hplc standard operating procedure
Contents
1. unreliable results A good understanding of the calibration process linear regression and Statistics is of great value see the Theory of Calibration in Chromeleon 6 80 User Manual starting on page 109 The manual is found on the reference library CD D library manuals software CM_680_V30 pdf Combining multiple analytes into a single standard vial is acceptable provided that there is good separation between peaks if in doubt make separate calibration samples Ensure concentration calculations are correct use the Certificate of Analysis CoA concentrations for the actual source bottle rather than the nominal concentration printed on the label If the CoA concentration is not on the source bottle it can usually be found on the supplier s website use the Lot Number and catalog number printed on the label as search parameters All of the calibration standards shall be run at the beginning and then unknown samples provide a blank a spiked sample and a duplicate unknown sample for each run or for every 15 to 17 unknown samples The three extra samples are used for quality control QC these 18 to 20 BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 10 11 samples represent a group of samples the unknowns plus the quality control samples The blank will be a sample that includes the background matrix i e any reagents used in sample preparation fresh microbial media etc but does not conta2. 2 December 2014 8 Now that the d 4 Create File Edi Yoew Jools Help instrument Is ready saat EEEE Hew ES HJ UNVERSITYORKEN 1 you can create a new papie kal instrument Onta i Stadia Gi Print Gup ineet Row EAI Dom Glock W Filtering Grouping find Next Aere Ri T Laval Position Volume u inatramentkdethod P maio 5 fir sequence or open an i Audit T a E ie i 0 Aica ma Bien rh s aa few 06 rT a Jke Barad abl niran GO WONO Sugar i puges DOC idle aL GA a wD existing sequence file from the data pane 7 A E Ate Pr 4 fr that has the same type of samples as yours aa and executing a save F hott ocean oe nents as YYYYMMDD_file on 2 Seen descriptor e g E O e o 20140613_JohnDoe_F Er ne ermentation For a T a ret eno e812 new sequence you a Bonen oP o A i sap nouson ty a Change the values in SS mmeona a T O name type and position columns to match your samples Use the appropriate injection volume C 9 above Use the existing instrument and processing methods unless you ve created new ones When using the UV open the instrument method and confirm that the channel amp wavelength are what you want Select the start drop down menu and select add sequence to the queue 9 Go to the queue tab and Back Ceeate Fle Ect View Tools Help a Instruments lawrch eWoetfiow Smat Startup amp Smart Shutdown Ga Relea
3. University of Kentucky Biosystems and Agricultural Engineering Dept HPLC STANDARD OPERATING PROCEDURE Aminex 87 H and 87 P analytical columns BAE 1 28 2015 BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 READ amp UNDERSTAND THE ENTIRE SOP BEFORE BEGINNING Note An SOP cannot cover all the needed information for proper instrument understanding and operation or cover all scenarios nor can an SOP eliminate the necessity of awareness attention to details and most importantly active thinking Please refer to the instrument manuals for the needed background information review literature and always keep the most powerful instrument the brain turned on When in doubt about something ask HPLC System Information 1 Hardware Dionex Thermo Fisher Ultimate 3000 w auto sampler HPLC 2 P 680 pump TCC 100 Column Compartment Variable Wavelength Detector UV Shodex R01 Refractive Index detector 2 Consumables Analytical column Bio Rad Aminex HPX 87H order 125 0140 ion exclusion Guard column is a Bio Rad Micro Guard Cation H cartridge order 1250129 column typically operates at 50 C 3 The column type is specific to the analytes The 87H column is a hydrogen form column used for analysis of carbohydrates in solution with carboxylic acids volatile fatty acids short chain fatty acids alcohols ketones and many neutral metabolic by products Most often used for organic acid ana
4. al flow through the column to prepare the system i Check the motor on box it will turn green ii Set the flow rate at 0 1mL min and let it flow for 30 minutes the system pressure should be stable after 30 minutes if not continue flow at 0 1mL until pressure is stable iii Set the flow rate at 0 2mL min for not less than 15 minutes this ensures that the column is full before heating begins 4 Goto the column tab Bon E Back Create File Edit Yw Tools Help adjust the desired instruments a Launch evvktion Smart Stamp Smart Shadown i Release Contro SI Monitor Baseline F Command C Detach 1E alast Used temperature set point frerusmeu smote s nma a a C a and turn on UNEASE emie D eae we o Temperature After B uaersrvonen sume comma 1 7 the column reaches noe Act Ve ea the temperature set Leok Sensor Satings point increase the pny Laak ced O flow in 0 100mL increments wait 15 20 minutes and me i repeat increase flow a e aie rate wait 15 20 min E L cue Tore up to the final desired flow rate Tima R ama rt 1131 24 AM Pomphicdule Pump Pomp has siopped purging Pirsse close the page wale 5 Goto the RI Tab and TERRA EER Back Create File pdit yaw Tools Help check the purge box Instruments a Launch eWorklow Smart Startup 48 smart Shutdown i Relate Control G Monitor Baseline Command Sj Detach alini heis O l Coumsihven Fi 5 Liv purge for not less t
5. appropriate SOP
6. dary the area definition is critical and requires intense focus The areal boundary lines base lines and drop lines may be defined manually using the HPLC software the area values exported to excel regressed and concentrations calculated without using the Chromeleon algorithms The data chromatogram file from every sample analysis event MUST be archived in MS excel format on a storage device other than the computer operating the HPLC The data shall be exported on a weekly basis or as needed such as prior to a HPLC software update The data in the archive MUST be maintained for a period of 10 years from the analysis date The data shall be archived in primary folders by experimenter s name then years and secondary folders months The MS Excel files shall include a primary identifier in the name so the analysis type can be readily identified The document properties dialog box is a convenient place to utilize keywords etc to facilitate a search function within the archive E ROUTINE INSTRUMENT MAINTANENCE Note Refer to the equipment manuals for in depth maintenance requirements including routine and periodic maintenance for the instruments and analytical columns See the attached Bio Rad Use amp Care guide for both analytical and guard columns BRUC HPLC Operation amp Maintenance Logbook A logbook shall be maintained by the operator for each HPLC system The logbook will be stored at each instrument s location and wi
7. der to yield a 5mM final concentration Vacuum filter the buffer from the graduated cylinder through a 0 2um x 47mm nylon filter Pall Corporation order 66602 to degas and clean buffer use a glass funnel support system Pall BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 Corporation order 4013 or 4012 Store filtered buffer into an appropriate container 1 2 or 4L glass bottle with 33mm threaded top The purity of the buffer and flush water is critical do not skip the filtration step Poorly prepared contaminated buffer can destroy a guard column and or the analytical column The working buffer and flush water can be degassed if desired however the HPLC has a de gassing pump so manual de gassing is not needed Clearly LABEL all solution bottles with the chemical composition of the contents the date prepared and the name of the preparer Dionex recommends replacing aqueous solvents at least every two weeks Do not top off solvent bottles due to potential buildup of unwanted components Do not let solvent bottles run dry Ifa solvent bottle looks like it will be empty before the analysis is complete stop the run between samples see section D 1 for the requirements to change solvent bottles C Standards amp Standard Curves NOTE Standard curves are developed by the operator using calibration standards and are used by the HPLC software to quantify the concentration of analyte in the samp
8. e supplier produced and investigator produced chromatographs should be dated and taped into the log book The HPLC pressure limit control a operating software setting shall be set so that a pressure increase of 15 greater than the pressure found in 3 operating pressure shall cause the pumps to stop The guard column shall be replaced if its pressure is greater than 150 of the pressure when new With a high pressure shutdown a flush amp regeneration cycle with the proper regeneration solvent shall be completed on the analytical column See the BRUC table 2 for specifics and troubleshooting The System will need to have buffer run through it for not less than hour once a week if column is in place when idle along with flushing pump line with dd water A gradual increase in column pressure is normal as the column ages and is proportional to the number of injections Lessons learned regarding column life amp performance shall be incorporated to this SOP by the operator F SOP MAINTANENCE 1 The HPLC operator shall review this standard operating procedure every 6 months and make all needed changes and revise the date of update Dionex amp Bio Rad technical support solutions to specific problems shall be incorporated into the SOP Best practices learned by the HPLC operator for sample preparation standards standard ranges etc shall be communicated by the operator to the owner of the SOP for incorporation into the
9. ecessity of understanding the calibration regression process The FIRST calibration standard injection is a waste the system uses this first injection as priming for the column so it never is as good as subsequent injections use any of calibration standards because it will not be incorporated into the standard curve The sequence could be S2 S1 S2 S3 S4 and so on where the first S2 injection is the waste injection The next three or more injections Shall be standards loaded in order of increasing concentration One standard from each concentration grouping shall be run after the last sample at the end of the run The total number of calibration standards verification samples QC samples and unknown samples has to be considered to determine the needed buffer volume Chromeleon will calculate the needed volume after the sequence has been set up For each group of unknown samples to be analyzed randomly select not less than three but not more than 10 samples and analyze using the previously selected standards Review the chromatograms to ensure that the correct analytes all the peaks from the unknown samples are accounted for and correct calibration standard concentrations have been chosen all the unknown sample concentrations lie within the calibration standard concentrations adjust as needed before analyzing the remaining samples The best practice is to inject the smallest amount possible for both standards and unknown sam
10. han rsss cns auaa Se eee 5 minutes toremove js a ou X E UNVERSITYOFKEN 1 i i 155s ieee S50 AAU air different solvents maen fee Midde Connect z etc UNCHECK THE c STARTING THE SAMPLE RUN comes SEQUENCE a r Pe Filtering 7 0 Find hia fe Fad Privai Date Tima ae Derice A014 126 Pi Rl Ai Purge On erat TEA PM Rl Al Purge Of WED 1255 PM Pamphicdube Purip Pampicsule Purp F kon Nominal 0 100 iy ir DVT 122008 Pd Ri Ri Purge On Pi mam ma b d aeda eT eaa ELTS weed eee eee a eee wie dhe oe ed BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 201 6 Ifusing the UV B creme Conca Bait Cese Ble Edit Yew Tools Help detector go to the UV tab and set the desired wavelength value in Deish E instruments a ze a e D Launch eWordliow Smart Situp gt 6 smat Shutdown Gi Release Control G Monitor Baseline E Command clan Hes Pumpblodhde Columetven Al Sampler UW Aude Stariop Gueun han boca imant coniro Sie en celta Serer Mndule Samiun l UNIVERSITY ORCEN B BAE 32100 E VERSIT YOFKEN 1 one of the 4 channels ji e UV1 to UV4 the ca E channel used is specified in the 2000 bie F 01 s Flatantesn Tore 7am 350am gt Milam Tam mvs uv viS 2 instrument method used in a sequence Turn on the UV lamp it will take at least 30 minutes for the lamp Acqusion Of Aadi Tand
11. in the analytes of interest the blank provides a chromatogram of the background matrix The spiked sample should be prepared by splitting an existing unknown sample and then adding a known quantity of each analyte to the sample the spike will show the background matrix effect on the analytes if any exists The duplicate sample is a split unknown sample that can come from any unknown it is often taken from the same sample that used in the spiking process the duplicate will show instrument variability The quality control samples shall be evenly spaced among the unknown samples so that the QC check occurs throughout the grouping Provide at least one calibration verification sample per group or two per run A calibration verification sample is a previously injected calibration standard that is injected again to check the accuracy and repeatability of the calibration and analytical results The sample must be designated as a validation sample in the type column when setting up the sequence run see D 1 8 Run not less than one standard from each analyte group several times during the run in order to determine if the peak is shifting in time and to provide additional calibration points or calibration verification points It is good practice to include more injections of the low end calibration standards to provide an average calibration point and some weight to balance the influence of the higher concentrations see item 2 about the n
12. is back to the desired value restart the sequence Results Quality Control amp Data Archival NOTE The area under the curve of each peak is used with the standard curve to generate a concentration value Thus any error in area delineation on the chromatogram and or calculation directly and significantly impacts the results di Standard Curves calibration curves shall be developed from the standards for each individual run do not re use standard curves The default regression options in Chromeleon MUST be reviewed for each analysis event to ensure the chosen regression options make sense The typical settings would be linear with offset averaging all response values for each calibration level before curve fitting and no weighting If any regression curve has an R squared value of less than 0 999 investigate the problem and repeat the process to achieve an acceptable regression See the Chromeleon 6 80 User Manual starting on page 109 The manual is found on the reference library CD D library manuals software CM_680_V30 pdf it provides excellent background information on calibration Chromeleon s automated tool can be used to establish baselines for each peak identify the peak and the drop lines for adjacent peaks However the software is only a starting point the chromatogram for each sample shall be reviewed manually and corrected to ensure the proper placement of the lines defining the peak s areal boun
13. le the standards allow the operator to establish the elution time of analytes and account for column drift or shifting elution times during analysis a normal occurrence Calibration standards MUST be used with every HPLC analysis event 1 Calibration standards prepared samples with known concentrations used to calibrate the instrument shall be prepared ahead of time for each analyte of interest The calibration standards prepared for each analyte shall include not less than a two 2 low end concentrations two 2 different mid range concentrations and a high end concentration for a total of five this range of concentration values should bracket the expected range of concentrations in the sample Typically the concentrations should be from 70 of the lowest to 130 of the highest expected in the sample note that blanks zeroes do not have a peak A starting point to develop the appropriate standard concentrations would be to use literature values from multiple sources to establish the bracketing concentrations with multiple points around the typical concentrations reported Ask the HPLC operator about the instrument detection limit s for your analytes Note the concentrations needed for the calibration samples will likely be different for each analyte The calibration is valid only within the concentration range of the standards used and not beyond it thus samples that are outside the range of concentrations in the standards will produce
14. ll be available for inspection The logbook entries shall include the initials of the person making the entry date time and notes regarding column guard amp analytical maintenance replacement and performance as well as BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 pertinent information about the samples being analyzed along with system performance All calls to Dionex and Bio Rad technical support shall also be detailed in the log Guard amp Analytical Columns Note The guard column maintenance and replacement frequency depends on the samples being analyzed and thus cannot be predicted In general replacement is warranted when standard samples illustrate a change in the measured data resolution 1 2a 3 10 All column procedures installation start up amp maintenance shall be done per the BRUC A new guard column shall be installed when a new analytical column is installed The total system analytical and guard column pressures shall be determined at normal flow rates when newly installed and recorded in the logbook Refer to the BRUC for the procedure to determine component pressures from system pressures Before any samples are analyzed all newly installed columns shall be tested and the results compared to the factory supplied chromatogram to ensure proper column and system performance Once the supplied and produced chromatograms match samples analysis can begin Both th
15. lysis this column is also useful for fermentation monitoring biological fluid analysis and acetylated amino Sugar separations See http www bio rad com for detailed information on other analytical column chemistry and purpose HPLC Operating Procedure A Mobile Phase also known as working buffer buffer or solvent 1 2 Flow rate is typically 0 4mL min constant flow rate isocratic typically a 20 70 minute run run time will be a function of the elution time of the analytes being quantified Mobile phase is 5mM sulfuric acid for the 87H column Fisher AC124645001 B Mobile Phase amp Flush Water Preparation for the 87H Column 1 The mobile phase is defined by the column type different columns may have a different mobile phase chemistry The volume of mobile phase buffer needed will be calculated by Chromeleon after you ve set up the run See D 9 below Confirm the amount needed check the existing volume in the buffer rack on the HPLC and make more if needed To make buffer prepare a 500mM stock solution of sulfuric acid using the certificate of analysis to define the purity of the concentrated acid Your calculations should yield a mixture of about 2 8mL of concentrated sulfuric acid into 97 2mL of double deionized water DI water note that the DI water unit has a 0 2 micron filter in line Add 10mL of the 550mM stock solution to 990mL of DI water using a clean DI water rinsed glass 1L graduated cylin
16. oy Pan Y Fiterng Dote Tema T1007 PA to warm up system will not proceed with Er B14 RI Purge Off the run until the lamp has reached operating temperature 7 Goto the sample tab ae Use the default tray control settings for each color the colors Create File Edit yaw Tools Help Deish E Instruments ai ig 7 E Launch eWordlow Smart Siactup 4 Smart Shutdown Gg Release Central G Monitor Baseline E Command eLan Led gt bkas Pumpitodule Columefiven A Sampler UW Aude Stare Gumus Sees ksi ii cenit kiul Statues Fi tinban Tata Sart Up Tray Gonbrod Tompu Chiral UNIVERSITY OF EN Temp or mi On SI BAE icS2100 E oneni YaF KEN_1 Nomra Temp Ao 4 represent sections of the tray Set the tray Modtukn Genn D n e temperature to the appropriate setting for your samples e g 8 C is typical however 4 may be appropriate for samples with microbes or higher if needed Use the start up and inject default values The injection volume value see C 9 above will be overwritten Y eworknows Fiore Ophis Vieira Savior Qusthitahon Relnywinputs irp Po Got Wo 20000 yal inpact Shop Inject Pustiet Tril WARDA ar WANA i 121007 PH 17 034E PM Trey To Fiom Fiun Sengeraend Trey To Frond by the value used in the sequence written for your samples Uninwiraty od kentut BAE HPLC Standard Operating Procedure 87H column Last Updated 1
17. ples it is suggested to start at 20 microliters and increase from there if there is not a sufficient response use the injections described in C 9 to assess the response of a given concentration and injection volume adjust as required The injection volume will determine how much sample standard volume should be in the vial e g a 100ul injection must have 600u l in the vial Typically the HPLC sample vials contain at least one mL If the available sample volume is limited there are inserts 250ul total volume that fit inside the standard vials If you re using an insert adjust the injection volume on the sample setup page in the HPLC software and change needle height Failure to adjust the software setting for needle BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 height for vials with inserts will result in broken inserts and or needles since the needle goes to the bottom of the vial when extracting the sample D Instrument Preparation amp Sample Analysis NOTES a The following screen shots are in the appropriate order of operations No Shortcuts The mobile phase must be flowing before heating the column or warming the UV lamp b The UV detector is not needed when analyzing only carbohydrates sugars on the 87H Both the UV amp RI detectors shall be used for fermentation analyses d Refer to the Chromeleon software manual for detailed information on creating new instrument T methods and
18. processing methods Getting Started 1 Open Chromeleon a rore SESA 100 PM ea 1 PM using the toolbar Icon It will open H to this screen a0000s ei at or E Ae oS aw G tm G Du E Ew E Het 4 E Mat Ej Me Ci Ms T E Tat G Te B va ow ch EW okiha Folder Dals plocipd Lini ot entice 2 Select the instrument tab in Obak O Cene file Edt View Tools Help Instruments ba CY Launch eWorklow Smart Startup gt 48 smart Shutdown i Release Control G Monitor Baseline Ci Command a Detach alisi neds the lower left z a Lome Fumpiicdive Cowman Sampha Hi LY Pure Sorig Butus Shon Goal EHATE ciir i thee J or T 54 Columinivwen_ Temp corner select LINTWERSITVOREEN Pe Pump Pressure Flee tszi Flt UNIVERSITYOFKE _ Se wrsrvonens N_1 in the left pane and select the Home tab for general status duns died urs p ui or yu Liri sap Pumpiiodule Pump Pump hes stopped purgeng Fiesso Ce The punji wishin 11 48 44 AM Pumpbtodube Pump Pumptodebe Fuaryp Pum ey 7327 Pu Calumet The compartirusnl door has been Tima Calumntven Daian A The bard hak bi n nerod ues Ubap igi Dama E gt eworknows i mune AN trent UNMMNERSITYOFEEN 1 selected Uninwenety of koniu BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 SB cen 3 On the pump ai mean u Edit Wew Tools Help d t b instruments a Launch eWorkilow Smart Startup 4
19. se Control Monitor Brine Command Ca Getach select the ready check mo gt sic tn r E A in the right pane The ee ready check will mpera calculate the needed mobile phase a k a buffer solvent volume needed and display at the bottom of the m a window on the right a side Ensure that there ere is more buffer in the sac bottle than the eee va ree ta te at Calculated volume if MOT reren unversnoreen s siara see B 2 B 9 once the E a w buffer volume supply is confirmed then click the start button The run will stop on its own once complete and use the Smart shutdown that is already programmed Remove the samples and dispose of the vials in the proper location 10 If there are errors during your run consult with the HPLC operator as the actions required depend on the nature of the problem After the error is resolved you can open the sequence change the last sample from interrupted to idle and then save the sequence Before restarting the run a Go to the console sampler tab select the wash buffer loop button This will clean the flow path to ensure there is no cross sample contamination Once the wash is complete the wash buffer BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 loop button will change back to gray restart the flow rate at 100uL and follow the flow rate incremental increase process discussed above in D 1 4 Once the flow rate
20. smart Shutdown i Releawe Control G Monitor Baseline Command Detach aliri ieis mo ule a you e veat id bjese Pampitoduin Columetven Sampler Al UF Audet Shore Queue Hop al aandar d Pine l can ensure that oo Ik E EEE the pump 1S m eiea i ze l 0 000 mma ops moo 00 k oos Soon s connected set the Saroa rana z ss TNE F TOARN flow rate choose i000 c raaa Tose the mobile phase bottle turn the pump on off and start a purge to Time set up a NeW a 82972014 31535 PM End of Shutderam solvent bottle P DTW 21S 28 FM 0 000 Ered If WaS A Tiat Pl 1 000 Pumpiiodube Pimp Pumphdicdule Pimp F iga omanal 0 OP WIJ a04 w Te PM O00 i 1 Yes a To purge the lines of air after installing a new bottle of mobile phase air in the lines is BAD open the pump module door and turn the knurled brass knob two full turns this bypasses the column and sends the flow directly to the waste container b Then puta check in the purge box and this window Fiesi check result Siencesstul hewarver there ane One oF ore WETEA will pop up Start the purge the purge process Set nO has a built in timer If there is still visible air in the 1 ae lines purge a second time Once the timer has elapsed the purge button will go from green back to gray Be sure to CLOSE THE PURGE VALVE BAE HPLC Standard Operating Procedure 87H column Last Updated 12 December 2014 c Now start initi
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