Part II
Contents
1. C across the oven are not uncommon even under the best c i rcumstances 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 temperaturesensor 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 tempera ture areas Upon elution they have experi enced an average temperature that is a com posite of the temperature at any defined point in the oven This is the normal situa tion and these slight temperature variations don t affect peak shapes or resolution significantly With a better understanding of the e ffects of column variability on retention times we are ina position to examine the requirements and e ffects of temperature and pressure calibration in laboratories that utilize multiple gas chromatographs Placing 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 often are near the inlets and detectors Shifting a column s position from the front of the oven to the back can have a notice able 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 e2. 0 psig pressure dro p would shift to 210 s on a 24 m column with the same pressure dro p The effect on later eluted peaks would be even larger If on the other hand the carrier gas lin ear velocity we re set the same for the 24 m column as the 25 m column by adjusting the inlet pressure downwardslightly 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 for both column lengths would require a further decrease in the shorter col umn spressure drop to around 13 8 psig This pressure corresponds to anaverage carrier gas linear velocity that is exactly the ratio of the two columns lengths times the original velocity In this case that s 24 0 25 0 X 34 0 32 64 cm s The other peaks follow suit in this case and have the same retention times on the shorter column under these lowe red inlet pressure condi tions as they do on the longer column at 15 0 psig Setting up by linear velocity In prac tice 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 T dy 4 wheres is the turns count and is the nominal column helical coil diameter Use a value of d that s close to the apparent average value for the coils of the column Next calc
3. 36 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JANUARY 2005 Connections In last month s installment of GC Connections John Hinshaw discussed how peak retention times depend upon relationships between pressure flow rate oven temperature column dimensions and stationary phase This concluding installment of a two part series discusses the effects that column variability has on isothermal capillary gas chromatography and explores instrument calibration with the goal of maximizing instrument to instrument similarity of retention times John V Hinshaw GC Connections Editor www chromatographyonline com Flow Pressure and Temperature Calibration Part Il ariations in the oven temperature and carrier gas pressure influence peak retention times significantly enough so that differences from column to column and instrument to insttument make method validation a necessity for ensuring consistent results Instriment to instrument variability can be brought under contro by implementing a few simple cali bration and set up procedure and valida tion is made more reliable as a result The first part of this series showed that analysts should understand the effects of paforming the same analysis on different gas chro matography GC systems on thevariability of their results in particular on retention times 1 The dependencies of peak reten tion times on oven temperature and inlet pressure can be large enough to cau
4. able tool Howe ver the pressure and related flow transducers in a GC should be ze roed at least eve ry three months as well as when ever 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 pressu re to ze ro and either disconnect the columns or remove the sep tum 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 Allowat least a half hour for the instru ment 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 ze roing pro cedure according to the user manual Finally reconnect the supply lines and establish a low pressure for long enough to purge air from the system before reconnect ing the columns or replacing the inlet sep tum nuts This also would be a good time to service the inlets if necessary Conclusion Some variability in results obtained on dif ferat instruments with different columns is always to be expected Analysts can mini mize instrument to instrument retention time variabilit
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6. d I dont know what the toler ance levels we re 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 technology that goes into producing capillary GC columns Two studies published 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 4 reveals retention factor standard deviations of 5 9 for 16 methylsilicone columns 11 4 for 7 phenylmethylsilicone columns and 30 4 for 9 Carbowax 20M columns 2 In the second report the authors meas ured 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 Car b owax 20M Nonpolar columns prepared with twice the stationary phase film thick ness had even smaller column to column relative retention variations 3 The vari ability of the column inner diameter does not affect retention factors under the isothermal conditions used and its range was given as d 270 20 wm 2 Today analysts rely upon column manu facturas to produce a consistent product from fused silica tubing Ad vances in tub ing production and chemical treatment stationary phase synthesis column coating 38 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JANUARY 2005 and conditioning have greatly reduced the var
7. fects both the ave r age carrier gas linear velocity and the reten tion factor given a constant stationary phase film thickness The linear velocity Table Retention times in seconds for three peaks across a wide range of column inner diameters operated isothermally at 100 C For conditions see Fig 1 Peak Retention Time s Inner Diameter um n Dodecane K100 C 522 1 200 415 3 210 363 8 220 320 7 230 284 4 240 253 7 250 227 4 260 204 8 270 185 2 280 168 1 290 153 2 300 140 1 www chromatographyonline com Reterimon Hm e Cal Raemon Time 5 ott litter damaar pri ae oot ou Figure 1 Effect of column inner diameter on retention times a Inner diameter 200 300 ym b close 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 K100 c 522 1 green n undecane K400 ec 270 2 and red n nonane K400 c 70 99 will decrease as the square of the inner stant column pressure length and temper ature as follows 1 se d 2 1 W H a 2 This type of square law relationship pre diameter decreases as equation 1 shows Ap jodi BLN We can compare the effect of different column inner diameters on velocity at con dicts a strong dependence of the linear velocity and thus retention times on the column diameter Table I shows the effect of chang
8. hermometers should all be calibrated to NIST standards by their manufacturers For single locations a single thermometer should work well enough with a simple ice bath re fe rence As long as the overall thermal environment is consistent the retention times will be as well It is ve ry important to place an external temperatureprobe consistently when meas uring oven temperatures for calibration purposes Bearing in mind that in any case the calibration will only reflect a single point temperature the best placement is as close as possible to the instrument s temper ature 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 center of the oven in the area where the column will be located In any case 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 chro matograph s oven door and that introduc ing the probe does not create an extra air leak from the outside See the user s manual or service manual for details on exactly how to calibrate the temperaturefor a specific instrument In general operate the GC system at a tem peraturein the middle of the method oper ating range or at 100 C A vell controlled room temperature also helps attain more consistent oven temperature calibrati
9. iability in column dimensions and reten tion as well as yielding much lower bleed levels higher stability and longer life It would be ve ry interesting to see similar data on populations of modern capillary columns that compares them with 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 ana lytical method Thezeare no technical re a sons to select or eliminate any paticular manufacturer but columns from the same company will be much more self consistent than would be the case for columns from differnt 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 before committing to any partic ular choice Batch to batch variations in stationary phase chemistry are important because they affect peak retention directly but in this article I will assume that the columns all are the same in this respect Working within the tolerances of the old column study which can be taken as larger than the absolute maximum range that would be encounter today what then are the effects of variability in column diameter and length on retention times Column Inner Diameter The column diameter af
10. ing the column inner diameter across a relatively wide range from 200 to 300 wm on the retention times of the same three exam n Undecane n Nonane ple hyd rocarbon peaks as used in the first Kio0c 270 2 Sure EAE part of this series and the data are pre ome 1382 sented graphically in Figu re la The effects 211 8 125 7 on retention time are large Table II gives ae 4 ee the same data for a narrower range of inner 153 2 94 5 diameters from 240 to 260 um as might 138 8 a be encountered in practice and the corre 115 3 73 6 sponding plot is shown in Figure 1b io pal According to this information to keep retention times within a maximum range of 40 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JANUARY 2005 15 s the inner diameter would have to fall within 6 wm or about 2 4 of the nominal 250 um inner diameter for the longest retained peak shown here within 10 um for the middle peak and within 20 um for the earliest eluted peak As peak retention increases the variabil ity 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 case for temperature p rogrammed 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 analys
11. is 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 Temperatureand pressure variability will add more uncertainty to the retention times Column Length Variations in column length also affect retention times Di ffe rences between col umn lengths on the order of 1 m or more a re not uncommon within a population of initially equal size columns that have been in use for some time Rem oval of a small portion of a column is part of good labora torypractices that call for the use of new inlet and detector ferrules with each instal lation Additional lengths can be removed from the column entrance as part of col umn reconditioning in order to remove nonvolatile sample residues that have accu mulated 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 case retention times will vary with the square of the column length as shown in equation 3 which was obtained by combining equa tions 1 2 and 3 from the first installment of this column series 1 This is not a desirable situation The peak with partition coefficient K 522 at 100 C n dodecane which is eluted in 227 s on a 25 m column with a 15
12. ither in the front or the 44 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JANUARY 2005 back position as dictated by the inlet detector configuration and the methodology As long as the overall thermal environment is consistent theretention times will be as vell Pressure Carrier gas inlet pressures are controled either by electronic pressure con t rollers or by manual regulators which may or may not have electronic pressure gauges For capillary columns with inner diameters less than 530 um a pressure contulled split splitless inlet system is the most com mon Even for EPC in the constant flow mode with this type of inlet the GC sys tem actually controls the inlet pressure and sets it as required to maintain the desired flowrate using relationships derived from equations 3 5 from the first installment of this column 1 Wide bore columns of 530 pm i d and up can use a true flow controlled carrier source instead If a Bourdon type mechanical pressure gauge is in use then there islitdle realistic need to calibrate it because it is inherently inaccurate and nonlinear compared to elec t ronic transducers In such cases chro matographers should rely on the gauge as an approximate pressure indicator and instead use carrier gas average linear veloc ity measurements for column setup pur poses I also have seen a digital electronic pressure device with an attached syringe needle which gives a fairly accurate reading when inser
13. n inner diameters operated isothermally at 100 C For conditions see Fig 1 Peak Retention Time s Inner Diameter um n Dodecane K100 c 522 1 240 253 7 242 248 1 244 242 7 246 237 4 248 232 3 250 227 4 252 222 6 254 217 9 256 213 4 258 209 0 260 204 8 n Undecane n Nonane K100 c 270 2 K100 c 70 99 169 8 103 5 166 3 101 6 162 9 99 8 159 5 98 0 156 3 96 2 153 2 94 5 150 1 92 9 147 2 91 3 144 3 89 7 141 5 88 1 138 8 86 7 42 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JANUARY 2005 Upon entering this corrected inner diam eter into the EPC column configuration the electronic pressure controller will adjust the pressure downwardsufficiently to pro duce the desired average carrier gas linear velocity Due to the variations in inlet pres sure from instrument to instnment how ever this procedure should berepeated wheneve 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 require ments and effects of temperature and pres s ure calibration in laboratories that utilize multiple gas chromatographs What to expect Gi ven that small changes in temperature or pressure can shift retention times significantly what should gas chromatographers expect from their instrumentation How much pressure and temperaturevariation is normal between gas chromatographs that are operati
14. ng within the manufacture r s specifications I read through a number of brochures speci fication sheets operator s manuals and service manuals I found that pressure and temperaturetolerances vary somewhat by manufacture and for some items there was little or no information Most GC systems p roduced in the past 10 15 years include p rovisions for oven temperature carrier pressure and flow calibration via the instuments 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 instru ments by as much as 2 C or slightly more When left uncalibrated larger devia tions can be expected between different models than between the same model gas chromatographs The apparent degree of variation also depends upon how the tem peratureis measured All GC ovens exhibit temperaturegradients between the internal temperaturesensor where the temperature is measured and other locations within the oven The degree of temperature gradients depends upon many factors such as the oven temperature setpoint whether the cooling flap or door is open or shut the www chromatographyonline com condition of the door closure and insula tion the temperatures of the inlets and detectors and any other material such as columns valves and other accessories that affect heat and air flow Gradients of as much as 2 4
15. on Let the instrument stabilize for at least 1 h and then access the instrument calibra tion routine Compare the resulting high www chromatographyonline com resolution reading with the probe reading and enter a corresponding temperature off set value on the keypad or in some cases 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 noware consistent to within a few tenths of a degree Make a note of the temperature p robe 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 pre s suremeasurement device can be used to obtain an independent pressure reading at or close to a single setpoint Suchreadings will be a valid indication of the relative pressures in multiple instruments for the purposes of setting up a method that uses constant inlet pressure However if the method calls out any type of pressure pro gramming including constant flow mode with temperature programming then these readings will only set the initial pressures and will have little bearing on subsequent control changes In the case of a mechanical pressure gauge then an external digital transducer becomes a very valu
16. se sig nificant deviations of 15 s or more between instruments when the individual oven tem perature differ by only 1 C or the pres sure by 1 psi The scope of these variations depends in turn upon the chromatographic conditions the column and the analytes under examination Although not a substi tute for validation and suitability testing instrument calibration can help to reduce the normal variability that will be encoun tered when working with multiple instru ments and multiple columns Column Effects Thereare three main column variables that affect retention times dimensional varia tions such as inner diameter and length stationary phase variations both in the chemistry and the film thickness and aging effects due to 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 I vividly recall spending many long nights in the graduate school lab drawing out borosilicate glass columns on a cantan ke rous machine that would have made car toonist Rube Goldberg proud see www tube goldberg com If I was lucky enough to obtain a single 10 m long piece of coiled tubing I then was faced with the tasks of coating the column with a station ary phase that I had synthesized from scratch and installing it intact into the gas chromatograph s oven I made no pretense of duplicating any of these handmade columns an
17. ted into an inlet Most of the electronic pressure transduc ers for EPC systems are specified to delive r accuracies of 2 of their full scale read ing For a 0 100 psig transducer that s about 2 psig while fora 0 30 psig trans ducer it s more like 0 6 psig These erro rs show up as deviations from a perfectly lin ear relationship between the actual pressure and the readout and according to our cal culations they are large enough to cause noticeable retention shifts from one instru ment to another The solid state pre s s u re transducers cannot be calibrated by the instrument for their nonlinear 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 Calibration Properly performed temperature calibra tion and inlet pressure ze ro compensation can help tremendously in attaining more consistent instument to instrument results However it also is necessary to consider carefully other variables such as column www chromatographyonline com dimensional variations and positions inside the column oven Oven temperature A precision tempera ture measuring device and appropriate probe are necessary for meaningful oven calibration The probe and meter combina tion should be capable of 0 2 C or better resolution If consistency of results betwe e n instruments that will be calibrated with dif fe rent thermometers is important then the t
18. ulate the ratio of the length of the column at hand to the nominal column www chromatographyonline com length for which the method conditions we re developed Third multiply by the desired average carrier gas linear velocity for the method to obtain the linear velocity goal for the specific column being installed as shown in equation 5 Finally as part of verifying the setup establish the operating or initial column temperatureand then adjust the inlet pres sureas required to produce the calculated velocity For GC systems with electronic pressure control EPC the EPC system will calcu late and set the correct pressure for the desired velocity if the measured column length is entered first However slight vari ations in the column diameter from the nominal diameter can produce a slight errorin this step The operator can approx i mate the apparent column diameter at this point by comparing the measured carrier gas linear velocity to the desired value as enteredinto 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 For the example given above if the desired velocity was 32 64 cm s for the 24 m X 250 um column but the observed velocity was 34 9 cm s then the corrected column inner diameter would be Table II Retention times in seconds for three peaks across a narrow range of colum
19. y by calibrating the oven tem perature installing the column in the same oven location and setting the carrier gas average linear velocity to compensate for slight column to column variations in length and inner diameter Good column maintenance practices also will help estab lish better repeatability Although not dis cussed in detail in this article with careful thermal calibration dimensionless retention measurements such as the retention factor relative retentions and retention indices inherntly rationalize interinstrument varia tions and intercolumn dimensional varia tions and effectively make such results more comparable JANUARY 2005 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 45 References 1 J V Hinshaw LCGC 22 12 1160 2004 2 M J Hartigan and L S Ettre J Chromatogr 119 187 206 1976 3 MJ Hartigan K Billeb and L S Ettre Chro matographia 10 571 579 1977 4 L S Etre personal communication November 2004 John V Hinshaw GC Connections editor John V Hin shaw is senior staff engineer at Serveron Corp Hillsboro Ore gon and a member of LCGC s editorial advisory board Direct correspondence about this column to GC Connections LCGG Woodbridge Corporate Plaza 485 Route 1 South Building F First Floor Iselin NJ 08830 e mail Icgcedit lcgcmag com For an ongoing discussion of GC issues with John Hinshaw and other chromatographers visit the Chromatography Forum dis
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