1 0 ul from vial 1 OPA M PA 4 Draw 0 0 ul from vial 0 water 5 Draw 1 0 yl fro
46. does show a higher S N ratio for these later PCBs As opposed to the full scan acquisition the SIM mode acquisi tion at higher source temperature does increase signal for the ions of interest and because there was no increase in background a useful S N increase was obtained As always the guiding principle that an increase in signal is only useful if 120 110 a xy c co c ec oc oo o o o Abundance l i i li i i i a ll ll ll t 1 1 i i i 1 li ll li I 1 1 li li i 1 1 t li i i i N tl 1 1 1 1 1 1 1 1 I 1 1 1 1 1 4 it exceeds the concomitant increase in background holds This is clearly illustrated by the third PCB the pentachlorobiphenyl Cl B Figure 5 shows the behavior of the signal and background for the two source temperatures for one of the pen tachlorobiphenyl confirming ions The higher source temperature raises the signal and the back ground for this ion of interest over the lower tem perature but fortunately signal increases faster than background In this case the background is due to column bleed components and is unavoid able but fortunately not very intense This may or may not be the case in sample analysis 300 C 230 C 0 d EEEE S EEEE HI ERE 5 90 6 00 6 10 6 20 6 30 6 40 6 50 6 60 Figure 4 6 70 6 80 6 90 7 00 7 10 7 20 7 30 7 40 7 50 Time Overlaid RTICC of six
47. earlier This application details some configurations and provides an example of backflushing Split splitless Pressure flow injection port controller vent 7890A GC A Z mL min E LEES 15 m HP 5 ms 0 25 mm id x 0 25 um mL min CFT Device Z mL min 5975C MSD El mode 15 m HP 5 ms 0 25 mm id x 0 25 um Figure 2 Schematic of pressure controlled tee arrangement for the GC MSD solid lines indicate the forward flow during GC MSD analysis and the dashed lines indicate backflushing flows Experimental A number of devices can be used in this approach and those arrangements will be cited later but for these experiments the instrument configuration was as follows e 7890A GC with split splitless ports in front and back and a 7683B ALS 5975C MSD with performance turbomolecular pump 2 HP 5ms 15 m x 0 25 mm id x 0 25 um film columns 19091S 431 CFT device 2 way unpurged splitter G3181 60500 with SilTite ferrules and nuts CFT GC mounting hardware dual wide mount ing bracket G2855 00140 or single wide mounting bracket kit 62855 00120 Deactivated 0 25 mm id column approximately 1mlong e 2 CFT blanking plugs G2855 60570 or as G2855 20550 with G2855 20593 As an overview of the configuration the 1 m column was connected to the back injection port and to the first position on the CFT splitter using the appropriate SilTite fittings This CFT device has three connect
48. ethopabat 16 ethylamine 48 excitation 96 97 excitation grating 95 extinction coefficients 87 extraction liquid liquid 65 solid phase 63 65 supercritical fluid 64 F fats 35 37 38 fatty acids 35 38 60 108 fertilizers III figs 21 22 fish 48 flavors Ill 12 flour 21 30 flow precision 79 ranges 76 rates 76 fluorescence detection 109 fluorescence detector 87 95 105 fluorescent tag 109 folic acid 43 folpet 27 Food and Drug Administration FDA V food colors 10 fragmentation 19 fructose 40 fruit juices 6 fruits 28 fumaric acid 2 fumonisins 19 fungi 15 21 furazolidone 15 G galactose 40 gel permeation chromatography GPC 27 66 glassy carbon 99 GLP GM P principles 114 glucose 40 glycerol 3 glyphosate 26 29 gold 99 good laboratory practice GLP 67 gradient elution 76 formation 77 high pressure 80 low pressure 78 guard column 59 67 H halogens 99 hesperidin 12 14 hexylamine 48 histamine 48 hormones III humulon 12 hydrazine 99 hydrogen peroxide 99 hydrolysis 38 hydroperoxides 35 36 131 indirect UV detection 33 injection volumes 70 injector automated 72 manual 71 program 110 inorganic anions 32 inorganic ions V instrument calibration 114 logbooks 114 parameters 115 integration events 115 parameters 113 integrators 112 113 interface M S moving belt 102 particle beam 102 thermospr
49. identical to that of a ref erence method In this way the speed of an analysis can be scaled pre dictably to accommodate the needs of a specific sample or instrument type The inlet pressure calculated for the new version of a method by the method translation software is based on the assumed or nominal dimen sions of the column As such the cal culated inlet pressure will provide a close but not exact match to the desired scaled retention times To match precisely the retention times of the scaled method to the desired scale factor the new method must be retention time locked Retention time locking RTL is a technique devel oped by Agilent Technologies whereby the inlet pressure required to match retention times precisely is calculated from a calibration curve of inlet pressure versus retention time Using method translation followed by RTL allows a method to be scaled by a precisely known factor Once the chromatography has been scaled a retention time table such as the RTL Pesticide Library can then be scaled by the same factor resulting in a new library whose retention times match those of the scaled method precisely The steps required to scale the method are l Determine the desired scale factor for the new method 2 Usethe method translation soft ware to calculate the inlet pres sure and oven temperature adjustments to obtain the desired scaling of the method The scale factor is the speed gain
50. in presweetened cereals AOAC Official M ethod 977 20 Separation of sugars in honey AOAC Official M ethod 979 23 Saccharides major in corn syrup AOAC Official M ethod 983 22 Saccharides minor in corn syrup AOAC Official M ethod 984 14 Sugars in licorice extracts n Vitamins Water soluble vitamins Fat soluble vitamins such as vitamins E D and A and water soluble vitamins such as vitamins C Bg B B and B have been analyzed Vitamins are biologically active compounds that act as controlling agents for an organism s normal health and growth The level of vitamins in food may be as low as a few micrograms per 100 g Vitamins often are accompanied by an excess of compounds with similar chemical properties Thus not only quantification but also identification is mandatory for the detection of vitamins in food Vitamins generally are labile compounds that should not exposed to high temperatures light or oxygen HPLC separates and detects these compounds at room temperature and blocks oxygen and light Through the use of spectral information UV visible diode array detection yields qualitative as well as quantitative data Another highly sensitive and selective HPLC method for detecting vitamins is electrochemical detection Sample preparation Different food matrices require different extraction procedures For simple matrices such as vitamin tablets water soluble vitamins can be extracted with water in an ul
51. in a water aspirator vacuum setup The use of chlorinated solvents is not recommended due to Two Stage On Off Gas regulator valve purifier m Of Main supply F 1 a on off valve Main gas supply Figure 1 General gas plumbing assembly The MS gas purifier must be installed diagonally or vertically a Agilent Technologies possible long term contamination of flow lines and controllers Another commonly used cleaning tech nique is to heat the copper tubing with a Bunsen burner propane torch or heat gun while helium is flowing through the tubing This is done after con necting the tubing to the helium supply but before connecting it to the GC This process bakes off all the volatile contaminants Take proper safety pre cautions while heating the tubing Laboratory manifold systems especially when new tend to have hydrocarbon contaminants Purging the new lines before connecting the clean tubing to analyti cal instruments is essential The supplies needed for the carrier gas line are 299 999 He Gas supplier Clean copper tubing 50 ft p n 5180 4196 Mass Spectrometer gas purifier p n RMSH 2 e Bracket to mount the gas purifier p n UMC 5 2 Splitless Inlet Consumables The capillary inlet Figure 2 has many consum able parts that should be kept on hand Many of these consumables such as liners 5 come in a variety of designs Appendix A The proper liner to use is dependent on the
52. injection port reaches the setpoint temperature retighten Linear velocity Column flow mL min 1 0 1 0 1 0 1 0 2 0 1 5 the column nut in case it loosened Check once again for column flow Remove the end of the column from the beaker and close the oven door Condition the column by slowly 5 C min ramp ing it to its maximum operating temperature Leave it at that temperature for least 2 hours overnight is preferable The maximum operating temperature for an HP 5MS column is 325 C Cool the oven to ambient and insert the interface nut and ferrule onto the column Properly cut off 5 10 cm of the column Properly place the column into the interface by following the directions in the MS hardware manual for the Agilent 5973 and for the Agilent 6890 series MSD Hand tighten the interface nut and then wrench tighten the nut The nut should be tightened only until you hear two squeaks This is a firm seal Pump down the detec tor as directed by the MSD manual for the Agilent 5973 and for the Agilent 6890 Series MSD Keep the oven at ambient temperature until the source is hot Check the interface connection after the interface is heated The interface nut may need additional tightening Tips for Better Method Performance Numerous splitless parameters need to be opti mized for the best splitless injection Those parameters include Injection port temperature Column flow Liner design Solvent Sample v
53. is recorded by a mass spectrometer Its appeal in HPLC however is limited owing to the cost of interfacing the mass spectrometer equipment If the spectra of the analytes differ markedly UV absorbance spectra can be used for identification using diode array technology Fluorescence and electrochemical detectors can be used only to identify compounds based on their retention times UV detectors Figure 55 shows the optical path of a conventional variable wavelength detector Polychromatic light from a deuterium lamp is focused onto the entrance slit of a monochromator using spherical and planar mirrors The monochromator selectively transmits a narrow band of light to the exit slit The light beam from the exit slit passes through the flow cell and is partially absorbed by the solution in the flow cell The absorbance of the sample is determined by measuring the intensity of the light reaching the photodiode without the sample a blank reference and comparing it with the intensity of light reaching the detector after passing through the sample Cut off filter Deuterium lamp Holmium oxide filter Mirror 1 Sample a Flow cell Mirror 2 Figure 55 Conventional variable wavelength detector Most variable wavelength detectors split off part of the light to a second photodiode on the reference side The reference beam and the reference photodiode are used to compensate for light fluctuations from the lamp For optimum sensiti
54. material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Printed in the USA April 8 2008 5989 8366EN Agilent Technologies Authors B D Quimby L M Blumberg M S Klee and P L Wylie Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 1610 USA Abstract Complete development of a gas chro matographic method often involves a significant amount of effort Once a method is completed retention time locking RTL can be used to implement the method and to obtain the same retention times on multiple systems This application note describes how to use method translation combined with RTL to implement precise time scaled versions of a method on multiple instru ment types This allows the original method to be re used with minimal effort while optimizing the method for a given sample type or instrument setup In this way the utility of the original method is extended greatly increasing the payback on the invest ment in its development and optimizing its use for specific analyses In this note the Agilent RTL Pesticide Library method is used as an example The steps involved in precise time scaling of the method to different speeds detec tors and columns are presented Precise Time Scaling of Gas Chromatographic Methods Using Method Translation and Retention Time Locking Application Gas Ch
55. method had a resulting pressure of 3 168 psig The change in locking pressure can over time provide guidance as to the extent at which column trimming can be undertaken with the need for full re locking of the method Abundance 1e 07 4 9000000 4 8000000 7000000 6000000 5000000 4000000 3000000 4 2000000 1000000 Figure 3 shows an overlay of the before and after column trimming and the extent of the retention time change Figure 4 presents an overlay of chro matograms one of the originals and one after column trimming and relocking The last two columns of Table 2 compare the relocked retention times of target compounds to the originals TIC RELOCK1 D data ms T1C 1802027 DNdata ms TIC RELOCK1 D data ms Abundance TIC 1902027 DNdata ms le 07 9000000 8000000 4 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 ee 0 Lr EIER Te 9 40 9 60 9 80 10 00 10 20 10 40 10 60 10 80 11 00 11 20 11 40 Time Time Figure 3 NU mi 16 00 18 00 Overlay of injection before and after column maintenance showing the extent of retention time variation including a zoomed area of the four peaks from phenanthrene to pyrene Abundance
56. multiple HPLC instruments A client server networked data system helps consolidate documentation and validation processes for multiple techniques and instruments from multiple vendors 118 Chapter 11 Factors that determine performance in HPLC 7 A Ll ad The analysis of food samples places high demands on HPLC equipment notably in the areas of performance stability and reliability Modern evaluation software enables you to determine the suitability of a particular piece of HPLC equipment for analysis The factors that influence the outcome of a measurement thus can be identified before results are published to confirm assumptions made during analysis or to draw attention to erroneous data In this chapter we focus on those instrument related parameters that strongly influence the limit of detection LOD and the limit of quantification LOQ We also discuss the accuracy precision and qualitative information that an HPLC system can provide Some vendors address the performance of specific instrumentation in technical notes Such notes include detailed performance test procedures and results for individual modules as well as for complete HPLC systems 120 Limit of detection and limit of quantification The principle determinant of the LOD in HPLC is the response of the detector to the compound of interest The response factor thus depends primarily on the choice of detection technique However regardless o
57. normalization and external and internal standard calcula tions are basic features of almost all modern integrators Annotated reports list amounts retention times calculation type peak areas or heights and integration parameters as well as the date and time of measurement Advanced fea tures may provide for automated drawing of the baselines during postrun replotting and for the plotting of calibration curves showing detector response For unattended analyses in which several runs are performed in series integrators normally are equipped with a remote control connected to the autosampler in the system Most models can also store raw data for replotting or reintegration at a later date Some instruments have computer programming capabilities and can perform more advanced customized statistical calcula tions using the BASIC programming language for example Multichannel integrators are available for some analytical methods requiring two or more detection signals Inexpensive Facilitates reporting of No instrument control or report retention times quantitative results and customization automatic baseline resets 113 Personal computers In recent years personal computers PCs have become increasingly popular as data analysis tools in analytical lab oratories PCs offer more flexibility and better data storage capabilities than traditional storage methods Moreover on line functions such as word processing spreadsheet ana
58. of the most popular derivatization techniques The reagent should be selected such that the absorption maximum of the reaction product exhibits not only improved sensitivity but also good selectivity This combi nation reduces matrix effects resulting from the reagent from by products or from the original matrix The following table lists common compounds and reactions In part one of this primer we give examples of compound derivatization including that of fatty acids and amino acids Target compound Reagent Alcohols OH phenylisocyanate 250 nm Oxidizable sulfur S0 2 2 dithiobis 5 nitro pyridine 320nm compounds Fatty acids COOH p bromophenacyl bromide 258 nm 2 naphthacyl bromide 250nm Aldehydes and CO COOH 2 4 dinitrophenyl hydrazine 365 nm ketones C 0 and CHO Primary amines NH o phthalaldehyde OPA 340 nm Primary and NHR 9 fluorenylmethyl chloroformate 256 nm secondary amines FM OC 108 Addition of a fluorescent tag Precolumn or postcolumn Pickering system Water Methanol Quaternary pump Auto Column Fluorescence vacuum sampler compart detector degasser ment LJ Control and Ss data evaluation oh Figure 75 Pickering postcolumn derivatization equipment for the analysis of carbamates Fluorescence is a highly sensitive and selective detection technique Adding fluorescent properties to the molecule of interest is of particular benefit in food analysis in which compone
59. other parameters that you specify Two case studies are given demonstrating some of the features and benefits of this software Introduction Agilent Lab Monitor amp Diagnostic LMD software is an innovative tool to help you manage your lab to ensure performance productivity and reliabil ity LMD software can monitor in real time a single Agilent GC or LC or all the Agilent GCs and LCs in your lab It automatically tracks supply usage monitors chromatographic quality and notifies you of maintenance needs before a problem occurs by keeping track of injections hours of operation and other parameters that you specify LMD software knows when it s time to replace consumables or perform basic upkeep It provides full diagnostic Diagnostic Software HPI Environmental Food Safety capabilities with an extended list of tests and cali bration procedures and automates basic diagnostic routines that help verify proper instrument perfor mance The software is coupled with an extensive suite of user information Help functions that pro vide quick easy access to maintenance informa tion such as manuals and videos so that you can get the information you need right when you need it LMD provides the following features and benefits ncreases your lab s uptime by alerting you to problems before they happen Provides intuitive help with diagnostic capabili ties and easy to follow repair procedures in case of a problem
60. preparation Sample preparation Hydrolyzation with HCl or enzymatic hydrolysis is used to break protein bonds Chromatographic conditions The HPLC method presented here was used in the analysis of secondary and primary amino acids in beer with precolumn derivatization and fluorescence detection 2 3 Fluores cence detector Control and data evaluation I Quaternary pump Auto Column Diode vacuum 77 sampler compart array degasser ment detector Water Acetonitrile 50 Sample preparation filtration Column 200 x 2 1 mm Hypersil ODS 5 um Mobile phase A 0 03M sodium acetate pH 27 2 4 0 596 THF B 0 1M sodium acetate ACN 1 4 Gradient at 0 min 0 B at 0 45 ml min flow rate at 9 min 30 96 B at 11 min 50 B at 0 8 ml min flow rate at 13 min 50 B at 14 min 100 96 B at 0 45 ml min flow rate at 14 1 min at 0 45 ml min flow rate at 142 min at 0 8 ml min flow rate at 17 9 min at 0 8 ml min flow rate at 18 0 min at 0 45ml min flow rate at 18 min 100 B at 19 min 0 96 B Post time 4 min Flow rate 0 45 ml min Column compartment 40 2C Injection volume 1 ul standard Detector UV DAD Fluorescence Excitation wavelength 230 nm Emission wavelength 450nm at 11 5 min Excitation wavelength 266 nm Emission wavelength 310 nm Photomultiplier gain 12 Response time 4s 338 nm and 266nm HPLC method performance Limit of detection DAD lt 5 pmol FLD lt 100 fmol Repeatability of RT
61. pressures calculated using mea sured and predicted 3x 0 1 mm id micro ECD calibration data The range of locking pressures from the measured data 66 03 to 65 93 only corresponds to a spread in retention times of about 0 004 minute How ever with the data calculated based on a 10 m assumed length the spread 66 38 to 63 18 is much larger and would correspond to a time range of 0 14 minute The locking pressures calculated using the 10 5622 value are much more consistent with the mea sured values The range in retention times would be 0 03 minute if all the calculated points are used and if the first value in column 5 is ignored the range drops to 0 005 minute The fact that the agreement in locking pressures is much improved by using 10 56 m instead of 10 m suggests that length is probably the largest contrib utor to the discrepancy These results should reinforce the recommendation that if a method is to be used exten sively it is prudent to obtain mea sured RTL calibration data It should be noted however that even with the RTL calibration from the 10 m assumed length the worst conse quence would be that the RT locking step would need to be repeated an extra time to get a more precise match Figure 8 compares the chromato grams of the RTL locking mixture from both the original and the 3 x 0 1 mm id micro ECD methods Table 5 RTL Calibration Points from Original GC AED Method
62. rate Post time Column compartment Injection volume Detector Carrez clearing and filtration for the salad dressing None for white wine 125 x 4 mm Hypersil BDS 5 ym A water 0 2 ml HS0 pH 2 3 B ACN start with 10 B at 3 min 60 B at 4 min 80 B at 6 min 90 B at 7 min 10 B 2 ml min lmin 40 C 2 yl UV DAD detection wavelength 260 40 nm HPLC method performance Limit of detection Repeatability of RT over 10 runs areas over 10 runs 10 ppm S N 2 0 1 96 396 Chromatographic conditions HPLC and UV visible diode array detection have been applied in the analysis of preservatives in white wine and salad dressing Spectral information and retention times were used for identification mAU 601 507 40 1 Absorbance scaled A Spectral library Sorbic acid PHB rrethyl 200 Wavelength nm 320 PHB propyl BHA m Standard Berzoic acid White wine 104 Salad dressing 0 4 iL 2 3 4 Time min Figure 5 Analysis of preservatives in white wine and salad dressing Co 4 Official M ethods of Analysis Food Compositions Additives Natural Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 AOAC Official M ethod 979 08 Benzoate caffeine saccharine in carbonated beverages Artificial sweeteners The following compounds are used as artificial sweeteners in food products acesulfam aspartame saccharin Nowadays low
63. requirements and on the degree of automation required Depending on individual requirements increasingly complex techniques are available to evaluate chromatographic data at the simplest level are strip chart recorders followed by integrators personal computer based software packages and finally the more advanced networked data systems commonly referred to as NDS Although official methods published by the U S Environmental Protection Agency EPA and by Germany s Deutsche Industrienorm DIN provide detailed information about calculation procedures and results they give no recommendations for equipment Strip chart recorders traditionally have been used in connec tion with instruments that record values over a period of time The recorder traces the measurement response on scaled paper to yield a rudimentary result In the age of electronic data transfer such physical records have been largely sur passed by data handling equipment preprogrammed to make decisions for example to reject peaks that lie outside a cer tain time window Inexpensive No record of retention times no quantitative results on line no automatic baseline reset between runs no electronic storage 112 Integrators Integrators offer several advantages over strip chart record ers and consequently are becoming the minimum standard for data evaluation Integrators provide a full scale chro matographic plot and multiple report formats Area percent
64. seen easily using three dimensional plots of data or as absorbance intensity plotted over time at different wavelengths that is as an isoabsorbance plot see figure 58 Figure 55 illustrates the optimization result for antibiotics The ability to acquire and store spectra permits the creation of electronic spectral libraries which can be used to identify sample compounds during method development 91 Figure 58 Isoabsorbance plot Figure 59 Multisignal detection of antibiotic drugs Multisignal detection yields optimum sensitivity over a wide spectral range However the spectral axis in figure 58 shows that no single wavelength can detect all antibiotics at highest sensitivity mAU 10 meticlorpindolmetronidazol nicarbazine 100 100 4 v E bi 80 4 0 260 300 340 380 60 1 Wavelength nm 275nm 5 40 6 11 7 315nm 8 20 9 10 360nm 11 0 T T T 1 10 20 Time min 30 40 92 Metronidazol Meticlorpinol Sulfapyridine Furazolidon Pyrazon Ipronidazol Chloramphenicol N Acetylsufapyridine Ethopabat Benzothiazuron Nicarbazin Figure 60 Peak purity analysis Purity Evaluation of Peak 8 at 6 83 min C NUC MDTP D E 3 8 Fy Signal DAD1 B Sig 270 40 Ref 450 80 NUCI 5 p 9 g Peaks 13 Date 27 May 99 16 25 06 UELI LJ Li JL A L W Peak Spectra Peak Signals Exit Help Next J Prev 210 0 nm 4020 6
65. system suitability 116 T table salt 34 tartaric acid 2 TBHQ mono tert butylhydroquinone 4 TDPA 3 3 thiodipropionic acid 4 tetracyclines 18 tetrahydrofurane 35 THBP 2 4 5 trihydroxybutyrophenone 4 thermal energy detector 86 thermal stability IV thin layer chromatography TLC 21 thiofanox 28 thiofanox sulfone 28 thiofanox sulfoxide 28 thiosulfate 99 tocopherols 4 45 47 tocotrienols 46 tolerance levels III total ion chromatography 53 54 toxicity 8 toxins 19 tracer 80 121 trend charts 114 triazines 26 triglycerides 35 39 trypsin 53 tryptamine 48 tungsten lamp 10 91 133 U ultrasonic bath liquid extraction 63 UV absorbance 86 UV detector 89 90 V validation processes 113 vanillin 12 13 variable volumes 70 variable wavelength detector 89 90 vegetables 26 28 vinclozolin 27 viruses 15 viscous samples 60 vitamins V 4 35 42 44 66 drink 43 fat soluble 42 46 natural III standard 46 synthetic III tablets 42 43 water soluble 42 43 vodka 2 W wavelength switching 96 wine 2 7 48 wool fiber method 10 working electrode 98 X xenon flash lamp 95 Z zearalenone 21 22 zinc sulfate 14 134 Authors Vince Giarrocco Bruce Quimby Matthew Klee Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 1610 USA Abstract The concepts and applications of reten tion time locking RTL are described RTL simp
66. the septum when the limit has been reached before the GC baseline goes bad After the septum is changed the expected baseline is obtained Figure 1 shows the baseline of the polar column after 400 runs top and after the septum has been changed bottom LMD can track not only the inlet septum but also other GC resources like the inlet o ring oxygen traps inlet gold seal and so on As Figure 2 shows LMD gives real time indicators and alerts of pre ventive maintenance needs pA After 400 runs the baseline polar column is getting worse because 10 4 of the septum leakage which may impact quantitative analysis especially for trace level analysis 10 3 l 10 2 10 1 10 0 0 2 3 6 8 10 12 14 min pA 6 2 Get the expected baseline after replacing the inlet septum based on LMD advice 0 2 B 6 8 Figure 1 been replaced 10 12 14 min Baseline chromatograms on polar column after 400 runs and after the inlet septum has A E DATASYSTEMODI Administrator Lab Monitor S E A yip Monitor zal Airani T x 78590 CN10734125 146 268 219 31 AA Lab at a Glance amp Diagnostic Software G Leb Montor Management Maintenance Indicators 4 Instrument Documentation Resource Unit value Progress Instrument R Front Inlet Septum Infections 45 300 E Jm Front Inlet Liner Injections 193 400 ES 48 Front Inlet Liner aga 54 10 56 21 1680 00 00 00 Wr Q System Information E Calender Ji Front Inlet Gold
67. transfer optimized both improving sensitivity A final note the choice of vials and septa will affect your results Screw cap vials are not recom mended for GC MS analyses Application note Effects of Vial Septa Used in GC ECD Analysis of Trace Organics Agilent Technologies publication 5091 8980E www agilent com chem will help you make the right choice References 1 Analyzing aromatics in reformulated gasoline by GC MS Agilent Technologies publication 23 5964 0116E 2 Philip L Wylie et al Improving the Analysis of Pesticides by Optimizing Splitless Injections 1995 Pittsburgh Conference paper number 347 3 Philip L Wylie and Katsura Uchiyama Improved Gas Chromatogaphic Analysis of Organophoshorous Pesticides with Pulsed Splitless Injection 1996 JAOAC 79 571 577 Appendix A Capillary Inlet System Liners Applications Linear seals Fast Slow amp injection manual Fluoro Configuration 7673 ALS injection carbon Graphite Glass ID wool Split Split Max 350 C Liner Part no Price volume Glass type Deactivated packing Split less Split less 350 C and above Single Taper liner 5062 3587 34 4mm 5180 4182 5180 4173 0 8 mm end Borosilicate YES YES e Q 5 12 pk 12 pk 900 L 12 48 5181 3316 4mm 0 8 mm end 900 L Single Taper liner Borosilicate 5181 3315 4mm 0 8 mm end 800 L Double Taper liner Borosilicate 19251 60540
68. was config ured as a stand alone GC all five chromatograms will be dis played From these displays you will select the compound that will be used for RTLocking of the method Selecting the RTLock Compound Use the mouse to select the com pound or peak that you would like used for locking For GC only mode you must select one peak from each of the five chro matograms for RTLocking Once you have made your selection you will be asked to allow the software to automatically find the remaining peaks You may choose to zoom the display for better visibility See Figure 2 Method METE view Abort Window Qualify Help EET Inlet Injection Types g gt GC Edit Parameters 7 gCPht E GC Monitors Run Time Detectors Select MS Tune File ES MS SIM Scan MS Monitors inje Edit MS Tune Parameters Perform MS Autotune RR Figure 1 GC Status Messages Total lon Volts zai Temperature em Flow Cal end From instrument control you select the mode of data acquisition for RTL E Enhanced RTL setup RTLPESTB M is LOCKED COL 12 TIC RTLOCK3 D 7T T7 144155 168180 197 212 228 243 258 271 50 100 150 200 250 300 Click Drag RIGHT Mouse under desired Peak for Lock Calculations Figure 2 From this panel
69. with 3 double bonds 10 ng Repeatability of RT over 10 runs areas over 10 runs gt 10 ug lt 0 7 lt 6 36 1405 A j g SA Hydroperoxides 7 204 0 fe E 8 0 8 215 nm 240 nm 20 25 0 1 Time min Figure 25 Triglyceride pattern of aged sunflower oil The increased response at 240 nm indicates hydroperoxides Olive oil muy Sigh 8 mau Sj 207 207 Good quality Poor quality 154 g 15408 104 104 g 54 d 215nm 54 JU Ue 0 730nm 0 280 nm 13 0 Time min 23 0 130 Time min 23 0 Figure 26 Analysis of olive oil The response at 280 nm indicates a conjugated double bond and therefore poor oil quality Triglycerides in olive oil Unsaturated triglycerides in olive oil have very characteris tic patterns Other fats and oils are also complex mixtures of triglycerides but with different patterns Sample preparation information Triglycerides can be extracted from homogenized samples with petrol ether Fats and oils can be dissolved in tetrahydrofurane Chromatographic conditions The presented HPLC method was used to analyze the unsaturated triglycerides LnLnLn LLL and OOO Sample preparation Samples were dissolved m in tetrahydrofurane Column 200 x 2 1 mm 1807 Hypersil MOS 5 um 1607 Mobile phase acetone ACN 30 70 Flow rate 0 5 ml min 1401 S 8 Column compartment 30 C 1207 E Injection volume 2
70. with a combi nation of other selective detectors like the electron capture detector ECD the nitrogen phosphorus detector NPD the flame photomet ric detector FPD or the electrolytic conductivity detector ELCD The GC AED technique can also be used to calculate element ratios and to quantitate unknown peaks that are detected because of its equimolar ele ment response factors The measured element ratios can be used to further distinguish between possible identi ties of detected heteroatomic com pounds often resulting in a single entry as the likely identity of a given peak With compound independent calibration the amount of the unknown can be calculated using ele ment response factors generated with a different standard compound Agilent Technologies Innovating the HP Way Once the element selective screen is completed samples that contain any suspect compounds are run on a GC with mass spectral detection GC MS system that is retention time locked to the pesticide method thus having the same retention times as the element selective detectors Using the possible identities generated from the element screen the GC MS data is evaluated to decide which if any of the possible identities for suspect peaks is correct The confirmation process is simplified greatly because the element screen usually yields only a few possibilities and because the retention time in the GC MS run is accurately known In practice
71. 0000 0 l 5 6 7 8 9 10 1 12 4 1 2e 07 6e 07 3 Initial PTV Temp 35 C E ge 06 Vent Flow 100 mL min E 4e 07 4 5e 06 4 2e 06 B 2e 074 8 20 8 60 9 00 i 5000000 4 i 0 T 5 6 7 8 9 10 1 12 Minutes Figure 5 Effect of PTV setpoints on peak shape sample pesticides at 1 0 ppm peaks identified in figure 3 column 30 m x 0 25 mm x 0 25 mm HP 5MS Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Copyright 2000 Agilent Technologies Inc Printed in the USA 2 2000 5965 7770E Agilent Technologies Innovating the HP Way
72. 008 3 31 4 Determining configuration suitability For selected test Front Split 5plitless Inlet 73 2 3 2008 3 31 4 Oven and inlet temperatures wil be set to 70C if originally OFF otherwise left alone Front Split Splitless Inlet 78 2 3 2008 3 31 4 Downloading test method to the selected GC Front Split Splitless Inlet 78 2 3 2008 3 32 2 Total Flow 14 312 Column flow 4 000 Leak rat L min Front Split Splitless Inlet 78 2 3 2008 3 32 4 Total How 14 354 Column How 4 000 Leak rat 354 ml min Figure 8 Procedure and result for SS inlet leak check test Test Name Front Split Splitless Inlet 7890 Pressure Decay Description The Split Splitless Pressure Decay Test checks for leaks in t i the inlet body Flow manifold and associated tubing The Approx Time 30 min test requires you to cool down the inlet cap fittings and remove the column The GC pressurizes the inlet then measures pressure decay to determine if there is a leak It takes approximately 30 minutes to complete the test For details click Run Test Now and refer to the available Help topics Status Test procedure Procedure Inspect the GC configuration Remove the column from the Inlet Cap the inlet column fitting Cap the septum purge fitting r E E A caes aed Test result shows there is Verify both caps are secure click Yes to continue Inlet to 25 psi then Pressure OFF for leak test leakage in inlet system Test resul
73. 10 runs areas over 10 runs Linearity 40 pg l 0 1 96 396 min 50 pg to 150 ng Chromographic conditions for electrochemical detection The HPLC method presented here was used for the analysis of iodide in table salt mV 180 1604 Standard 1401 Table salt 1207 100 1 2 4 6 8 10 12 14 Time min Figure 24 Analysis of iodide in table salt Control and data evaluation m we nm xm O AA mb m m ecur A ER ater 17 A G Huesgen R Schuster Analysis of selected anions with HPLC and electrochemical detection Agilent Application Note 5091 1815E 1991 Lipids Triglycerides and hydroperoxides in oils Both saturated and unsaturated triglycerides have been analyzed Fats and oils are complex mixtures of triglycerides sterols and vitamins The composition of triglycerides is of great interest in food processing and dietary control Owing to the low stability of triglycerides containing unsaturated fatty acids reactions with light and oxygen form hydroperoxides which strongly influence the taste and quality of fats and oils Adulteration with foreign fats and the use of triglycerides that have been modified by a hardening process also can be detected through triglyceride analysis The HPLC method presented here was used to analyze triglycerides hydroperoxides sterols and vitamins with UV visible diode array detection UV DAD Spectra were evaluated in order to trace hydr
74. 2 44 anions 33 inorganic 32 antibiotics IIl antioxidants III 4 63 apples 21 22 artificial sweeteners Ill 8 ascorbic acid 4 aspartame 8 atmospheric pressure chemical ionization APCI 102 104 autoincrement mode 100 automated injector 72 autosampler 72 109 B backflash valve 67 bacteria 15 BASIC programming language 112 beer 48 50 Beer s law 121 benzoic acid 6 benzothiazuron 16 BHA butylated hydroxyanisole 4 BHT butylated hydroxytoluene 4 biogenic amines 48 biotin 43 biphenyl 84 bisphenol A BADGE 24 130 bitter compounds 12 14 bromophenacyl bromide 38 butocarboxim 28 butocarboxim sulfone 28 butocarboxim sulfoxide 28 butter 38 butyric acid 38 C calibration curves 113 settings 115 tables 116 capacity factor 121 capillary electrophoresis V capillary liquid chromatography 52 carbamates 26 103 109 carbaryl 28 carbendazim 27 carbofuran 28 carbohydrates III 40 41 Carrez 7 14 cell design electrochemical porous flow through 99 100 thin layer 99 100 wall jet 98 100 cellobiose 39 cereals 19 20 cheese 48 chemical residues 16 chemotherapeutics 16 chewing gum 5 chiral drug V chloramphenicol 15 chlorite 99 chlorpyripho ethyl 27 chromophore 38 108 citric acid 2 3 43 cleanup 54 client server based software 114 cognac 13 collision induced dissociation CID 19 colorants III 10 column guard 59 67 narrow bore
75. 3004 without mixer 380 ul with mixer 850 ul gt a 200 1004 at5 min start of gradient quaternary pump 07 950 ul T T T T T T T T 3 4 5 6 7 8 9 10 Time min Figure 52 Delay volume of high and low pressure gradient pumps 81 Degassing mAU 3004 2504 200 5 1504 1004 0 08 0 15 50 4 T T T T T T 0 80 100 120 Time min Figure 53 Overlay of six repetitive runs of a tryptic digest of myoglobin in RSD of RT is as low as 0 07 0 5 Degassing removes dissolved gases from the mobile phase before they are pumped over the column This process prevents the formation of bubbles in the flow path and eliminates volumetric displacement and gradient mixing which can hinder performance Instable flow causes retention on the column and may increase noise and drift on some flow sensitive detectors Most solvents can partially dissolve gases such as oxygen and thereby harm detectors Detrimental effects include additional noise and drift in UV detectors quenching effects in fluorescence detectors and high background noise from the reduction of dissolved oxygen in electrochemical detectors used in reduction mode in oxidation mode the effect is less dramatic 82 Fluorescence z Signal heights 104 for selected PNAs 10 11 12 13 14 Time min m Agilenton line degassing W Helium degassing No degassing Figure 54 The loss of response due to quenching can be recovered with either h
76. 5 14 time min Figure 2 Plot of calibration data as displayed by RTL software Re Lock current method Retention time Enter current retention time of alpha methylstyrene fi 8 099 Minutes Then select button Update Method to calculate a new pressure and enter it in the method Method Information Current Method D5135 M 1 Pressure used fi 8 20 psi Desired RT 17 778 Minutes Update current HP6890 Method Figure 3 Dialog box used to calculate locki 6890 method ng pressure and update the flow sensitive detectors one can set constant column flow makeup via the 6890 keyboard or ChemStation In this mode the makeup flow is increased as the column flow decreases to keep the sum of the two constant The underlying theory of RTL pre dicts that constant pressure mode of EPC provides the closest matching of retention times If one desires to com pare data from systems with very dif ferent configurations such as GC FID to GC MSD it is best to use constant pressure mode As can be seen from the styrene analysis data herein retention time matching between sys tems of the same configuration GC FID in this case is still quite good in the constant flow mode This application note shows the use of RTL to lock retention times between multiple chromatographic instruments columns and detector types and demonstrates RTL in both constant flow and constant pr
77. 5 GC Ready Wakin Front Split Soliiess Iniet 78 2 3 2000 4 59 4 Pressure OFF Mor Front Splt Spltiess Inet 78 2 3 2009 5 09 3 Evaluating PASSIF aaa Source Front Split Spitiess Inlet 78 2 3 2008 5 09 3 Restoring original ne baton thine Front Spit Sclitiess Iriet 78 2 3 2008 5 09 3 Request Disc ront Spit Spltiaee Irist 76 2 3 2008 5 3210 Front Solt cities Iniet 78 2 3 2008 09 4 Concudina Pres Pont Spit Spitiees Init 76 12 2008 5 32 90 Front Split Scitess Inlet 78 2 3 2008 5 32 1 Front Split Spitless Inlet 78 2 3 2008 5 32 1 Front Spit Spltiess Inlet 78 2 3 2008 5 32 1 Front Spit Spltiess Inlet 76 2 3 2008 5 32 3 Front Spit Splitiess Inlet 78 2 3 2008 532 3 Inlet leak check test a Use the Splt Spliiess Leak Check to quicHy test for leaks in forming v Both inlet leak check and pressure decay test were passed after the inlet septum and liner O ring were replaced v The leakage problem is fixed Message Downloading test method to the selected GC Total Flow 6 078 Column Flow 4 000 Leak ra Total flow 6 058 C Total Flow 6 058 C Total flow 6 070 Total flow 6 070 Restoring orignal Method to Instrument 7690 U5 10652005 Request Disconnection from 7850 Instrument 7890 US 10652005 Colugin Flow 4 000 Leak rate 0 000 mi jmin Figure 11 The result passed both inlet leak check and pressure decay test after troubleshooting 8 Fro
78. 59 standard bore 59 temperature 60 compressibility 79 computer networks 114 computing equipment 112 conductivity detector 86 copper 99 corn 41 coulometric detection 98 counter electrode 98 cross sample reports 114 D dairy products 22 dansyl chloride 49 data evaluation 112 generation 112 storage 114 dead volume 59 71 121 DEG 3 degassing 82 helium 83 ultrasonic 83 vacuum 83 84 derivatization 73 chemical 62 108 postcolumn 109 110 precolumn 109 110 detection amperometric 98 coulometric 99 detector 86 105 conductivity 86 diode array 86 90 105 electrochemical 86 87 98 105 electroconductivity 32 fluorescence 87 95 105 mass spectrometer 86 88 101 105 refractive index 86 87 104 response 88 thermal energy 86 UV 89 90 variable wavelength 89 90 105 deuterium lamp 10 90 92 DG dodec yl gallate 4 diagnostic test 114 diethylamine 48 diode array detector 87 91 diquat 26 direct solvent extraction 45 drift 87 drift trigger 101 drinking water 26 33 dual lamp design 10 dual piston mechanism 78 dyes V dynamic range 88 E eggs 16 17 electrochemical detector 86 87 98 105 electroconductivity detector 32 electrospray ionization 102 103 elution order 60 emission 96 97 emission grating 95 enzymatic hydrolysis 45 essential oils 12 ethanol 3 ethanolamine 48 ethiofencarb 28 ethiofencarb sulfone 28 ethiofencarb sulfoxide 28
79. 7890 CN10734125 e 146 208 219 31 for Front Inlet Septum has been reached Figure3 Alert email for when the front inlet septum limit has been reached Four alert actions are available Set Not Ready Set An email alert is configured in only three steps Service Due Email and Text Message For exam ple when the email alert action is selected LMD will send an alert email to the users you specify 2 Manage the alerts see Figure 5 Figure 3 shows an example email alert informing a user that the front inlet septum s limit has been reached 1 Manage the users see Figure 4 3 Configure the maintenance indicators b Monitor Management Dres Procedure Select Lab Monitor Management gt Users M OELSE RIRA Add a graphic for the user if you like as SON Select Email from the Method pull down menu E Instrument Groups Enter the first email address for your distribution list in the Parameter field Select E seuss Add Add as many email addresses to your distribution list as you like W He Select Apply Changes when you have added all the email addresses Help A Q Managing users 9 Leb Monitor Management Print List Remove Current Selection Add New User Useful Links Q9 Lab at a Glance me yun zou indows Login ID ag enticoiumn User is an Administrator Title ATS Company AE Contact Methods Method Parameter Email yun_zou adgilent com Email chun xiao_wang agient com Add Contact
80. 8 556 0 006 0 997 9 621 9 692 0 010 0 996 9 671 9 746 0 006 0 996 11 041 11 116 0 003 0 996 11 308 11 383 0 005 0 996 12 719 12 799 0 009 0 997 12 774 12 849 0 006 0 995 14 168 14 277 0 008 0 995 14 201 14 306 0 008 0 996 14 686 14 807 0 008 0 996 16 890 17 074 0 014 0 995 16 903 17 082 0 016 0 997 17 508 17 709 0 016 4500000 4000000 3500000 3000000 2500000 1 xX 2000000 Response 1500000 1000000 500000 T T 3000 4000 Concentration ppb T 2000 5000 6000 Figure 1 GCMS calibration curves for 16 PAHs using 320 pm id column with low head pressure Figure 2 presents overlayed chromatograms for the replicate sample injections showing the excellent precision of the replicate injections summarized in Naphthalene Acenapthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Chrysene Benz a anthracene Benz b fluoranthene Benz k fluoranthene Benz a pyrene Indeno 1 2 3 cd pyrene Dibenz a h anthracene Benzo ghi perylene Table 2 Abundance 3000000 2600000 TIC 1802024 D data ms TIC 1801023 D data ms 2200000 TIC 1803025 D data ms 1800000 1400000 1000000 6000004 J J Lae 20000044 JV a A M CLE 9 60 9 80 10 00 10 20 10 40 10 60 10 80 11 00 11 20 11 40 Time Abundance 3800000 3400000 3000000 2600000 E A 2200000 p Hesk 1800000 1400000
81. 900 yl beginning at 50 pl min with mixer Pressure pulse lt 2 amplitude typically The same tracer gradient used to determine composition S ue em mr precision and accuracy also was used to determine the x P e F ripple of the binary pump see figures 50 and 52 The delay Composition volume was measured by running a tracer gradient Large precision 0 2 at 0 1 and delay volumes reduce the sharpness of the gradient and 1 0 ml min therefore the selectivity of an analysis They also increase the run time cycle especially at low flow rates Damper To sampling unit and column Inlet Inlet valve valve ms 2 Figure 51 Schematics of the high pressure gradient Agilent 1100 Series pump 80 When working at the lowest detection limits it is important to use a mixer to reduce mixing noise especially at 210 220 nm and with mobile phases containing solvents such as tetrahydrofuran THF Peptide mapping on 1 mm columns places stringent demands on the pump because small changes in solvent composition can result in sizeable changes in retention times Under gradient conditions at a flow rate of 50 l min the solvent delivery system must deliver precisely 1 l min per channel A smooth baseline and nondistorted gradient profiles depend on good mixing and a low delay volume Figure 53 shows six repetitive runs of a tryptic digest of myoglobin with a retention time precision of 0 07 0 596 RSD mAU binary pump
82. BAKE MAC in the MSDCHEMMMSEXE directory The indicates a comment line which is not executed Note that the temperature limits which reside in the tune file must be edited to allow the higher settings ChemStation Macro for Baking the Source and Quadrupole After Source Maintenance this macro sets the source and quad temps to their maximum and holds for a set period parameter hours def 6 msinsctl mstemp QUAD 200 synchronize msinsctl mstemp SOURCE 300 synchronize SLEEP hours 60 60 msinsctl mstemp QUAD 150 synchronize msinsctl mstemp SOURCE 230 synchronize return bakes for set period default setting is 6 hours this is customizable sets the quad temperature to bake at 200C sets the source temperature to bake at 300C sets the quad temperature to operating temp at 150C sets the source temperature to operating temp at 230C Usually a source cleaning is executed at the end of the working day and the system pumped down overnight for operation the next day In this case a pumpdown sequence is useful After the system is confirmed to be leak tight this sequence is loaded and executed which bakes the source and quad overnight then executes an Autotune and then makes a few injections of a checkout standard to confirm system performance In this way the analyst returns the next day to review data about the system prior to beginning new analyses An example of this is give
83. CI source was set up for the experiments The following process was used to tune the MS 1 Perform the CI autotune at the normal methane reagent gas flow rate typically at a mass flow controller MFC setting of 20 2 Reduce the CI flow to 2 3 Set the emission current to 250 ua ge In Manual Tune ramp the repeller from 0 5 volts for the mass 69 ion Set the repeller voltage to the maximum value Turn off the CI gas Save tune file 0 N Oo c Associate tune file with method Data was acquired in positive CI PCI and EI modes Figure 1 shows the CI and EI total ion chromatograms using the CI source The major and minor peaks are easily comparable in the two chromatograms Figure 2 shows the CI spectrum for Hexadecanolide MW 254 with the expected adduct ions for methane Note the relatively large response for the 255 ion As expected there is little fragmentation due to the soft ionization a Agilent Technologies ini xi aha File Method Chromatogram Spectrum Calbrate Quantitate Tools wew Help la x agisBesanBgoe msusnmummi2o22 AE ERA X os fm isii ku 0iT TIC BSBT PERFME PCI C Fragrance Mixture PCI Methane Fragrance Mixture El mote with CI source Figure 1 PCI and El total ion chromatograms using the Cl source fg Enhanced Data Analysis FLAY_RTLM PEREMIX EL COMBO D MS Data Not Quantitated 15 xj File Method Chromatogram Spectrum Cal wate
84. D and DDE products of DDT and the ketone and aldehyde products of Endrin The situation is complicated here as the degradation products can be generated in both the injection port and the CFT tee How o l c EJ c 24 lt r e _ _ 6 00 8 00 10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00 Time 7 18 30 1850 18 70 18 90 ue o e i e kl E S 2 st l LI h A NU LL 6 00 8 00 10 00 12 00 14 00 16 00 18 00 20 00 22 00 24 00 Time Figure 7 Reconstructed total ion chromatogram RTIC of a multicomponent FAMES standard using pulsed splitless injection with CFT tee upper and the reconstructed extracted ion chromatogram REIC for m z74 The enlarged panel is for octadecanoic methyl ester ever because those products formed in the injec tion port and those formed at the CFT device will have different retention times due to differing lengths of column the degradation contributions from the two origins should be discernable By analyzing these known breakdown products in the PCT and then injecting the DDT and Endrin agents themselves an estimate of the activity contributed by the CFT device can be calculated The upper panel of Figure 8 presents the reconstructed total ion current RTIC for the selected ion monitoring SIM signals of the four breakdown products These were acquired in SIM sc
85. Endrin identifying degradation products Those with an asterix are attributed to the injection port and due to the CFT device activity such as from Endrin 5 as ketone and from 4 4 DDT 6 as DDE Note 7 is tenatively identified as DDMU source unknown Figure 8 of all components but the verdict is likely the same the CFT device has some activity but is com parable to that of other elements for example in the inlet and liner It is worth noting that this CFT device has a very long path compared to others see the Alternative Configurations section and that air intrusion in any part of the system will be a major issue in considering activity problems Adding Backflush Figures 9A 9B and 9C show the GC parameters for adding backflush They are quite simple The oven temperature can remain the same as the tem perature at the end of the oven program or can be raised to the isothermal or programmed tempera ture limits in Post Run for backflushing Raising the column temperature during Post Run helps condition the column and removes some column bleed but is not necessary The front column column 1 flow is dropped to 0 3 mL min and the back column column 2 flow is raised to 4 mL min To quickly estimate the duration of the Post Run time parameter notice that the back column column 2 in Figure 9C cites the column Holdup Time at a given flow At the 1 25 mL min shown the Holdup Time is roughly 0 4 minutes When the
86. FID NPD ECD FPD and other atmospheric detector methods Note that this would not always be the case If for example a method is being scaled that uses a very low inlet pressure the 1 5 psi difference in outlet pressure could become signifi cant It is best to check the method with method translation and see if the inlet pressure will change by gt 10 If it does it would be advisable to col lect or translate a new RTL calibra tion centered around the translated nominal inlet pressure Gaining Speed in the Same Instrument Setup In the analysis of pesticide residues in food there are usually only a few compounds encountered in any one sample Because the screening method uses selective detectors it makes sense to consider trading speed for chromatographic resolu tion Selective detectors respond to only those compounds containing a specific heteroatom s and the chro matography only needs to resolve those compounds from each other not from every other compound in the matrix This approach can save a significant amount of analysis time In this example of scaling the RTL Pesticide Library the method will be increased in speed at the expense of chromatographic resolution The first consideration is by what factor to increase the speed The method trans lation software is useful for determin ing this A candidate speed gain in this example threefold is entered into the method translation software The resu
87. For example flavonoids from plant material can be cleaned fractionated and enriched on alumina Other examples are given in chapter 5 The advent of narrow bore columns F 250 x 2 1 mm id column 250 x 4 6 mm id column 5 10 15 20 Time min Figure 44 Effect of bore dimensions on separation Discussions of HPLC methods often revolve around the internal diameter id or bore of the column to be used Standard bore columns have an internal diameter of approximately 4 or 5 mm whereas narrow bore columns have an internal diameter of approximately 2 mm When packed with the same materials as the standard bore column the narrow bore column can achieve the same resolving power with less solvent because the analytes can be eluted at a lower flow rate 0 5 ml min than the 2 3 ml min required for standard bore columns In addition narrow bore columns are 4 6 times more sensitive using the injection volume required for a standard bore column see figure 44 Narrow bore columns nonetheless place higher demands on the equipment used than standard bore columns First the HPLC pump must yield these low flow rates in a way that is both reproducible and precise Second all capillary connec tions that is from injector to column and from column to detector must be kept to a minimum Third because column frits block more often guard columns are recommended An HPLC system designed for narrow bore columns low dead volume
88. Liquids uu ERE GO EE SER aia ERG Ra Meg Liquid liquid extraction 020 eee eee Solid phase extraction 0 0 esee eee Gel permeation chromatography Guard columns 0 cee tenes Chapter 6 Injection techniques Characteristics of a good sample introduction device 70 Manual injectors 00 0 c eee eee eee eee 71 Automated injectors 0 00 cece eee eee 72 Autosampler with sample pretreatment capabilities 72 Derivatization 0 0 cee eens 73 Chapter 7 Mobile phase pumps and degassers Characteristics of amodern HPLC pump 76 Flow ranges 226 e ee eee ew dea ee Baad 76 Gradient elution 00 0 cece eee eee 76 Gradient formation at high pressure 77 Gradient formation at low pressure 77 VII Pump designs for gradient operation 78 Low pressure gradient Agilent 1100 Series pump 78 High pressure gradient Agilent 1100 Series pump 80 Degassing cx RARO EN ERU T ee Au heh onde us 82 Helium degassing 0 cece eee eee eee 83 Vacuum degassing 0 ce eee eee eee 84 Chapter 8 Detectors Analytical parameters 00 0 cece e eee eee 87 Limit of detection and limit of quantification 87 Selectivity issu RE Rex hr Rx RES HUESP US ene 87 Linearfily 2i m RR Yee ee ele ee 88 Qualitative information 0000 000s 88 UV detec
89. MS flow rates A pulsed splitless injection transfers more of the sample onto the column and allows for increased injection volumes up to 5 uL When the injected volume is flash vaporized the required expansion volume for the solvent is greatly increased solvent choice also effects expansion volume The use of higher initial inlet pressures reduces the volume PV P2V2 so the entire injected volume can move to the column The higher pressure also decreases the likelihood that highly volatile compounds will escape out the top of the injection port through the septum purge vent Figures 4 and 5 In the case of thermally labile compounds the faster they leave the hot injection port the less likely they are to degrade 2 3 The flow rate is then reduced to the value desired for the chromatographic separation This flow is held constant by increasing the pressure as the oven temperature increases Figure 6 is a graphical representation of the pulsed splitless technique with constant flow Pulsed splitless injections should always be used when sensitivity and or repeatability are critical Refer to the GC operating manual for how to set up a pulsed splitless injection Electronic pressure and flow control of carrier gases not only help with larger volumes they also help to decrease run times and maintain stable MS sensitivity by keeping the carrier gas flow con stant These lead to shorter analyses higher sensi tivity and higher reprod
90. Method 3 Parameter chun xiao_wang aglient cem o E Figure 4 Procedure for managing users x Y O1 I Manage Manage Alerts 3 users A Send Email Access Rights Send an email through the specified server to the recipients selected in the checkbox below P Instruments Server f03 Ifs agilent c Port 25 3 23 Instrument Groups Recipients chun xiao_wang agilent com yun zou Licenses ym luanwei yun_zou agilent com yun zou 7 wei luan amp agilent v Help R Managing alerts Send Text Message Send a text message to a mobile device This option requires email server configuration above Useful Links A Recipients Lab Monitor Management ppc Q9 Logs and Results Procedure Select Lab Monitor Management Manage gt Alerts Enter the address for the email server of the LMD PC network this needs to be provided by the customer IT department Use the default Port 25 unless the IT department instructs you otherwise Select the email recipients from the distribution list that was created in the Users menu Select Apply to save these entries Figure 5 Procedure for managing alerts Agilent GC Method Instrument 1 NOG 10D X 7890 Counters Indicate which counters should be monitored when this method is run wi Front Inlet Septum iw Front Inlet Liner Y Front Inlet Gold Seal iw Front Inlet Split Vent Trap wi Front Inlet Top W eldme
91. PCBs acquired in SIM at source temperatures of 230 C and 300 C From left to right or earlier to later in the chromatogram the PCBs consist of a Cl Biphenyl Cl B Cl B Cl B another Cl B and a Cl B 6000 5500 3 300 C 230 C Figure 5 temperatures of 230 C and 300 C Time Overlaid extracted ion current chromatograms of one ion M 70 for the pentachlorobiphenyl acquired in SIM at source The detection limits for many late eluting high boiling compounds that will improve by imple menting higher source temperatures for example PAHs terphenyls etc As an illustration of the enhancement for very high boiling compounds consider the 6 ring benzenoid hydrocarbon PAH coronene CAS 191 07 1 This compound is diffi cult to determine due to low response and poor chromatography although it is present in many sediment samples Figure 6 shows overlaid RICCs for acquisitions of coronene at 230 C and 300 C Although the peak area is the same the enhanced Gaussian peak shape achieved at 300 C improves detection 1800000 1600000 Lers iiy R e e e e e 1200000 7 1000000 300 C Abundance 800000 230 C e e c ce c e L 400000 200000 Tar aea acr Ei e 1 1 1 1 1 1 1 1 1 1 1 i 1 rI rarr rarere rrr rrr ror a TI TOT T C 9 38 9 40 9 42 944 946 9 48 9 50 9 52 Time U 9 54 Hi TT
92. actors that determine performance in HPLC Limit of detection and limit of quantification 121 Accuracy and precision 0 0 cece eee eee 122 Qualitative information 0 00 00 e eens 123 References 0 0 cece eee e esee 125 Index iere ze Rats Ceased heed ieee d E ii 129 Part One The HPLC Approach A demonstration of liquid chromatographic separations in food analysis Chapter 1 Analytical examples of food additives Acidulants Water Isocratic Detector pump Auto Column VWD DAD vacuum sampler compart or refractive degasser ment index JB Control and data evaluation CAN t Sorbic acid and citric acids are commonly used as acidulants and or as preservatives Acetic propionic succinic adipic lactic fumaric malic tartaric and phosphoric acids can serve as acidulants as well Acidulants are used for various purposes in modern food processing For example citric acid adds a fresh acidic flavor whereas succinic acid gives food a more salty bitter taste In addition to rendering foods more palatable and stimulating acidulants act as e flavoring agents to intensify certain tastes and mask undesirable aftertastes buffering agents to control the pH during food processing and of the finished products preservatives to prevent growth of microorganisms synergists to antioxidants to prevent ran
93. al sweeteners Ill Nernst equation 98 networked data systems NDS 112 118 nicarbazin 16 nitrites 32 nitro compounds 27 nitrofurans 16 NOGA nordihydroguaiaretic acid 4 noise 87 122 normalization 113 normal phase column 46 47 nuts 21 22 0 oat seedlings 52 53 ochratoxin A 21 22 0G octyl gallate 4 oils 35 38 one lamp design 10 online spectral measurements 96 o phthalaldehyde OPA 9 108 orange juice 14 organic acids V oxalic acid 3 oxamyl 28 oxidizable sulfur compounds 108 oxytetracycline 18 P pantothenic acid 43 paprika 27 paraquat 26 partition phases 58 Patent blue 10 patuline 21 22 p bromophenacyl bromide 108 peak co eluting 123 dispersion 121 122 elution 93 identity 93 94 purity 93 94 Peltier control 60 peptides 52 performance test 122 personal computers 114 pesticides Ill V 26 58 petrol ether 35 37 PG propyl gallate 4 PHB ethyl 6 PHB methyl 6 PHB propyl 6 phenethylamine 48 phenylisocyanate 108 phenylurea herbicides 26 phosphoric acid 2 photodiode 89 array 91 photomultiplier tube 97 photoreceptor protein 52 phytochrome proteins 52 pistachio nuts 23 platinum 99 polycyclic aromatic hydrocarbons 96 pork muscle 18 postcolumn derivatization 28 29 109 110 potassium ferrocyanide 14 precision 120 122 precolumn derivatization 109 110 precolumns 65 preservatives Ill 6 7 63 procymidon 27 pr
94. als designed to hold microliter volumes can be used to inject as little as 1 pl of a 5 1 sample Even the way the needle enters the vial can be controlled with computer software deep down to aspire from the denser of two layers or a shallow dip into the supernatant phase With this technique even viscous samples can be analyzed if the right equipment is used The time required to extract the syringe plunger is simply protracted permitting meniscus motion of higher reproducibility Highly reproducible Can be fully Equipment can be costly automated Flow maintained over all parts in contact with sample preventing inaccuracies from intermingling between runs Autosamplers can provide online precolumn derivatization dilute small volumes of sample and add internal standards You may need to protect unstable species by keeping the sample chilled with a cooling device connected to a flow of refrigerated water or more conveniently by Peltier elements Alternatively you might want to induce reactions using heat applied within the injection device of the autosampler Commercially available autosamplers offer all these features 72 Derivatization As discussed later in chapter 8 derivatization may be required if the analytes lack chromophores and if detection is not sensitive enough In this process a chromophore group is added using a derivatization reagent Derivatization can occur either in front of or behind the analytical col
95. ammed to yield results on peak purity and identification by spectra or for more complex analyses to generate system suitability reports Any computer generated report can be printed or stored electronically for inclusion in other documents PCs are well suited for the modification of calibration tables and for the reanalysis of integration events and data The software must record such recalculation procedures so that the analysis can be traced to a particular set of parameters in accordance with GLP GMP principles A computer can automate entire sequences of unattended analyses in which chromatographic conditions differ from run to run Steps to shut down the HPLC equipment also can be programmed if the software includes features for turning off the pump thermostatted column compartment and detector lamp after completion of the sequence If the HPLC equipment malfunctions the software reacts to pro tect the instrumentation prevent loss of solvents and avoid unnecessary lamp illumination time A good software appli cation should be able to turn off the pump thermostatted column compartment and detector lamp in the event of a leak or a faulty injection System suitability tests also can be incorporated in a sequence When performed on a regular 116 basis such tests can validate assumptions about perfor mance of the analytical system and help verify results Enables control of multiple instruments Requires more bench space for Ad
96. ams and suggests appropriate HPLC equipment In addition we list chromato graphic parameters as well as the performance characteris tics that you can expect using the methods shown In part 2 we examine sample preparation and explain the principles behind the operation of each part of an HPLC system sam pling systems pumps and detectors as well as instrument control and data evaluation stations In the last of 11 chap ters we discuss the performance criteria for HPLC which are critical for obtaining reliable and accurate results Part 3 contains a bibliography and an index Contents Part One The HPLC Approach Chapter 1 Analytical examples of food additives Acid lants ii chad Hie ce oka Oe RR ET UPC dan AntioxidantS cles Preservatives eel REA dean EINER COE A PE eg Artificial sweeteners 0 000000 Colorants 4 4 te eb Sala PEN ES GMa dees PAV OVS ese prp PRO E sane Beas he Sau ex qu Vanillin 52 vd EE e Ra PS We UE dur Bitter compounds hesperidin and naringenin Chapter 2 Analytical examples of residues and contaminants Residues of chemotherapeutics and antiparasitic drugs etracyclines 222v e e ex gH RE REESE Fumonisins eeseeeeeee eee MyGCOLOXIIIS osse RR ev RR X ERES EN Bisphenol A diglydidyl ether BADGE Pesticides zesssest os EU REq UEM OPEN MERE HE PES SES Carbamates 00 cece eee eee Glyphosate esios oe em Re ERU e ERE ES Ch
97. an mode with a single SIM group composed of one or two major ions for each compound so there was no time DDD Abundance 7 DDE EA selection for the compounds appearance On the basis of summed areas the total breakdown for Endrin is less than 13 with the CFT device con tributing less than 10 of the total breakdown area or less than 1 2 to the area total The DDT breakdown is less than 4 for the system how ever the CFT device contributes about 46 of the total observed breakdown and is about double the breakdown generated by the port It is possible some DDD breakdown is hidden under the DDT peak On the basis of the DDT to DDE contribution from the CFT tee however it is likely to increase the breakdown perhaps less than about another 2 A better study would use on column injection EK Time Endrin Abundance DDD EA DDE 20 00 20 20 2040 20 60 2080 21 00 21 20 21 40 21 60 21 80 22 00 22 20 22 40 22 60 22 80 4 4 DDT EK 20 00 20 20 2040 20 60 20 80 21 00 21 20 2140 21 60 Time 21 80 22 00 22 20 2240 22 60 22 80 CFT tee activity A the REIC of a GC MS SIM acquisition using pulsed splitless injection with the PCT configuration of the expected degradation products of DDT and Endrin at 0 2 ng on column 44 DDE DDE 4 4 DDD DDD Endrin alde hyde EA and ketone EK B REIC for an injection of 2 0 ng of 4 4 DDT and
98. and Calculated Points for 3x 0 1 mm id Micro ECD Method Assuming 10 m Column Length GC AED RTL Calibration 3x Micro ECD RTL Calibration Calculated Calculated Pressure Ret Time Pressure Ret Time psi min psi min 33 1 15 346 71 03 5 115 30 4 15 919 64 90 5 306 27 6 16 578 58 51 5 526 24 8 17 338 52 11 5 779 22 1 18 242 45 91 6 081 Table 6 RTL Calibration Points from Original GC AED Method and Calculated Points for 3x 0 1 mm id Micro ECD Method Assuming 10 5622 m Column Length GC AED RTL Calibration 3x Micro ECD RTL Calibration Calculated Calculated Pressure Ret Time Pressure Ret Time psi min psi min 33 1 15 346 80 03 5 115 30 4 15 919 73 13 5 306 27 6 16 578 65 95 5 526 24 8 17 338 58 74 5 779 22 1 18 242 51 75 6 081 Table 7 Comparison of Locking Pressures Calculated Using Measured and 3x Micro ECD Locking Runs Predicted 3x 0 1 mm id Micro ECD Calibration Data Locking Pressures Measured 3x Micro ECD RTL Using Measured Using 10 m Calculated Using 10 56 m Calculated Cal Points RTL Cal Points RTL Cal Points RTL Cal Points Pressure Ret Time Pressure Pressure Pressure psi min psi psi psi 48 81 6 323 65 95 66 38 65 30 52 66 6 041 66 03 65 77 65 85 58 51 5 797 65 95 65 12 65 96 64 36 5 585 65 93 64 36 65 95 70 22 5 396 66 00 63 18 65 90 Note that while the most of the References chromatographic resolution is pre 1 P L Wylie and B D Quimby A served the speed is increased by a Met
99. and high performance pumping system can achieve solvent economies of more than 60 96 as well as improve detection limits with the same injection volume Moreover under the same conditions a standard bore column may have higher resolving power 59 Influence of column temperature on separation In brief Many separations depend not only on the column material and mobile phases but also on the column temperature In such cases column temperature stability is the dominating factor for the elution order A thermostatted column compartment using Peltier control with good ambient temperature rejection ensures stable chromatographic conditions Periodic fluctuations in room temperature during 24 hour use influence these conditions Figure 45 shows the advantage of Peltier control over conventional air cooling Retention time Agilent 1100 Series thermostatted column compartment 71 207 ee e me Le ee 11 00 Conventional column oven 70 804 Day 70 604 70 404 1 2 3 4 5 6 7 8 9 10 Run number Figure 45 Comparison of Peltier and conventional cooling as demonstrated using retention time fluctuations of a peptide peak over a sequence of 10 consecutive tryptic peptide maps Reversed phase stationary phases are the most popular LC media for the resolution of food mixtures The use of narrow bore columns can result in gains in sensitivity and reduced solvent consumption For example these columns have been applied succ
100. apter3 Analytical examples of natural components Inorganic anions leeeeeeeeeeeenn Lipids seats RR cR RE REEPIEEE Re ERR E Triglycerides and hydroperoxides in oils Triglycerides in olive oil 20 202 e eee Fatty acids 4 269 e taeda sdviebee tend sa I Rd Carbohydrates 0 cc eee eee eee Vitamins ss casee sneep a a blee wade neles TEX Water soluble vitamins seeeeeee Fat soluble vitamins 0 20 eee eee eee Analysis of tocopherols on normal phase column Biogenic amines 0 0 ce eee eee eee ences Amino acids 0 eee eee eee Peptides o wies RR RPEX OH SEE GU ea bee dees VI Part Two The Equipment Basics Chapter 4 Separation in the liquid phase Separation mechanisms 0 cece eee enee Reversed phase materials 0 0000 ee eee Ion exchange materials 0 0 c cece e eee Size exclusion gels 00 00 e eee eee eee eee Adsorption media 00 cece e eee eee The advent of narrow bore columns Influence of column temperature on separation Chapter5 Sample preparation Sample preparation steps 00 c cece eee eee Automation 64 6 bose ee ee aed bb ea E IR E RES SOlldS nac Hered haa ae ee ex pae Ultrasonic bath liquid extraction Steam distillation 0 cece eee eee eee Supercritical fluid extraction 0 000000
101. arbofuran 3 butoncarboximsulfone 15 aldicarb 10 thiofanox sulfone 21 1 naphthol 4 aldicarb sulfone 17 propoxur 11 3 hydroxycarbofuran 22 thiofanox 6 methomyl 19 carbaryl 12 methiocarb sulfoxide 23 methiocarb it ethiofencarb sulfoxide 20 ethiofencarb 13 methiocarb sulfone Figure 21 Analysis of two different carbamate standards Control and data evaluation Quaternary Pickering i pump Auto post column rd mm vacuum sampler derivatiza fiam A degasser tion system i ABN Water Methanol Cy 15 A new approach to lower limits of detection and easy spectral analysis Agilent Primer 5968 9346E 2000 28 Glyphosate Sample preparation none Column 150 x 4mm cation exchange K form from Pickering 8 um Mobile phase A 5 mM KH PO pH 22 0 B 5mM KOH Flow rate 0 4 ml min Gradient at 15 min 0 B at 17 min 100 B Column compartment 55 C Injection volume 50 ul standard Fluorescence detector Excitation wavelength 230 nm or 330 nm Emission wavelength 425 nm Photomultiplier gain 12 Response time 4 s Derivatization reagent pump flow rate for hydrolization agent 0 3 ml min OCI flow rate for derivatization agent 0 3 ml min OPA HPLC method performance Limit of detection 500 ppt Repeatability of RT over 10 runs lt 0 8 of areas over 10 runs 2 2 96 Chromatographic conditions The HPLC method presented here was used for the direct analysis of glyphosate i
102. arget compound is run at GC Method Translation r Column Length Internal Diameter Ix 30 Ix 250 0 Film Thickness Phase Ratio Unlock C 0 250 250 0 Carrier Gas Helium Enter one Setpoint Head Pressure psi Flow Rate Outlet Velocity cm sec Average Velocity cm sec Hold up Time mn f mL nZmin Outlet Pressure absolute psi Ambient Pressure absolute psi Unlock 27 6 C 17 934 2 7153 C 1 8938 96 64 50 20 0 996060 50 20 0 996060 16 2 14 696 r Oven Temperature 3 ramp Program Ramp Final Final Rate Temp Time L C min C min Ramp inal Rate emp 0 25 150 3 200 0 8 280 Figure 1 Method translation software showing scaling HP RTL Pesticide Method from GC AED conditions to GC MS with a scale factor of 1 the nominal method pressure and the retention time is observed The pres sure and resulting retention time are then entered into the Re Lock New Column menu item of the RTL soft ware to calculate the correct pressure for obtaining locked retention times Normally the RTL calibration for a new method is determined by actually making the five calibration runs In the current example methyl chlor pyrifos would be run at e 17 93 psi 20 14 34 psi e 17 93 psi 10 16 14 psi 17 98 psi nominal method pressure e 17 93 psi 10 19 72 psi 17 93 psi 20 21 56
103. ated no glass wool p n 5181 3316 e Viton O ring 12 pk p n 5180 4182 Gold plated seal p n 18740 20885 Washer to go with seal p n 5061 5869 e 10 uL Blunt needle syringe p n 9301 0713 Column Consumables The optimal choice of column is once again depen dent on the application For trace level high sensitivity applications a column with a thin film and low bleed is best A 30 m 0 25 mm id 0 25 um film 5 phenyl 95 methyl silicone column is a versatile column used for many applications Spe cial low bleed MS columns cost more but give better results The proper column nut and ferrule combination are critical for a leaktight seal Newer column nuts may not be compatible with all ferrules The proper ferrule will be dependent on column outer diameter od and is specified below The ferrule should only be slightly larger than the column od The use of 100 graphite ferrules is not recom mended as they are easily over tightened causing graphite to extrude into the injection port This will be apparent when disassembling the injection port If there are pieces of graphite in the bottom of the injection port the ferrule s was were over tightened The presence of graphite in a hot injec tion port can cause thermally labile compounds to degrade It can also affect the chromatography and cause tailing Thus 10 graphite 90 Vespel fer rules are highly recommended Vespel ferrules will shrink as they are heated Con
104. atile sample components and degradation prod ucts remain behind in the inlet mini mizing column contamination There is evidence that inlet contamination in PTVs influences subsequent injec tions less than in hot inlets If conta mination becomes an issue the inlet liner is easily changed Thus PTV is a better choice for dirty samples than cool on column and split splitless inlet For a PTV inlet to work well inlet temperature must be programmed independently from the column oven The 6890 provides this function For example the inlet can be heated with the split flow off to transfer the sample to the column before the oven temperature program begins After sample transfer the inlet can be heated further to bake off contami nants with a high split flow to mini mize inlet contamination LVI by PTV is not a good choice when the target compounds include highly volatile species because these low boiling compounds are vented along with the solvent The lowest boiling target compound should boil at least 100 C above the solvent to have a rea sonable chance of success Table 2 lists the advantages and dis advantages of LVI by solvent elimina tion PTV Experimental A 6890 GC with electronic pneumat ics control EPC was used A G1916A automatic liquid sampler ALS with a G1513A controller per formed sample injection An Agilent ChemStation version A 04 02 con trolled the instruments and acquired and processe
105. atively high molecular weight and contain one or more oxygenated alicyclic rings The analysis of individual mycotoxins and their metabolites is difficult because more than 100 such compounds are known and any individual toxin is likely to be present in minute concentration in a highly complex organic matrix Most mycotoxins are assayed with thin layer chromatography TLC However the higher separation power and shorter analysis time of HPLC has resulted in the increased use of this method The required detection in the low parts per billion ppb range can be performed using suitable sample enrichment and sensitive detection Sample preparation Samples were prepared according to official methods Different sample preparation and HPLC separation conditions must be used for the different classes of compounds The table on the next page gives an overview of the conditions for the analysis of mycotoxins in foodstuffs Chromatographic conditions The HPLC method presented here for the analysis of myc otoxins in nuts spices animal feed milk cereals flour figs and apples is based on reversed phase chromatography multisignal UV visible diode array detection and fluores cence detection UV spectra were evaluated as an additional identification tool 21 Column class Matrix Sample preparation Chromatographic conditions Aflatoxins nuts G G B B spices M M animal feed milk dairy products gt ex
106. ay 102 intermediate precision 122 iodide 34 ion exchange chromatography 10 ion exchange phases 58 ionox 100 4 hydroxymethyl 2 6 di tert butyl phenol 4 ion pairing reversed phasechromatography 10 11 ipronidazol 16 isoabsorbance plot 91 92 isobutylamine 48 isopropylamine 48 K ketones 108 L lactic acid 2 lactose 42 LC M S 52 101 102 LC M SD 19 24 lemonade 41 light intensity 91 imit of detection LOG 87 120 121 imit of quantification LOQ 87 120 121 inearity 87 88 ipids 35 iquid liquid extraction 65 ocal area network LAN 117 ong term variability 123 uminescence 95 upulon 12 M malic acid 2 maltose 40 mannitol 40 manual injector 71 margarine 38 47 mass spectra 53 54 mass spectrometer V 86 88 101 105 meat 16 63 memory effect 70 mercaptobunzothiazol 26 mercapto propionic acid 9 mercury 99 mercury gold 99 methabenzthiazuron 26 methanol 3 methiocarb 28 methiocarb sulfone 28 methiocarb sulfoxide 28 methomyl 28 methylamine 48 meticlorpindol 16 metronidazol 16 microbial growth 6 microorganism 6 microsampling 70 milk 16 21 22 mixing noise 81 molecular weight 66 monochromator 89 morpholine 48 moving belt interface M S 102 multichannel integrators 113 multisignal 97 multisignal detection 92 mycotoxins 21 132 N N acetyl metabolite 15 naringenin 12 14 narrow bore column 59 natur
107. bove can cause retention time differences between identically configured GC systems of as much as 0 4 minute It would be impractical to control all of the instrument and column vari ables to a degree where retention time differences between similarly configured GC systems are removed There is however a means of greatly reducing these differences By making an adjustment in the inlet pressure the retention times on a given GC setup can be closely matched to those of a similarly con figured GC system RTL is based on this principle The process of RTL is to determine what adjustment in inlet pressure is necessary to achieve the desired match in retention times Agilent RTL software G2080AA which integrates into the Agilent GC ChemStation version A 05 02 or later provides the tool required to determine the correct inlet pressure quickly and simply There are several advantages gained by using RTL in the laboratory Peak identification becomes easier and more reliable It is easier to compare data both between instruments and over time Comparison of data when using different detectors for analyte identification is simplified Transfer ring methods from instrument to instrument or laboratory to labora tory is easier because calibration time windows normally will not require readjustment Validation of system performance is easier With locked GC methods the development and use of retention time data bases for unk
108. ce example of the Purged Ultimate Union For More Information For more information on our products and services visit our Web site at www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Printed in the USA June 24 2008 5989 8664EN Low Pressure Retention Time Locking with the 7890A GC e e 8 e e e ee a o0 e Application 050 e 9 e e 9 e HPI e e Authors Russell Kinghorn and Courtney Milner BST 41 Greenaway Street Bulleen VIC 3105 Australia Matthew S Klee Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 USA Abstract Retention time locking was introduced over a decade ago with the Agilent 6890 gas chromatograph The next gener ation of GC from Agilent the 7890A has enhanced and extended the functionality previously available including a new electronic pneumatic control system capable of pressure control to the third decimal place This applica tion demonstrates the ability of the EPC system to be used for retention time locking at low pressures in this case with a 320 pm column on a 5975 GC MS Introduction The introduction of retention time locking RTL with the 6890 GC gave us
109. ce indi cators see Figure 6B The next case study demonstrates how LMD can provide intuitive help and easy to follow proce dures in case there is a problem with diagnostic capabilities It also illustrates how the complete searchable user information enhances the diagnos 4 tic and troubleshooting functionality of LMD Problem The trace level peak was lost or its response reduced on the 7890A GC with FID and capillary column In this scenario the response of ethylben zene is reduced and m oxylene is lost as shown in 5 Figure 8 Designing Diagnostics and Tests to Solve Specific Problems This problem may be caused by inlet leakage The diagnostic tests of the LMD software simplify com plex troubleshooting tests for the user by automati cally performing specific troubleshooting related tasks For example the inlet leak and inlet decay tests helps the user with quick diagnostics and the FID checkout test helps verify proper performance with convenient guidance The problem can be fixed efficiently with the intuitive help of LMD When an LMD test is running the GC Remote light comes on indicating control of the GC At this time the ChemStation must be closed to run the LMD tests 1 Run SS inlet 7890A leak check Result Failed 8 The procedures and result are shown in Figure 8 Run SS inlet 7890A pressure decay test to con firm a leakage Result Failed The above tests indicate that there is leakage in t
110. changeover of mobile phases Less expensive to use and maintain than Increases dead volume and may result in helium degassing The composition of ghost peaks depending on the type of premixed solvents is unaffected and tubing and type of solvent used removal of oxygen is highly efficient Several channels can be degassed simultaneously In brief The choice of pump depends on both elution mode isocratic or gradient and column diameter narrow bore or standard bore Although an isocratic system often is sufficient gradient systems are more flexible Moreover their short analysis times make gradient systems ideal for complex samples sharp peaks resolution of multiple species and automatic system cleansing with additional online solvent channel Agilent 1100 Series pumps are best suited for flow ranges from 0 05 ml min up to 10 ml min and can therefore be used with columns that have an inner diameter of 1 mm to 8 mm Although many officially recognized methods are based on standard columns and flow rates the trend is toward narrow bore columns These consume less solvent which also reduces waste disposal thus lowering operating costs 84 Chapter 8 Detectors Most detectors currently used in HPLC also can be applied in the analysis of food analytes Each technique has its advantages and disadvantages For example diode array UV absorbance detectors and mass spectrometers provide additional spectral confirmation
111. ciation CID show no difference between fumonisins B2 and B3 Consequently it was necessary to separate these compounds chromatographically for quantitative analysis However in crude corn extracts the CID fragment ions provide important confirmatory information In order to obtain spectra of the fragment ions as well as the pseudo molecular ions in a single scan operating at maximum sensitivity the fragmentor voltage was set to 230 V while scanning from 150 amu to 680 amu and then to 100 V when scanning from 690 amu to 800 amu Sample preparation Extraction according to 35 LMBG 8 Chromatographic conditions The Agilent 1100 Series LC MSD proved to be capable of detecting and quantifying fumonisins at 250 picograms per component regardless of their chemical structure and without the need for derivatization during the sample preparation procedure The Agilent 1100 Series LC MSD provided optimum sensitivity in the selected ion monitor ing mode Even when operating in scan mode 150 amu to 800 amu the Agilent 1100 Series LC MSD still provided sensitivity more than a factor of 10 better than reported for a fluorescence detector LC MS conditions Colum Mobile phase A M obile phase B Gradient Flow rate Injection vollume Column compartment lonization mode Nebulizer pressure Dryng gas temp Drying gas flow Vcap Fragmentor Scan range Zorbax Eclipse XDB C18 2 1 mm x 150 mm 5 um 5mM ammonium acetate pH3
112. cidity and browning viscosity modifiers in baked goods melting modifiers in cheese spreads and hard candy meat curing agents to enhance color and flavor Sample preparation Sample preparation depends strongly on the matrix to be analyzed but in general steam distillation and solid phase extraction techniques can be used Chromatographic conditions High performance liquid chromatography HPLC with UV visible diode array detection UV DAD has been applied in the analysis of citric acid in wine and in a vodka mixed drink Retention time and spectral data were used as identification tools Sample preparation filtration Column 300 x 7 8 mm BioRad HPX 87 H 9 um M obile phase 0 0035 M H 50 isocratic Flow rate 0 6 ml min Column compartment 65 C Injection volume 10 yl Detector UV VWD detection wavelength 192 nm or 210 nm Conditions as above except M obile phase 0 007 M H550 isocratic Detector UV DAD HPLC method performance Limit of detection 100 ng injected amount S N 2 equivalent to 2 ppm with 50 pl injected volume Repeatability of RT over 10 runs lt 0 1 areas over 10 runs lt 3 EU 1 Oxalic acid 7 Lactic acid 2 Citric acid 8 Glycerol 3 Tartaric acid 9 DEG 300 4 Malic acid 10 Acetic acid 5 Sulfur trioxide 11 Methanol 200 4 6 Succinic acid 12 Ethanol 100 White wine 03 Standard 0 5 10 15 20 25 Time min Figure 2 Analysis of acidulants in white wine
113. cles hexane isopropanol 95 5 as isocratic mixture flow rate 0 6 ml min at 30 C DAD 270 20 nm HPLC method performance Limit of detection 1 5 ug kg Repeatability ofRToverl0runs lt 0 12 of areas over 10 runs 1 5 96 Linearity of UV visible DAD 1 500 ng of fluorescence 30 pg to 2 ng Water Methanol Quaternary pump Auto Column vacuum sampler compart degasser ment Control and data evaluation Fluores cence detector Diode array detector mAU 201 15 FLD aem 365 nm M25ng Bi5ng dex 455 nm A DAD 365 nm T T V s Time min 6 Figure 15 Analysis of aflatoxins with UV and fluorescence detection mAU 54 Pistachio nut 2 4 6 8 Time min Figure 16 Analysis of aflatoxins in pistachio nuts with UV and fluorescence detection L 13 Lebensmittel und Bedarfsgegenst ndegesetz Paragraph 35 Germany 4 Official M ethods of Analysis Food Compositions Additives Natural Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 AOAC Official Method 980 20 aflatoxins in cotton seed products AOAC Official M ethod 986 16 Aflatoxins M M in fluid milk AOAC Official M ethod 985 18 a zearalenol 23 Bisphenol A diglycidyl ether BADGE Bisphenol A diglycidyl ether BADGE is present in the three most common coatings epoxy lacquer organosol lacquer and polyester lacquer used to protect the inside surfac
114. d RTL Cal Points Locking Pressures Using Calculated RTL Cal Points Pressure Ret Time Pressure Pressure psi min psi psi 20 16 127 17 73 17 75 18 16 326 17 72 17 73 18 16 536 17 72 17 72 17 16 760 17 74 17 74 16 16 988 17 72 17 74 deviation is clearly largest in the isothermal hold region which starts at 31 87 minutes This effect is seen with GC MS but not with scaling to other atmospheric pressure detectors While the cause is not yet clearly understood it appears related to the vacuum outlet pressure of the GC MS column Although this level of match ing is very good the table includes both the GC FID and GC MS retention times so that smaller time windows can be used in searching unknowns Locking GC AED with Other GC Detectors When the method translation step is done to scale the GC AED method to other atmospheric pressure detectors the only different parameter to enter is the outlet pressure The outlet pres sure for the GC AED method is 16 2 psi and that for the others is 14 696 psi The method translation calculates that the nominal GC AED inlet pressure of 27 6 psi would be changed to 26 29 psi for the other atmospheric detectors This differ ence lt 5 is so small that it can be neglected because corrections in this range are compensated easily by the retention time locking step Thus the method conditions and RTL calibra tion points used with GC AED are interchangeable with
115. d data Table 3 lists the hardware and soft ware revisions that support multiple injections postdwell time and the slow plunger mode required for LVI PTV Experimental conditions for the GC methods are given with the chromatograms Table 2 Advantages and Disadvantages of LVI by Solvent Elimination PTV Advantages Most flexible LVI technique Good for late eluting compounds such as pesticides PAHs etc Inlet protects column so one can use dirty samples Disadvantages Loss of volatile sample components Possibility of sample decomposition although less than with split splitless More difficult to use than conventional inlets like split splitless Table 3 Software Hardware and Firmware Versions that Support LVI PTV Item ChemStation software G1513A injector G1512A ALS 6890 GC Software Firmware A 04 02 or higher A 09 10 A 01 08 A 02 01 or higher Results and Discussion Multiple Injections Multiple injections are a straight forward and reliable way to introduce large sample volumes into the inlet Figure 1 shows the linearity obtained from two sets of multiple injections using a standard G1513 ALS Depending on solvent type and injec tion volume liquid sample may run down the liner and enter the column If this occurs the column may over load with solvent causing cata strophic peak splitting and possible damage to the stationary phase To minimize this possibility a packed liner should
116. d on reversed phase chromatography UV spectra were evaluated as an additional identification tool mAU Naringenin A Hesperidin Standard Orange juice 0 5 1 L5 2 25 Time min Figure 10 Analysis of bitter compounds in orange juice LE 8 Official M ethods of Analysis Horwitz W Ed 14th ed AOAC Arlington VA 1984 secs 12 018 12 021 14 Chapter 2 Analytical examples of residues and contaminants Residues of chemotherapeutics and antiparasitic drugs In addition to several other drugs nitrofurans and sulfonamides such as sulfapyridine N acetyl metabolite ethopabat chloramphenicol meticlorpindol metronidazol ipronidazol furazolidone and nicarbazin are frequently fed to domestic cattle Modern intensive animal breeding demands permanent suppression of diseases caused by viruses bacteria protozoa and or fungi A number of chemotherapeutics are available for the prevention and control of these diseases After application residues of these drugs can be found in foods of animal origin such as milk eggs and meat These chemotherapeutics can cause resistancy of bacteria Because of the toxic nature of chemotherapeutics for example choramphenical government agencies in many countries including the United States Germany and Japan have set tolerance levels for residues of these drugs Simple and reliable analysis methods are necessary in order to detect and quantify
117. ditional software can be used for peripherals such as printers or plotters many other tasks Provides for better data storage and archival Local area networks A laboratory running food analyses frequently requires multiple instruments from multiple instrument vendors for sample analysis Although the integrators and PC systems described above can evaluate data at analytical instrument stations throughout the laboratory this data must be col lected centrally over a network for example in order to generate a single report for multiple analytical techniques Local area networks LANs offer several advantages in addition to shared data processing see figure 78 Central ized printing saves bench space and reduces equipment 3 expenses and centralized file security through a single RI computer the server accelerates data backup Standard im network software and hardware cannot handle data files from diverse analytical instrument vendors The analytical software therefore should have file conversion utilities H B based on the Analytical Instrument Association ANDI file format cdf Figure 78 connecting instruments and collating analytical results Integrates multiple techniques and Data processing features may not match instruments from multiple vendors Saves those of dedicated data analysis software bench space and computer processing applications resources Access to network utilities such as e mail ToU Sha
118. ditioning them for 4 hours in a 250 C oven will preshrink them before use Alternatively the column nuts Figure 2 number 16 can be retightened after the column oven cycles a few times The column column nut and ferrules supplies include e 30 m column 0 25 mm id 0 25 um low bleed HP 5MS p n 190918 433 Column nuts wrench tighten only 2 pk p n 5181 8830 Ferrules for 0 2 mm id columns 10 pk p n 5062 3516 Ferrules for 0 25 mm id columns 10 pk p n 5181 3323 Ferrules for 0 32 mm id columns 10 pk p n 5062 3514 Ceramic scoring wafer column cutter 4 pk p n 5181 8836 A sharp column cutting tool is needed for making clean cuts The ceramic scoring wafers or sapphire square edge pens are desirable The diamond point pens are harder to use Ceramic scoring wafers are extremely sharp They should be used with care An X ACTO or Swiss Army knife is not a column cutting tool Use a 10x magnifier to assure that the cut is clean and no column shards are lodged inside the column Interfacing the Column to the MS The column is connected to the MS through an interface that is sealed with a column nut and fer rule The specific ferrule used depends on the column diameter Never use a 100 graphite ferrule Similar to the injection port pieces of graphite may extrude into the interface and contaminate the MS The ferrules required are 15 graphite 85 Vespel The column nut listed is brass stain
119. ductivity and data quality This solution can also be implemented on the Agilent 6890 GC Of course the PCT tee configura tion is not confined to the Agilent GC MS detector but is suitable for other detection schemes as well Future software releases will contain a key com mand that will allow more functionality and greater ease of use it will allow the user to apply the IGNORE READY TRUE condition to the EPC device controlling the CFT tee This will prevent the pressure pulse or other flow conditions from producing a not ready condition for the instru ment d Agilent Technologies www agilent com chem References 1 The 5975C Series GC MSD Agilent Technolo gies publication 5989 7827EN 2 Frank David and Matthew S Klee Analysis of Suspected Flavor and Fragrance Allergens in Cosmetics Using the 7890A GC with Column Backflush Agilent Technologies publication 5989 6460EN 3 Frank David and Matthew S Klee GC MS Analysis of PCBs in Waste Oil Using the Back flush Capability of Agilent QuickSwap Acces sory Agilent Technologies publication 5989 760 1EN These references are available in the Literature Library at www chem agilent com Acknowledgements The author is very grateful to Bruce Quimby Wes Norman and Matthew Klee for several informative and encouraging discussions Also a special thanks to Wes Norman for providing superb CFT devices tailored to the needs of the GC MSD and an advan
120. e and flow simplifies 5 X 5 uL Injections oe 5 Chlorothalonil 9 Imazalil 3 Map 6 Chlorpyrifos Methyl 10 Ethion i Diazi 7 Chlorpyrifos Tt Phosmet TRUM 8 Thiabendazole 12 Azinphos methyl j 45 8 7 a v 78 12 4 2 E 3 9 0 5 6 7 8 9 10 Tl 12 Minutes Figure 3 LVI with solvent elimination PTV Pesticides 0 01 ppm PTV conditions vent flow 300 mL min vent pressure 0 until 1 min purge flow 50 mL min at 3 50 min gassaver on at 4 70 min PTV initial temperature 20 C PTV initial time 1 1 min PTV rate 700 C min PTV final temperature 300 C injection delay 0 00 min column 30 m x 0 25 mm x 0 25 um HP bMS pA 1 j D C23 m PIN Sap 100 Recovery 2000 C20 C16 1000 Bora C14 Solvent C10 C12 0 a G 6 8 10 12 pA C10 3000 eig C23 OPIV C107C 2000 C1 CIs C20 1000 0 0 2 4 6 8 10 12 Minutes Figure 4 PTV with cryocooled inlet PTV use LVI with solvent elimination UsingCOC SVE Agilent PTV requires careful method develop Technologies Application Note ment for maximum accuracy and 228 377 Publication Number reproducibility 23 5965 7923E March 1997 References 2 J Staniewski and J Rijks HRC 16 1993 182 1 B Wilson et al Large Volume Injection for Gas Chromatography Abundance ge 05 9e 06 Initial PTV Temp 20 C 5e 05 Vent Flow 300 mL min 7e 06 Be 08 3 2e 05 7 60 8 00 8 40 8 70 3e 06 A 50
121. e and can be used for sample cleanup and enrichment Alternatively a backflush valve can be used to enrich the sample on a precolumn Reversing the direction of flow transfers compounds concentrated from the precolumn to the analytical column Highly reproducible good automation More complex and more expensive if a possibilities valve is used Many food analyses are governed by officially recognized methods which often include details on sample preparation Recent trends toward automated sample preparation increase precision by eliminating operator variances Should you adopt a newly developed sample preparation technique however please be aware that the method must comply with existing good laboratory practice GLP regulations and with accreditation standards 9 67 68 WO WD a e ka C2 Injection techniques Characteristics of a good sample introduction device Figure 46 A typical 6 port injection valve After the sample has been prepared for introduction onto the LC column analysis can begin Judgements based on analyte concentration require a reliable quantity of sample volume The process of introducing the sample onto the column with precision syringes can be automated for increased throughput 39 The main requirements for any sampling device are good precision of injection volumes low memory effects carry over of material from one injection to another and the ability to draw
122. e colors and filtration the solution can be injected directly into the HPLC instrument 10 Sample preparation Column Mobile phase Gradient Stop time Post time Flow rate Column compartment Injection volume Detector injection without further preparation 125 x 3 um Hypersil BDS 3mm A 20 01 M NaH PO 0 001 tetrabutyl ammoniumdihydrogen phosphate pH 2 4 2 B A CN start with 15 in 10 min to 40 96 in 14 min to 90 until 19 min at 90 96 in 20 min to 15 ACN 20 min 4 min 0 8m 40 C Tul min UV DAD signa optim A 254 50 nm for ization of separation signa signa B 350 20 nm C 465 30 nm signa HPLC method performance Limit of detection for UV DAD Repeatability of RT over 10 runs of areas over 10 runs D 600 40 nm 2 ng injected amount S N 22 0 2 96 396 Chromatographic conditions The HPLC method presented here for the analysis of dyes is based on ion pairing reversed phase chromatography UV spectra were evaluated as an additional identification tool Woodruff lemonade mAU 127 Chinolin yellow Patent blue 10 81 i 465 nm 30 nm 2 600 nm 40 nm 0 2 4 6 8 10 12 14 Time min Spectra of different colors Amaranth Patent blue red A PN Brilliant Norm fTartrazine blue yellow 500 600 Wavelength nm 400 Figure 7 Analysis of synthetic colors in lemonade Overlay
123. e controlled tee arrangement for the GC MSD GC Configuration The GC can be configured in several ways How ever for instructional purposes and those of these experiments the GC was configured as follows Column 1 30 m x 0 25 mm id x 0 25 um column Inlet Front injection port pulsed splitless mode split flow 15 mL min Outlet MSD vacuum Mode Constant flow Column 2 15m x 0 25 mm id x 0 25 um column Inlet Back injection port split mode split flow 15 mL min Outlet MSD vacuum Mode Constant flow The flows were set to 1 2 mL min all zones were left cold and the MSD power was turned on With the MSD and GC zones still cold the MSD back ground was checked to be sure m z 28 was decreasing indicating that the system was tight Only after there was confidence that there was no leak were other zones brought up to temperature Mii GC Edit Parameters Oven Temperature 45 0 C Wi 2 F w SSL Front 55L Back Split Splitess Inlet Operating with Pressure Pulsed Splitless Injection Figures 4A and 4B show screen captures of the 7890A GC configuration for a standard pressure pulsed splitless injection with constant flow mode operation they show the front and back injection port parameters Remember the arrangement is set up such that the front port into which the sample will be injected is configured as if a 30 m column were installed into the MSD Typical pres sure pulse conditions ar
124. e is a shoulder evident on the front side of the phenylacetylene peak in figure 4 It would simplify locating the impu rity in the GC MSD data if the reten tion times closely matched that of the GC FID Because constant pressure mode is preferred when comparing data from FID and MSD systems constant pres sure mode was chosen and the styrene sample was re run on GC system 1 at 18 2 psi for reference The next step was to determine the chromatographic conditions to be used on the GC MSD The Agilent method translation software tool was used to calculate the conditions nec essary to have the peaks elute in the identical order on the two systems Because the retention times need to match the dead time and tempera ture program used for running the GC MSD must be the same as the GC Table 1 Peak Identities for Figure 4 Peak Name Peak Name 1 Nonaromatics 8 p m Ethyltoluene 2 Ethylbenzene 9 Styrene 3 p Xylene 10 a Methylstyrene 4 m Xylene 11 Phenylacetylene 5 i Propylbenzene 12 D Methylstyrene 6 o Xylene 13 Benzaldehyde 7 n Propylbenzene pA 215 Ethylbenzene o Methylstyrene 25 6 10 318 min 17 778 min 22 5 10 658 min E pes 18 099 min 20 17 5 4 15 1 Original GC system 1 column 1 12 5 1 10 5 CE EE Scouting GC system 2 column 2 T T 5 7 5 10 12 5 15 17 5 20 22 5 min Figure 5 Comparison of original chromatogram on GC system 1 with GC system 2 before retention time locking pA ot Methylst
125. e middle of the analytical column essentially splitting the column in half For example a 15 m column pre ceeds and follows a CFT tee Schematically this arrangement is illustrated in Figure 2 The auxil iary EPC device adds just enough pressure flow to match the flow pressure from the first column so there is little flow addition and therefore less dilution and loss in the GC MSD signal Back flushing is similarly simple the pressure or flow is dropped in the first column section while the second section column flow is increased 1 to 4 psi Aux EPC 0 8 to 2 5 mL min MSD 17 1 cm MSD Onti timum Quickswap lt L5mL min 10 to 75 psi Aux EPC 10 to 25 mL min E MSD 17 1 cm Quickswap Advantages of this pressure controlled tee PCT approach are similar to those of QuickSwap such as Service of injection port liner and septum with out venting the MSD Column cutback or replacement of the front or first column without venting the MSD But additional advantages of the PCT arrangement over QuickSwap are Minimal or no signal loss in EI or CI MS is obtained because of the very small additional makeup gas flow Constant flow mode and pressure pulsed injections are straightforward This configuration is suitable for diffusion pumped systems and allows backflushing in diffusion pumped systems Backflushing is more rapid and can be initiated
126. e new RT vs P calibration curves The new RT vs P calibration curve equa tion will be displayed on the screen along with the correlation coefficient Select Yes to either create a new or update an exist ing RT vs P calibration file See Figure 3 Next you can enter the name and retention time of the RTLock compound See Figure 4 and Figure 5 OK Cancel 0 9 Enter or confirm the name of the RTLock compound Figure 4 TIC RTLOCKS D Enter or confirm the retention time of the RTLock compound Figure 5 You will then be asked to con firm the RTLock pressure that has been calculated and will be used for that method Select Yes to confirm the new RTLock pressure and save the method See Figure 6 TIC RTLOCKS D Figure 6 By selecting Yes you RTLock and save the method View Current RTLock Method Set points and Report Once the system is calibrated and locked you can view and confirm the RTLock setpoints by selecting View Current Method Setpoints See Figure 7 Le EN HT Pressure psi Figure 7 Viewing the RTLock retention times and corresponding pressures for the RTLock method A report is also available that provides detailed information regarding the RTLock method See Figure 8 The report includes Method name Calibration date Instrument name Operator name Status of method on or off RTLock compound name Tabular retention time pressure cal
127. e set for these parameters a 25 psi pulse for 0 5 minutes split flow on at 0 75 minutes at 50 mL min with gas saver on at 2 minutes at 15 mL min The general rules apply for pressure pulsed splitless injections given a particular liner inlet temperature injection volume and solvent the expansion of the solvent is confined to a fraction of the interior volume lt 0 75 of the liner by the pressure applied Figure 4B shows that the back injection port is in split mode at 120 C to remove water back ground with split flow and gas saver set at 15 mL min flow Ix Gas Saver Jv on M Heater 250 er 5 mL min After E min M Pressure 9 4667 psi Total Flow pez mL min Septum Purge Flaw IE mL min Septum Purge Flow Mode Standard Mode Pulsed Splilless Injection Pulse Pressure 5 psi until 0 5 min r Purge Flow to Split Vent so mL mit at 0 75 min gel Apply OK Figure 4A upper panel front injection port Cancel Help Typical pressure pulsed splitless injection parameters for constant flow E Gc Edit Parameters Oven Temperature 45 0 C Split Splitess Inlet M Heater mc I Pressure 30 Total Flow 20 875 mL min Septum Purge Flow mmn Septum Purge Flow Made Standard Gas Saver V On 5 mL min After E min Mode f plit Split Ratio D E fi 5
128. e solvent effect The solvent effect focuses the analytes on the head of the column The oven temperature should typically be set to 210 C below the boiling point of the solvent used Table 2 On the other hand the MS interface temperature should be hot enough to avoid loss of analytes on cold spots The interface should be set to the maxi mum oven temperature for the analysis or 10 C 15 C higher if the upper temperature limit for the column is not exceeded The default method temperature is 280 C the interface tem perature should be optimized as part of method development Finally the rate of auto injection of a sample has been studied for splitless injections It has been found that fast injections such as with the ALS tend to give the most repeatable and non discriminating results Table 2 Boling and Initial Oven Temperatures for Common Solvents Solvent Boiling point Initial oven temperature C C Diethyl ether 36 10 to 25 n Pentane 36 10 to 25 Methylene chloride 40 10 to 35 Carbon disulfide 46 10 to 35 Acetone 56 25 to 45 Chloroform 61 25 to 50 Methanol 65 35 to 55 n Hexane 69 40 to 60 Ethyl acetate 71 45 to 65 Acetonitrile 82 50 to 70 n Heptane 98 70 to 90 Isooctane 99 70 to 90 Toluene 111 80 to 100 Using Pulsed Splitless Injections Pulsed splitless injections are the best way to do splitless injections EPC of the splitless inlet allows for high flow rates initially followed by more typi cal GC
129. e up Hand tighten the reducing nut back into place within the retaining nut and then wrench tighten until very tight Replace the insulation cup Insert a new liner and O ring The single taper liners are installed with the taper down toward the column end of the inlet Hand tighten the insert assembly Figure 2 number 3 Add the Merlin Microseal or proper preconditioned septum and septum nut The molded septum is installed with the hole up Follow the directions supplied with the Merlin Microseal to insure proper installation If the green septum nut is used wrench tighten the weldment and septum nut with the septum nut wrench until the C ring lifts off the top of the green septum nut At this point the inlet should be leak checked Follow the directions in the GC maintenance and troubleshooting section of the manual Column Installation Working with fused silica columns may be danger ous Wear proper eye protection Inspect the column for damage or breakage Unweave 0 5 1 coil of the column from its basket to make it easier to install Push a septum onto the inlet end of the column about 10 cm Put the column nut and appropriate ferrule on the column Cut 5 10 cm off the inlet end of the column Check the cut with a 10x magnifier the cut should be straight not jagged with no column shards within the column If the cut is jagged or shards are inside try again After a clean cut is obtained mark the proper column position wit
130. ead Pressure psi 27 6 87 852 Flow Rate mL n min z 2 7153 7 18 2422 Outlet Velocity cm sec 96 64 649 27 Average Velocity cm sec 50 20 C 150 59 Hold up Time miri E 0 996060 C 0 33202 Outlet Pressure absolute psi 16 2 r Sample Information Figure 4 Method translation software showing scaling RTL Pesticide method scaled to threefold faster method those in the 3 3 minute hold time at the end of the run Gaining Speed with a Small Bore Column In the previous example speed was gained at the expense of resolution In this example speed will be gained while maintaining most of the resolu tion but sacrificing capacity This is done by scaling the original method to a 0 1 mm id column In scaling to columns of a different diameter there are two important considerations that must be obeyed to obtain precise matching to a library or reference method The first is that the stationary phase composi tion must be the same as that used in the original method The second is that the phase ratio of the column being scaled to must be the same as that of the reference method Columns of the same phase ratio have the same ratio of inner diameter to film thickness Because the reference method was developed on a column with 0 25 mm id x 0 25 um film thick ness scaling to a 0 1 mm id column will require a 0 1 1um film thickness A 10 m column of these dimensions was chosen for this exa
131. ed on the electrical charge transfer that occurs when electrons are given up by a molecule during oxidation or absorbed by a molecule during reduction This oxidation or reduction takes place on the surface of a so called working electrode Whether a compound is reduced or oxidized and the speed of the reaction depend on the potential difference between the working electrode and the solution containing the compounds From the activation energies and redox potentials expressed by the Nernst equation reaction speed can be determined The resulting current is proportional to the number of reactions occurring at the electrode which in turn is an indicator of the concentration of the compound of interest at the surface In the detection process three electrodes are used the working electrode in which the reaction takes place the counter electrode which applies the potential difference between mobile phase and the working electrode and the reference electrode which compensates for any change in eluant conductivity see figure 64 The reference electrode readings feed back to the counter electrode in order to keep the potential difference constant during peak elution as current flows through the working electrode 98 Current Optimum potential 04 06 08 10 Potential V Figure 65 Current voltage relationship Electrode materials Flow cell aspects Detector response results from amplification of the electron flow and
132. ed pressure and save the method 4 Validate the retention time lock by injecting the standard at the new pressure and compare the reten tion time obtained to the desired retention time 5 Repeat steps 2 to 4 if necessary A Note on Constant Flow versus Constant Pressure Modes of EPC Operation Many GC chromatographers prefer to use the constant flow mode of EPC operation In this mode inlet pressure increases automatically to maintain constant outlet flow rate as the oven temperature increases during the run Constant flow mode reduces run time and ensures that flow sensitive detec tors see a constant column effluent flow The constant pressure mode of EPC operation is also popular In this mode the pressure remains constant during the run outlet flow will decrease as temperature increases For those wishing to reduce run time in constant pressure mode a higher pressure can be chosen For Retention Time Locking Calibration Pressure Ret Time Run 1 14 2 19 735 Run 2 16 2 18 679 Runa 82 075 Run4 202 zo Run 5 22 2 16 312 Pressure Units Psg al Desired Ret Time Min relock pressure t Ti Max relock pressure Column Compound Name alpha methylstyrene Figure 1 Dialog box used for entering retention time locking calibration data RTL Calibration Data Max departures 0 008 minutes and 0 02 psig PIEI F psig 24 20 19 18 17 16 1
133. elium or vacuum degassing Helium degassing The oxygen effect is most apparent in the analysis of polycyclic aromatic hydrocarbons PNAs with fluorescence detection as shown in figure 50 The less oxygen present in the mobile phase the less quenching occurs and the more sensitive the analysis In general one of three degassing techniques is used on or offline vacuum degassing offline ultrasonic degassing or online helium degassing Online degassing is preferable since no solvent preparation is required and the gas concentration is held at a constant minimal level over a long period of time Online helium and online vacuum degassing are the most popular methods In helium degassing gas is constantly bubbled through the mobile phase reservoir This process saturates the solvent and forces other gases to pass into the headspace above Requires only a simple regulator Several Expensive Evaporation of the more channels can be purged simultaneously volatile components can change without additional dead volume composition over time Oxygen is better purged by vacuum degassing 83 Vacuum degassing In vacuum degassing the solvent is passed through a membranous tube made of a special polymer that is permeable to gas but not to liquids under vacuum The pressure differences between the inside and outside of the membrane cause continuous degassing of the solvent New online degassers with low internal volume 1 ml allow fast
134. emove less polar impurities can be done through solid phase extraction on C18 columns Control and data evaluation Isocratic C Ge pump Auto Column ioe pem I index vacuum sampler compart R degasser ment detector AEL ES Water 40 Chromatographic conditions The HPLC method presented here was used to analyze mono di and trisaccharides as well as sugar alcohols Sample preparation Samples were directly injected Konm Citric acid Lactose Column 300 x 7 8 mm Bio Rad HPXP 9 um 8007 Mobile phase water Raffinose Column compartment 80 C Flow rate 0 7 ml min 600 Detector refractive index Standard 400 Lemonade HPLC method performance 2007 Limit of detection lt 10 ng with S N 2 Repeatability of T T RT over 10 runs 0 05 5 Time min 10 15 areas over 10 runs 2 Figure 30 Analysis of carbohydrates in lemonade Norm B TE 180 4 SIN a n S g Q 160 1 2 pi 9 8 a t 140 8 E 120 4 Standard 100 4 Masc Corn extract 5s Cellbiose vA Sucrose Standard 5 10 15 20 Time min Figure 31 Analysis of carbohydrates in corn extract 4 Official M ethods of Analysis Food Compositions Additives Natuaral Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 AOAC Official M ethod 980 13 Fructose glucose lactose maltose sucrose in milk chocolate AOAC Official M ethod 982 14 Glucose fructose sucrose and maltose
135. ent GC ChemStation software rev A 05 04 Both the GC AED and the GC micro ECD ChemStations contained RTL soft ware for GC ChemStation G2080AA and the Retention Time Locking Pes ticide Library for GC ChemStation G2081AA The GC MS system G1723A used consisted of an 6890 Series GC equipped with an Agilent 5973 mass selective detector MSD The process for retention time locking the GC MS system is described in reference 2 All systems except the micro ECD instrument used 30 m x 0 25 mm id x 0 25 um HP 5MS columns part no 19091S 433 The Agilent micro ECD instrument used 10 m x 0 1 mm id x 0 1 um HP 5 column part no 19091J 141 RTL measurements were made with a solution of dichlorvos methyl chlor pyrifos and mirex each at 10 ppm concentration in acetone All injec tions were 1 uL splitless except for the micro ECD experiments which were 1 uL split 100 1 In all methods inlets were operated at 250 C and detectors at 300 C Method translation requires inlets to be run in constant pressure mode to obtain precise scaling of retention times Thus all methods discussed in the note were run in this mode Results and Discussion Locking GC MS with Other GC Detectors When using selective GC detectors in conjunction with GC MS one prob lem that is encountered is knowing the relationship between retention times on the selective detector and that of the GC MS In GC MS the outlet pressure of the colum
136. ent maintenance requirements limited the effective ness of these interfaces More recently two atmospheric pressure ionization API interfaces electrospray and atmospheric pressure chemical ionization APCT have replaced almost completely thermospray and particle beam techniques These interfaces have a broad range of analyte molecular weights and polarities high sensitivity improved usability and reduced maintenance needs Selection of the appropriate LC MS interface for an application depends on factors such as the polarity molecular weight and thermal lability of the analyte In electrospray effluent is directed through a nebulizing needle into a high voltage field where charged droplets are formed see figure 71 The charged droplets are then dried HPLC inlet _ Skimmers Octopole lt jim n Nebulizer l y Fragmentation zone CID Quadrupole Figure 71 API electrospray LC MS interface 102 Abundance i 8 1 Aldicarb sulfoxide 2 Aldicarb sulfone 3 Methomyl 4 3 hydroxy carbofuran 5 Aldicarb 6 Carbofuran 7 Carbaryl 8 Methiocarb 0 10 Time min 20 Figure 72 Carbamate analysis and as they shrink analyte ions are desorbed The ions are transported to the mass analyzer through a series of vacuum stages and ion focusing elements Electrospray ionization can produce multiply charged ions of macromolecular analytes such as proteins and peptides Because mass analy
137. epro ducible results and loss of sample With the new LVI technique good chromatography can be obtained with injection volumes of 5 to 500 uL or more Table 1 summarizes several common ways to lower detection limits Agilent Technologies Innovating the HP Way Table 1 Approaches to Lowering Detection Limits Concentrate the Sample Column Extraction followed by evaporation of solvent Solid phase extraction SPE Solid phase micro extraction SPME Headspace sampling Purge and trap P amp T sampling Large volume injection LVI Transfer More Sample into the Cool on column COC injection Splitless injection Large volume injection LVI Detectors ELCD Atomic emission detector AED Selected ion monitoring mass spectrometry SIM MS Use More Sensitive or Selective Electron capture detector ECD Electrolytic conductivity detector Decrease System Noise Selective detectors Sample cleanup to reduce interferences Use headspace or purge and trap sampling Decrease column bleed Nitrogen phosphorus detector NPD Flame photometric detector FPD In LVI a large volume of sample is injected The bulk of the solvent is evaporated before transfer of the sample to the analytical column and the start of the analytical sepa ration There are two primary techniques to eliminate solvent PTV and cool on column injection with solvent vapor exit COC SVE COC SVE is most appropriate for clean sample
138. ers a new way of improv ing productivity by eliminating the need to con stantly update compound retention time data whenever a column was trimmed or replaced It also allowed the same methods to be run on multi ple systems with the same retention times The introduction of eMethods further enhanced the portability of these methods Most RTL based methods were established on GC MS systems where a typical head pressure for a 30 m x 0 25 um id column is gt 10 psi With setability to two decimal places four digit pressure setpoints for such columns for example 11 54 psig result in excellent inter instrument and intra instrument RTL precision Amongst the many optimization tasks in GC method development is deciding on the best column dimension to select This can realistically only be determined with full knowledge of the sample characteristics and analysis goals that is components of interest complexity detection limits required and the matrix of the sample Larger diameter columns have the advantages of ruggedness and sample capacity over smaller dimension columns The larger the diameter of the column the less pressure is needed to establish optimal flows However for the most precise RTL one needs the ability to set pressure very precisely With two decimal place precision at low pressures for example 1 28 psig locking a system to a target retention time is less precise for column dimensions such as 0 32 um columns
139. es and times with the calculated pres sures and times for the GC MS method To test the accuracy of using a predicted RTL calibration file for GC MS a real calibration set was measured on the GC MS system The data is shown in the first two columns of table 2 Note The calibration points are spaced 596 apart in pres sure instead of the typical 1096 A GC MS RTL calibration file was con structed with these measured points For each point the locking pressure required to lock the method was cal culated and is shown in column 3 of table 2 The locking pressure is the pressure determined by the RTL software that would make methyl chlorpyifos have a retention time of 16 596 minutes This is determined by entering the pressure and retention time for each point into the Re Lock New Column menu item of the RTL soft ware If the calibration is done cor rectly the locking pressures determined from each point should be very similar as they are in column 3 of table 2 Column 4 of table 2 shows the locking pressures for the same set of runs but determined using the GC MS RTL cal ibration points calculated using method translation The calculated data provide locking pressures that agree well with those based on mea sured data The range in locking pres sures pressure is only from 17 72 to 17 75 psi This range of 0 03 psi corre sponds to only about a 0 006 minute range in the retention time of methyl chlorpyrifos
140. es of cans used for food packaging In canned foods containing a high proportion of fat BADGE tends to migrate into the fatty phase where it remains stable whereas in water it is hydrolyzed BADGE was originally determined to be mutagenic during in vitro tests but a later re assessment using in vivo tests led to a different conclusion While further tests are being performed a maximum concentration of 1 mg BADGE per kg of food has been agreed Sample preparation Extracted with water alcohol 50 50 or n heptane at reflux temperature for six hours Chromatographic conditions A fast separation was developed by using the enhanced specificity provided by the Agilent 1100 Series LC MSD in CID collision induced dissociation mode allowing the detection of BADGE via the molecular ion combined with confirmation using the most abundant fragment ion 24 LC MS conditions Colum Mobile phase A Mobile phase B Gradient Flow rate Injection volume Column compartment Detector lonization mode Nebulizer pressure Dryng gas temp Vcap Fragmentor Scan range Scan speed Zorbax Eclipse XDB C8 2 1 mm x 50 mm 5u 5 mM ammonium acetate in water pH3 acetonitrile 0 min 25 B 5 min 5096 B 300 ul min Tul 40 C UV DAD 210 nm 6 nm ref 360 60 nm 254 nm 6 nm ref 360 60 nm API ES positive 50 psig 350 C 3500 volts 70 volts m z 250 400 2 s scan mAU 2d 04 x23 EA 64 84 104 SPE 8
141. essfully in the analysis of aflatoxins and fatty acids 60 Chapter 5 Sample preparation Sample preparation steps Automation The isolation of analytes from other matrix constituents is often a prerequisite for successful food analysis The broad selection of cleanup and enrichment techniques takes into account the many matrices and compound classes under study Sample preparation for HPLC can be broken down into the following main steps 1 Sampling Collection Storage 2 Cleanup enrichment offline Homogenization centrifugation precipitation hydrolyzation liquid liquid extraction solid phase extraction ultrasonic bath liquid extraction supercritical fluid extraction concentration 3 Cleanup enrichment online Guard columns Online solid phase extraction Gel permeation chromatography GPC 4 Chemical derivatization Precolumn online or offline see also discussion of postcolumn derivatization chapter 9 Manual extraction cleaning and concentration of the sample prior to transfer to the HPLC instrument is time consuming and can drain resources Sample preparation therefore should be automated where possible Nowadays the sample can be fractionated and or derivatized automatically 62 Supercritical fluid extraction SFE systems and automated solid phase extraction equipment also have been interfaced directly to liquid chromatographs Equipment used to auto mate preparation of HPLC sample
142. essure modes Experimental Two 6890 Series GC systems were used Each system was equipped with Electronic pneumatics control EPC e Split splitless inlet 250 C He carrier gas split 80 1 e Automatic liquid sampler e GC ChemStation version A 05 02 e Flame ionization detector FID e 60m x 0 32 mm 0 5 mm HP INNOWax column part no 19091N 216 e Temperature program 80 C 9 min 5 C min to 150 C The inlet pressures flows used are indicated with each chromatogram A third 6890 Series GC was also used This system was equipped with an Agilent 5973 mass selective detector MSD and was used for peak identifi cation The GC MSD chromato graphic parameters used were the same as the GC systems noted above except for the inlet pressures as indicated Results and Discussion GC FID to GC FID Locking Figure 4 shows the original chromatogram GC system 1 obtained from running a styrene sample under the conditions specified in ASTM D 5135 Many of the typical impurities found in styrene are found here The phenylacetylene peak rep resents about 60 ppm The peaks are identified in table 1 pA 2 28 26 24 22 20 The sample was then run at four other pressures to collect the five data pairs for RTL calibration Because this method was run in con stant flow mode the pressures entered into the RTL software were the initial pressures The o methyl styrene peak peak 10 was chose
143. ethod was 7 9 psi and was entered into the GC MSD Figure 8 shows the resulting matched chromatograms from the GC FID and GC MSD As seen in table 3 the reten tion times are now closely matched within 0 02 minute Figure 9 shows the MSD first choice of library search result of the impu rity that created the shoulder on the front side of the Phenylacetylene peak RTL ensured that this shoulder remained separated on the MSD system and eluted at the same time Table 2 GC FID Retention Times Before and After Locking for Styrene Impurities Constant Flow Conditions Chromatograms Shown in Figures 4 5 and 6 Original Run Scouting Run Locking Run GC 1 Column 1 GC2 GC1 GC 2 Column 2 GC2 GC1 GC 2 Column 2 Component 18 2 psi Before RTL 18 2 psi After RTL 19 0 psi Ethylbenzene 10 318 0 340 10 658 0 020 10 298 p Xylene 10 616 0 333 10 949 0 026 10 590 m Xylene 10 858 0 337 11 195 0 022 10 836 i Propylbenzene 11 985 0 359 12 344 0 005 11 990 o Xylene 12 533 0 345 12 878 0 012 12 521 n Propylbenzene 13 360 0 364 13 724 0 016 13 376 a Methylstyrene 17 778 0 321 18 099 0 002 17 776 Phenylacetylene 18 806 0 275 19 081 0 040 18 766 B Methylstyrene 20 248 0 310 20 558 0 006 20 242 Benzaldehyde 24 097 0 279 24 376 0 069 24 028 Average A 0 326 0 028 Used in locking calculation Ju GC Method Translation Criterion C Translate Only C Best Efficiency C Fast Analysis None Speed gain 1 00000 Original Method Trans
144. etic colors using HPLC and diode array detection at 190 950 nm Agilent Application Note 5964 3559E 1995 A Herrmann et al Rapid control of vanilla containing products using HPLC J Chromatogr 1982 246 313 316 Official M ethods of Analysis W Horwitz Ed 14th ed AOAC Arlington VA 1984 secs 12 018 12 021 H Malisch et al Determination of residues of chemotherapeutic and antiparasitic drugs in food stuffs of anomaly origin with HPLC and UV Vis diode array detection J Lig Chromatogr 1988 11 13 2801 2827 EC Guideline 86 428 EW G 1985 M H Thomas J Assoc Off Anal 1989 72 4 564 Farrington et al Food Additives and Contaminants 1991 Vol 8 No 1 55 64 Lebensmittel und Bedarfsgegenstandegesetz Paragraph 35 Germany me A 15 16 17 18 19 20 21 22 23 24 W Specht Organochlor und Organo phosphor Verbindungen sowie stickstoffhaltige sowie andere Pflanzenschutzmittel DFG M ethoden sammlung 1982 19 A new approach to lower limits of detectionand easy spectral analysis Agilent Primer 5968 9346E 2000 R Schuster A comparison of pre and post column sample treatment for the analysis of glyphosate Agilent Application Note 5091 3621E 1992 A G Huesgen R Schuster Analysis of selected anions with HPLC and electrochemical detection Agilent Application Note 5091 1815E 1991 Determ
145. etonitrile 53 Voltages Scan Threshold Sampling Stepsize Drying gas Nebulizer Vcyl 5500 Vend 3500 Vcap 4000 CapEx 150 400 1800 m z 150 1 0 15 amu nitrogen 150 C gas nitrogen 20 psi The Agilent 5989B M S engine was equipped with an Iris Hexapole lon Guide MS data was used for further evaluation Some of the tryptic mass fragments of the phytochrome are signed As an example figure 42 shows two mass spectra jris 160000 140000 77 S g 120000 3 100000 3 80000 60000 40000 20000 40 50 60 70 80 90 100 110 Time min Figure 42 Capillary LC MS of a phytochrome tryptic digest 17 5 pmol total ion chromatography TIC 415 4 796 6 10000 T12 MW 828 5 6000 T58 MW 2387 2 u 8000 5000 6000 g 4000 E 3000 4000 829 7 3900 1194 7 2000 1000 l d 450 550 650 750 850 m z 500 700 900 1100 1300 m z Time min Time min Figure 43 Mass spectra of T12 and T58 54 55 Part Two The Equipment Basics An overview of the hardware and the software components needed for successful HPLC and an introduction to the analytical techniques that have become routine in food analysis Chapter 4 Separation in the liquid phase Separation mechanisms Reversed phase materials lon exchange materials Liquid chromatography offers a wide variety of separation modes and mobile phases for optimizing your separation system Sta
146. extracted ion chromatograms for characteristic ions of each possible compound are used to determine the identity of suspect compounds This screening method minimizes false negatives even in dirty samples by using element selectivity and time in the initial screen With element selective detection all compounds containing chlorine phosphorus nitrogen etc are detected Even if a detected heteroatomic compound is not in the table its presence is known and it can be marked for fur ther GC MS evaluation By using GC MS for confirmation false positives are also minimized The RTL Pesticide Library method is a good example of a method in which a substantial investment of time and material has been made As with many methods intended for use in multiple laboratories it would be desirable to be able to scale the method for use in different situations of sample type and instrument setup Because the method relies on the measured retention times of 567 com pounds it would be impractical to re measure all the retention times whenever the method is modified for example to increase its speed Method translation is a calculation technique developed at Agilent Technologies that allows a capillary column GC method to be translated to different chromatographic condi tions The technique calculates the required changes in inlet pressure and oven temperature ramp rates and hold times required to maintain peak elution order
147. f the quality of the detector the LOD or LOQ remains a function of peak height This height can sink if the peak is allowed to disperse within the surrounding liquid in the flow path AII parts of the flow path in front of the detector therefore must be designed to limit broadening and flattening of the response A minimum of narrow capillaries between injector and column and from column to detector helps keep dead volume low With low injection volumes separation efficiency of the column can be utilized to the maximum thereby improving peak height In other words the lower the column volume the lower the peak volume eluted Other factors that influence peak dispersion include pump performance degassing efficiency capacity factor k and column particle size Any improvements can be registered by calculating the S N of the analyte Indeed the noise of the detector should be tested regularly in this way to ensure that performance is maintained Dead volume of the complete injection system can be determined by first injecting a tracer mobile phase additive into the flow path with the column disconnected and then recording the time this additive takes to reach the detector at a particular flow rate The flow cell volume of the detector should be as low as possible whereas its pathlength should be as long as possible according to Beer s law Maximizing analyte response is not sufficient to ensure good results however since the level
148. fatty acid composition of chro matographic peaks Full scan methods allow easy identifi cation at the low nanogram level If more precise quantitation is required selected ion mode SIM can be used to obtain detection limits at the low picogram level Polar and semipolar compounds up to Data analysis for complex heterogeneous 150 000 daltons can be analyzed Highly mixtures of multiply charged analytes is sensitive Strong molecular ions not straightforward M atrix can interfere Fragments depending on in source CID with the ionization process parameters Refractive index RI detection is based on the difference in RI between the solution in the sample cell and the pure mobile phase solution in the reference cell Because the composition of the eluents must remain fixed throughout the analysis this detector is not suitable for gradient analysis Four main types of RI detectors are available deflection according to Snell s law reflection according to Fresnel s law interference and Christiansen effect The first which uses the dual cell design is by far the most popular However the nearly designed Agilent 1100 Series refractive index detector allows detection limits to the low ng range Because RI detectors lack sensitivity and exhibit a tendency to drift owing to temperature changes they are used prima rily in the analysis of carbohydrates and nonaromatic acids Universal detector Low sensitivity no gradient operation 104
149. from the RTL runs Table 3 RTL Data Pressure Retention Deviation Run psi time mins seconds RTLOCK1 D 3 01 11 212 6 018 RTLOCK2 D 3 38 11 166 3 264 RTLOCK3 D 3 76 11 112 0 000 RTLOCK4 D 4 14 11 070 2 508 RTLOCK5 D 4 51 11 020 5 508 Maximum deviation 6 018 seconds Correlation co efficient 0 999 Even though pressure setpoints used for RTL calibration need only be to be to two decimal places the ability to precisely set locking pressures based on the calibra tion curve requires setability to the third decimal place Figure 1 shows the calibration curves correspond ing to the linearity metrics summarized in Table 2 Calibration RT before Relocked ART when linearity r relocking RT relocked 0 997 5 809 5 876 0 008 0 997 7 676 7 146 0 007 0 997 7 893 7 959 0 009 0 997 8 486 8 556 0 006 0 997 9 621 9 692 0 010 0 996 9 671 9 746 0 006 0 996 11 041 11 116 0 003 0 996 11 308 11 383 0 005 0 996 12 719 12 799 0 009 0 997 12 774 12 849 0 006 0 995 14 168 14 277 0 008 0 995 14 201 14 306 0 008 0 996 14 686 14 807 0 008 0 996 16 890 17 074 0 014 0 995 16 903 17 082 0 016 0 997 17 508 17 709 0 016 4500000 4000000 3500000 Naphthalene 3000000 Acenapthylene Acenaphthene Fluorene Phenanthrene Anthracene 2500000 2000000 Fluoranthene Pyrene Chrysene 1 x Response 1500000 Benz a anthracene Benz b fluoranthene Benz k fluoranthene Benz a pyrene x Indeno 1 2
150. gs in eggs milk and meat is based on reversed phase chromatography and multisignal UV visible diode array detection UV DAD UV spectra were evaluated as an additional identification tool Sulfapyridine taz 12 2 min match 997 Pyrazon taz 9 min match 998 offset 250 300 350 400 Wavelength nm 250 300 350 400 Wavelength nm Standard w Time min 20 m 1 metronidazol 5 pyrazon 9 3 ethopabat 2 meticlorpindol 6 ipronidazol 10 benzothiazuron 3 sulfapyridine 7 chloramphenicol 11 nicarbazin 4 furazolidone 8 N acetyl metabolite of 3 Figure 11 Analysis of residues in an egg sample Identification through spectra comparison 9 H Malisch et al Determination of residues of chemotherapeutic and antiparasitic drugs in food stuffs of anomaly origin with HPLC and UV Vis diode array detection J Liq Chromatogr 1988 11 13 2801 2827 14 10 EC Guideline 86 428 EWG 1985 17 Tetracyclines Tetracyclines are used worldwide as oral or parenteral medication in the form of additives in animal feed In food producing animals these drugs exhibit a high degree of activity toward a wide range of bacteria 1 Sample preparation After homogenization or mincing and addition of mineral acids to dissociate tetracyclines from proteins the samples were extracted using liquid liquid extraction followed by degreasing and or deproteinization purification and 12 Sample preparation 1g sample wa
151. h the septum Figure 3 The septum will hold the column nut and ferrule in place Place the column on the column hanger Insert the column nut into the inlet reducing nut and finger tighten Wrench tighten the column nut The column should be stationary in the ferrule Carefully slide the septum down away from the nut without disturbing the column positioning The septum can be left in place if desired Using the GC keypad or the MS ChemStation software input the Table 1 Head Pressures and Calculated Flowrates for a Splitless Inlet at an Oven Temperature of 25 C with the Outlet Pressure set to Vacuum Column id Length Head pressure mm meters psi cm s 0 20 12 6 0 57 0 20 25 15 0 39 0 20 50 28 0 28 0 25 30 6 2 36 0 32 30 3 4 50 0 32 50 5 5 34 Column hanger position Marking the column position Capillary column 4 Capillary inlet 2 C ma Inlet reducing nut 3 Ferrule Column nut Septum Capillary Septum column Figure 3 Proper installation of capillary columns in a capillary inlet Column Dimensions set Outlet Pressure to Vacuum and Column Flow between 1 and 1 5 mL min helium Table 1 The Split Vent Flow should be approximately 50 mL min These parameters are accessed through the inlet and column screens Place the detector end of the column into a beaker of water and check for bubbles to show helium flow Heat the injection port When the
152. he furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Microseal is a trademark of the Merlin Instrument Company Swagelok is a registered trademark of the Swagelok Complany Snoop is a registered trademark of the Nupro Company Vespel is a registered trademark of E I du Pont de Nemours Co Inc X Acto is a registered trademark of the Hunt Corporation Agilent Technologies Inc 2003 Printed in the USA November 14 2003 5988 9944EN Agilent Technologies HPLC for Food Analysis A Primer Copyright Agilent Technologies Company 1996 2001 All rights reserved Reproduction adaption or translation without prior written permission is prohibited except as allowed under the copyright laws Printed in Germany Www agilent com chem September 01 2001 Publication Number 5988 3294EN Agilent Technologies A Primer HPLC for Food Analysis The fundamentals of an alternative approach to solving tomorrow s measurement challenges Angelika Gratzfeld H sgen and Rainer Schuster Acknowledgements We would like to thank Christine Miller and John Jaskowiak for their contributions to this primer Mrs Miller is an application chemist with Agilent Technologies and is responsible for the material contained in chapter 5 Mr Jaskowiak who wrote chapter 7 is a product manager fo
153. he inlet system The procedures and result are shown in Figure 9 Solve the inlet leak problem by tightening the column fittings base gold seal fitting split vent trap housing septum nut and the latch to the SSL inlet Result These actions don t work Replace the inlet septum and the liner O ring User document search capability makes it easy to find instructions on how to change the con sumables including the inlet septum and liner O ring Also detailed consumable information including part number helps you select the right part see Figure 10 Run the SS inlet pressure decay and leak check tests Result Passed See Figure 11 Run the FID checkout test to confirm that the GC system is operating properly Before the detector checkout test can run you have to install the appropriate consumable parts that is an evaluation column HP 5 30 m x 0 32 mm x 0 25 um p n 19091J 413 an FID performance evaluation checkout sample p n 5188 5372 is also needed The software will prompt you when you are required to per form a task or answer a question When the FID detector checkout test is finished restore the instrument to normal operating conditions Result Passed See Figure 12 Run trace level aromatic sample The expected result is obtained as shown in Figure 13 after diagnostics and troubleshooting The GC system is fixed Column INNOWax 60 m 320 um 0 5 um pA 4 R 1 Benzene 4 54 2 Toluene j 3 Et
154. he raw data files can store more than just signal data A binary check sum protected file stores instrument parameters system pressure temperature flow and solvent percent as well as all aspects of the analytical method including integration events calibration settings and a date stamped logbook of events as they occurred dur ing the run Additionally with spectral libraries compounds can be identified not only on the basis of their elution pro file but also according to their spectral characteristics Such procedures can be fully automated to reduce analysis time and user interaction F1 Heip F3 Recali Figure 77 Maintenance and diagnosis screen 115 A single PC running the appropriate chromatography soft ware can process data from several detectors simulta neously This feature is particularly useful in analyses in which sensitivity and selectivity must be optimized to differ ent matrices and concentrations For example in the analy sis of polynuclear aromatic hydrocarbons UV absorbance and fluorescence detection are applied in series The PC displays graphically the chromatographic signals and spec tra enabling detailed interpretation of the data Software purity algorithms can be used to help determine peak homo geneity even for coeluting peaks Flexible software programs can report data in both stan dard and customized formats For example some chroma tography software can be progr
155. his resulted in a calculated column length of 10 5622 m A new set of cal culated RTL calibration points were calculated using 10 5622 m as the length of the 0 1 mm column The results are shown in table 6 Difference min o rm 3 Pe e e e 5 e c Re 0 1 0 15 0 2 Retention Time min Figure 6 Difference plot of RTL Pesticide Library GC FID retention times divided by 3 minus 3x GC AED retention times for 36 compound subset of the library GC Method Translation Column Length Internal Diameter Film Thickness Phase Ratio C 10 100 0 C Unlock 0 100 250 0 Carrier Gas Enter one Setpoint Head Pressure psi Flow Rate mLn min Outlet Velocity cm sec Average Velocity cm sec 50 20 Hold up Time min 0 996060 Helium Unlock 58 514 0 7027 172 31 50 20 0 332020 Outlet Pressure absolute psi 16 2 Ambient Pressure absolute psi 14 696 14 696 r r Oven Temperature 3 ramp Program L C mn C mi 150 200 280 mas Temp Time Rate Temp L C mn C mi 70 0 667 75 000 150 0 000 9 000 200 0 000 24 000 280 3 333 Figure 7 Method translation software showing scaling RTL pesticide method scaled to a threefold faster method on a 10 m x 0 1 mm id column Table 7 shows a comparison of lock ing
156. hod Used to Screen for 567 factor of 3 Pesticides and Suspected Endocrine Disrupters After being locked the three peaks in Hewlett Packard Company Appli the 3x micro ECD method had reten cation Note 228 402 Publication tion times of 1 924 5 533 and 9 963 5967 5860E April 1998 minutes respectively These values are very close to the RTL Pesticide 2 M Klee and V Giarrocco Pre dictable Translation of Capillary GC Methods for Fast GC Hewlett Packard Company Appli cation Note 228 373 Publication 5965 7673E March 1997 Library retention times for the three compounds divided by 3 1 932 5 532 and 9 949 The fact that the largest difference between the scaled table and the 3x micro ECD method is only 0 014 minute again demonstrates the 3 V Giarrocco B D Quimby and precision of retention time matching M S Klee Retention Time Lock achievable with the scaling technique ing Concepts and Applications Hewlett Packard Company Appli cation Note 228 392 Publication 5966 2469E December 1997 described here Conclusions 4 Capillary Column Method Transla Using method translation combined tor user contributed software with retention time locking provides a free download from means of extending the usefulness of www hp convgo mts existing capillary GC methods The ability to precisely scale a method to meet the needs of different samples and instrument types greatly reduces the effort requi
157. hylbenzene 2 44 4 M oxylene 434 Concentration about 0 2 ppm anm inet T 4 24 h 1 A perl 41 p meet Theresponse of ethylbenzene is reduced and m oxylene is lost 4 04 dont 7 Inlet leakage may be the reason Intuitive help for diagnostics f Vea Pn is available from LMD 3 9 M d ud 4 5 6 7 8 8 Figure7 Trace level peak lost or response reduced on 7890GC with FID and capillary column 6 Test Name Front Spit Spitless Inlet 7890 Leak Check Description Use the Solit Splitless Leak Check to quickly test for leaks in the inlet after z performing maintenance or if a gross leak is suspected It provides a fast result Approx Time 5 min and requires no disassembly It is not as definitive as the pressure decay test Status Running Test Procedure Inspect the GC configuration Establishing control of the selected GC Downloading Leak Check method to selected GC 7890 SS Inlet Leak Check Total flow 14 413 Column flow 4 000 Leak rate 7 413 mL min Test complete Rerun Leak Check Entering Prep Run state System vail attempt to establish pneumatic condtions Restoring original method and disconnecting from GC Test procedure Test result shows there is leakage in inlet system Categor Source Time Message Front Split Splitless Inlet 78 2 3 2008 3 31 3 Establishing contro of Instrument 7890 U510652005 Front Split Splitless Inlet 73 2 3 2008 3 31 4 Saving original Method Front Split Splitless Inlet 78 2 3 2
158. ial damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Printed in the USA May 15 2008 5989 8613EN i Agilent Technologies Capillary Flow Technology for GC MS A Simple Tee Configuration for Analysis at Trace Concentrations with Rapid e ra 2 Backflushing for Matrix Elimination e e e p 1 e p 2 e Application e o e e e Environmental Drug Testing and Forensics e e e e Author chromatographic peaks of consistent width time and allows optimization of MS cycle times to meet Harry Prest either qualitative or quantitative requirements Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Abstract Capillary Flow Technology devices offer the potential to enhance GC MSD operation and robustness In operation they can allow rapid service of the GC column and inlet including liner and septum without venting or subjecting the MSD to air In terms of robustness late eluting com poundscan be removed from the column by backflush ing which forces components to retreat through the column into the injection port before they damage the MSD source or compromise the next analysis This leads to higher analytical integrity as both the column phase and the MSD can be protected This application describes a simple arrangement for Capil
159. ibration table Maximum deviation RTLock curve equation and correlation coefficient data Locked retention time infor mation file name acquisition date instrument name and operator name Report date MultiVu F XHPCHEMNSIXMETHODSSRTLPESTB MNXRTLREP TXT Oy x EB Fie Edit Search Window Retention Time Locking Data Report Retention Locked Method Retention Locked Cal Date Instrument Operator Method Lock is currently On Compound Chloropyrifos methyl Retention Time Calibration psi Time File Pressure min RTLOCK1 D 12 66 17 345 RTLOCK2 D 14 24 16 940 RTLOCK3 D 15 82 16 567 RTLOCK4 D 17 40 16 214 RTLOCK5 D 18 98 15 885 Maximum Deviation RTL Curve R Terms of Curve Fit Constant 1 35686e 007 Linear 1 20506e4006 Quadratic 27275 2 Coefficient Locked Retention Time informati Retention Locked File cq Date Instrument Operator Spec Xcor 000 000 000 000 000 Herer 46 650 seconds 2 73e 004 A A 1 21e 006 A 1 36e 007 on la x F NHPCHEHN15NMETHODSNRTLPESTB H 11 May 1998 GC MS Ins 7 35 pm Deviation Seconds 46 650 22 356 0 000 21 192 40 968 0 999997 Good Fit HP Demo RTLOCK3 D 11 May 1998 HP Demo 7 35 pm Measured Retention Time 16 567 Pressure 15 82 pei Locked Retention Time 16 596 Pressure 15 69 psi Locked RT and Pressure Within Calibrated Limits Leck run spectrum XCor 1 0000 Rep
160. ighly retained components that elute after the target compounds of analytical interest by reversing the carrier flow direction through the column in what is called backflushing With the oven temperature elevated and the flow reversed these very high boiling interferences can be pushed off the column into the split vent and thereby prevent degradation of the column phase or the detector A schematic representation of the arrangement that makes this possible is shown in Figure 1 Agilent Technologies During Split Vent GC Z mL min run S SL Inlet C After Split Vent GC ji i S SL Inlet 2 psi j Figure 1 Schematic of QuickSwap arrangement Every new approach has a downside and for QuickSwap it is the additional makeup flow required to purge the QuickSwap device during analysis which dilutes the signal in the GC MSD This is not an issue for many users since the sensi tivity of the MSD is usually more than adequate However analysis at trace concentrations has more stringent requirements and maintaining a signal closely comparable to that of a single contin uous column is essential Another CFT configuration for GC MSD applica tions designed specifically for trace GC MS analy sis where customers do not wish to surrender signal is possible using the QuickSwap or any of several other CFT devices In this alternate config uration the CFT device is located in th
161. ilent com chem The author Chris Sandy is a GC MS Applications Specialist for Agilent Technologies in the UK Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2004 Printed in the USA January 30 2004 5989 0595EN Agilent Technologies Optimizing the Agilent Technologies 6890 Series GC for High Performance MS Analysis e e Technical Overview eo o e 0 o o ee 050 amp o e o e e e Author The Carrier Gas Line Linda Doherty Agilent Technologies Inc Life Sciences and Chemical Analysis 5301 Stevens Creek Blvd Santa Clara California 95051 USA Abstract Trace level GC MS analysis requires a system that is performing at its best Without a properly optimized GC the mass spectrometer may not give the sensitivity expected In other words when more of the sample gets from the injection port to the ion source the more likely a detector will produce a signal Also if the chemical noise from the GC is too high the signal to noise ratio and ability to detect small analyte concentrations will be reduced This note is a how to guide for improving the GC performance This will in many instances improve the overall performance of a GC MS system
162. iling components because volatile compounds evaporate with the solvent as shown in Figure 2A with PTV However for volatile sam ples that are too dirty for COC SVE sampling the PTV inlet can be cryocooled below ambient tempera ture resulting in much better recovery of the low boilers Figure 4 compares the recovery of C10 using PTV with the inlet temperature at 40 C and 10 C during the injection step There is 100 recovery of C10 with cryocooling Much less solvent was eliminated at the lower temperature which helped retain the early eluting compounds It is important to determine carefully the best injection parameters for LVI methods Figure 5 shows the improve ment in peak shape obtained for a sample containing pesticides when the initial PTV temperature vent flow and injection delay are opti mized Inlet temperature and vent flow influence the speed and extent of solvent removal The chro matogram in figure 5B indicates that insufficient solvent was vented thereby increasing the chance of carry over degrading peak shape and increasing ghost peaks Conclusion GC using LVI is useful for lowering detection limits It has broad applica bility for applications that require more sensitivity than can be obtained with standard injection volumes PTV inlets are appropriate for LVI of late eluting samples for dirty samples and for any cold split splitless injec tion Total automation of tempera ture pressur
163. in Procymidon 7 Chlorpyripho ethyl Paprika Spain Paprika Turkey 10 20 30 40 50 Time min Figure 20 Analysis of pesticide residues in two paprika samples 21 Carbamates Sample preparation none Column 250 x 4mm C18 phase from Pickering 5 um water methanol M eOH 88 12 at 2 min 12 MeOH at 42 min 66 MeOH at 46 min 66 MeOH at 46 1 min 100 MeOH at 49 min 100 M eOH Flow rate 0 8 ml min Column compartment 37 C Injection volume 10 yl standard Fluorescence detector Excitation wavelength 230 nm or 330 nm Emission wavelength 425 nm Photomultiplier gain 12 Response time 4 s Derivatization reagent pump flow rate for hydrolization agent 0 3 ml min NaOH flow rate for derivatization agent 0 3 ml min OPA Mobile phase Gradient HPLC method performance Limit of detection 100 ppt S N 22 Repeatability ofRToverl runs 0 196 of areas over 10 runs 0 5 5 96 Chromatographic conditions The HPLC method presented here was used for the direct analysis of carbamates in water with postcolumn derivatization Fruits and vegetables must be extracted at neutral pH with water prior to HPLC analysis F 6 D Sample A 15 0 15 20 25 30 3 40 45 Time min F 55 Sample B 5 10 ll 10 15 20 25 30 35 40 45 Time min Sample A Sample B 1 butocarboxim sulfoxide 9 ethiofencarb sulfone 5 oxamyl 16 3 ketocarbofuran 2 aldicarb sulfoxide 14 butocarboxim 8 thiofanox sulfoxide 18 c
164. ination of triglycerides in vegetable oils EC Regulation No L248 28ff L M Nollet Food Analysis by HPLC New York 1992 A G Huesgen R Schuster Analysis of selected vitamins with HPLC and electrochemical detection Agilent Application Note 5091 3194E 1992 O Busto et al Solid phase extraction applied to the determination of biogenic amines in wines by HPLC Chromatographia 1994 38 9 10 571 578 Sensitive and reliable amino acid analysis in protein hydrolysates using the Agilent 1100 Series Agilent Technical Note 5968 5658E 2000 R Schuster Determination of amino acids in biological pharmaceutical plant and food samples by automated precolumn derivatisation and HPLC J Chromatogr 1988 431 271 284 Capillary Liquid Chromatography with the Agilent 1100 Series M odules and Systems for HPLC Agilent Technical Note 5965 1351E 1996 25 26 27 28 29 30 33 34 35 36 37 R W Frei and K Zech Selective sample handling and detection in HPLC J Chromatogr 1988 39A D R Gere et al Bridging the automation gap between sample preparation and analysis an overview of SFE GC GC M SD and HPLC applied to several types of environmental samples J Chromatogr Sci 1993 J uly M A Schneidermann et al J Assoc Off Anal Chem 1988 71 815 R Schuster A comparison of pre and postolumn sample treatment for the analysis of gl
165. iner 1 Insulation 8 Capillary inlet body 9 Retaining nut 10 Inlet base seal 11 Washer 12 Reducing nut I 13 Insulation 14 Insulation cup 15 Ferrule 16 Column nut Figure 2 Capillary inlet assembly Electronic pneumatic control EPC is an integral part of the Agilent 6890 Series GC The EPC ver sion of the Agilent 6890 is required when using an Agilent 5973 MSD The manual version of the Agilent 6890 will not work with the Agilent 5973 MSD Electronic control gives the best repeatability in retention time and area counts Using the elec tronic pulsed splitless mode allows for complete transfer of larger volume injections up to 5 uL onto the column Even larger volume injections 75 uL may result in more inlet maintenance especially with dirty samples The Agilent 6890 does have an inlet that accommo dates injections up to 250 uL It is called the pro grammable temperature vaporizer PTV It works with the ALS to deliver large volume injections LVI The inlet works by venting the solvent before analysis The analyte is trapped and concentrated It is then delivered to the column as a single plug The list of splitless inlet consumables is Molded Septa 11 mm red 25 pk p n 5181 3383 Or High Pressure Merlin Microseal starter kit p n 5182 3442 Merlin Microseal septum p n 5182 3444 Merlin Microseal septum nut p n 5182 3445 Liner single taper deactiv
166. ion of an external or internal stan dard calibration procedure ensures accuracy in food analysis The precision of a method is the degree of agreement among individual test results when an analysis is applied repeatedly to multiple samplings Precision is measured by injecting a series of standards and then calculating the rela tive standard deviation of retention times and areas or peak heights Precision may be measured at three levels repeat ability intermediate precision and reproducibility Repeat ability is associated with an analysis performed in one laboratory by one operator using a single piece of equipment over a relatively short time period Intermediate precision is 122 the long term variability of the measurement process for a method performed within one laboratory but on differ ent days Reproducibility applies to an analysis performed in more than one laboratory Any HPLC method used in food analysis should be tested for both repeatability and reproducibility The precision of a method is strongly influenced by the performance of the HPLC instrumentation Repeatability of flow rates gradient formation and injection volumes can affect precision as can response stability of the detector aging of the column and temperature stability of the column oven The equipment should be inspected on a regular basis using the test methods recommended by the supplier to ensure reliability high performance and good analytical re
167. ion points and is really best thought of as a simple tee reminiscent of glass Y or T connectors and will be referred to as a CFT device or CFT tee from here forward One of the 15 m HP 5ms columns was connected at the uppermost position on the CFT tee and the other end through the transfer line into the MSD as usual The other 15 m HP 5ms column was con nected to the midpoint of the CFT device and the front injection port In detail the arrangements were as follows The CFT tee was attached to the forward position on the mounting hardware on the right side in the GC oven The 1 m long section of guard column was wound on a spare column cage and hung on the column hanger in the back of the oven This could simply be added to one of the 15 m HP 5ms column cages to avoid the extra cage Using a Vespel graphite ferrule one end was connected to the back injection port and the other end to the lowest connection of the CFT device with a SilTite ferrule and nut The other two CFT tee connections were sealed with CFT blanking plugs and the back injec tion port was pressure tested as described in the 7890A Advanced User Guide part number G3430 90015 One of the 15 m columns was then hung on the cage carrying the 1 m column and installed with one end through the MSD transfer line Since this column column 2 can be expected to have a rather long life as it will be protected by the upstream column a SilTite ferrule is recom me
168. is flow rate is optimal for capillary columns with an internal diameter of 300 pm 52 Sample Capillary column Mobile phase Gradient Flow rate Column compartment Injection volume Detector tryptic digest of phytochrome from oat seedlings 7 pmol ul 300 um x 25 cm C18 A 0 025 TFA in water B 0 02 TFA in ACN 0 35 B min 100 ul min split to 4 yl min 25 2C 2 5 ul UV VWD wavelength 206 nm with a 35 nl 8 mm flow cell HPLC method performance Limit of detection Repeatability of RT over 10 runs areas over 6 runs 1 pmol 0 7 96 196 Chromatographic conditions Capillary HPLC with UV and MS detection has been used in the analysis of phytochrome protein from dark grown oat seedlings Figures 41 42 and 43 show the UV and total ion chromatogram together with two mass spectra of selected fragments The Agilent 1100 Series LC system was used without mixer All tubings were as short as possible with an internal diameter of 75 120 pm id Sample preparation The extracted protein was reduced and alkylated prior to digestion with trypsin mAU 1204 1004 80 1 60 1 40 4 20 40 60 80 100 Time min Figure 41 Capillary LC MS of a phytochrome tryptic digest 17 5 pmol UV trace Control and data evaluation l Binary Mass pump Flow Auto Column spectrome vacuum split sampler compart terorVWD gt degasser device ment detector ZS Water Ac
169. its subsequent conversion to a signal Extremely low currents representing analyte quantities in the picogram range and below can be measured with today s advanced electronics Although electrochemical detection can detect only those substances that can be electrolyzed this limitation is actually an advantage when applied to complicated food matrices because it improves selectivity To determine the optimum working electrode potential the relationship between detector response current and potential applied voltage must be plotted for each compound as a current voltage CV curve as shown in figure 65 At a potential less than E1 oxidation cannot occur because the supply of energy is insufficient Increasing the potential to E1 2 will electrolyze 50 of all molecules at the surface of the electrode Maximum response requires a potential just above E2 This potential is known as the limiting current because any further increase in voltage will limit detection by raising noise Several materials are used in working electrodes the most common of which is glassy carbon These materials also include gold for sugars and alcohols platinum for chlo rite sulfite hydrazine and hydrogen peroxide silver for halogens copper for amino acids mercury in reductive mode for thiosulfate and combined mercury gold in reductive mode for nitrogenous organic compounds Numerous cell designs have been described in the literature The majorit
170. l contributed to the ability to measure compounds at decreasing levels Concentrating samples is an estab lished approach for increasing method sensitivity For many environ mental analysis this involves extrac tion followed by solvent evaporation which generates large volumes of waste solvent and increases sample preparation time significantly Recent advances such as supercritical fluid extration SFE solid phase extrac tion SPE solid phase microextrac tion SPME and pressurized fluid extraction are making inroads on liquid liquid extractions but these still involve additional sample prepa ration time Detection limits can be reduced by lowering system background and interferences This can be done through sample cleanup such as florisil column chromatography SPE and SPME and by using selective detectors that do not respond to the background How ever the cleanup requires time and the need for reduced limits has surpassed the sen sitivity of even the best detectors Large volume injection LVI is another approach to lower detection limits The typical injection volume for capillary column analysis is 0 5 to 2 uL Agilent 6890 Series and 5890 gas chromatographs GCs allow approxi mately two times the normal injection volume up to 5 uL depending on the solvent using pulsed splitless injec tion Injecting still larger volumes with standard techniques can lead to contamination of the system irr
171. l is 10096 B without degassing for standard needed for mobile phase compositions applications At low pressure mixing of the gradient solvents occurs early in the flow path before the pump applies pressure as in the two examples below Less expensive than gradient elution Can Degassing is necessary for highest mix more than two channels Low mixing reproducibility noise without a dedicated mixer Pump designs for gradient operation Low pressure gradient In food analysis pump performance is critical In the Agilent 1100 Series pump examples we describe a low pressure gradient system and a high pressure gradient system both of which perform according to food analytical requirements The former has a single dual piston mechanism for low pressure gradient formation whereas the latter has a double dual piston dnm mechanism for high pressure gradient formation After A passing the online vacuum degasser the mobile phase enters the first pump chamber through an electronically activated vum inlet valve see figure 48 Active valves resolve the problem V aros IE gu of contaminated or sticky ball valves by making the pump goes easy to prime Output from the first piston chamber flows mm through a second valve and through a low volume pulse DEUM dampener with pressure transducer into a second piston eed n SEE chamber Output from the second chamber flows onto the us sampling unit and c
172. lary Flow Technology devices that provides ventless maintenance features with highly accelerated backflushing and minimal losses in the MSD signal This solution supports GC analysis in con stant flow mode with pressure pulsed injections and is recommended for all MSD users in both electron impact or chemical ionization modes including those with diffu sion pump systems Introduction The introduction of Electronic Pressure Control EPC was a major advance for GC and especially GC MS analysis EPC allowed development of the constant flow mode of analysis which generates Also splitless injections gained pressure pulsing or ramped flow modes which lowered the ana lytes residence time in the hot injection port and confined the expansion of the injection solvent avoiding overfilling of the liner The power of this approach lead to continued evolution of EPC tech nology with the present state of the art represented in the new 7890A GC The recent addition of Capillary Flow Technology CFT devices has reinvigorated and recast Deans switching and other pressure control approaches to GC analysis One such CFT device the Quick Swap 1 3 provides two important capabilities to GC MS 1 The ability to service and or replace the entire analytical column or the injection port liner and septum without venting the MSD yet still retaining high vacuum integrity 2 The ability to remove from the column late elut ing h
173. lated Method Column Length M B0 Internal Diameter w 320 Film C Unlock Thickness C 0500 Phase Ratio 160 0 Carrier Gas Unlock 8 443 Enter one Setpoint Head Pressure psi mL n min cm sec cm sec min 18 200 Flow Rate 1 9611 1 2122 48 44 28 53 3 50562 Outlet Velocity Average Velocity Hold up Time Very large 28 53 3 50562 14 696 0 14 696 Outlet Pressure absolute psi Ambient Pressure absolute psi Oven Temperature 1 ramp Program Ramp Final Rate Temp Ci min C Figure 7 Method translation software provides scaled conditions for GC systems with different configurations for easy comparison to the FID instrument from column to column results and from detector to detector by locking retention times The retention times of a styrene sample analyzed Conclusions T nique according to ASTM D 5135 matched Retention time locking facilitates to within 0 06 minute after locking replicating results from instrument to References 1 ASTM D 5135 95 Analyses of Styrene by Capillary Gas Chro matography Annual Book of Standards Volume 06 04 ASTM 100 Bar Harbor Drive West Conshohocken PA 19428 USA 2 M Klee and V Giarrocco Pre GC FID dictable Translation of Capillary GC Methods for Fast GC Agilent Technologies Inc Appli cation Note 228 373 Publication 5965 7673E March 1997 3 GC Pressure Flow Calculator fo
174. ld Seal Injections 14 1000 1 qj Meintenenice Indicators A Back Inlet Gold Seal age Time 220 4440 1 000000 i 100 Logs and Results Back Inlet O Ring injections Injections 0 500 0 Back Inlet O Ring age Time 9 21 27 44 360 00 00 00 E 3 Tasks R 49 To monitor a resource and set a lenit Procedure 3 Select Maintenance Indicators 4 Select counters that you want to use within LMD TE r z 5 Setup the same counters within LMD Maintenance Indicators 49 Managing instruments 6 Reset Value sets the count back to 0 Instrument Documentation 7 Save changes accepts your selections for Warning and Limit values Q9 To configure an alert notification 49 To view completed tasks 49 To print a status report 350 Warning Injections Limit 400 Injections Alert Actions Set Not Ready Eat Service Due Emad C Text Message Figure 6B Procedure for configuring maintenance indicators Set up the same counter within LMD In this example a warning email will be sent at 350 injections on the front inlet liner At 400 injections the GC will be set to Not Ready thus stopping the sequence and an email will be sent to the distribution list that was set up in the previous procedure see Figure 4 For the 7890A GC the resource counters must first 2 be enabled from ChemStation before they will count within LMD see Figure 6A Next the same counters are set up within LMD maintenan
175. le D pump Auto Column Wave L Emm vacuum sampler compart length A degasser meni detector Z Water ACN 32 Chromatographic conditions The HPLC method presented here was used for the analysis of anions in drinking water Sample preparation filtration mAU Column HP IC modifiers for the 100 HCO mobile phase are p included 80 4 Mobile phase water acetonitrile ACN 60 Cl 215 ppm Drinking water 86 14 adjusted to 1 Fo pH 8 6 with 40 NO 0 9 ppm ia A E carbonate free NaOH Raae 2 Flow rate 1 5 ml min 20 NO NO HP 0 gt Oven temperature 402C 0 Br 3 S04 Injection volume 25 yl Standard Detector UV VWD 20 detection wavelength 2 4 6 8 10 266 nm Time min Figure 23 HPLC method performance Analysis of anions in drinking water with indirect UV detection Limit of detection for UV VWD 0 1 1 ppb with S N 22 and 25 ul injected volume Repeatability of RT over 10 runs 0 8 96 areas over 10 runs lt 1 33 Sample preparation Column Mobile phase Flow rate Oven temperature Injection volume Detector Electrode Table salt was dissolved in water 200x 4mm Sperisorb ODS2 5 um water with 5 2 g l K HPO 3 g l tetrabutylammoniumdi hydrogenphosphat A CN 85 15 1 ml min ambient 24 9C 0 1 ul electrochemical ECD glassy carbon Working potential 1 V Operation mode amperometry HPLC method performance Limit of detection for ECD Repeatability of RT over
176. le Road Wilmington DE 19808 USA Abstract Retention time locking was introduced over a decade ago with the Agilent 6890 gas chromatograph The next gener ation of GC from Agilent the 7890A has enhanced and extended the functionality previously available including a new electronic pneumatic control system capable of pressure control to the third decimal place This applica tion demonstrates the ability of the EPC system to be used for retention time locking at low pressures in this case with a 320 pim column on a 5975 GC MS Introduction The introduction of retention time locking RTL with the 6890 GC gave users a new way of improv ing productivity by eliminating the need to con stantly update compound retention time data whenever a column was trimmed or replaced It also allowed the same methods to be run on multi ple systems with the same retention times The introduction of eMethods further enhanced the portability of these methods Most RTL based methods were established on GC MS systems where a typical head pressure for a 30 m x 0 25 um id column is gt 10 psi With setability to two decimal places four digit pressure setpoints for such columns for example 11 54 psig result in excellent inter instrument and intra instrument RTL precision Amongst the many optimization tasks in GC method development is deciding on the best column dimension to select This can realistically only be determined with full know
177. ledge of the sample characteristics and analysis goals that is components of interest complexity detection limits required and the matrix of the sample Larger diameter columns have the advantages of ruggedness and sample capacity over smaller dimension columns The larger the diameter of the column the less pressure is needed to establish optimal flows However for the most precise RTL one needs the ability to set pressure very precisely With two decimal place precision at low pressures for example 1 28 psig locking a system to a target retention time is less precise for column dimensions such as 0 32 u m columns The 7890 GC s fifth generation EPC provides excel lent low pressure control and with third decimal place control of the pressure providing the precision demanded for RTL at low pressures This application explores the suitability of the 7890 for RTL at low pressures with a 320 um column and uses a translation of the method described in Agi lent Technologies publication 5989 6569EN Reli able transfer of existing Agilent 6890 5973GC MSD methods to the new 7890 5975 GC MSD a Agilent Technologies Experimental The method used for this example was translated using the Agilent Method Translator software to convert to a 250 um id column method to a 320 um method A series of standards was run from 10 ppb to 5 ppm to illustrate the performance of the method followed by trimming approximately 45 cm off
178. less steel nuts should never be substituted Stain less nuts may damage the threads on the interface Damaged threads cause air leaks and replacement of the entire interface The MS interface supplies include Brass column nut p n 05988 20066 Ferrules for 0 2 and 0 25 mm id columns 10 pk p n 5062 3508 Ferrules for 0 32 mm id columns 10 pk p n 5062 3506 Installation of Consumables This section assumes that you are going to perform preventative maintenance PM on your Agilent 6890 Series GC If this is a new gas chro matography mass selective detector GC MSD system many of these steps will be completed by a Customer Engineer during installation Before beginning the PM please read this section care fully The necessary manuals will be referenced frequently These manuals can be downloaded from our Web site at www agilent com chem The manuals necessary include Agilent 6890N GC User Information Manual G1530 90210 Agilent 6890 Series GC Service Manual G1530 90220 Agilent 5973 Series MSD Hardware Installation Manual G2589 90006 Agilent 5973 inert MSD Hardware Manual G2589 90071 Agilent 5973 Series MSD Site Preparation Guide G2589 90070 With all of the consumable supplies previously mentioned at hand a proper PM can be completed To begin a PM it is necessary to cool the GC zones oven inlet MS interface Vent the Agilent 6890 Series MSD Please refer to the MS hardware manuals f
179. lifies the process of transfer ring methods from chromatographic instrument to chromatographic instru ment column to column and detector to detector The analysis of impurities in styrene according to ASTM D 5135 is used to demonstrate the efficacy of the approach Using RTL the retention times matched within an average of 0 16 0 02 0 03 minute in constant pressure modes Retention Time Locking Concepts and Applications Application Gas Chromatography December 1997 Key Words Retention time locking method vali dation styrene analysis ASTM D 5135 capillary gas chromatography laboratory productivity Introduction Retention time is the fundamental qualitative measurement of chro matography Most peak identification is performed by comparing the reten tion time of the unknown peak with that of a standard It is much easier to identify peaks and validate methods if there is no variation in the retention time of each analyte However shifts in retention time occur frequently Routine mainte nance procedures such as column trimming alter retention times In a multi instrument laboratory running duplicate methods the retention times for each instrument will differ from each other even when run under nominally identical conditions These differences in retention times mean that each instrument must have a separate calibration and integration event table making it time consuming to transfer methods fr
180. llin extract mAU SM alkene NET 50 Vanillin allic aci ringaaldehyde 504 os y z Match 991 Vanillin E30 404 2520 10 al 0 2 licyl 217 Wavelength nm 400 4 Salicyl a aldehyde 103 1 1L Standard 0 Cognac 0 2 4 6 8 10 Time min Figure 9 Analysis of vanillin in cognac Identification of vanillin through spectra comparison using HPLC J Chromatogr 1982 246 313 316 E 7 Herrmann A et al Rapid control of vanilla containing products 13 Bitter compounds hesperidin and naringenin Sample preparation The orange juice was prepared according to Carrez 1 and 2 Column 125 x 4 mm Hypersil BDS 5 um Mobile phase A zwater 0 15 ml l H5SO conc pH 224 B ACN Gradient start with 20 B at 3 min 20 B at5 min 90 B at 6 min 20 B Flow rate 2 ml min Post time lmin Column compartment 40 C Injection volume Tul Detector UV DAD detection wavelength 260 80 nm reference wavelength 380 80 nm HPLC method performance Limit of detection 1ng injected amount for DAD S N 2 Repeatability ofRTover10runs 0296 of areas over 10 runs 1 96 Sample preparation for bitter compounds in orange juice The samples were prepared according to Carrez 1 and 2 This method uses potassium ferrocyanide and zinc sulfate for protein precipitation Chromatographic conditions The HPLC method presented here for the analysis of hesperidin and naringenin is base
181. llowing Replacement O ring See Co Parts for the Snlit nlitlass Replacement BE RL qj TableG Other consumables and parts for the split sphtless inlet E 2 Load 3 Description quantity Part number GC tos Septum rotainor nut for headspace 18740 60830 Septum rotainor nut 18740 60035 11 mm soptum high temperature low 5183 4757 WARNINY blood S0 ok 114m septum prepierced long life 5183 4761 S0 pk 2 MerlnMicroseal septum high pressure 5182 3444 Merlin Microseal septum 30 psi 5181 6815 the Nonstick fluorocarbon liner O ring for 44519053265 the septum assembly straight up and away from to avoid chipping or breaking the hm Figure 10 Searchable complete user information capability allows for enhanced diagnostics and troubleshooting functionality Inlet pressure decay test Test Name Front Spit Splitiess Inlet 7890 Pressure Decay Description Approx Ti 20 min z CELIE 25 75 i 3s 1 245 2x5 d The Spit Splitiess Pressure Decay Test checks for leaks in associated tubing The fittings and remove the column The GC pressurizes the inlet then measures pressure decav to determine there is a leak It Categor Source Tene Message Front Splt Soltiess Iniet 78 2 3 2008 4 54 1 Inlet to 70C oven Front Spit Spiitiess Inlet 78 2 3 2008 4 54 1 Preparing test met Front Split Spltiess Inlet 76 2 3 2000 4 57
182. lting inlet pressure and oven tem perature ramp rates are then inspected to see if the instrument on GC MS 3 2 5 10 15 20 25 30 35 GC AED T T T T T T T T T 0 5 10 15 20 25 30 35 40 min Figure 2 GC AED chlorine and GC MS TIC chromatograms of three component locking mix ture Peak identifications 1 dichlorvos 2 methyl chlorpyrifos 3 mirex 0 300 0 200 0 100 Difference mm 0 100 0 5 10 e Ibe RA i 40 45 0 200 0 300 Retention Time min Figure 3 Difference plot of GC MS and GC FID retention times in RTL Pesticide Library which the new method will be run is compatible with those parameters Figure 4 shows the method transla tion software with the data entered for a speed gain of 3 Note that columns for Original Method and Translated Method are set up as in the previous example with two excep tions Because the scaling is from GC AED to GC AED the outlet pres sure in both columns is entered as 16 2 psi The second and most signifi cant difference is the holdup time The desired speed gain is 3 To set the speed gain the calculated value of hold up time in the first column 0 996060 minute is divided by exactly 3 This value 0 33202 minute is entered for the hold up time in the second column This will force the speed gain to exactly 3 The inlet pressure and oven tempera ture ramp for the new threefold speed method are no
183. lyses and database operations can be performed simul taneously see figure 76 Through computer networks lab oratory instruments can be interconnected to enable the central archival of data and the sharing of printer resources Client server based software extends these capabilities by distributing the processing across multiple processing units and by minimizing the time spent validating software With PCs all aspects of the HPLC system can be accessed using a single keyboard and mouse Parameters for all mod ules including pump detector and autosampler can be entered in the software program saved to disk and printed for documentation Some HPLC software programs include diagnostic test procedures instrument calibration proce dures and extensive instrument logbooks all of which can facilitate the validation processes of various regulatory agencies Such complementary functions although not al Y FT DB aj e mJ eum 3 ej Figure 76 Cross sample reports regression analyses trend charts and other calculations consolidate sample data enhancing the overall productivity and efficiency of the laboratory 114 directly related to the control of the equipment are more easily built into a software program than into the equipment itself In fact many GLP GMP features are added to every new version of the software programs sold with HPLC equipment see figure 77 For example in some chroma tography software t
184. m sample 6 Mix 7 ul 6 cycles 7 Inject Detectors UV DAD fluorescence detection wavelength 338 20 nm or excitation wavelength 230 nm emission wavelength 445 nm Chromatographic conditions The HPLC method presented here for the analysis of aspartame is based on automated on column derivatization and reversed phase chromatography UV spectra were evaluated as an additional identification tool 5 mAU 60 50 40 Aspartame spectra original scaled derivatized 30 250 30 350 400 Wavelength nm Aspartame Time min Figure 6 Chromatogram and spectra of derivatized and non derivatized aspartame HPLC method performance Limit of detection for fluorescence 200 pg injected amount S N 2 for DAD 1 ng injected amount S N 2 Repeatability ofRToverl0runs 0 196 of areas over 10 runs 5 96 JAA 5 A M Di Pietra etal HPLC analysis of aspartame and saccharin in pharmaceutical and dietary formulations Chromatographia 1990 30 215 219 4 Official M ethods of Analysis Food Compositions Additives Natural Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 Official M ethod AOAC 979 08 Benzoate caffeine saccharin in soda beverages Colorants Water Acetonitrile Quaternary pump Auto Column vacuum sampler compart degasser ment Diode array detector Control and data evaluation Ex F We have selected the food color E104 Quin
185. mall quantities of organic Samples high in fat cannot be extracted solvents thereby reducing costs and facilitating disposal Extraction times are in minutes rather than hours Steam distillation is only used to extract volatile compounds from solid homogenized matrices For example biphenyl and 2 phenylphenol pesticides can be extracted from citrus fruits with this technique S x Enables selective extraction of volatile Extraction times are long and offline compounds Narrow range of use Until now supercritical fluid extraction SFE was rarely used in food analysis However the input of modern SFE instruments can be automated with sampling devices This method is used primarily for GC although LC coupling also has been performed with SFE 2 S x Uses small quantities of organic solvents Weak solvating power limits range of thereby reducing costs and facilitating analytes Ultrapure fluids for trace disposal Extraction times are in minutes analysis are not always available rather than hours Can be automated Liquids Liquid liquid extraction Solid phase extraction Liquid liquid extraction on and offline solid phase extraction and GPC are used in the analysis of liquid samples or extracts from solid samples Liquid liquid extraction is the most common extraction method It requires an appropriate solvent and a separating funnel or a continuous or counter current distribution apparatus Sim
186. metimes defined as equal to the noise level However the LOD depends not only on the detector but may also depend on the oxygen content of the mobile phase the injection system peak broadening on the col umn and temperature differences among system compo nents Taking these factors into account the LOD is defined as 2 to 3 times the noise level The LOQ is defined as 10 to 20 times the noise level A UV detection system can be used to measure quantitative amounts down to 500 pg per injec tion The LOD can be as low as 100 pg for food compounds such as antioxidants if detection wavelengths have been optimized to match the extinction coefficients of as many compounds as possible Fluorescence and electrochemical detectors operate in the very low picogram range The LOD of a mass spectrometer connected to HPLC equipment depends on the type of interface used Instruments with electrospray interfaces can detect down to the picogram range Refractive index detectors normally are appropriate above 500 ng We define the selectivity of a detection system as the ability to select only those compounds of interest in a complex matrix using specific compound properties A detector is selective if it does not respond to coeluting compounds that 87 Linearity Qualitative information could interfere with analyte quantification A UV absorbance detector can be made selective by setting an appropriate wavelength with a narrow bandwidth for the co
187. metry 0 9V 0 5 pA AgCI KCI 8s HPLC method performance Limit of detection Repeatability of RT over 10 runs areas over 10 runs Linearity 80 pg injected amount S N 2 0 5 96 lt 5 30 pg to 1ng Analysis of tocopherols on normal phase column Chromatographic conditions for electrochemical detection The HPLC method presented here was used in the analysis of a vitamin standard Standard tocopherol Time min Figure 36 Analysis of a fat soluble vitamin with electrochemical detection Control and data evaluation Isocratic Electro pump Auto Column i om vacuum sampler compart chemical ZE degasser ment detector pee Bes Water Tocopherols cannot be separated completely using reversed phase chromatography However normal phase chromatography can separate isocratically all eight tocopherols T and tocotrienols T naturally occurring in fats oils and other foodstuffs Fluorescence detection is recommended for the analysis of total lipid extraction because UV absorbance detection is not selective enough to prevent detection of coeluting peaks 46 Sample preparation Column Mobile phase Stop time Flow rate Column compartment Injection volume Detector UV DAD Fluorescence 20 g sample dissolved in 15 ml hexane 100 x 2 1 mm Hypersil SI 100 5 um hexane 2 isopropanol 8 min 0 3 ml min 25 2C 0 5 ul 295 80 nm excitatio
188. mple The micro ECD for the 6890 GC is extremely sensitive with detection limits in the low femtogram range for polyhalogenated pesticides These detection limits are so low that it is reasonable to consider using split mode for a rapid screening method Using split mode with a split ratio of 100 still gives a detection limits in the range of a few picograms The split is also more compatible with the rela tively low capacity of the column Table 3 RTL Calibration Points from Original GC AED Method and Calculated Points for Threefold Speed Gain 3x Method GC AED RTL Calibration 3x GC AED RTL Calibration Calculated Calculated Pressure Ret Time Pressure Ret Time psi min psi min 33 1 15 346 106 21 5 115 30 4 15 919 97 23 5 306 27 6 16 578 87 86 5 526 24 8 17 338 78 44 5 779 22 1 18 242 69 31 6 081 Table 4 Comparison of Locking Pressures Calculated Using Mea sured and Predicted 3x GC AED RTL Calibration Data 3x GC AED Locking Runs Measured 3x GC AED RTL Cal Points Locking Pressures Using Measured RTL Cal Points Using Calculated RTL Cal Points Pressure Ret Time Pressure Pressure psi min psi psi 97 5 319 87 99 87 99 92 5 433 87 94 87 95 87 5 557 87 99 87 99 82 5 689 87 99 87 96 77 5 832 87 97 87 88 GC AED 1x 2 0 5 10 15 20 25 30 35 40 min GC AED 3x o 2 4 6 8 10 12 min Figure 5 Chlorine chromatograms from original and 3x GC AED methods of
189. mpound of interest However the selectivity of detectors based on such a universal feature is low compared with the selectivity of detectors based on fluorescence and electrochemistry Response characteristics are very selective shown by a limited number of compounds Mass spectrometers can be applied selectively or universally in total scan mode depending on the analysis to be performed RI detectors are universal by definition Detector response can be expressed both as dynamic range and as linear dynamic range Dynamic range is the ratio of the maximum and the minimum concentration over which the measured property absorbance current and so on can be recorded However in practice linear dynamic range the range of solute concentration over which detector response is linear is more commonly used Plotting the response of injections of different analyte concentration against their concentrations should give a straight line over part of the concentration range Response often is linear for only one tenth of the full dynamic range UV detectors are linear over a range of a maximum of five orders of magni tude whereas fluorescence and electrochemical detectors are linear over a range of two orders of magnitude Mass spectrometers are usually linear over three orders of magni tude and RI detectors are linear over a maximum of four orders of magnitude A classical identification tool in chromatography is the mass spectrogram which
190. n By making an adjustment to the inlet pressure the retention times on one system can be closely matched to those on another system using the same nominal column The ability to very closely match retention times from one system to another can greatly reduce the time it takes to develop and transfer methods RTLocked methods can also compen sate for degradations in chromatographic perfor mance The ability to correct for degrading chro matographic performance optimize lab resources and still provide the correct answer saves time money and results in significant productivity gains Using GC MS it is also possible to screen samples for the presence of target compounds using a mass spectral database of RTL spectra The RTL mass spectral database provides additional confirma tory information in spite of changes to the chro matographic system One such database is the G1672AA Pesticide RTL Library This database allows for quick and easy screening of pesticide samples When Should I Lock My Methods Locking or relocking your methods should be done whenever you make any changes to the chromato graphic system or move methods from one system to another GC or GC MS To establish and main tain a locked method RTL should be performed whenever The column is changed or trimmed The method is installed on a new instrument A detector of different outlet pressure is used GC vs MS e System performance is validated
191. n and relocking process to be appropriate to achieve the target retention time of 11 112 min for the locking compound fluoranthene Table 3 shows the calibra tion data from the RTL runs Table 3 RTL Data Pressure Retention Deviation Run psi time mins seconds RTLOCK1 D 3 01 11 212 6 018 RTLOCK2 D 3 38 11 166 3 264 RTLOCK3 D 3 76 11 112 0 000 RTLOCK4 D 4 14 11 070 2 508 RTLOCK5 D 4 51 11 020 5 508 Maximum deviation 6 018 seconds Correlation co efficient 0 999 Even though pressure setpoints used for RTL calibration need only be to be to two decimal places the ability to precisely set locking pressures based on the calibra tion curve requires setability to the third decimal place Figure 1 shows the calibration curves correspond ing to the linearity metrics summarized in Table 2 Average retention RSD time 96 Naphthalene 5 884 0 055 Acenapthylene 7 153 0 045 Acenaphthene 7 968 0 044 Fluorene 8 562 0 048 Phenanthrene 9 702 0 089 Anthracene 9 752 0 044 Fluoranthene 11 119 0 031 Pyrene 11 388 0 090 Chrysene 12 808 0 094 Benz a anthracene 12 855 0 046 Benz b fluoranthene 14 285 0 045 Benz k fluoranthene 14 314 0 069 Benz a pyrene 14 815 0 035 Indeno 1 2 3 cd pyrene 17 088 0 051 Dibenz a h anthracene 17 098 0 066 Benzo ghi perylene 17 725 0 089 Calibration RT before Relocked ART when linearity r relocking RT relocked 0 997 5 809 5 876 0 008 0 997 7 676 7 146 0 007 0 997 7 893 7 959 0 009 0 997 8 486
192. n as the target compound The calibration data are shown in figure 1 The method conditions and RTL cali bration were then moved to GC system 2 a different GC and column The sample was run at the original method inlet pressure of 18 2 psi The chromatogram obtained using this scouting run is overlaid on the origi nal chromatogram in figure 5 The retention times shifted about 0 3 minute on the second GC This is a typical result obtained when trying to replicate an analysis on a second instrument or with a second column The retention time of o methylstyrene was entered into the RTL software 12 2 5 5 7 5 10 T T T 12 5 15 17 5 20 22 5 min Figure 4 Styrene sample run on GC system 1 at 18 2 psi initial pressure constant flow mode dialog box on GC system 2 as shown in figure 3 The RTL software indi cated the initial pressure should be modified from 18 2 psi to 18 96 psi The new initial pressure was entered into the method and saved Figure 6 compares the chromatograms obtained from the original run and after locking reten tion times using the o methylstyrene Table 2 compares the retention times before and after using this approach The retention times are now closely matched GC FID to GC MSD Locking A second experiment was conducted to lock the original method from GC system 1 to the GC MSD This is useful for identification of unknown impurities that show up in the FID chromatogram For example ther
193. n in Figure 7 i Sample Log Table Data Path CAMSDchem T dete Browse al Sample Method Keyword Method Path EAMSDCHEMM METHODS DEFAULT M Ex Data File Command Command Tune 1 Checkout sample CHECKOUT 1 Checkout sample CHECKOUT 1 2 3 4 5 6 7 8 9 Insert Row Repeat Row 5 times Figure 7 Pumpdown sequence table using source bakeout Line 1 Loads the Bake macro Line 2 sets the bake time to 10 hours After the bake Line 3 an auto tune is executed Lines 4 and 5 run the system per formance method CHECKOUT M on the system checkout standard Note after the system has been cleaned and leak checked the CHECKOUT M method should be loaded THEN this sequence should be run Checkout1 Checkout2 Macro Bake mac Bake 10 Auto test of system performance test of system performance rep Read Barcode OK Cancel Help xi www agilent com chem Conclusions The increased source temperature limit available on the 5973N inert MSD can provide improved detection limits for common late eluting recalci trant compounds such as the POPs when properly applied A requirement that must be explored is that the higher source temperatures do not increase compound fragmentation or reduce the intensity of the useful higher mass ions These improvements are most likely to be realized in SIM acquisitions where the increased background that must result from higher source temperatures i
194. n is vacuum while with most other GC detectors the outlet pressure of the column is at or near atmospheric pressure This difference in outlet pressures results in large differences in retention time between GC with MS detection and GC with other detectors Comparison of GC FID a general detector with GC MS is rea sonably straightforward because the total ion chromatogram TIC of the GC MS system has similar response to the FID Retention times on the GC MS system corresponding to those on the GC FID can be deter mined by looking for similar patterns of response With selective detectors this is much more difficult because the response patterns from selective detectors usually do not resemble the TIC For this reason matching the retention times of selective detectors precisely with the GC MS system sim plifies data analysis greatly In this first example of scaling the RTL Pesticide Library the method will be scaled from the GC AED method to the GC MS method In this case the desired scale factor is exactly 1 that is the GC MS retention times are desired to be exactly the same as those of the GC AED The first step is to use the method transla tion software to determine the GC conditions to use for GC MS Figure 1 shows the method transla tion software The original method conditions for the GC AED pesticide method are entered in the column labeled Original Method The column dimensions carrier gas type i
195. n water with postcolumn derivatization 6 Norm 83 74 Glyphosate 6 5 4 6 4 5E DES 25 5 US 10 125 Time min Figure 22 Analysis of glyphosate standard Quaternary Pickering pump Auto post column vacuum sampler derivatiza degasser tion system Water KOH Control and data evaluation Fluores mm cence detector L 16 R Schuster A comparison of pre and post column sample treatmentfor the analysis of glyphosate Agilent Application Note 5091 3621E 1992 29 30 Chapter 3 Analytical examples of natural components Inorganic anions Anions containing halogen nitrogen and sulfur are used as additives in food industries For example nitrites act as preservatives in smoked sausage Nowadays dedicated instrumentation such as special columns and electro conductivity detectors are used in the analysis of inorganic anions Because specialized equipment has a very limited application range a method was developed for analyzing anionsusing reversed phase chromatography and indirect UV detection Another more selective and sensitive approach for the analysis of selected anions is electro chemical detection Sample preparation Excepting filtration sample preparation normally is unnecessary if the sample is aqueous Other matrices can be extracted with hot water followed by filtration Control and m data evaluation Isocratic Variab
196. n wavelength 295 nm emission wavelength 330nm HPLC method performance Limit of detection 10 20 ng S N 22 for diode array Limit of detection 0 5 2 ng S N 22 for fluorescence Repeatability of RT over 10 runs lt 2 areas over 10 runs lt 2 Fluores cence detector Hexane Isocratic Diod pump Auto Column ms En vacuum sampler compart E degasser ment SE Control and IE data evaluation FI NN Chromatographic conditions for analysis of tocopherols on normal phase column The HPLC method presented here was used in the analysis of margarine mAU y toc opherol 20 151 6 tocopherol 104 a tocopherol B tocopherol 5 2 i DAD 1 i ee Time min Figure 37 Analysis of tocopherols on normal phase using UV and fluorescence detection a B tocopherol tocopherol 90 704 6 tocopherol a tocopherol _ Standard 71356 9 5 96 301 Margarine 101 1 2 3 4 5 6 Time min Figure 38 Analysis of tocopherol concentration in margarine fat extract with fluorescence detection 47 Biogenic amines The following amines were analyzed ammonia amylamine 1 butylamine 1 4 diaminobutane 1 5 diaminopentane diethylamine ethanolamine ethylamine hexylamine histamine isobutylamine isopropylamine methylamine 3 methylbutylamine morpholine phenethylamine propylamine pyrrolidine and tryptamine Free amines are present in various food products a
197. nd beverages including fish cheese wine and beer High concentrations of specific amines can have toxic properties As a result several countries have set maximum tolerance levels for these compounds in foodstuffs HPLC is now preferred for the analysis of amines in food matrices because of its shorter analysis time and relatively simple sample preparation Sample preparation Amines can be extracted from different matrixes using liquid liquid extraction or solid phase extraction followed by derivatization Control and data evaluation Quaternary a pump Auto Column Variable L z vacuum 7 sampler compart wavelength EL ment detector LER degasser Water Acetonitrile g 48 Chromatographic conditions for UV detection The HPLC method presented here was used to analyze amines in wine Sample preparation mAU 1 93 04 25 ml wine was decolored with polyvinylpyrrolidoine After filtration the 8 0e4 amines 5 ml sample pH 10 5 were Sj derivatized with 2 ml dansyl chloride 14 Standard solution 196 The reaction solution was 6 0e cleaned with solid phase extraction using C18 cartridges 500 mg After elution 4 0e with 2 ml ACN the solution was concentrated to 100 ul Column 250 x 4 6 mm 2 0e Spherisorb ODS2 5 um T r Mobile phase A water 5 ACN 20 Time min 40 60 0 Lm 25 1 ethanolamine 8 pyrrolidine 15 amylamine 2 ammonia 9 i butylamine 16 1
198. nd photosynthesis occurs Phytochrome proteins are present in very low concentrations in potato clod and sample volume and concentration of these proteins is rather low following sample preparation In this case columns or capillaries with a small internal diameter are preferred because sensitivity increases with decreasing internal diameter of the column The use of capillaries with an internal diameter of 100 300 pm enables flow rates as low as 0 5 4 0 pl min which reduces solvent consumption Such flow rates are well suited to liquid chromatography mass spectroscopy LC MS electrospray ionization In our experience the appropriate conversion of standard HPLC equipment to a capillary HPLC system is cost effective and yields the highest performance for running capillary columns For conversion a flow stream split device a 35 nL capillary flow cell for the detector and capillary con nections between system modules are required System delay volume should be as low as possible To meet the demands of such a system the Agilent 1100 Series binary pump which has inherently low delay volume was selected as a pumping system The flow splitter the capillary flow cell for the detector and the column were purchased from LC Packings in Amsterdam 4 With this design a standard flow rate for example 100 or 50 pl min can be set for the pump This flow then can be reduced by calibrated splitters between 0 5 and 4 pl min for example Th
199. nded for the transfer line seal These ferrules do not develop leaks as the transfer line temperature is cycled however the Vespel graphite ferrules can shrink and develop leaks Note that if the surface of the transfer line is very worn it may fail to seal well in which case the Restek Agilent interface cleaner P N 113450 can be used to resurface the sealing surface if very carefully employed The Figure 3 other end of this GC column was connected to the uppermost connection on the CFT tee with the SilTite ferrule The upstream 15 m GC column column 1 was hung on the other 15 m column cage and installed in the front split splitless injection port with a Vespel graphite ferrule liner and BTO septum as usual The other end was connected to the CFT tee middle post and after temporarily removing the other connected columns blanked off and pressure tested as above All connections were then re established to the CFT tee with the 1 m column in the lower position the front first column 1 connected in the middle position and the rear second or MSD column 2 in the uppermost connection Helium was supplied to both the front and back ports and a helium leak detector was used to check for any leaks A picture of the arrangement is shown in Figure 3 To MSD second column From front inlet first column From back inlet flow control Picture of the installed pressur
200. njection volume Detector Working electrode Operation mode Working potential Range Reference electrode Response time Vitamin preparation was diluted with water 1 100 125 x 4mm Lichrospher RP 18 5 um water 0 02 M KH PO 0 03 M tetrabutylammo niumhydrogensulfat 0 03M heptanesulfonic acid 2 ACN 15 min 0 8 ml min 30 9C 1 yl standard 0 5 ul sample electrochemical glassy carbon amperometry 12V 0 5 pA AgCI KCI ls HPLC method performance Limit of detection Repeatability of RT over 10 runs areas over 10 runs Linearity 30 pg injected amount S N 2 0 5 96 lt 5 30 pgto 1ng Chromatographic conditions for electrochemical detection The HPLC method presented here was used in the analysis of vitamins in animal feed 2 mV 240 2207 200 1807 1601 140 120 Vitamin C Vitamin B c Standard yh 3 Time min Figure 34 Analysis of vitamin B in a vitamin preparation Control and data evaluation Isocratic pump Auto Column Electro BEES vacuum sampler compart degasser ment etector Water g 20 A G Huesgen R Schuster Analysis of selected vitamins with HPLC and electrochemical detection Agilent Application Note 5091 3194E 1992 44 Fat soluble vitamins Column 100 x 2 1 mm Hypersil M OS 5 um Mobile phase A z water B ACN 70 96 Gradient at 15 min 90 B at16min 95 B Post time 3 min Flow
201. nlet pressure outlet pressure ambi ent pressure and oven temperature program are entered here Note that the inlet pressure is in psi gauge while the outlet pressure and ambient pressure are psi absolute The origi nal method here is being used on a GC AED system so the outlet pres sure is entered as atmospheric pres sure plus 1 5 psi the operating pressure of the GC AED The Criterion parameter is set to None which allows the user to select a specific value of speed gain by adjusting the value of hold up time for the translated method see figure 1 In the column labeled Translated Method the parameters of column dimensions carrier gas type outlet pressure and ambient pressure for the GC MS method are entered Note that the inlet pressure and oven program are not entered they are calculated by the program To set the speed gain to a desired value take the calculated value of hold up time in the first column 0 996060 minute and divide it by the scale factor Because in this case the desired scale factor speed gain is 1 the same hold up time for both the GC AED and the GC MS methods is required Clicking the radio button next to the hold up time in the Trans lated Method column will do this automatically The method translation indicates that to obtain the same retention times on the GC MS system as on the GC AED use all the same method parameters except inlet pressu
202. nown identification is much more straightforward To maintain a locked method RTL should be performed whenever The column is changed or trimmed e The method is installed on a new instrument e A detector of different outlet pres sure is used e System performance is validated Troubleshooting chromatographic problems To lock a given method for the first time or for the reasons below one must first develop a retention time versus pressure RT vs P calibration Even when using columns with the same part number same id station ary phase type phase ratio and same nominal length separate different locking calibration curves are needed when using e Systems with different column outlet pressures FID atmos pheric MSD vacuum AED elevated e Columns differing from the nomi nal length by more than 15 e g due to trimming e Systems where the predicted lock ing pressure falls outside the range of the current calibration A specific solute usually one found in the normal method calibration standard must be chosen and then used for both developing the locking calibration and locking all future sys tems The solute or target peak should be easily identifiable symmet rical and should elute in the most critical part of the chromatogram Solutes that are very polar or subject to degradation should be avoided Once the target solute has been chosen and all other chromatographic parame
203. nt FID 7890 Checkout Test Instrument 7890 0S10652005 senesi fS Check the Signals to view the chromatogram Test Name Front FID 7690 Checkout Test Approx Ti D Y The test is passed Y The system is confirmed as proper To Measure FID Baseline Output E To Measure FID Leakage Current 49 To Bakeout the FID To Perform Maintenance on the FID Downloading test method to connected GC and wating for readiness 25 1 Performing pre run injection steps od RIRO 6 252 Started theruny Front FID 7890 Checkout T 2 3 2008 6 32 Front FID 7890 Checkout T 2 3 2008 6 3215 Front FID 7890 Checkout T 2 3 2008 6 32 5 Front FID 7890 Checkout T 2 3 2008 6 32 5 Front FID 7890 Checkout T 2 3 2006 6 33 0 E 7 Figure 12 The result has passed FID checkout test pA 5 0 Benzene Toluene Column INNOWax 60 m 320 um 0 5 pm 3 Ethylbenzene 4 C 48 M oxylene oncentration about 0 2 ppm 4 6 44 42 40 About 0 2 ppm of ethylbenzene and m oxylene has an excellent signal to noise ratio after the inlet septum and liner O ring are replaced 3 8 Figure 13 Expected result for trace level aromatic sample is obtained after troubleshooting with intuitive help from LMD Conclusions Agilent Lab Monitor amp Diagnostic LMD software is a new tool to help ensure the productivity per
204. nt o ring iw Back Inlet Septum iw Back Inlet Liner iw Back Inlet Gold Seal V Back Inlet Split Vent Trap II Back Inlet Top W eldment o ring Front Detector Insert Liner Back Detector Insert Liner C Column 1 Injection Cycle Procedure 1 Edit parameters and click the icon that allows for selection of diagnostic counters 2 Selectthe counters that you want to use within LMD Figure 6A Procedure for configuring maintenance indicators Set up the resource counter within ChemStation Workstation Instrument Documentation Unit Value mit Progress Instrument Injections 51 300 17 QURE IND A Front Inlet Liner Injections 199 400 50 ff Calendar Front Inlet Liner age Tene 56 00 13 28 160 00 00 00 I 0 31 T Front Inlet Gold Seal Injectors 160 200 I mn Et JS chromatographic Abitur Front Inlet Gold Seal age Tme 5519 28 02 90 000000 BEBE ex Front Inlet O Ring injections Injections 0 500 0 3 Front Inlet O Ring age Time 56 00 13 28 190 00 00 00 EE 31 TI Status Roper i Front Inlet Spit Vert Trap Injections 160 160 sy 10 Mg Loos amp Results Back Inlet Septum Injections 14 100 E 14 E Tools i Back Inlet Liner Injections 14 500 iu M J 396 A Back Inlet Liner age Time 2 47 47 28 2 00 00 00 G oo e 2 Back Inlet Go
205. nts must be detected at very low concentrations The following table lists common fluorescent tags In part one of this primer we give examples for carbamates and glyphosate 2 Target compound Tagging reagent Alcohols OH phenylisocyanate Aex 230 nm Aem 315 nm Primary amines NH o phthalaldehyde Aex 230 nm em 455 nm OPA Primary and NHR 9 fluorenylmethyl Aex 230 nm em 315 nm secondary amines chloroformate FM OC Precolumn techniques can be run either offline or online but postcolumn techniques should be run online for maximum accuracy In postcolumn derivatization reagents can be added only through supplementary equipment see figure 75 such as pumps Mixing and heating devices also may be required Increasing the dead volume behind the column in this way will result in peak broadening Although this broadening may have no effect on standard bore columns with flow rates above 1 ml min postcolumn derivatization is not suitable for narrow bore HPLC 109 Automatic derivatization Both pre and postcolumn derivatization techniques can be automated with modern HPLC equipment The single step mechanical functions of an autoinjector or autosampler can be programmed prior to analysis and stored in an injector program see left These functions include aspiration of the sample and of the derivatization agent and mixing 1 Draw 10y from vial 12 Precolumn derivatization is fully compatible with 2 Draw O
206. of a high pressure pump left and of a low pressure pump right In this design gradients are formed by a high speed proportioning valve that can mix up to four solvents on the low pressure side The valve is synchronized with piston movement and mixes the solvents during the intake stroke of the pump The solvents enter at the bottom of each chamber and flow up between the piston and the chamber wall creating turbulences Compared with conventional multisolvent pumps with fixed stroke volumes pumps with variable stroke volumes generate highly precise gradients even at low flow rates see figure 50 79 High pressure gradient The Agilent 1100 Series high pressure gradient pump is Agilent 1100 Series pump based on a double dual piston mechanism in which two pumps are connected in series in one housing This con figuration takes up minimal bench space and enables very short internal and external capillary connections Both pistons of both individual pumps are servocontrolled in order to meet chromatographic requirements in gradient formation see figure 51 Three factors ensure gradients with high precision at low Farfonmencs of high pressure pump flow rates a delay volume as low as 180 480 pl internal design i Py age Flow precision lt 0 3 volume without mixer maximum composition stability Flow range 0 05 5 ml min and retention time precision and a flow range typically Delay volume 180 480 ul 600
207. of background noise from the detector can counter any gains made In particular the performance of the pump in combination with certain solvents can increase detector noise level as described in chapter 7 Degassing is necessary in order to avoid gas 121 Accuracy and precision bubbles which can cause noise or spikes or oxygen quenching in fluorescence High k values result from higher elution volume or from longer retention time These values are accompanied by broader peak width and smaller peak height that is peaks with longer retention times have poorer S N The use of different columns different mobile phases and different flow rates can improve S N Packing material also directly influences peak dispersion for example smaller sized particles reduce peak dispersion Accuracy is the degree of agreement between test results and true values It is influenced by the analytical method the extraction procedure used and the choice of column or detector Prior to the adoption of any HPLC method for rou tine use the degree of agreement with an established refer ence method should be determined or a control run should be performed with a known quantity of spiked sample matrix In practice however the degree of agreement will never reach 100 96 This mismatch can be corrected by cali bration with standards of known concentration and based on these results by calculating the accurate results from an unknown sample Inclus
208. of spectra of yellow red blue and black colors ELI 4 Official M ethods of Analysis Food Compositions Additives Natural Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 Official M ethod AOAC 981 13 Cresidine sulfonic acid in FD amp C Red No 40 Official Method AOAC 982 28 Intermediates and reaction by products in FD amp Y Yellow No 5 Official M ethod AOAC 977 23 44 Diazoamino dibenzene sulfonic acid DAADBSA in FD amp C Yellow No 6 Official M ethod AOAC 980 24 Sulfanilic acid in FD amp C Yellow No 6 6 A G Huesgen R Schuster Sensitive analysis of synthetic colors using HPLC and diode array detection at 190 950 nm Agilent Application Note 5964 3559E 1995 11 Flavors Vanillin The following compounds are examples of flavoring agents used in food products e Jupulon and humulon hop bittering compounds vanillin naringenin and hesperidin bittering compounds Three major classes of compounds are used as flavoring agents essential oils bitter compounds and pungency compounds Although the resolution afforded by gas chromatography GC for the separation of flavor compounds remains unsurpassed HPLC is the method of choice if the compound to be analyzed is low volatile or thermally unstable Sample preparation Turbid samples require filtration whereas solid samples must be extracted with ethanol After filtration the solution can be injected directly into the HPLC instrument Cont
209. olin yellow and E131 Patent blue as application examples Synthetic colors are widely used in the food processing pharmaceutical and chemical industries for the following purposes to mask decay to redye food to mask the effects of aging The regulation of colors and the need for quality control requirements for traces of starting product and by products have forced the development of analytical methods Nowa days HPLC methods used are based on either ion pairing reversed phase or ion exchange chromatography UV absorption is the preferred detection method The UV absorption maxima of colors are highly characteristic Maxima start at approximately 400 nm for yellow colors 500 nm for red colors and 600 700 nm for green blue and black colors For the analysis of all colors at maximum sensitivity and selectivity the light output from the detector lamp should be high for the entire wavelength range However this analysis is not possible with conventional UV visible detectors based on a one lamp design Therefore we have chosen a dual lamp design based on one deuterium and one tungsten lamp This design ensures high light output for the entire wavelength range Sample preparation Whereas turbid samples require filtration solid samples must be treated with 0 1 ammonia in a 50 96 ethanol and water mixture followed by centrifugation Extraction is then performed using the so called wool fiber method After desorption of th
210. olume Sample volatility Splitless valve time Injection speed The proper inlet temperature is needed to evapo rate high boiling point compounds without thermally degrading other compounds Normally the inlet temperature is a compromise between these two factors A good starting point is 250 C Liner design is one of the most difficult choices simply because of the variety of liners available The features that are most important in a liner are the volume whether it is deactivated or not and whether or not it contains deactivated glass wool As a general choice for high sensitivity work a 4 mm single tapered deactivated liner with no glass wool is recommended For large volume injec tions 22 uL and for the highest repeatability especially with small volume injections of lt 0 5 uL 1 deactivated glass wool is necessary For dirty samples deactivated glass wool helps to keep the nonvolatiles from getting to the column but too much deactivated glass wool can greatly decrease sensitivity Often the most appropriate liner must be determined through experimentation Please note Removing and or breaking deactivated glass wool creates active sites Splitless valve timing is critical The ON time split less mode should to be long enough to assure that all of the injected sample reaches the column A textbook splitless injection has the liner volume swept at least two times during the ON time A 4 mm liner ha
211. olumn The pistons in the pump chambers are motor driven and operate with a fixed phase igure Low pressure gradient pump 0 08 mAU 0 09 y 0 08 0 09 0a 0 10 504 404 304 204 10 1 2 2 Time min 5 o 2 Figure 49 Retention time precision RSD of 10 injections of a polycyclic aromatic hydrocarbon PNA standard sample 78 Performance of low pressure pump design Flow precision Flow range Delay volume Pressure pulse Composition precision 0 3 typically 0 15 96 based on retention times of 0 5 and 2 5 ml min 0 2 9 999 ml min ca 800 1100 ul lt 2 amplitude typically lt 1 1 ml min propanol at all pressures 0 2 SD at 0 2 and 1 ml min difference of 180 so that as one delivers mobile phase the other is refilling The volume displaced in each stroke can be reduced to optimize flow and composition precision at low flow rates With solvent compressibility compensation and a low volume pulse dampener pulse ripple is minimal resulting in highly reproducible data for retention times and areas see figure 49 A wide flow range of up to 10 ml min and a delay volume of 800 1100 pl support narrow bore standard bore and semipreparative applications Four solvents can be degassed simultaneously with high efficiency mAU mAU 801 601 404 204 04 JUNE 0S 107 G A Time min Time min Figure 50 Results of a step gradient composition 0 7
212. om one instru ment to another Differences in reten tion time also complicate comparison of data between instruments and over time Retention time locking RTL is the ability to very closely match chro matographic retention times in any Agilent 6890 gas chromatograph GC system to those in another 6890 GC system with the same nominal column There are several subtle effects that combine to cause retention time dif ferences between similarly config ured GC systems Columns of the same part number can vary slightly in length diameter and film thickness Agilent Technologies Innovating the HP Way GC pneumatics can have small varia tions in the actual inlet pressure applied at a given setpoint The actual temperature of the GC oven also has minute but real deviations from the indicated value The sum of these and other effects result in the observed retention time differences between similarly configured GC systems The pneumatics and oven tempera ture control of the 6890 GC have advanced the state of the art in GC hardware accuracy and precision Agilent s advances in fused silica cap illary column technology have resulted in highly reproducible column to column retention charac teristics With these advances reten tion time precision for a given peak in a single GC setup is usually better than 0 01 minute However even with these advances in columns and instru ment hardware the sum of the effects mentioned a
213. operoxides and to differentiate saturated from unsaturated triglycerides Unsaturated triglycerides in olive oil have a very distinctive pattern Other fats and oils are also complex mixtures of triglycerides but exhibit an entirely different pattern Adulteration with foreign fats and the use of refined triglycerides in olive oil also can be detected through triglyceride analysis Sample preparation Triglycerides can be extracted from homogenized samples with petrol ether Fats and oils can be dissolved in tetrahydrofuran 7 Control and data evaluation Quaternan gt pump y Auto Column Diode mm vacuum sampler compart a 5 GEE degasser ment elector i Water Acetonitrile 35 Sample preparation Samples were dissolved in tetrahydrofuran THF Column 200 x 2 1 mm Hypersil M OS 5 um M obile phase A 2 water B ACN methyl tert butylether 9 1 Gradient at 0 min 87 96 B at 25 min 100 B Posttime 4 min Flow rate 0 8 ml min Column compartment 60 9C Injection volume 1 ul standard UV absorbance 200 nm and 215 nm to detect triglycerides 240 nm to detect hydroperoxides 280 nm to detect tocopherols and decom posed triglycerides fatty acids with three conjugated double bonds HPLC method performance Limit of detection for saturated triglycerides for unsaturated triglycerides fatty acids with 1 double bond 2150 ng fatty acids with 2 double bonds gt 25 ng fatty acids
214. opionic acid 2 6 propoxur 28 propylamine 48 protein precipitation 14 proteins 18 protozoa 16 pulse ripple 79 pump high pressure gradient 80 low pressure gradient 78 pumps 76 84 pungency compounds 12 pyrazon 15 pyrrolidine 48 Q qualitative information 87 88 120 123 quenching effects 82 Quinolin yellow 10 R raffinose 40 redox potentials 98 reference electrode 98 refractive index detector 86 87 104 regression analysis 114 reintegration 113 repeatability 122 reproducibility 122 residues 16 26 reversed optics 91 reversed phase 58 riboflavin 5 phosphate 43 S saccharin 8 43 salad 27 salad dressing 7 sample cleanup 59 preparation 62 pretreatment 72 110 volume 72 sampling device 70 scanning 96 selected ion mode SIM 105 selectivity 87 separation 58 sequences 116 silver 99 size exclusion chromatography 66 slit 91 smoked sausage 32 soft drinks 8 solid phase extraction 65 sorbic acid 2 6 sorbitol 39 spectral libraries 115 resolution 91 spices 21 22 27 spikes 122 spreadsheet 114 standard external 113 internal 113 standard bore column 59 steam distillation 64 sterols 35 strip chart recorders 112 succinic acid 2 sucrose 40 suitability reports 110 sulfapyridine 16 sulfite 99 sulfonamides 16 sulfur dioxide 6 supercritical fluid extraction SFE IV 63 64 surfactants V sweeteners 8 switching valves 63
215. or venting instructions Installation of Gas Supplies Follow the directions in the GC site preparation installation manual and install the gas line supplies Take care in making the Swagelok connections The trap can break if too much stress is placed on the connection during tightening Leak check all connections with a helium leak detector No Snoop please Make sure to purge all lines with helium before connecting them to the GC Installation of Capillary Inlet Supplies Before handling any of the injection port supplies wash hands and or wear lint free gloves Oils on the hands will be transferred to these parts and become background in the system requiring extra bakeout time Following the instructions in the GC operating manual remove the septum nut septum and liner Discard the septum liner and liner O ring Open the oven door loosen the 1 16 inch column nut and remove the column and nut Remove the insulation cup and any necessary insu lation Figure 2 number 14 to provide access to the reducing nut Figure 2 number 12 If the lower insulation cup was not in place find it because this piece improves the inlet temperature profile With a 1 2 inch wrench remove the reducing nut Figure 2 number 12 Due to heat cycling of the GC the reducing nut will be very tight Remove the seal and the washer Figure 2 number 10 11 and discard Place a new washer in the reducing nut and a new seal flat side with groov
216. ort created Wed Sep 23 15 35 55 1998 Figure 8 Example RTLock Report Running an RTLock Method Once the RTL method is created you can analyze and process new samples This requires that you unlock and relock the methods without editing the quantitation calibration data The following example demonstrates how retention times might change when column maintenance is performed or a method is moved to a new GC MS system See Figure 9 Once the method is RTLocked new samples are ana lyzed and retention times cor rected See Figure 10 Unlocking and Relocking a Method Once a method is locked you may unlock or relock it using the same or different compounds or after additional maintenance To define a new compound for RTLocking select RTLock Setup from the View menu in Data Analysis See Figure 11 The nominal RTLock sample that rep resents the method you are work ing on will be displayed Once again use the mouse to identify a new RTLock compound FE window 2 Abundance After cut 400000 y Locked TT TIC RTLAFTER D Tic RTLOCK D 300000 200000 100000 0 Time gt 14 00 16 00 18 00 Figure 9 Overlay of an RTLocked data file labeled Locked and the resulting data file after clipping one meter off the column labeled After Cut FE window 2 Abundance BD JC NIE TIC RTLPOIST D 400000 300000 200000 100000 0 Time gt 16 00 18 00
217. ource temperature of 230 C is commonly applied in electron impact EI ioniza tion on the 5973 MSD platforms The new Inert Source when used with the new revision of the ChemStation software rev DA allows ion source temperature to be set to a maximum of 300 C As with all advances there are advantages and disad vantages in operating at higher source tempera tures This note will address several general aspects in EI operation Source Temperatures Tuning Figures 1 and 2 show the results for autotuning the Inert Source at the standard 230 C ion source temperature and the 300 C temperature limit of the new source quadrupole temperature 200 C The higher temperature for the source produces a perfluorotributylamine PFTBA spectrum that shows lower abundances of the higher mass frag ments which is not entirely unexpected The m z 219 fragment has dropped to an abundance comparable to the m z 69 ion and the ion at m z 502 has dropped about 50 This is to be expected as the internal energy of the calibrating gas has increased Note however that the isotopic ratios are maintained The user should also expect to see a higher back ground in the higher temperature tunes A portion of the background will be due to ions associated with column bleed Bleed which usually condenses in the source now is volatized and will appear as an increase in background and baseline a Agilent Technologies
218. over 6 runs areas over 6 runs Linearity DAD lt 1 lt 5 1 pmol to 4 nmol mAU 70 4 60 50 40 4 30 7 20 4 10 4 dSVv Figure 40 AD SIH LD NSV uds NE z q LAT 8 rime min 10 12 Analysis of amino acids in beer after online derivatization Injector program for onli 1 Draw 3 0 yl from via 2 Draw 1 0 yl from via 3 Draw 0 0 ul from via ne derivatization 2 borate buffer 0 OPA reagent 100 water 4 Draw 1 0 ul from sample 5 Draw 0 0 yl from via 6 Mix 7 0 yl 6 cycles 7 Draw 1 0 from vial 1 8 Draw 0 0 ul from via 9 Mix 8 0 ul 3 cycles 10 Inject 100 water FM OC reagent 100 water Cy 22 Sensitive and reliable amino acid analysis in protein hydrolysates using the Agilent 1100 Series Agilent Technical Note 5968 5658E 1999 23 R Schuster Determination of amino acids in biological pharmaceutical plant and food samples by automated precolumn derivatization and HPLC J Chromatogr 1988 431 271 284 51 Peptides Peptide mapping of phytochrome from dark grown oat seedlings using capillary liquid chromatography The analyzed phytochrome is a photoreceptor protein that controls light dependent morphogenesis in plants For example potato clod forms pale long sprouts if it germi nates in a dark cellar However if this process takes place in the light a normal plant with green leaves grows a
219. pidity of backflushing In other words rapid backflushing suggests a shorter upstream column 1 So another arrangement is at the two thirds mark or a 10 m column then the CFT tee and then a 20 m column to create a 30 m analytical column Here Purged Ultimate Union 13 backflushing would be nearly 10 times faster than the arrangement with QuickSwap and more than twice as fast as the 15 m column for the same pres sure This would be the best arrangement for the MSD with a diffusion pump Also in terms of ana lytical time this approach would provide even higher efficiency since 10 column volumes could be flushed in about 2 minutes If backflushing begins before the analytical run ends as shown in Advance Techniques and in Figure 10 then in many cases the Post Run time would be very short or entirely unnecessary yet still provide sufficient backflushing This would further reduce total cycle times The joined columns need not match in many aspects For example a 0 32 mm id may be the first column and a 0 25 mm id the second column In this situation it will be better to have the columns configured and described as they actually exist in the 7890A For example column 1 inlet is the splitless port and the outlet is the PCM module A column 2 inlet is the PCM module A and the outlet is the MSD Considerations of capacity reso ution robustness etc can be entertained in sev eral innovative ways to enhance pro
220. pl from vial 0 narrow bore HPLC and can result in fivefold improvements EM m ne a in S N with much lower solvent consumption than that 5 Draw Ow from sample from standard bore methods The analysis of fatty acids in 6 Draw Ol from vial 0 part one of this primer illustrates this principle 7 Mix 8 cycles 8 Draw 10u from vial 12 9 Inject Derivatization improves detectability of Additional investment in equipment trace species It can be automated and integrated online within the analysis Many chemistries have been developed for routine use both pre and postcolumn In brief Derivatization offers enhanced analytical response which is of benefit in food analysis Chemical modifications can be automated either before or after separation of the compounds under study In precolumn derivatization autoinjectors with sample pretreatment capabilities See chapter 6 are used whereas in postcolumn derivatiza tion additional reagent pumps are plumbed to the chro matograph upstream of the detector The latter approach adds dead volume and therefore is not suitable for the narrow bore column technique described in chapter 4 110 Chapter 10 Data collection and evaluation techniques Strip chart recorders Regardless which detection system you choose for your laboratory the analytical data generated by the instrument must be evaluated Various computing equipment is available for this task The costs depend on the reporting
221. ple with highly selective modifiers Requires large amounts of toxic solvents pH salts or ion pairing reagents can emulsify and is difficult to automate Suitable for cleaning clear liquids such as filtered bever ages solid phase extraction SFE is simple in principle The sample is first sucked through a preconditioned car tridge or disk filled with adsorbents The solid then traps the compounds of interest which can be extracted later with a small amount of an organic eluant A variety of mate rials provides a choice of selectivities for use as a fraction ing tool Two or more separate cartridges filled with specific adsorption materials can trap individual fractions of the sample SPE is one of the fastest growing sample preparation and cleanup techniques 4 Attempts have been made to auto mate both the procedure and its interface with the chro matograph Systems based on robotics and valves are available Pumping a certain volume of water sample through one or more precolumns filled with extraction materials will extract and concentrate the compounds of interest After desorption with a suitable solvent the ana lytes can be introduced into a liquid or gas chromatograph for identification and quantification The precolumns are 65 Gel permeation chromatography exchanged automatically between analyses to prevent clogging and memory effects So far this system has been used only to extract pesticides and polynuclear
222. postolumn sample treatment for the analysis of glyphosate Agilent Application Note 5091 3621E 1992 H Godel Performance characteristics of the HP 1100 Series modules and systems for HPLC Agilent Technical Note 5965 1352E 1996 127 128 Index 129 Numerics 1 4 diaminobutan 48 15 diaminopentane 48 1 butylamine 48 1 naphthol 28 2 2 dithiobis 5 nitro pyridine 108 2 4 dinitrophenyl hydrazine 108 2 naphthacyl bromide 108 2 phenylphenol 58 3 hydroxycarbofuran 28 3 ketocarbofuran 28 3 methylbutylamine 48 9 fluorenylmethyl chloroformate FM OC 108 A absorption spectrum 91 accreditation standards 59 accuracy 120 122 acesulfam 8 acetic acid 2 acids 3 3 thiodipropionic 4 acetic 2 adipic 2 amino 50 ascorbic 4 benzoic 5 butyric 38 citric 2 3 43 fatty 35 38 60 108 folic 43 fumaric 2 lactic 2 malic 2 mercapto propionic M PA 9 nordihydroguaiaretic 4 oxalic 3 panthothenic 43 phosphoric 2 propionic 2 5 sorbic 2 6 succinic 2 tartaric 2 acidulants 2 3 additives Ill adipic acid 2 adsorption chromatography 59 adulteration 35 aflatoxins 21 22 23 60 alcohols 108 alcoholysis 45 aldehydes 108 aldicarb 28 aldicarb sulfone 28 aldicarb sulfoxide 28 alducarb 28 alkaline hydrolysis 45 amines 48 primary 108 secondary 108 amino acids V 50 99 ammonia 48 AM PA 29 amperometric detection 98 amylamine 48 animal feed 18 21 2
223. powered on during most of the detector operating time it offers a lifetime of several thousand hours No warmup time is needed to get a stable baseline A holographic grat ing is used as a monochromator to disperse the polychro matic light of the Xenon lamp The desired wavelength is then focused on the flow cell for optimum excitation To minimize stray light from the excitation side of the detec tor the optics are configured such that the emitted light is recorded at a 90 degree angle to the incident light beam Another holographic grating is used as the emission mono chromator Both monochromators have optimized light throughput in the visible range A photomultiplier tube is the optimum choice to measure the low light intensity of the emitted fluorescence light Since flash lamps have inherent fluctuations with respect to flash to flash intensity a reference system based on a 95 Cut off filter Signal spectral mode Online spectral measurements and multisignal acquisition photodiode measures the intensity of the excitation and triggers a compensation of the detector signal Since the vast majority of emission maxima are above 280 nm a cut off filter not shown prevents stray light below this wavelength to enter the light path to the emis sion monochromator The fixed cut off filter and band width 20 nm avoid the hardware checks and documenta tion work that is involved with an instrument that has exchangeable filte
224. psi However because the new GC MS method is scaled from an existing GC AED method that already has RTL calibration data method translation can be used to calculate the new RTL calibration points This is useful when you want to try a scaled method rapidly and save the time required in making the five runs Note For methods that will be used exten sively the five runs approach may provide a somewhat better calibra tion It is recommended that for these methods the standard calibration be performed To calculate the five RTL calibration pairs of pressure and retention time for the GC MS method from those of the GC AED method e Take the inlet pressure used for each original GC AED RTL cali bration run and enter it into the method translation software for the inlet pressure of the original method Make sure the hold up times are locked giving a speed gain of 1 e The inlet pressure calculated in the Translated Method column will now change to a new value corresponding to the pressure that would be obtained if the calibration run were made on a GC MS system This pressure is used with the retention time obtained for the corresponding GC AED calibration run as a cali bration point for the GC MS method When all five points have been calculated in this way they are entered into the RTL calibration dialog box for the GC MS method and saved with the method Table 1 lists the original RTL calibration pressur
225. ptumless head for the inlet can eliminate the inlet problems Figure 2A shows septum cap extract that contami nated the sample after the vial was pierced 40 times during several multiple injection experiments 100 Teflon septa minimize sample conta mina tion such as this but once punc tured Teflon septa do not reseal The PTV inlet can be considered a temperature programmable split splitless inlet with the same basic configuration While it can be used hot for split and splitless appli cations this is not recommended because the volume of vaporized solvent may exceed the low internal volume of the PTV inlet PTV is ideal for cold split or splitless applications avoiding most of the problems associ ated with hot inlets such as sample discrimination liner overload and sample decomposition For large volume injections the PTV is used in a solvent vent or solvent elimination mode Sample is intro duced into the inlet with the inlet temperature near the boiling point of the solvent and with a relatively high split ratio The solvent and low boiling solutes is vented while the higher boiling solutes more than about 100 C above the solvent boiling point remain and are concentrated in the inlet After a preset time the split vent is closed and the inlet tem perature increased to transfer the solutes and any residual solvent to the column for separation Because the sample is evaporated from the inlet nonvol
226. r SE MODI Windows Version 2 0 and Method Translation Tool Version 2 0 I I I I I I AU i a GET Available at http www 10 30 50 70 90 110 130 15 0 170 190 210 230 25 0 min chem agilent com servsup Figure 8 Comparison of chromatogram on GC system 1 with GC MSD system after retention usersoft main html time locking Constant Pressure Mode Table 3 GC FID vs GC MSD Method Translated then Locked Retention Times Constant Pressure Conditions GC FID RT Original GC MSD Difference Component 18 2 psi 7 9 psi min Ethylbenzene 10 315 10 338 0 023 p Xylene 10 620 10 642 0 022 m Xylene 10 869 10 890 0 021 i Propylbenzene 12 038 12 053 0 015 o Xylene 12 613 12 630 0 017 n Propylbenzene 13 492 13 508 0 016 a Methylstyrene 18 276 18 267 0 009 Phenylacetylene 19 406 19 389 0 017 b Methylstyrene 21 008 20 987 0 011 Benzaldehyde 25 475 25 415 0 060 Average 0 021 Used in locking calculation Phenylacetylene T Ethenyl 3 methyl benzene 4 VT EEGLEPRET RR BO ns Re Ce Ee CARR nS Be R E I ET 17 6 18 0 18 4 18 8 19 2 19 6 20 0 20 4 20 8 min Figure 9 GC MSD identification of impurity in shoulder of phenylacetylene peak Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Copy
227. r liquid chromatography products at Agilent Technologies O Copyright Agilent Technologies Company 1996 2001 All rights reserved Reproduction adaption or translation without prior written permission is prohibited except as allowed under the copyright laws Printed in Germany September 1 2001 Publication Number 5988 3294EN Preface Modern agriculture and food processing often involve the use of chemicals Some of these chemicals and their func tions are listed below Fertilizers increase production of agricultural plants Pesticides protect crops against weeds and pests Antibiotics prevent bacteria growth in animals during breeding Hormones accelerate animal growth Colorants increase acceptability and appeal of food Preservatives and antioxidants extend product life e Natural and artificial sweeteners and flavors improve the taste of food e Natural and synthetic vitamins increase the nutritive value of food Carbohydrates act as food binders Such chemicals improve productivity and thus increase competitiveness and profit margins However if the amounts consumed exceed certain limits some of these chemicals may prove harmful to humans Most countries therefore have established official tolerance levels for chemical additives residues and contaminants in food products These regulations must be monitored care fully to ensure that the additives do not exceed the pre scribed levels To ens
228. rate 0 5 ml min Column compartment 40 9C Injection volume 2 5 ul Detector UV DAD detection wavelengths 230 30 nm 400 100 nm reference wavelengths 280 40 nm 550 100 nm HPLC method performance Limit of detection 1ppbwith S N 22 Repeatability of RT over 10 runs 0 82 areas over l 0runs 2296 Water Methanol Quaternary pump Auto Column Diode vacuum 77 sampler compart T degasser ment Bor Control and data evaluation Sample preparation Different food matrices require different extraction procedures These procedures include alkaline hydrolysis enzymatic hydrolysis alcoholysis direct solvent extraction and supercritical fluid extraction of the total lipid content Chromatographic conditions for UV detection The HPLC method presented here was used in the analysis of a vitamin standard mAU 700 600 500 400 300 200 100 Standards Vitamin D 6 tocopherol B and y tocopherol a tocopherol 2 4 6 8 10 12 14 Time min Figure 35 Analysis of fat soluble vitamins with UV detection 45 Column Mobile phase Stop time Flow rate Oven temperature Injection volume Detector Working electrode Operation mode Working potential Range Reference electrode Response time 125 x 4mm Lichrospher RP 18 5 um methanol 5 g l lithiumperchlorate 1 g l acetic acid 20 min 1ml min 30 9C 1 yl standard electrochemical glassy carbon ampero
229. re Instead of using 2 1 6 psi as is used on the GC AED method translation calculates that 17 93 psi on the GC MS system will result in matching retention times As mentioned above this inlet pressure is calculated on the assumed dimen sions of the column in the GC MS system To get the retention times to match precisely RTL is used To retention time lock the GC MS method to the GC AED method in this example it is necessary to construct an RTL calibration file for the GC MS system Construction of this file only needs to be done once All subse quent users of the GC MS method will then be able to use this calibration file for a similarly configured GC MS instrument The RTL calibration file is con structed by running five calibration runs of the target compound in this case methyl chlorpyrifos at five dif ferent inlet pressures The runs are made at conditions identical to the nominal method except that four of the runs are made at different pres sures The pressures used are typically Target pressure 20 Target pressure 10 Target pressure nominal method pressure Target pressure 10 e Target pressure 20 The retention time of the target com pound is determined for each run The resulting set of five pressures and corresponding retention times is then entered in the RTL calibration dialog box for the method and saved with the method To lock the method on the GC MS setup the t
230. red printing peripherals ak E Em s fhe aie F B Bale 117 Networked data systems The Agilent ChemStation remote access and data storage modules combine isolated islands of data into a powerful client server networked information system Each Agilent ChemStation becomes a network client It is possible to oversee and control all laboratory operations securely and easily from any computer on the network The progress of each analysis is monitored to ensure the quality of the results the first time the sample is analyzed Appropriate action can be taken with the access remote capability from wherever you happen to be if the performance looks suspect Laboratory data is automatically stored on one centralized and secure server system In brief Which data handling technique is most effective and eco nomical for your laboratory depends on several factors the size of the laboratory the role of the laboratory in the organization industrial testing public safety testing and so on the demands on sample throughput the range of analytes under study For laboratories with few instruments and low sample throughput integrator systems normally suffice although a PC may be more appropriate for automated operation of
231. red to re use methods thus saving time and money GC AED 1x 2 0 5 10 15 20 25 30 35 40 min GC micro ECD 3x 0 2 4 6 8 10 12 min Figure 8 Chlorine chromatogram from 1x GC AED method top and 3x micro ECD method bottom of three component locking mixture Peak identifications 1 dichlorvos 2 methyl chlorpyrifos 3 mirex Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Microsoft and Windows NT are U S registered trademarks Copyright 2006 Agilent Technologies Inc Printed in the USA 1 2006 5967 5820E Agilent Technologies Innovating the HP Way The 5973N inert MSD Using Higher lon A Application e Authors Harry Prest and Charles Thomson Agilent Technologies Inc 5301 Stevens Creek Boulevard Santa Clara CA 95052 8059 Abstract The new 5973N inert MSD and ChemStation software G1701DA offers the capability of operating the ion source at higher temperatures This feature combined with the improved inertness of the source can provide the user with improvements in analysis if exploited coher ently This application note provides advice and examples of how to explore the utility of ion source temperature Introduction The default ion s
232. residues of chemotherapeutic and antiparasitic drugs in food products Malisch et al have developed an HPLC method to determine 11 of these compounds The internal standard ISTD comprises benzothiazuron and pyrazon Sample preparation After homogenization or mincing and pH adjustment the samples were extracted using liquid liquid extraction followed by degreasing purification and concentration Control and data evaluation Quaternary pump Auto Column Diode L iml sampler compart array REA inem ment detector degasser Water Acetonitrile 16 Sample preparation Column Mobile phase Gradient Flow rate Injection volume Detector Sample preparation was done according to reference 250 x 4 6 mm Spherisorb ODS 2 5 um A z sodium acetate buffer 0 02 M pH 24 8 B ACN water 60 40 start with 8 B at5min 896 B at 7 min 20 B at 14 min 23 B at 16 min 33 B at 19 min 40 B at 21 min 50 B at 26 min 60 B at 30 min 80 B at 33 min 90 B at 43 min 90 B at 55 min 8 B 1 5 ml min 20 ul UV DAD detection wavelengths 275 80 nm 315 80 nm and 360 80 nm reference wavelength 500 100 nm HPLC method performance Limit of detection Repeatability of RT over 10 runs 0 001 0 05 mg kg 0 12 96 of areas over 10 runs 1 5 96 Chromatographic conditions The HPLC method presented here for the analysis of residues of dru
233. right 2000 Agilent Technologies Inc Printed in the USA 3 2000 5966 2469E Agilent Technologies Innovating the HP Way Authors Bill Wilson Philip L Wylie and Matthew S Klee Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 1610 USA Abstract Reduced sample detection limits is a continuing goal in gas chromatography Large sample injection volume is one possible approach New inlets and injec tion techniques supporting large volume injection LVI have been developed in recent years This paper discusses the uses and limitations of LVI describes LVI with a programmable temperature vaporizer PTV inlet and reviews the results of LVI analysis of pesticides and straight chain hydrocarbons Key Words Large volume injection LVI pro grammed temperature vaporizer PTV gas chromatography GC pesti cide analysis hydrocarbon analysis Large Volume Injection for Gas Chromatography Using a PTV Inlet Application Gas Chromatography March 1997 Introduction The demand for lower detection limits is important in environmental pharmaceutical food analysis and other gas chromatography GC applications This demand has driven instrument manufacturers to provide more sensitive instrumentation and procedures which has prompted reg ulators to reduce allowable limits and so on in a never ending cycle Improvements in sample handling sample injection techniques and detectors have al
234. rmation and that designed for flow rates between 0 05 and 5 ml min high pressure gradient formation In separating the multiple constituents of a typical food sample HPLC column selectivity with a particular mobile phase is not sufficient to resolve every peak Changing the eluant strength over the course of the elution by mixing increasing proportions of a second or third solvent in the flow path above the column improves peak resolution in two 76 Gradient formation at high pressure Gradient formation at low pressure ways First resolution is improved without extending the elution period which prevents long retention times peaks that have been retained on the column for a longer period of time tend to broaden and flatten through diffusion lowering the S N and therefore detection levels Second gradient elution sharpens peak widths and shortens run time enabling more samples to be analyzed within a given time frame The solvents that form the gradient in front of the column can be mixed either after the pump has applied high pressure or before at low pressure If mixing takes place after pressure has been applied a high pressure gradient system results this is most often achieved by combining the output of two isocratic pumps each dedicated to one solvent Ability to form sharp gradient profiles and Expensive An additional mixer for low est to change solvents rapidly 10096 A to mixing noise at flow rates below 200 u
235. rol and data evaluation Quaternary T pump Auto Column Diode L vacuum 7 sampler compart array EL degasser ment detector a LEE Water Acetonitrile 12 Sample preparation Column Mobile phase Gradient Flow rate Post time Column compartment Injection volume Detector injection without further preparation 100x 4mm Hypersil BDS 3 um A water 0 15 ml H550 conc pH 223 B ACN start with 10 B at 3 min 40 B at 4 min 40 B at 6 min 80 B at 7 min 90 B 0 8 ml min 3 min 30 C 5 yl UV DAD detection wavelength 280 80 nm reference wavelength 360 100 nm Conditions as above except Column Mobile phase Gradient Flow rate 100 x 2 1 mm Hypersil ODS 5 um A water 5 mM NaH PO B 2 methanol at 10 min 70 B 0 4 ml min HPLC method performance Limit of detection Repeatability of RT over 10 runs of areas over 10 runs 0 2 5 ng injected amount S N 22 02 96 196 Chromatographic conditions The HPLC method presented here for the analysis of vanillin is based on reversed phase chromatography UV spectra were evaluated as an additional identification tool Norm Vanillin 400 4 hydroxy benzoic acid 4 hydroxybenzaldehyde 300 Ethyl Coumarin vanillin N 200 Vanillin alcohol Standard 100 Vanillin extract Time min Figure 8 Determination of the quality of vani
236. romatography May 1998 Key Words Pesticides GC GC AED retention time locking RTL method transla tion scalable RT libraries Introduction Interest in the analysis of pesticide residues has been increasing recently in part due to the discovery that some of these compounds act as endocrine disrupters Agilent Technologies has responded to the need for rapid accu rate and comprehensive screening analysis for pesticides by developing a method to screen for 567 pesticides and suspected endocrine disrupters The method uses element selective detection and a retention time locked library of retention times to find and identify pesticides in a sample In the method sample extracts are run with element selective detection using a prescribed set of chromato graphic conditions and with the column retention time locked to the retention times in a table If any peaks containing heteroatoms are observed the section of the table corresponding to a small time window around the observed peak is searched The time search results are further sorted using the observed element content of the peak The combination of time and element content narrows rapidly the possible compounds that could have produced the heteroatom response to a few pesticides The element selective detection is done with either gas chromatography atomic emission detection GC AED which can screen for all the individual elements found in pesticides or
237. romide Control and data evaluation Quaternan pump 1 Auto Column Variable LJ z vacuum sampler compart Wavelength EA pes degasser ment detector EEEN Water Acetonitrile 38 Sample preparation 0 215 g fat was hydrolyzed with 500 ul M EOH KOH at 80 9C for 40 min in a thermomixer After cooling 1 5 ml ACN THF 1 1 was added and the mixture was shaken for 5 min The mixture was then filtered through a 0 45 um M Satorius Column Mobile phase Gradient Flow rate Column compartment Detector Derivatization inisart RNML from 200 x 2 1 mm M OS 5 um A water 70 B ACN 1 THF 30 at 5 min 30 B at 15 min 70 B at 17 min 70 B at 25 min 98 B 0 3 ml min 50 C variable wavelength 258 nm 60 mg ml bromophenac yl bromide was dissolved in ACN Injector program for online derivatization 1 Draw 2 0 yl from v 2 Draw 1 0 ul from ai 3 Draw 1 0 ul from vi agent 4 Draw 0 0 ul from vi ACN THF 50 50 5 Draw 1 0 yl from sample 6 Draw 0 0 ul from vi 7 Draw 1 0 ul from vi agent 8 Draw 0 0 ul from v 9 Draw 1 0 ul from vi 596 TEA 10 Draw 0 0 ul from 11 Mix 9 ul in air 30 12 Wait 2 0 min 13 Inject ia ial 2 ACN 3 al 3 derivatization D wash bottle qp wash bottle derivatization a a w 4p la a wash bottle acetonitrile Un vial 4 wash bottle yl min speed 10 times
238. rs and slits The excitation and emission monochromators can switch between signal and spectral mode In signal mode they are moved to specific positions that encode for the desired wavelengths as with a traditional detector This mode offers the lowest limits of detection since all data points are generated at a single excitation and emission wave length A scan of both the excitation and the emission spectra can be helpful in method development However only detectors with motor driven gratings on both sides can perform such a scan Some of these detectors also can transfer this data to a data evaluation computer and store spectra in data files Once the optimum excitation and emission wavelength has been determined using scanned spectra detectors with grating optics can be programmed to switch between these wavelengths during the run The spectral mode is used to obtain multi signal or spec tral information The ignition of the flash lamp is synchro nized with the rotation of the gratings either the excita tion or emission monochromator The motor technology for the gratings is a long life design as commonly used in high speed PC disk drive hardware Whenever the grating has reached the correct position during a revolution the Xenon lamp is ignited to send a flash The flash duration is below two microseconds while the revolution of the grat ing takes less than 14 milliseconds Because of the rotat ing monochromators the loss in
239. rt array degasser ment detector Water Acetronitrile 14 Specht W Organochlor und Organophosphor Verbindungen sowie Stickstoffhaltige sowie andere Pflanzenschutzmittel DFG M ethoden sammlung 1982 19 26 Sample preparation Column Mobile phase Gradient Flushing time Post time Flow rate Oven temperature Injection volume Detector Salad was homogenized and then extracted with liquid liquid extraction The extract was cleaned with gel permeation chromatography using cyclohexane ethyl ace tate Spices were pre pared according to Specht with gel per meation chromatography 100 x 3mm Hypersil BDS 3 um water ACN 95 5 at 10 min 25 ACN at 26 min 42 ACN at 34 min 60 ACN 10 min at 100 ACN 6 min 0 5 ml min 42 C 3 10 yl UV DAD detection wavelengths 214 15 nm 230 20 nm and 245 20 nm reference wavelength 400 80 nm HPLC method performance Limit of detection Repeatability of RT over 10 runs 0 01 ug l 02 96 of areas over 10 runs 196 Chromatographic conditions The HPLC method presented here was used for the analysis of pesticides in salad samples and spices mAU 3 different Vinclozolin salad samples 1204 804 Folpet im 40 Carbendazim 10 i 2 25 30 i 40 Time min Figure 19 Analysis of pesticide residues in three different salad samples Carbendazim has a low recovery rate of only approximately 40 96 Vinclozol
240. s an approximate volume of 1 mL With a GC MS flow rate of 1 mL min a 2 min splitless injection would be necessary However this long splitless time has not been common There are two reasons for this Conventional manually controlled capillary inlets were pressure regulated constant pres sure regardless of oven temperature and not flow regulated changing pressure with oven temperature so a higher than optimal flow was set initially so that the flow did not go to zero at high oven temperatures Thus a typical splitless or OFF time has been between 0 5 to 1 5 min Liner volumes smaller than the textbook exam ples have typically been used Since a 2 mm liner 250 uL volume was more commonly employed the splitless time was proportionally shorter Finally with the programmable control afforded by EPC flows can be reliably pulsed during the injection process With pulsed splitless injections flows during the splitless time can be 2 6 mL min resulting in splitless times less than 2 min for a 4 mm id liner The pulsed splitless injection mode on the Agilent 6890 is recommended for GC MS work After the injection pulse the system returns to analytical flow rates of 1 4 mL min He The highest flow allowable depends on the MSD Refer to the appropriate MS hardware manual for your detector s limit Unless all analytes have high boiling points the ini tial oven temperature should be set to take advan tage of th
241. s and test with a sample followed by a solvent blank to see if this is sufficient Experimentation with particular samples will enable setting these requirements more efficiently Conclusions Alternative Configurations The CFT is very rich and allows many possible arrangements these are only a few suggestions or alternatives The CFT tee used here can be replaced by a purged two way splitter with one channel plugged 63180 61500 or even the QuickSwap itself can be moved back from the MSD interface and suspended in the oven Figure 11 Purged Ultimate Union However the best CFT tee device appears to be the new Purged Ultimate Union G3186 60580 Figure 11 As the name describes this is essen tially a union with a gas purging line making it a very low dead volume tee It occupies very little space and can be suspended from the column cage the oven wall or through the upper GC wall Pre liminary tests of this Purged Ultimate Union using DDT and Endrin have shown very little breakdown Chromatographic behavior is also very good Similarly the carrier control need not be the back injection port split splitless module a Pressure Control Module PCM or EPC module can be used Of the two the Pressure Control Module may be more convenient Most importantly the CFT tee position itself does not need to be exactly in the middle The best arrangements can be considered on the basis of selection against components and the ra
242. s includes Valves Valves are used to switch to guard columns and online solid phase extraction techniques Switching valves are common in HPLC and some instruments even have built in column compartment valves With a six port valve for example the eluant stream can be switched from one column to another to cut out a peak This peak is then analyzed on the second column e SFE interfaces This technique is rather new and online systems are under development An offline procedure has been used successfully in the analysis of vitamin K in infant formula Precolumn derivatization This well accepted and commercially available technique has been applied in the analysis of amino acids in beer see page 50 ff Reagents also can be used postcolumn see page 28 e Automated solid phase extraction This relatively new technique is used to analyze bittering compounds in beer Solids Ultrasonic bath liquid extraction Solid samples for example chocolate or meat should be homogenized before such techniques as steam distillation SFE or ultrasonic stimulated liquid extraction are applied Ultrasonic bath liquid extraction is a very simple extraction method Selectivity is achieved through the use of appropriate solvents Antioxidants and preservatives can be extracted with this technique if the matrix is low in fat 63 Steam distillation Supercritical fluid extraction S x Uses relatively s
243. s mixed concentration with citric acid 100 mg i adiri gtric aid Chromatographic conditions 2 p T or OEM oxalic The HPLC method presented here for the analysis of meat is add 4 ml methanol based on reversed phase chromatography and UV visible 5 min in the ultrasonic diode array detection UV spectra were evaluated as an bath additional identification tool add water up to 10 ml total volume gt centrifuge gt inject Oxytetracycline 1 8 ng 3 Oxytetracycline Column 100x 4mm 61 Hypersil BDS 3 um 5 2 Mobile phase A water pH 2 1 with 1 sulfuric acid 44 B ACN 34 Wavelength nm Gradient start with 15 B at 10 min 60 B 24 370 ppb Flow rate 0 5 ml min Pork muscle Column compartment 25 C 1 Detector UV DAD 04 detection wavelength 355 nm 20 nm 2 s min 6 8 reference wavelength 600 100 nm Figure 12 Trace analysis of tetracycline residues in meat Identication of oxytetracycline through spectra comparison HPLC method performance Limit of detection for UV DAD 100 ppb Repeatability ofRToverl runs 0296 of areas over 10 runs 2 96 18 Fumonisins Fumonisins are characterized by a 19 carbon aminopoly hydroxyalkyl chain which is diesterified with propane 1 2 3 tricarboxylic acid Analogues B 1 3 in figure 13 show a difference only in the number and position of the hydroxyl groups present on the molecule Fragmentation experiments using collision induced disso
244. s not as likely to affect the signal This application note also describes a programmed bake out of the source and quadrupole that can be automatically implemented after source cleaning This bake out provides a rapid lowering of the air water background and can be used within the sequence table as part of the instrument performance checkout For More Information For more information on our products and ser vices visit our Web site at www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2004 Printed in the USA February 10 2004 5989 0678EN Agilent Technologies Combined El and CI Using a Single Source Technical Overview e e e e e e e e o o e e 0 o o o 0 0 e o e e Chris Sandy e Agilent Technologies e Introduction The Agilent 5973x gas chromatograph mass selec tive detectors GC MSDs come with sources opti mized for electron ionization EI and chemical ionization CI However there are occasions where another ionization mode is desired without chang ing sources This note demonstrates the capability of acquiring high quality EI spectra with the CI source Data Acquisition An Agilent 5973 inert MSD with a
245. s of the sample loop into the mobile phase fronts in front of and behind the column So called low dead volume fittings with the minimum required internal capacity are available These fittings have no dead ends or unnecessary spaces where solvent and sample can mix Simple manual injectors remain popular in some laboratories because they are inexpensive and because their operation requires little previous experience which is important if the equipment is used infrequently With a precision syringe the operator can fill the sample loop at atmospheric pressure by injecting the contents into the injection port A switch of the rotor attached to the valve realigns the valve ports to the inject position Solvent from the pump then flushes the contents of the sample loop onto the column Continual flushing during the run keeps the injection port and valve clear of remnants of previous samples Inexpensive No automation and no provision for online derivatization Syringe must be cleaned manually offline n Automated injectors Autosampler with sample pretreatment capabilities Automated injectors contain a mechanically driven version of the same six port valve found in manual injectors Pneumatic or electrical actuators control the valve as it switches between steps in the injection cycle A metering device can handle injection volumes of 0 1 1500 pl With sample loops of larger capacity such a device can inject up to 5 ml Vi
246. s with volatile early eluting compo nents such as extracts of drinking water The COC SVE technique is discussed elsewhere PTV LVI with PTV is ideal for trace analy sis of later eluting solutes boiling points approximately 100 C higher than the solvent and for dirty sam ples Typical injection volumes for solvent elimination PTV are 25 to 100 uL Injection volumes up to 1 mL have been demonstrated The large sample volumes are injected by manual injection by multiple sample injections from a standard automatic sampler or by LVI with a variable speed injector An automatic injector is recom mended for maximum reproducibility A standard automatic sampler making repeat injections is more cost effec tive than purchasing a variable speed sampler and requires less solvent for syringe cleaning The Agilent 6890 Series GC uses a standard automatic sampler with syringe sizes up to 50 uL A 50 uL syringe can inject up to 25 uL Multi ple injections can be used with the PTV inlet when even larger volumes are required With the 6890 GC system delay between injections can be controlled as well as the number of injections or the total injection volume Injection parameters are set through the Agilent ChemStation A problem with multiple injections is the increased number of punctures of the GC inlet and vial septa This reduces septum life and increases the possibility of contamination of sample and inlet Using a se
247. sensitivity in the spectral 96 Multisignal mode is much lower compared to conventional dual wave length detection with UV detectors PNA analysis for example can be performed with simulta neous multi wavelength detection instead of wavelength switching With four different wavelengths for emission all 15 PNAs can be monitored figure 63 T excitationWlbat260 nin 1 Naphthalene 8 Benz a anthracene 4 emission WL at 350 420 2 Acenaphthene 9 Chrysene 440 and 500 nm 3 Fluorene 10 Benzo b fluoranthene 4 Phenanthrene 11 Benzo k fluoranthene 5 Anthracene 12 Benz a pyrene 6 Fluoranthene 13 Dibenzo a h anthracene LU 7 Pyrene 14 Benzo g h i perylene 5 15 Indeno 1 2 3 cd pyrene Reference chromatogram with switching events Ex 275 Em 350 TT Time min Figure 63 Simultaneous multi wavelength detection for PNA analysis The upper trace was received with traditional wavelength switching Ex Em 260 420 nm Q Ex Em 270 440 nm Ex Em 260 420 nm Q Ex Em 290 430 nm Ex Em 250 550 nm 97 Electrochemical detectors Reference electrode Counter Working electrode electrode Figure 64 Three electrode electrochemical detector Highly specific Flash lamps eliminate Fluorescence spectra are not commonly drawback of baseline drift from heat used to confirm peak identity transfer Fluorescent tagging improves detection limits Electrochemical detection techniques are bas
248. sults Qualitative information HPLC analytes can be identified on the basis of their retention times and either their UV visible or mass spectra Compounds on the other hand are identified primarily according to the degree of agreement between retention times recorded using calibration standards and those obtained from the sample Unfortunately co eluting peaks can falsify results obtained with samples containing unknowns especially for food matrices such as meat vegetables or beverages In such cases samples often can be identified using UV visible spectral information A diode array detection system enables online acquisition and a number of software packages offer automatic evaluation for example for the analysis of polynuclear aromatic hydrocarbons PNAs and pesticides 123 References and Index Part Three References 10 11 12 13 126 D N Heiger High Performance Capillary Electrophesis An Introduction Agilent Primer 5968 9936E 2000 CD ROM CE Partner Agilent publication 5968 9893E CD ROM CE Guidebook Agilent publication 5968 9892E Official M ethods of Analysis Food Compositions Additives Natural Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 A M Di Pietra et al HPLC analysis of aspartame and saccharin in pharma ceutical and dietary formulations Chromatographia 1990 30 215 219 A G Huesgen R Schuster Sensitive analysis of synth
249. ters of the method have been determined five calibration runs are performed The runs are made at con ditions identical to the nominal method except that four of the runs are made at different pressures The pressures used are typically Target pressure 20 e Target pressure 10 e Target pressure nominal method pressure e Target pressure 10 e Target pressure 20 The retention time of the target com pound is determined for each run The resulting five pairs of inlet pres sures and corresponding retention times are entered into the ChemStation software to generate an RTL calibration file Figure 1 shows the dialog box used to enter the calibration data After the data is entered a plot is displayed as shown in figure 2 The maximum departure of the fitted curve from the data is given for both time and pres sure If the fit is acceptable the reten tion time versus pressure calibration is stored and becomes part of the GC method This calibration need only be generated once Subsequent users of the method can use this calibration when running the method on a similar instrument setup regardless of location To relock a system or lock a new one 1 Setup the method conditions and run a standard containing the target compound 2 Enter the actual retention time of the target compound into the Re Lock current method dialog box see figure 3 9 Update the 6890 method with the new calculat
250. the column and relocking the method Table 1 Method Conditions Column HP 5 25 m x 0 32 pm x 0 52 um p n 19091J 112 Carrier gas Helium constant pressure mode nominal 3 761 psi RTL peak Fluoranthene at 11 112 min Split splitless inlet 300 C pulsed splitless 7 psi for 0 3 in 50 mL min purge at 0 75 min Oven 55 C 1 1 min to 320 C 6 5 min at 22 88 C min Sample 1 pL injection PAH 0 01 to 5 ppm concentration range MSD Scan 45 400 u Samples 2 Autotune EM 200V Source 230 C Quad 150 C Transfer line 280 C Results and Discussion The test sample with 16 polynuclear aromatic hydrocarbons PAHs was chosen for this example as it covers a wide range of physical properties and provides several challenging separations This requires the retention time precision to be as tight as possible to ensure correct identification of the Table 2 Retention Time Precision of Low Pressure RTL peaks of interest and relocking must be effective or peak identification will fail on the different column Samples were injected in triplicate to measure the retention time precision of the locked method at different concentrations from 0 01 to 5 ppm Table 2 shows the performance metrics for the retention time and also the correlation co efficient for the analysis The initial retention times in Table 2 were achieved at a head pressure of 3 761 psig This setpoint was determined from the retention time calibratio
251. three component locking mixture Peak identifications 1 dichlorvos 2 methyl chlorpyrifos 3 mirex Figure 7 shows the method transla tion from the GC AED method to the 0 1 mm id column with a scale factor of 3 A speed gain of 3 was again chosen based on oven and inlet limi tations as described above The same scaling process as used above is followed The RTL calibration points for the new 3x 0 1 mm micro ECD method were both calculated with method translation and measured Table 5 shows the calculated values When the locking pressures from the measured and calculated values were examined the calculated values pro vided much poorer predictions of locking pressure than expected The pressure required to actually lock the column was confirmed to be 65 95 psi as predicted by the mea sured RTL calibration data Method translation had predicted the inlet pressure would be 58 514 psi for an assumed 10 m column length Because the actual locking pressure was noticeably higher this suggests that the actual column length was longer and or the column diameter was smaller and or the film thickness larger than the assumed values As an experiment it was assumed that the problem was in the assumed length of the column used in calculat ing the RTL calibration points The column length entry for the 0 1 mm column was iteratively adjusted until the calculated inlet pressure matched the actual locking pressure 65 95 psi T
252. tion This application demonstrates the ability of the For more information on our products and services 7890 GC system to perform RTL at low pressures visit our Web site at www agilent com chem sub 5 psi such as those experienced when using a 320 um column in a GC MS system The average retention time variation before and after column maintenance for a 16 PAH mixture is less than 0 5 sec providing high confidence in peak assignments even with critical separations www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Printed in the USA July 11 2008 5989 8366EN Agilent Technologies Case Study for Agilent Lab Monitor and e e Q o eo mE o0 Q e Application 050 o o e e e e 2 e Author Chunxiao Wang Agilent Technologies Shanghai Co Ltd 412 Ying Lun Road Waigaoqiao Free Trade Zone Shanghai 200131 China Abstract Agilent Lab Monitor amp Diagnostic LMD software can monitor in real time all the Agilent GCs and LCs in your lab It automatically tracks supply usage monitors chro matographic quality and notifies you of maintenance needs before a problem occurs by keeping track of injec tions hours of operation and
253. tionary phases can be classified according to the mechanism by which they separate molecules partition phases adsorption phases e ion exchange phases size exclusion phases Nowadays the most popular column material is reversed phase in which separation is achieved through partition and through adsorption by unprotected silanol groups In reversed phase chromatography the stationary phase is nonpolar or less polar than in the mobile phase and the analytes are retained until eluted with a sufficiently polar solvent or solvent mixture in the case of a mobile phase gradient Reversed phase materials have wide application and a long lifetime Moreover these media have good batch to batch reproducibility low equilibration times high mechanical stability and predictable elution times and elution order Reversed phase chromatography is frequently used in food analysis as shown in part one of this primer Compared with reversed phase media ion exchange materials have a shorter lifetime are less mechanically stable and take longer to equilibrate These columns have limited application in food analysis and are used primarily for inorganic cations and anions or for glyphosate 58 Size exclusion gels Adsorption media Size exclusion chromatography is used for sample cleanup and fractionation and is described in more detail in chapter 5 Sample preparation Adsorption chromatography is used for sampling and cleanup
254. tive results for the analysis of antibiotic drugs Peak Purity Check and Identification Part 1 General information REPORT Operator Name BERWANGER 0 1 s0B Date amp Time 10 Sep 86 9 17 am Data File Name LH LETAAO0A Integration File Name DATA DEFAULT I Calibration File Name DATA ANTI Q Quantitation method ESTD Sample Info DOTIERUNGSVERSUCHE Misc Info Method File Name ANTIBI M Library File Name DATA ANTIBI L Reference Spectrum Apex Time window from 6 0 amp to 2 0 amp Dilution Factor 1 0 Sample Amount 0 0 s1B Vial Inj No calibrated by Area response Wavelength from 230 to 400 nm Library Threshhold 950 Peak Purity Threshold 950 Smooth Factor i Resp Fact uncal peaks None Part 2 Quantitation peak purity check and peak identification Name Amount Peak Ret Cal Ret Lib Ret Purity Library Res ng 1 min min min Matchfactor Sulfapyridine 10 31 A 12 183 12 143 12 159 999 1000 0 9 Furazolidone 4 54 A 16 096 16 024 16 028 992 984 1 3 Pyrazon 13 72 A 19 024 18 987 19 000 1000 1000 1 7 N Acetylsul fapyidine 14 66 A 23 307 23 282 23 282 976 1000 1 1 Ethopabat up 13 40 A 23 874 23 840 23 848 911 996 2 3 Benzthiazuron 12 80 A 24 047 24 024 24 029 998 1000 0 7 Nicarbazin up 3 00 A 32 733 32 722 32 709 336 984 1 2 Enables maximum peak purity and identity measurement of multiple wavelengths acquisition of absorbance spectra and spectral library searches
255. tors 2 0 ccc ccc eee n 89 Diode array detectors 0 0 eee eee eee eee 90 Three dimensions of data 000 0e ee eeee 91 Fluorescence detectors 0 e eee eee eens 95 Cut off filter o errire eiiie ea cee enn ens 96 Signal spectral mode 0 0c eee eee eee 96 Online spectral measurements and multi signal acquisition 0 0 0 00 96 Multisignal 2 0 eee eee 97 Electrochemical detectors 000 cece eee 98 Electrode materials 0 00 0 cece eee eee 99 Flow cell aspects 00 0 0 eee ee eee eee 99 Automation features 0 0 ccc eee es 100 Mass spectrometers 20 20 c eee cece eee eee 101 API interfaces 2 0 0 0 ccc cece eens 102 Refractive index detectors 0 0 e ee ee eee 104 vill Part Three References and Index Chapter9 Derivatization chemistries Addition of UV visible chromophores 108 Addition of a fluorescent tag 2 2 005 109 Precolumn or postcolumn 000000 109 Automatic derivatization 00 0202 eee eee 110 Chapter 10 Data collection and evaluation techniques Strip chart recorders 00 0 e eee cece eens 112 Integrators i69 edad Piney ad ERN UE Cases 113 Personal computers 0 0 2 cece cece eee eee 114 Local area networks 000 cece eee eee eee 117 Networked data systems 02020e eee eeee 118 Chapter 11 F
256. traction according to Para 35 LMBG 92 Hypersil ODS 100 x 2 1 mm id 3 um particles water methanol ACN 63 26 11 as isocratic mixture flow rate 0 3 ml min at 25 C DAD 365 20 nm Fluorescence detector FLD excitation wavelength 365 nm emission wavelength 455 nm Ochratoxin A cereals flour figs gt extraction according to Para 35 LM BG acidify with HCI gt extract with toluene SiO cleanup elute toluene acetic acid 9 1 Lichrospher 100 RP 18 125 x 4 mm id 5 um particles water with 2 acetic acid ACN 1 1 flow rate 1 ml min at 40 C FLD excitation wavelength 347 nm emission wavelength 480 nm Zearalenone cereals gt extract with toluene c Sep pak cleanup elute toluene ace tone 95 5 gt AOAC 985 184 a zearalenol and zearalenone in corn Hypersil ODS 100 x 2 1 mm id 3 um particles water methanol ACN 5 4 1 isocratic mixture flow rate 0 45 ml min at 45 C DAD 236 20 nm FLD excitation wavelength 236 nm emission wavelength 464 nm Patuline apple products Lebensmittel und Bedarfsgegenst ndegesetz Germany 100 B is recommended for cleaning the column 22 c Cleanup on Extrelut Superspher RP 18 125 x 4 mm id c silica gel cleanup gt elute toluene ethylacetate 3 1 4 um particles water 5 95 ACN flow rate 0 6 ml min at 40 C DAD 270 20 nm or Lichrospher diol 125 x 4 mm id 5 um parti
257. trasonic bath after homogenization of the food sample Control and data evaluation Quaternary a pump Auto Column Diode L Lees vacuum 7 sampler compart array ment detector EEN degasser Water Acetonitrile 42 Sample preparation Column filtration 100x 4mm Hypersil BDS 3 um Mobile phase Azwater with pH 22 1 H S0 99 B zACN 196 Gradient at 3 5 min 196 B at 11 min 25 B at 19 min 90 B Post time 6 min Flow rate 0 5 ml min Column compartment 30 9C Injection volume 2 5 yl Detector UV DAD detection wavelength 220 30 nm reference wavelength 400 100 nm HPLC method performance Limit of detection 500 pg injected amount S N 22 Repeatability of RT over 10 runs 0 2 96 areas over 10 runs lt 2 Chromatographic conditions for UV detection The HPLC method presented here was used to analysis vitamins in a vitamin drink Folic acid d in Norm S E E c 1500 y E z c 5 c 1000 n t ats a Standard Saccharin Vitamin tablet 8 10 12 Tine min Figure 32 Analysis of water soluble vitamins in a vitamin tablet Norm Norm 800 Riboflavin Folic acid 400 400 200 0 T T T T 0 nm T T T T 2 us 0 250 350 450 550 nm Norm 1000 M Vitamin B1B cB 1 600 200 250 350 450 550 nm Figure 33 Spectra of water soluble vitamins Sample preparation Column Mobile phase Stop time Flow rate Column compartment I
258. ts are plotted and found on the Signals Tab Remove cap from septum purge fitting Remove cap from column inlet Reinstall column in Inlet amp amp amp S amp SSA Z Categor Source Time Front Split Splitless Inlet 78 2 3 2008 4 13 2 Front Split Splitless Inlet 78 2 3 2008 4 13 2 Front Split Splitless Inlet 78 2 3 2008 4 13 2 Front Split Splitless Inlet 78 2 3 2008 4 16 5 Front Split Splitless Inlet 78 2 3 2008 4 18 4 Unable to attain a pressure of at least 24 5 psi aborting Front Split Splitless Inlet 78 2 3 2008 4 18 Restoring original Method to Instrument 7890 US10652005 Front Split Splitless Inlet 78 2 3 2008 4 18 4 Request Disconnection from 7890 Instrument 7890 US106S2005 Figure 9 Procedure and result for SS inlet pressure decay test inlet liner All Available Books Restrictio esi 4 Poor Peak Resolution 3 To Perform a PTY Pressure Decay Test Maintaining your GC 100 To 98 To Change the Liner on the PTV Inlet Procedure User document search capability gives the instructions 1 Type inlet liner then click Go for searching 2 Select Maintaining your GC gt 100 then you can get the instructioins 3 Instructions on how to change the inlet septum and liner O ring 4 Click Replacement O ring for detailed information on consumables and parts for SS inlet including part number Gather the fo
259. ucibility The benefits for the analyst are more chromatographic analyses per shift better data and higher revenues per instrument Septum purge vent 9 Flow Carrier gas in Inlet purge off Low inlet pressure Low column flow o More volatile compound Liner e Less volatile compound Column Figure 4 A low initial inlet pressure causes loss of volatile compounds Septum purge vent Flow Carrier gas in Inlet purge off High inlet pressure High column flow Liner o More volatile compound e Less volatile compound Column Figure 5 With the correct inlet pressure there is no loss of volatile compounds 30 psi m 400 C 1 5 min 7 320 C yd 300 C 98 psi min d 16 psi m 200 C r 100 C 5 4 psi 90 C s 2 0 min 30 m x 0 25 mm id column vacuum compensation on constant flow rate 0 8 mL min TJ 5 10 15 20 25 30 Injection Run time Inlet pressure Oven temperature Figure 6 Pulsed splitless injection technique employing con stant flow with EPC This technique allows larger injection volumes and inhibits the loss of volatile compounds out of the septum purge vent Summary Following the instructions in this guide will improve analytical results with your GC MSD system Contamination interferring with the deter mination of your analytes will be minimized and sample
260. ul 5 m Detector refractive index 100 E 804 Rape oil 604 Olive oil HPLC method performance Standard Limit of detection 40 6 8 2 4 Ti A for ECD 50 ug l with S N 22 ime min Repeatability of RT over 10 runs 0 396 Figure sd dosar Mme 5 Analysis of the triglyceride pattern of olive and rape oil Control and data evaluation Miete Auto Column EE E E ler compar EE a degasser SEMIS ment detector E Acetronitrile 18 Determination of triglycerides in vegetable oils EC Regulation No L248 28ff 37 Fatty acids Saturated and unsaturated fatty acids from C through C have been analyzed Fatty acids are the primary compo nents of oils and fats and form a distinctive pattern in each of these compounds For example butter and margarines can be differentiated by the percentage of butyric acid in the triglycerides To determine the fatty acid pattern of a fat or oil free fatty acids first are obtained through hydrolysis Derivatization is then performed to introduce a chro mophore which enables analysis of the fatty acids using HPLC and UV visible detection Sample preparation The triglycerides were hydrolyzed using hot methanol and KOH followed by derivatization Chromatographic conditions The HPLC method presented here was used in the analysis of the fatty acid pattern of dietary fat The method involves hydrolysis with hot KOH methanol and online derivatization with bromophenacyl b
261. um sampler compart ae degasser ment etector Water Acetonitrile 4 Official M ethods of Analysis Food Compositions Additives Natural Contaminants 15th ed AOAC Arlington VA 1990 Vol 2 AOAC Official M ethod 983 15 Antioxidants in oils and fats Preservatives The following compounds are used as preservatives in food products benzoic acid sorbic acid propionic acid methyl ethyl and propylesters of p hydroxy benzoic acid PHB methyl PHB ethyl and PHB propyl respectively 4 Preservatives inhibit microbial growth in foods and beverages Various compound classes of preservatives are used depending on the food product and the expected microorganism PHBs are the most common preservatives in food products In fruit juices in addition to sulfur dioxide sorbic and benzoic acid are used as preservatives either individually or as a mixture Sample preparation Sample preparation depends strongly on the matrix to be analyzed For samples low in fat liquid extraction with ultrasonic bath stimulation can be used For samples with more complex matrices solid phase extraction liquid liquid extraction or steam distillation may be necessary Control and data evaluation Quaternary CJ pump Auto Column Diode L a sampler compart array ge PR i ment detector LEE degasser Water Acetonitrile B Sample preparation Column Mobile phase Gradient Flow
262. umn and is used to improve sensitivity and or selectivity Precolumn derivatization is preferable because it requires no additional reagent pump and because reagents can be apportioned to each sample rather than pumped through continuously Automated precolumn derivatization yields excellent precision Moreover it can handle volumes in the microliter range which is especially important when sample volume is limited The principles involved are illustrated in figure 47 Sample Reagent M etering device From pump gt To column To waste l Figure 47 Automated precolumn derivatization The robotic arm of the autosampler transports in turn a sample vial and several reaction vials under the injection needle The needle is extended by a length of capillary at the point at which the derivatization reaction takes place 73 In brief The injector draws distinct plugs of sample and derivatization reagent into the capillary The back and forth movement of the plunger mixes the plugs With the right software the autoinjector can be paused for a specified length of time to allow the reaction to proceed to completion If the reaction requires several reagents the autosampler must be programmable that is it must be able to draw sequentially from different reagent vials into one capillary In this complex sample manipulation the needle must be cleaned between vials for example by dipping into
263. un Time B min Tryo F on Quick Coal Initial 45 i 225 Ramp 1 8 Cryo Use Temperature J Timeout Detection o min Fault Detection Post Run 2o C Apply Cancel Help Figure 9A upper panel Adding backflushing in Post Run oven parameters 10 Bill GC Edit Parameters x D z i X Oven Temperature 151 0 C ra ke di yZ RJ A 7 ae SS E 2e Control Mode M on Flow 2 mL min Initial 0 min C Pressure 5 4667 psi HS eae Agilent 190217 431 325 C 15 mx Average Velocity 33 824 cm sec 30 m x 250 pm 0 25 250 um x 0 25 um Holdup Ti 3 pm In Back 55 Inlet He a Dni 12565 mn Out Vacuum Aux Pressure 1 NZ peie Value Hald Time Run Time Aux Pressure 2 NZ mLimin per mL min min i Aux Pressure 3 N2 mm PCM A 1 NZ nna PCM 4 2 Nz f Final value will be extended by GC run time Post Run na mL min Apply Cancel Help Figure 9B middle panel Adding backflushing in Post Run front column column 1 parameters MB GC Edit Parameters E x Oven Temperature 45 0 C a oe ee Description Agilent 190915 433 325 C 30 m Control Mode jv On x 250 um x 0 25 pm ob lem In Front 55 Inlet He Pressure fi 25 mL min Initial 0 min He 45 C Oven zn 3psi Out Vacuum 57 48 cm sec 15m x 250 um x 0 25 0 43483 min d Average Velocity Holdup Time
264. undance for ce e overlaid reconstructed total ion current chro matograms RTICCs suggest that the higher source temperature increases the total response for the later eluting PCBs but produces little enhancement for the early eluters This could be due to more frag mentation and may not necessarily be useful if the increase in the RTIC is due to lower mass fragments since these lower mass ions are usually compro mised by interferences A calculation of the signal noise S N for the RTICCs shows that while there is an increase in signal at the source higher temperature there is also an increase in the back ground noise and the result is a lower S N ratio for the higher source temperature 300 C 230 C x 6 00 6 10 6 20 6 30 6 40 6 50 6 60 6 70 6 80 6 90 7 00 740 7 20 7 30 7 40 7 50 Time Figure 3 Overlaid RTICC of six PCBs acquired in full scan 50 505 amu at source temperatures of 230 C and 300 C From left to right or earlier to later in the chromatogram the PCBs consist of a Cl Biphenyl Cl B Cl B Cl B another Cl B and a Cl B Figure 4 shows the same analytes acquired in selected ion monitoring mode SIM using three ions for each component M M 2 or M 2 and M 70 The same trend appears with an enhance ment apparent in signal for the later eluting PCBs but little increase for the earlier PCBs Now how ever the RTIC for the SIM acquisition
265. ure compliance with these regulatory requirements analytical methods have been developed to determine the nature and concentration of chemicals in food products Monitoring of foodstuffs includes a check of both the raw materials and the end product To protect consumers public control agencies also analyze selected food samples High performance liquid chromatography HPLC is used increasingly in the analysis of food samples to separate and detect additives and contaminants This method breaks down complex mixtures into individual compounds which in turn are identified and quantified by suitable detectors and data handling systems Because separation and detec tion occur at or slightly above ambient temperature this method is ideally suited for compounds of limited thermal stability The ability to inject large sample amounts up to 1 2 ml per injection makes HPLC a very sensitive analysis technique HPLC and the nondestructive detection tech niques also enable the collection of fractions for further analysis In addition modern sample preparation tech niques such as solid phase extraction and supercritical fluid extraction SFE permit high sensitivity HPLC analysis in the ppt parts per trillion range The different detection techniques enable not only highly sensitive but also highly selective analysis of compounds x Amino acids Inorganic ions Volatile Synthetic carboxylic ES Glyphosate food dyes Sugar A alcohols Sugars
266. viscous samples and inject variable vol umes Modern sampling systems can further increase pro ductivity with features such as online precolumn derivatization for selective detection heating and cooling for improved stability and microsampling of material in low supply Some analyses may require corrosive solvents or mobile phase additives such as 0 1 N HCl or 60 96 formic acid Some vendors supply devices of corrosion resistant titanium to solve this problem Injection systems often are based on a six port valve which is put through several steps for each injection as illustrated in figure 46 In the first step denoted here as load the sam ple is either aspired by a vacuum in automated systems or expressed by a syringe plunger in manual systems into a sample loop where it rests until the valve is switched to inject This second step connects the pump and the mobile phase with the column The contents of the sample loop then move into the solvent flow path and onto the analytical column Because all parts of the system are constantly flushed during analysis the remnants of a previous injec tion are removed before the next injection occurs 70 Manual injectors The quality of the separation on the column depends on the quality of the injection a short sharp injection increases the likelihood of short sharp peaks The use of a minimum number of fittings between the injector and the column reduces the diffusion of the content
267. vity 89 the UV detector can be programmed for each peak within a chromatographic run which changes the wavelength automatically The variable wavelength detector is designed to record absorbance at a single point in the spectrum at any given point in time However in practice different wavelengths often must be measured simultaneously for example when two compounds cannot be separated chromatographically but have different absorbance maxima If the entire spectrum of a compound is to be measured the solvent flow must be stopped in order for a variable wavelength detector to scan the entire range since scanning takes longer than elution Sensitive can be tuned to the wavelength Single wavelength measurement is not maxima of individual peaks Some always sufficient Without spectra peaks instruments are equipped with scanning cannot be identified mechanisms with stopped flow operation Diode array detectors Figure 52 shows a schematic diagram of a photodiode array detector DAD An achromatic lens system focuses poly Tungsten lamp Holium oxide Deuterium lamp Achromatic lens 1024 element diode array Standard flow cell Programmable slit Figure 56 Diode array detector optics 90 0 7 mAU 0 I 240 260 280 nm Figure 57 High resolution spectrum for benzene in the low absorbance range Conventional DAD Signal il 8 acquisition Spectra stop flow on line acquisition
268. w calculated The cal culated inlet pressure is 87 862 psi which is compatible with the EPC module on the current system maxi mum 100 psi Note that the helium source supplying the GC must be capable of reaching 100 psi of helium An optional 150 psi EPC module is available for the HP 6890 GC to pro vide additional inlet pressure if necessary The oven temperature program calcu lated for the new method has the first ramp listed as 75 C min This ramp rate is compatible with the 240 V oven option on the current instru ment but would not work with a 120 V oven which is limited to about 50 C min in this temperature range With a 120 V oven the speed gain would be limited to about 2 The next step is to calculate the RTL calibration points from the original GC AED method This is done by the same process as shown in the GC MS scaling above In this case when one of the original method RTL calibra tion pressures is entered the result ing holdup time must be divided by 3 and entered for the holdup time in the Translated Method column This will force the speed gain back to 3 The resulting inlet pressure is then paired with the retention time of the corresponding original GC AED cali bration run but divided by 3 as a cali bration point for the new method Table 3 shows the RTL calibration points from the original GC AED method and calculated points for the threefold speed gain 3x method When the
269. wash vials of distilled water Automated sampling systems offer significant advantages over manual injectors the most important of which is higher reproducibility of the injection volume Sample throughput also can be increased dramatically Modern autosamplers are designed for online sample preparation and derivatization For food analysis an automated injection system is the best choice 74 Chapter 7 Mobile phase pumps and degassers Characteristics of a modern HPLC pump Flow ranges Gradient elution The pump is the most critical piece of equipment for successful HPLC Performance depends strongly on the flow behavior of the solvent mixture used as mobile phase varying solvent flow rates result in varying retention times and areas Conclusions from a calibration run for peak identification or quantification depend on reproducible data In this chapter we discuss multiple aspects of pump operation including solvent pretreatment and its effect on performance A modern HPLC pump must have pulse free flow high precision of the flow rates set a wide flow rate range and low dead volume In addition it must exhibit control of a maximum operating pressure and of at least two solvent sources for mobile phase gradients as well as precision and accuracy in mixing composition for these gradients We discuss two gradient pump types that constructed for flow rates between 0 2 and 10 ml min low pressure gradient fo
270. y can be classified as one of three principal types thin layer design wall jet design and porous flow through design see figure 66 The porous flow through cell design differs significantly from the other two in that coulometric detection ensures 100 96 reaction yield on the surface of the electrode The other designs allow an efficiency of only 1 10 96 by amperometric detection However amperometric detection is usually the more sensitive technique and is preferred over coulometric 99 detection electrochemical detectors can employ 1 pl flow cells and are well suited to narrow bore HPLC Thin layer Wall jet Porous flow through Reference Auxilliary Reference Auxilliary Reference Auxilliary electrode electrode electrode electrode electrode A Ji Workin electrode 7 R e Working electrode Working electrode Figure 66 Thin layer design wall jet design and porous flow through design Automation features oxidative cleaning 1 3 V working potential 1 2 V Potential V reductive cleaning 0 1 V Figure 67 Cleaning of working electrode 3 N itrophenol p Chloro m cresol 4 6 8 10 12 Time ms Figure 68 Autoincrement mode 100 Current Falling current Current steady detectornotready detector triggers next injection Threshold set by drift trigger parameter 4 Time min Baseline Figure 69 Drift trigger Mass spectrometers Molecular weight
271. y or as combinations in foodstuffs maximum levels for these compounds have been set Sample preparation Sample preparation depends strongly on the matrix to be analyzed For samples low in fat liquid extraction with ultrasonic bath stimulation can be used For samples with more complex matrices solid phase extraction liquid liquid extraction or steam distillation may be necessary Sample preparation ultrasonic liquid extraction with acetonitrile ACN Column 1 100 x 4mm BDS 3 um Mobile phase A water 0 2 ml HSO pH 22 54 B ACN Gradient start with 10 B at3 min 60 B at4 min 80 B at11min 9096 B Flow rate 0 5 ml min Post time 4 min Column compartment 30 C Injection volume 5 ul Detector UV DAD detection wavelength 260 40 nm reference wavelength 600 100 nm HPLC method performance Limit of detection 0 1 2 ng injected amount S N 2 Repeatability of RT over 10 runs lt 0 2 areas over l 0runs lt 1 Chromatographic conditions HPLC and UV visible diode array detection have been applied in the analysis of antioxidants in chewing gum Spectral information and retention times were used for identification mAU 1 Vitamin C 2 PG 1500 3 THBP 4 TBHQ 5 BHA 1000 6 4 hydroxy 7 BHT 2 8 ACP Chewing gum extract 500 E 8 Standard 0 Y TR 2 4 6 8 10 12 Time min Figure 4 Analysis of antioxidants in chewing gum Control and T 44 data evaluation MEUM Auto Column Diode vacu
272. yphosate Agilent Application Note 5091 3621E 1992 M Verzele et al J Am Soc Brew Chem 1981 39 67 W M Stephen Clean up techniques for pesticides in fatty foods Anal Chim Acta 1990 236 77 82 J E Farrow etal Analyst 102 752 32 H Schulenberg Schell et al Poster presentation at the 3rd International Capillary Chromatography Conference Riva del Garda 1993 S K Poole et al Sample preparation for chromatographic separations an overview Anal Chim Acta 1990 236 3 42 R E Majors Sample preparation perspectives Automation of solid phase extraction LC GC Int 1993 6 6 E R Brouwer et al Determination of polar pollutants in river water using an on line liquid chromatographic preconcentration system Chromatographia 1991 32 445 McM urrough et al J Am Soc Brew Chem 1988 K K Unger Handbuch f r Anf nger und Praktiker 1989 Git Verlag Germany 38 39 40 41 42 43 W O Landen J r J Assoc Off Anal Chem 1985 68 183 L Huber Good laboratory practice for HPLC CE and UV Visible spectroscopy Agilent Primer 5968 6193E 2000 R L Grob M A Kaiser Environmental problem solving using gas and liquid chromatography J Chromatogr 1982 21 A G Huesgen et al Polynuclear aromatic hydrocarbons by HPLC Agilent Application Note 5091 7260E 1992 R Schuster A comparison of pre and
273. yrene 215 Ethylbenzene yisty 256 10 318 min vs 10 298 min 17 778 min 22 5 vs 17 776 min 20 17 5 15 Original GC system 1 column 1 12 5 10 Locked GC system 2 column 2 5 7 5 10 12 5 15 17 5 20 22 5 min Figure 6 Comparison of original chromatogram on GC system 1 with GC System 2 after retention time locking method The pressure used however will be different due to the difference in column outlet pressure The GC MSD inlet pressure is calculated using the none mode of the method translation software figure 7 In this mode the holdup time between the two columns was forced to be identi cal to the GC FID This gives a speed gain of 1 The pressure calculated for use on the GC MSD was 8 44 psi Note that this calculated pressure is only the nominal pressure required to get similar retention times not the exact locking pressure A different RTL calibration is required for GC MSD because the outlet pres sure is vacuum and that of the FID is atmospheric pressure Five runs were made on the GC MSD system bracket ing the 8 44 psi nominal method pres sure Because the GC MSD used in this study was not equipped with RTL software a dummy method was cre ated in GC system 1 and the GC MSD RTL calibration data was entered into it A scouting run of the Styrene sample was made on the GC MSD and the o methylstyrene retention time was used for locking The lock ing inlet pressure calculated with the dummy m
274. zers separate ions based on mass to charge ratio m z lower cost mass spectrometers with mass ranges of several thousand nvz can be used to analyze compounds in excess of 150 000 daltons The primary use of electrospray has been the analysis of compounds of higher molecular weight However this technique also has been applied successfully to small polar molecules Fig 72 shows a separation of carbamate pesticides using electrospray HPLC inlet Nebulizer Skimmers Octopole m a Fragmentation Lenses E zone CID Quadrupole Capillary Corona needle Figure 73 APCI LC MS interface APCI also can be used to analyze moderate polarity analytes As in electrospray APCI ionization occurs at atmospheric pressure via a chemical ionization process see figure73 103 Abundance 100000 80000 60000 40000 20000 lt M NHJ w 639 n m z 400 600 800 100 Figure 74 Mass spectrum of the fatty acid triolein 18 1 cis 9 molecular weight 884 781 molecular formula C H 0 Refractive index detectors APCI requires some compound volatility and is less suitable for highly thermally labile compounds Figure 74 shows a typical triglyceride mass spectrum Both the degree of unsaturation and the length of the fatty acid side chains can be determined from the M NH ion which corresponds to mass M 18 In source CID experiments also can be helpful in determining the

3 - Agilent Technologies

Contents

Download Pdf Manuals

Related Search

Related Contents