Large Vol Injection Tutorial - G3510-90020

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Notices Agilent Technologies, Inc. 2009No part of this manual may be reproducedin any form or by any means (includingelectronic storage and retrieval or transla-tion into a foreign language) without prioragreement and written consent from Agi-lent Technologies, Inc. as governed byUnited States and international copyrightlaws.

Manual Part NumberG3510-90020

EditionFirst edition, May 2009Printed in USA and ChinaAgilent Technologies, Inc. 2850 Centerville RoadWilmington, DE 19808-1610 USA

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Agilent Multimode Inlet Large Volume Injection Tutorial

Agilent Technologies

Multimode Inlet TutorialOverview 4Hot Splitless 5Cold Splitless 6Solvent Vent 7Tutorial 8


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4 Large Volume Injection Tutorial

Overview

A growing number of researchers are exploring large

volume injection (LVI) techniques to improve existinganalyses. With traditional injection approaches incapillary gas chromatography, most inlets and columnscan only handle 1-2 L at a time. Attempts to increasethe injection volume lead to broadened and distortedanalyte peaks, large and long solvent peak tails, andsaturated or damaged detectors.

The desire to increase the injection volume is normallyto improve trace analysis. By introducing more of thesample to the system, the mass of analyte reaching thedetector will be proportionately increased, resulting inlarger peak areas and peak heights. If the baseline noiseis kept constant, larger peak heights mean greatersignal to noise ratios and lower system detection limits.An additional benefit of LVI is the ability to reduce the

amount of sample originally processed. For example,suppose a water sample contains 1000 ng/L ofpollutant. If the current method extracts the pollutantand reconstitutes it in 1 mL of solvent, theconcentration of analyte in the extract is 1000 ng/mL. A1 L injection of this extract puts 1 ng onto the column.Now assume that LVI allows a 10 L injection volume.The researcher could start with 100 mL of sample,extract it with less solvent, and reconstitute it in 1 mL.A 10 L injection puts 1 ng onto the column as before,but starts with an order of magnitude less sample (andlikely, an order of magnitude less extraction solvent).Another advantage of using LVI is the decrease insolvent that actually reaches the detector. Usually, only10-30% of the injection solvent actually enters the

column and makes it to the detector.LVI can be applied to injection volumes ranging from afew microliters up to 1 mL or more. In most LVIapproaches, the sample solvent is selectivelyevaporated and removed from the inlet system beforethe analytes are transferred to the separation column.In this way, LVI is similar to nitrogen evaporation orrotary evaporation of the solvent, with the added benefit


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Large Volume Injection Tutorial 5

of being performed in the GC inlet rather than in a fumehood. Analytes that would be lost during nitrogenevaporation may be retained in the inlet andsuccessfully analyzed via LVI. Furthermore, the LVIprocess can be automated and is reproducible. As in theother evaporation techniques, the LVI approach is afunction of the solvent type, the inlet temperature, thevent flow of evaporation gas, and the analyte boilingpoint. In addition, the inlet pressure during evaporationand the inlet liner have an impact on the rate of solventremoval and analyte recovery. These parameters will be

described in more detail in the tutorial.

Hot Splitless

For most researchers considering LVI, their currentmethods are using hot splitless injection. This provenand reliable sample introduction method has worked

well for almost 40 years; however, it does present somechallenges to the sample integrity and to the methoddeveloper. First, the inlet must be hot enough to flashvaporize the solvent and analytes so that the resultingvapor cloud can be transferred to the column. The inletliner volume must be sufficiently large to contain thisvapor cloud. If the liner volume is too small, thevaporized sample can exit the liner and reach reactivesurfaces, leading to analyte loss. In addition, thepressure wave generated by the vaporized sample canpush back against the incoming carrier gas and entersensitive pressure and flow control systems. Using theAgilent pressure/flow calculator, a 1 L injection ofacetone into an inlet at 240 C and 14.5 psig expands to288 L of gas. Most inlet liners for standardsplit/splitless inlets have a nominal volume of 1 mL. Anincrease of injection volume to only 3.5 L under theseconditions creates a vapor cloud of 1 mL which couldeasily overflow the inlet liner.

Hot splitless injection also creates a challengingenvironment for thermally unstable analytes.Compounds such as the organochlorine pesticides DDTand endrin can rearrange to form breakdowncompounds. This process is accelerated with the inlet


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temperatures normally used to analyze them. Effectivechemical deactivation of the liner can minimize analytebreakdown. However, high inlet temperatures candecrease the lifetime of deactivated liners.

Another challenge created by hot splitless injection isthe opportunity for needle fractionation or analytediscrimination. The needle temperature increases asthe sample is being transferred from the syringe to theinlet because the needle is in contact with the septum.The rise in needle temperature can cause the solvent toboil away and deposit high boiling analytes inside theneedle. To avoid this fractionation problem, someresearchers load a solvent plug into the syringe first andthen draw up the desired sample volume. The thought isthat the solvent plug will wash any deposits into theinlet. An effective way to address this problem is tomake a high speed injection. This minimizes the timethe needle is in contact with the septum and the timethe sample touches the needle. Even with these issues,hot splitless injection can be made to work well. Analternative approach, such as cold splitless can addressthese concerns and improve the analysis results.

Cold Splitless

The Agilent Multimode Inlet (MMI) uses the sameliners and consumables as a standard split/splitlessinlet, making it compatible with existing hot split andsplitless methods. However, its temperatureprogrammability allows it to perform cold split andsplitless analyses as well. In cold splitless mode, theMMI is cooled to a temperature below the normalboiling point of the sample solvent so that when thesample is injected, no vaporization takes place. Theinjection is simply a liquid transfer from the syringe tothe inlet. Once the syringe is removed from the inlet, theinlet is heated to vaporize the sample and transfer it tothe column. The solvent vaporizes first and moves tocolumn, allowing analyte focusing to take place as innormal hot splitless injections. The analytessubsequently vaporize and move to the column. Themain advantage is that the analytes vaporize at the


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lowest possible inlet temperature, rather than at aconstant high temperature, minimizing thermaldegradation while still allowing a wide range ofanalytes to vaporize. Cold splitless operations also donot thermally stress the liner as harshly as hot splitlessdoes, prolonging its usable life. Cold splitless can alsoextend the amount of sample that can be injected insome cases. If a slow inlet temperature program isused, the solvent can be vaporized slowly and notoverflow the liner volume. As long as the analytes canbe refocused on the column, slow inlet temperature

programs cause no detrimental effects to thechromatography.

Solvent Vent

The solvent vent mode is how the MMI is able to do LVI.In solvent vent mode, the inlet is kept at a low initial

temperature during sample application. Pneumatically,the inlet is in split mode with a low inlet pressure. Theflow of gas through the inlet liner and out to ventremoves the evaporating solvent. The sample isinjected so that the incoming liquid is deposited on theliner wall and the solvent evaporates at a similar rate.Once the entire sample has been injected, the inletswitches to a splitless mode for analyte transfer. Theinlet is then heated to vaporize the concentratedsample and any remaining solvent and they aretransferred to the column. After a sufficient period toensure the sample transfer, the inlet is then switched toa purge mode to allow any remaining material in theinlet liner to be removed to waste. During the sampleinjection and solvent venting period, the GC oven hasbeen held at an appropriate temperature to allow thesolvent to refocus the analytes on the column. Whenthis refocusing is complete, the oven is thenprogrammed to perform the separation.


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Tutorial

You can choose to use a current hot splitless method to

follow this tutorial or the checkout sample that camewith your instrument. The tutorial will use the FlameIonization Detector (FID) MDL checkout sample (p/n5188-5372) to demonstrate the method developmentprocess. This sample contains four hydrocarbons (C13,C14, C15, and C16) in isooctane. Flame ionizationdetection is used as this will show you more of the LVIbehavior for analytes that elute closely to the solventand the solvent itself.

Step 1 - Hot Splitless

In order to calibrate your system for recoverycalculations, you will need to run your current method.For your first step, simply run your sample by yourexisting hot splitless method or use the conditionsbelow for the FID MDL alkane mix.

Column and sample

Type HP-5, 30 m x 0.32 mm x 0.25 m(19091J-413)

Sample FID MDL Checkout (5188-5372)

Column flow 4 mL/min

Column mode Constant flow

MMI

Mode Splitless

Inlet temperature 250 C

Initial time 5 min

Rate 1 0 C/min

Purge time 2 min

Purge flow 60 mL/min

Septum purge 3 mL/min


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*Wash solvent Isooctane

FID

Temperature 300 C

H2 flow 30 mL/minAir flow 400 mL/min

Makeup flow (N2) 25 mL/min

Lit offset Typically 2 pA

Oven

Initial temperature 50 C

Initial time 2 min

Rate 1 20 C/min

Final temperature 200 C

Final time 0 min

ALS

Sample washes 2

Sample pumps 6

Injection volume 1 L

Syringe size 10 L

PreInj Solvent A washes 3PreInj Solvent B washes 3

PostInj Solvent A washes 3

PostInj Solvent B washes 3

Viscosity delay 0

Plunger speed Fast

PreInjection dwell 0PostInjection dwell 0

Data system

Data rate 20 Hz


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You may want to run the sample 2-3 times to get anaverage for the peak areas. Figure 1 shows the typicalresults for the FID MDL sample under these conditions.

Step 2 - Cold Splitless

To make a cold splitless analysis, you will need tochange the inlet temperature. Set the inlet initialtemperature to 5-10 C below the normal boiling pointof your sample solvent. Hold this temperature for 0.1minutes, then program the inlet at 720 C/min up to theinlet temperature for the hot splitless method. See theconditions below for the FID MDL method (only theMMI conditions are given, the rest are all the same asfor hot splitless).

Figure 1 Typical hot splitless FID MDL sample results

pA

500

400

300

200

100

00 2 4 6 8 min

C13 C14

C15 C16


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Compare the peak areas, peak widths, and peak shapesfor the hot and cold splitless modes. Figure 2 shows thetypical cold splitless results for the FID MDL sample. For

this sample, the results are almost identical betweenhot and cold splitless.

MMI

Mode Splitless

Inlet temperature 90 CInitial time 0.1 min

Rate 1 720 C


Final time 5 min

Purge time 2 min

Purge flow 60 mL/min

Septum purge 3 mL/min

Figure 2 Typical cold splitless FID MDL sample results

min0 2 4 6 8

pA

0

100

200

300400

500

C13 C14

C15 C16


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Step 3 - Solvent Vent

Now change the MMI mode to PTV Solvent Vent.Notice that the Solvent Elimination Calculator buttonappears ( Figure 3).

This calculator was designed to help you determinereasonable starting conditions for your LVI method.Click the Solvent Elimination Calculator button to startthe calculator. In the first screen ( Figure 4 on page 13 ),

you are asked for several pieces of information. Youshould be able to provide the sample solvent and yourdesired injection volume. The third piece of informationis the boiling point of the earliest eluting analyte. If youknow this, select the temperature closest to the value;otherwise, you can leave it at 150 C as this will helpretain a wide range of analytes. For the FID MDL

Figure 3 Accessing the Solvent Elimination Calculator


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sample, set the solvent to isooctane, the injectionvolume to 5 L, and the boiling point to 200 C. ClickNext to go to the calculation screen.

Figure 5 on page 14 shows the calculation screen.Taking the information that you provided, the calculatorhas used an initial set of instrument conditions todetermine the solvent elimination rate according tofundamental theory. This Elimination Rate does notaccount for other factors specific to LVI and is normallytoo fast as determined from practical experience. TheSuggested Injection Rate does consider these factorsand is designed to leave a small amount of solvent inthe liner at the end of the venting period. This solventserves as a liquid trap for the more volatile analytesand promotes their recovery. The Suggested VentTime is determined by dividing the injection volume bythe Suggested Injection Rate.

Figure 4 Solvent Elimination Calculator


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NOTEFor more information, see the LVI Method Help in yourinstrument control software.

Figure 5 Solvent Elimination Calculator variables


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The variables for determining elimination rate areuser-settable in the lower portion of the window. Toillustrate how these parameters interact, try thefollowing changes (marked in black ) and record theElimination Rate value in Table 1.

Note that a small change in inlet temperature has asignificant impact on elimination rate. Vent flow has alinear effect such that a decrease by a factor of two invent flow gives an equal decrease in elimination rate.As the vent pressure decreases, the elimination rateincreases. Bear in mind that the vent pressure also

impacts how much solvent reaches the column duringventing. As the vent pressure is increased, more solventis loaded onto the column before the analytes aretransferred. Finally, the solvent type, specifically itsnormal boiling point, has a substantial impact on theelimination rate.

To continue with the tutorial, change the calculator

values back to those shown in Figure 5 on page 14 andlisted below. Click Next to move to the method changesscreen ( Figure 6 on page 16 ). The calculator knowsthe syringe that is currently installed and will only allow50% of that volume to be injected. If you ask for more,the calculator will warn you that the system cannotmake the injection and give you a choice as to how toproceed.

Table 1 Elimination Rate worksheet

Inlettemp(C)

Vent Flow(mL/min)

InjectVol (L)

VentPress(psig)

OutletPress(psig)

Solvent EliminationRate(L/min)

60 100 5 5 0 Isooctane 137.6440 100 5 5 0 Isooctane

60 50 5 5 0 Isooctane



60 100 5 5 0 Hexane


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This screen shows you all of the method changes thatwill be downloaded to the Edit Parameters screen. You

can choose to accept or reject any of these parameters.

MMI

Mode Solvent Vent

Inlet temperature 60 CInitial time 0.07 min

Rate 1 720 C


Final time 5 min

Vent flow 100 mL/min

Vent pressure 5 psig

Vent time 0.07 min

Figure 6 Method changes to download to the Method Editor(Solvent Elimination Calculator)


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The Oven initial temperature and hold times are notautomatically checked in case your method requiresthese values to be unchanged (e.g. you have aRetention Time Locked method). For the FID MDLsample, click Confirm and Copy and then Ok in the EditParameters screen.

Run the analysis and compare the peak areas betweenthis run and your original hot splitless analysis. Figure 7 shows an overlay of these two runs. The dotted trace isthe original hot splitless injection result and the solidtrace is the solvent vent result.

Table 2 on page 18 compares the resulting peak areasfor the two runs. The peak widths for the analytes areessentially the same for both runs. The result is that thepeaks are five times taller and show an increase of fivefold in signal to noise ratio.

Figure 7 Overlay of the original hot splitless injection result (dottedline) and the solvent vent result (solid line)

min6.5 7 7.5 8 8.5 9

pA

0

500

1000

1500

2000

C13 C14

C15 C16


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In Table 2, solvent vent recovery was calculated bydividing the solvent vent run areas by five times the hotsplitless areas. For the analytes, the recoveries arealmost 100% and are almost identical, indicating thatthe solvent vent conditions gave essentially a five-foldimprovement in instrument detection limits. Notice thatthe solvent recovery is only 44%. This means that fromthe 5 L sample injection, only 2.2 L entered thecolumn.

Let's extend this to larger injection volumes. Install alarger syringe into the autosampler such as a 25 or50 L. Increase the injection volume to 10 L and usethe Solvent Elimination Calculator to determine initialconditions. For the FID MDL sample, the MMI

conditions are now:

Table 2 Resulting peak areas for hot splitless and solvent ventruns

Inlet mode Solventarea

C13 area C14 area C15 area C16 area

1 L Hot splitless 17113114 56 56 555 554

5 L Solvent vent 36859256 261 268 2622 2596

Solvent ventrecovery

44% 93% 96% 94% 94%

MMI

Mode Solvent Vent


Initial time 0.15 min

Rate 1 720 C


Final time 5 min

Vent flow 100 mL/min

Vent pressure 5 psig

Vent time 0.15 min


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Figure 8 shows a close-up of the analyte peaks with a10 L injection. The first two peaks are still reasonablysymmetrical but the last two peaks clearly showfronting. This is due partially to column overload and tothe amount of solvent transferred to the column.

Table 3 shows the recovery from the initial hot splitlessrun. Notice that the 10 L Solvent Vent recoveries areslightly lower than the 5 L Solvent Vent run shown inTable 3. This is confirmed by the lower solvent recovery.To improve this, the inlet temperature could be loweredwhile keeping all other parameters the same or byshortening the vent time slightly. In both cases, moresolvent would be left behind to help trap the C13. Ofthese two approaches, the inlet temperature has alarger effect on trapping the early eluting analytes.

Figure 8 Analyte peaks using a 10 L injection

min6.5 7 7.5 8 8.5 9

pA

0

1000

2000

3000

C13 C14

C15 C16

Table 3 Recovery from the initial hot splitless run

Inlet mode Solventarea

C13 area C14 area C15 area C16 area

1 L Hot splitless 17113114 56 56 555 554

10 L Solvent vent 59579040 493 510 5106 5208

Solvent ventrecovery

35% 88% 91% 92% 94%


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To scale up to larger injection volumes, the easiest wayis to increase the vent time proportionally. You can usethe Solvent Elimination Calculator to explore thisrelationship. For a 50 L injection, a vent time of 0.75minutes is needed. The injection parameters for the FIDMDL sample are given below. In order to avoidoverloading the column, the FID MDL sample wasdiluted 1:10 in isooctane.

The resulting chromatogram is shown in Figure 9 onpage 21 . The peak shapes are obviously distorted, a

result of too much solvent being transferred to thecolumn. You can fix such a problem in several ways.Simply increasing the vent time even more will reducethe amount of solvent in the column. Figure 10 onpage 22 shows the resulting chromatogram with a venttime of 0.90 minutes instead of 0.75 minutes. The peakshapes are greatly improved and are very similar to the5 L chromatogram shown in Figure 7 on page 17 . Otherapproaches to reduce the solvent being transferred tothe column are to increase the vent flow, decrease thevent pressure, or increase the inlet temperature duringthe vent period. The Solvent Elimination Calculator canshow you how much a change in the parameters willaffect the elimination rate. Two other approaches canalso help improve analyte recovery and peak shape.

Using a retention gap will aid in refocusing the analyte

MMI

Mode Solvent Vent


Initial time 0.75 min

Rate 1 720 C


Final time 5 min

Vent flow 100 mL/minVent pressure 5 psig

Vent time 0.75 min


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peaks and improve peak shape. A second method is toinclude some retaining material in the liner such asglass wool or packing. Material in the liner aids inholding the analytes during solvent venting and allowsmore solvent to be vented. When you use retainingmaterial in the liner, you need to be aware of losinganalytes due to irreversible adsorption.

For more information and sample applications, pleaserefer to the Agilent Web site(http://www.chem.agilent.com/en-US/Products/Instruments/gc/multimodeinlet/pages/default.aspx ).

Figure 9 50 L injection of FID MDL sample diluted 1:10 inisooctane

min6.5 7 7.5 8 8.5 9

pA

0

500

1000

1500

2000

2500

C13 C14

C16C15
http://www.chem.agilent.com/en-US/Products/Instruments/gc/multimodeinlet/pages/default.aspxhttp://www.chem.agilent.com/en-US/Products/Instruments/gc/multimodeinlet/pages/default.aspxhttp://www.chem.agilent.com/en-US/Products/Instruments/gc/multimodeinlet/pages/default.aspxhttp://www.chem.agilent.com/en-US/Products/Instruments/gc/multimodeinlet/pages/default.aspx


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Figure 10 50 L injection resulting chromatogram with a vent timeof 0.90 minutes instead of 0.75 minutes

min6.5 7 7.5 8 8.5 9

pA

0

500

1000

1500

2000

2500

C13 C14

C15

C16


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Large Volume Injection Tutorial

Agilent Technologies, Inc. 2009

G3510-90020

Large Vol Injection Tutorial - G3510-90020

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