Flow, Pressure and Temperature Calibration: Part 2
Contents
1. by manufacturer and for some items there was little or no information Most GC systems produced in the past 10 15 years include provisions for oven temperature carrier pressure and flow calibration via the instruments keyboard display and firmware Thus gas chromatographers can bring these variables under some degree of control Temperature In my experience average oven temperatures vary between instruments by as much as 2 C or slightly more When left uncalibrated larger deviations can be expected between different models than between the same model gas chromatographs The apparent degree of variation also depends upon how the temperature is measured All GC ovens exhibit temperature gradients between the internal temperature sensor where the temperature is measured and other locations within the oven The degree of temperature srodien s depends upon many Ieis that affect Keat and air flow Gradients of as rtiuch asy2 4 C actossthe overrare not tinc mmon v n und f the best circumstances One very important consideration is the distinction between the oven temperature setpoint as displayed on the GC calibrated or not and the actual temperatures along the column Columns are not located at the temperature sensor which measures only a single point anyway As peaks move through the column they circle around with the column tubing and run through slightly hotter and slightly cooler temperature areas2. Flow Pressure and Temperature Calibration Part 2 John V Hinshaw Serveron Corp Hillsboro Oregon USA In last month s instalment of GC Connections Js upon relationships between pressure flow rate phase This concluding instalment of a two parts ries ieee the effects that column variability has on isothermal capillary gas chromatography and explores instrument calibration with the goal of maximizing instrumenfete strument si Variatifns in th oven tenipetdture and carrier gas pressure influence peak retention times significantly enough so that differences from column to column and instrument to instrument make method validation a necessity for ensuring consistent results Instrument to instrument variability can be brought under control by implementing a few simple calibration and set up procedures and validation is made more reliable as a result The first part of this series showed that analysts should understand the effects of performing the same analysis on different gas chromatography GC systems on the variability of their results in particular on retention times The dependencies of peak retention times on oven temperature and inlet pressure can be large enough to cause significant deviations of 15 seconds or more between instruments when the individual oven temperatures differ by only 1 C or the pressures by 1 psi The scope of these variations depends in turn upon the chromato
3. for both column lengths would require a further decrease in the shorter column s pressure drop to around 13 8 psig This pressure corresponds to an average carrier gas linear velocity that is exactly the ratio of the two columns lengths times the original velocity In this instance that s 24 0 25 0 X 34 0 32 64 cm s The other peaks follow suit in this instance and have the same retention times on the shorter column under these lowered inlet pressure conditions as they do on the longer column at 15 0 psig Setting up by linear velocity In practice it s fairly easy to set up a column in this manner First measure the approximate length of the column by counting the turns include any fractional first or last turn and multiplying by the average length of a single turn as in Equation 4 L t Tah 4 where t is the turns count and dp is the nominal column helical coil diameter Use a _ _ h 5 Up Uy D 5 Finally as part of verifying the set up establish the operating or initial column temperature and then adjust the inlet pressure as required to produce the calculated velocity For GC systems with electronic pressure control EPC the EPC system will calculate and set the correct pressure for the desired velocity if the measured column length is entered first However slight variations in the column diameter from the nominal diameter can produce a slight error in this step The operator can approximate the app
4. Upon elution they have experienced an average temperature that is a composite of the temperature at any defined point in the oven This is the normal situation and these slight temperature variations don t affect peak shapes or resolution significantly acing a column too close to the oven wall will increase this effect because the coolest areas in the oven tend to be nearest the walls Conversely the hottest areas are often near the inlets and detectors Shifting a column s position from the front of the oven to the back can have a noticeable effect on retention times as well Thus for the best consistency it is wise to install columns close to the central axis of the oven and always either in the front or the back position as dictated by vu www Icgceurope com GC Connections With a better understanding of the effects of column variability on retention times we are in a position to examine the requirements and effects of temperature and pressure calibration in laboratories that use multiple gas chromatographs the inlet detector configuration and the methodology As long as the overall thermal environment is consistent the retention times wilkbe as well Pressure Carrier igas inlet pressures are controlled ithen Hy iectronicy pressure constant fis mode with this type of inlet the GC system actually contrdls the inlet i a Bourdon type madhanieslt pressure i Gauge isiin use than th
5. an external digital transducer becomes a very valuable tool However the pressure and related flow transducers in a GC should be zeroed at least every three months as well as whenever the instrument is moved or serviced If the pressure readout is not 0 0 when the pressure is off and no column is attached then the associated transducer should be zeroed To zero the transducers first cool down the column oven then turn the carrier gas off or set the pressure to zero and either disconnect the columns or remove the septum nuts from the inlets Most EPC equipped instrument models also monitor the incoming carrier gas supply pressure so disconnect the carrier gas supply at the instrument bulkhead being careful to cap off the supply tubing to protect carrier gas filters from air incursion Allow at least half an hour for the instrument to warm up if it is not already warmed then select the pressure transducer zeroing portion of the keyboard display user interface and execute the zeroing procedure according to R fererices e the user manual Finally reconnect the supply lines and establish a low pressure for long enough to purge air from the system before reconnecting the columns or replacing the inlet septum nuts This would also be a good time to service the inlets if necessary Conclusion Some variability in results obtained on different instruments with different columns is always to be expected Analysts can minimiz
6. arent column diameter at this point by comparing the measured carrier gas linear velocity to the desired value as entered into the EPC system Because according to Equation 2 the ratio of the velocities is equal to the square of the ratio of the diameters a corrected diameter can be calculated in this manner 6 For the example given above if the configuration the electronic pressure controller will adjust the pressure downward sufficiently to produce the desired average carrier gas linear velocity Due to the variations in inlet pressures from instrument to instrument however this procedure should be repeated whenever a column is set up Temperature and Pressure Calibration With a better understanding of the effects of column variability on retention times we are in a position to examine the requirements and effects of temperature and pressure calibration in laboratories that use multiple gas chromatographs What to expect Given that small changes in temperature or pressure can shift retention times significantly what should gas chromatographers expect from their instrumentation How much pressure and temperature variation is normal between gas chromatographs that are operating within the manufacturer s specifications read through a number of brochures specification sheets operator s manuals and service manuals found that pressure and temperature tolerances vary somewhat LC eGC Europe 18 3 138 144 2005
7. e instru ent to instrument retention tinge v ri i ity by calibrating the l istalling ihe column in variations in tat Good col amp mn also help esk article ae thermal catibration dimensignless retention measurements oe effctvely mi ae 1 J V Hinshaw LCeGC 22 12 1160 2004 2 MJ Hartigan and L S Ettre J Chromatogr 119 187 206 1976 3 MJ Hartigan K Billeb and L S Ettre Chromatographia 10 571 579 1977 4 LS Ettre personal communication November 2004 GC Connections editor John V Hinshaw is senior staff engineer at Serveron Corp Hillsboro Oregon USA and a member of the Editorial Advisory Board of LCeGC Europe Direct correspondence about this column to GC Connections LCeGC Europe Advanstar House Park West Sealand Road Chester CH1 4RN UK e mail dhills advanstar com For an on going discussion of GC issues with John Hinshaw and other chromatographers visit the Chromatography Forum discussion group at http Wwww chromforum com LC eGC Europe 18 3 138 144 2005
8. ere is little realistic 3 i nged to catiprate it because it is inf fently inaccurate and non linear compared to electronic transducers In such instances chromatographers should rely on the gauge as an approximate pressure indicator and instead use carrier gas average linear velocity measurements for column set up purposes have also seen a digital electronic pressure device with an attached syringe needle which gives a fairly accurate reading when inserted into an inlet Most of the electronic pressure transducers for EPC systems are specified to deliver accuracies of 2 of their full scale reading For a 0 100 psig transducer that s about 2 psig while for a 0 30 psig transducer it s more like 0 6 psig These errors show up as deviations from a perfectly linear relationship between the actual pressure and the readout and according to our calculations they are large enough to cause noticeable retention shifts from one instrument to another The solid state pressure transducers cannot be calibrated by the instrument for their non linear full scale errors but all of these GC systems support a zero offset adjustment which can be as large as an additional 0 5 psig or so Pressure and sets it as required to maittain Calibration Properly performed temperature calibration and inlet pressure zero compensation can help tremendously in attaining more consistent instrument to instrument results However it is a
9. graphic conditions the column and the analytes under examination Although not a substitute for validation and suitability testing instrument calibration can help to reduce the normal variability that will be encountered when working with multiple instruments and multiple columns ita ity of refentign tim s connie ects S There are three main column aie that affect retention times dimensional variations such as inner diameter and length stationary phase variations both in the chemistry and the film thickness and ageing effects caused by gradual contamination with sample residue as well as phase loss due to overheating This month s column addresses some of the issues related to dimensional variations vividly recall spending many long nights in the graduate school lab drawing out borosilicate glass columns on a cantankerous machine that would have made cartoonist Rube Goldberg proud see www rube goldberg com If was lucky enough to obtain a single 10 m long piece of coiled tubing was then faced with the tasks of coating the column with a stationary phase that had synthesized from scratch and installing it intact into the gas chromatograph s oven made no pretense of duplicating any of these handmade columns and don t know what the tolerance levels were on their inner diameters or film thicknesses Fortunately this had no bearing on my work However it did leave me with an appreciation of the technol
10. h the older studies Gas chromatographers who want to ensure the best consistency from column to column should choose one manufacturer as their column source for each specific analytical method There are no technical reasons to select or eliminate any particular manufacturer but columns from the same company will be much more self consistent than would be the situation for columns from different manufacturers with the same nominal dimensions and stationary phase types Of course some column companies proprietary stationary phases and column chemistry might be better suited for certain applications Careful evaluation of multiple examples of a specific column is always a good idea betipre committing to any GC Connections Table 1 Retention times in seconds for three peaks across a wide range of column inner diameters operated isothermally at 100 C For conditions see Figure 1 Peak Retention Time s Inner Diameter um n Dodecane Kacore 522 1 200 415 3 210 363 8 220 320 7 230 284 4 240 2537 250 227 4 260 204 8 270 185 2 280 168 1 290 1532 300 140 1 n Undecane n Nonane Kioo 270 2 Kyggec 70 99 270 4 155 9 238 6 139 6 Zales 257 189 1 113 8 169 8 103 5 1532 94 5 138 8 86 7 126 3 USI 1153 73 6 105 7 68 1 97 2 63 2 are aff the same in BRE eBbpect eno within the tolerances of a olg column Figure 1 Effect of column inner diameter on retention times a Inner diameter 200 300 um b c
11. lose up view inner diameter 240 260 um Column 25 m X 250 um column temperature 100 C pressure drop 15 psig column outlet at room pressure Key blue n dodecane K400 c 522 1 green n undecane K 99 c 270 2 and red n nonane Kj99 c 70 99 2 v i f 2 pb i D D 7 a T T T 1 200 220 240 260 280 300 300 4 b 250 m 200 4 150 100 lt _ a 50 4 0 T T T 240 245 250 255 260 Inner diameter um Retention time s www lcgceurope com i The columnidiam ter aff ets both the 4 average api length dp rafention t igs column inner Diameter tier gag linear velocity l the retention factor given a constant stationary phase film thickness The linear velocity will decrease as the square of the inner diameter decreases as equation 1 shows g Aji a 32L 4 We can compare the effect of different column inner diameters on velocity at constant column pressure length and temperature as follows 2 d Bah Ga 2 This type of square law relationship predicts a strong dependence of the linear velocity and thus retention times on the column diameter Table 1 shows the effect of changing the column inner diameter across a relatively wide range from 200 to 300 um on the retention times of the same three example hydrocarbon peaks as used in the first part of this series and the data are presented graphically in Fig
12. lso necessary to consider carefully other variables such as column dimensional variations and positions inside the column oven Oven temperature A precision temperature measuring device and appropriate probe are necessary for meaningful oven calibration The probe zang meter gombinakion shauld be capable Ot 0 2 C Sfbetter eSolutiont If Pnsistepicy af result etw n instruments that wii be calibrat d different siherngiometers ig importartts then the fherifometers shaul all be Glibrated to NIST standards by their manufacturers For singte locatioris a sirigle thermometer shdaild w ik yvell endugh with asimple ice bath reference It is very important to place an external temperature probe consistently when measuring oven temperatures for calibration purposes Bearing in mind that in any situation the calibration will only reflect a single point temperature the best placement is as close as possible to the instrument s temperature sensor If variations between different instrument models are a concern then slightly better results might be obtained by positioning the temperature probe close to the centre of the oven in the area where the column will be located In any situation the probe should never be placed close to the oven walls or directly in a line of sight with the oven heater coils Be sure that the probe cable does not interfere with the gas chromatograph s oven door and that introducing the probe does no
13. ogy that goes into producing capillary GC columns Two studies sublishied in the 1970s recorded the state of the art of commercial glass capillary GC column production at the time 2 3 A statistical evaluation of the data published in the two papers reveals retention factor standard deviations of 5 9 for 16 methylsilicone columns 11 4 for 7 phenylmethylsilicone columns and 30 4 for 9 carbowax 20 M columns 2 In the second report the authors measured the relative retention of several peak pairs and found not surprisingly much smaller standard deviations 0 28 0 37 for methylsilicone and 0 32 for carbowax 20 M Non polar columns prepared with twice the stationary phase film thickness had even smaller column to column relative retention variations 3 The variability of the column inner diameter does not affect retention factors under the isothermal conditions used and its range was given as d 270 20 um 2 Today analysts rely upon column manufacturers to produce a consistent product from fused silica tubing Advances in tubing production and chemical treatment stationary phase synthesis column coating and conditioning have greatly reduced the variability in column dimensions and retention as well as LCeGC Europe 18 3 138 144 2005 yielding much lower bleed levels higher stability and longer life It would be very interesting to see similar data on populations of modern capillary columns that compares them wit
14. t create an extra air leak from the outside See the user s manual or service manual for details on exactly how to calibrate the temperature for a specific instrument In general operate the GC system at a temperature in the middle of the method operating range or at 100 C A well controlled room temperature also helps attain more consistent oven GC Connections temperature calibration Let the instrument stabilize for at least 1 hour and then access the instrument calibration routine Compare the resulting high resolution reading with the probe reading and enter a corresponding temperature offset value on the keypad or in some instances enter the probe temperature reading itself as instructed in the manual Allow some time for the new temperature level to settle in and then verify that the probe and oven are now consistent to within a few tenths of a degree Make a note of the temperature probe make and model its position in the oven how it was calibrated and the offset value in effect after calibration Inlet pressure As mentioned earlier pressure calibration is not practical to better than 2 of the full scale reading If desired however an external digital pressure measurement device can be used to obtain an ore pressure reading mode with temperature programming then hese readings will oily Set thes tle subsequent control changes In the situation of a mechanical pressure gauge then
15. ure 1 a The effects on retention time are large Table 2 gives the same data for a narrower range of inner diameters from 240 to 260 um as might be encountered in practice and the lenges fined uk shown Aes within 10 um far the mid le peak arid within 29 um f r tHe earliestieluted_geak As peak retention increases the variability in inner diameter required to keep peaks within a defined range decreases rapidly However with isothermal elution the widths of the peaks increase with longer retention and the effect of the variability becomes less significant This is not the situation for temperature programmed elution but this topic lies outside of the discussion being presented here Thus it appears that for columns of the same type used for the same isothermal analysis if the inner diameters from one column to the next lie within less than 2 of the nominal diameter then peaks will be eluted within a fairly tight window Remember though that other variables are also at play here Temperature and pressure variability will add more uncertainty to the retention times Column Length Variations in column length also affect retention times Differences between column lengths on the order of 1 m or more are not uncommon within a population of initially equal size columns that have been in use for some time Removal of a small portion of a column is part of good laboratory practices that call GC Connections for the
16. use of new inlet and detector ferrules with each installation Additional lengths can be removed from the column entrance as part of column reconditioning in order to remove non volatile sample residues that have accumulated at the beginnings of columns The effects of varying column lengths depend upon how the analyst sets up the columns One approach would be to choose the same pressure drop for all columns of a particular type In this instance retention times will vary with the square of the column length as shown in Equation 3 which was obtained by combining Equations 1 2 and 3 from the first instalment of this column series 35125 Apj d tg 1 k 3 This is not a desirable situation The peak with partition eae K 522 at Table 2 Retention times in seconds for three peaks across a narrow range of column inner diameters operated isothermally at 100 C For conditions see Figure 1 Peak Retention Time s Inner Diameter um n Dodecane Kagorc 522 1 240 258 242 248 1 244 242 7 246 237 4 248 2323 250 227 4 252 222 6 254 217 9 256 213 4 258 209 0 260 204 8 n Undecane n Nonane Kroge 270 2 K4oo c 70 99 169 8 OSES 166 3 101 6 162 9 99 8 159 5 98 0 156 3 96 2 1532 94 5 150 1 92 9 147 2 91 3 144 3 89 7 141 5 88 1 138 8 86 7 slightly to 14 4 psig for the shorter column then the same peak would shift by about half as much to around 218 s Keeping the retention time the same
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