LC•GC Europe - May 20012 LC troubleshooting

This column is the first of a two-part seriesabout autosamplers used in liquidchromatography (LC) systems. Today,nearly all chromatographers useautosamplers daily. Autosamplers reducemanual labour, increase sample throughputand improve precision for routine work.This month’s “LC Troubleshooting” looksat three common autosampler designs,how they work, and some of their strongand weak points. The next instalment ofthis series will consider common problemsencountered with autosampler use.

Injection ValvesMost autosamplers use six-port injectionvalves as the mechanism to deliver samples tocolumns. This type of valve comprises a fixedstator and a movable rotor. The stator isconnected to the valve body in a manner thatallows the attachment of tubing, loops,injection ports and other peripheralconnections. The stator is generally made of awear-resistant material, such as stainless steelor ceramic. The rotor is held closely againstthe stator and, as the name implies, rotatesduring operation. The rotor is usually made ofa softer material than that used in the stator,such as a fluorocarbon polymer.

In autosamplers, the rotor is driven by anelectric motor, although some older unitsuse compressed air; manual injectors havea lever handle attached to the rotor. Therotor contains small passages that connectthe various stator passages as needed. Inthe drawings used in this discussion, thestator and associated connections areoutside the main circle of the valve,whereas the rotor with its U-shapedconnecting channels is drawn inside thecircle. Figure 1 shows a typical injectionvalve configuration. Figure 1(a) shows the

valve in the load or fill position, in whichthe injection loop is filled with sample.Meanwhile, the pump delivers mobilephase directly to the column and bypassesthe loop. Next, the rotor is turned to theinject position (Figure 1(b)), and theconnecting passages direct the pump flowthrough the loop and flush its contentsonto the column.

Filled- and Partial-Loop InjectionConventional six-port valves can deliversample to a column in the filled-loop orpartial-loop mode. In the filled-loop mode,the volume of sample injected is dictatedby the volume of the loop. Figure 2(a)shows this function schematically. Forexample, a 20 µL loop might be fitted tothe valve and then sample would bedelivered to the loop until the excesssample exited the waste port. The loopcontents (the cross-hatched portion) arethen delivered to the column after therotor is moved to the inject position.Because of the fluid dynamics involved,analysts should overfill the loop by at leasttwice its volume to ensure uniform loop

filling. To change the injection size, userswould mount a different volume loop onthe injector.

An alternative injection technique is thepartial-loop injection. In this technique,illustrated in Figure 2(b), a carefullymeasured volume of sample is delivered tothe loop. For example, 20 µL might beplaced in a 100 µL loop. Once again, theloop contents are flushed onto the columnwhen the rotor is moved. For the mostreproducible injections, it is best to keepthe injection volume at less than half theloop volume. This technique is moreflexible than the filled-loop injectionbecause it allows chromatographers toinject different volumes without having toinstall another loop. However, the partial-fill technique requires accurate and precisedelivery of sample to the loop for goodinjection reproducibility.

Note that the loop is backflushed ontothe column. That is, the sample is loadedinto the loop in one direction and flushedout of the loop in the opposite direction.Backflushing does not affect the results inthe filled-loop mode, but it is important for

Autosamplers are more than just automated injection valves.

John W. Dolan, LC Resources Inc., Walnut Creek, California, USA.

Autosamplers, Part I —Design Features

Figure 1: Schematics of a six-port injection valve shown in (a) load and (b) inject positions.Arrows show direction of flow through the loops.

(a)

Sample

Waste

Column

Pump

(b)

Sample

Waste

Column

Pump

3LC•GC Europe - May 2001 LC troubleshooting

the partial-fill method. Consider thesituation in which a large loop is fitted tothe valve, as might be the case for someautosamplers, for example, a 1 mL loop. Ifan analyst placed 10 µL of sample in the 1 mL loop and backflushed it onto thecolumn, the sample would be delivered ina discrete plug followed by the remainderof the loop contents (mobile phase), andwe would expect good chromatographicperformance. With forward flushing, aswould be the case if the pump and columnconnections were reversed in Figure 2(b),the 10 µL plug would have to flow through1 mL of tubing and would be broadened inthe process. A broad peak at the point ofinjection will continue to broaden duringthe separation, and all the peaks will bewider than expected, especially in isocraticseparations. Therefore, it is important thatthe loop is plumbed to ensure that theloop is backflushed onto the column. Thispractice is generally not a problem unless auser dismantles a valve to service it andreattaches the connections in the wrongpositions.

Manual injectors operate as illustrated inFigures 1 and 2, in which the sample isintroduced with a syringe and the valve is

rotated by hand (or by pressing an Injectbutton). Most autosamplers use one ofthree configurations, in which the sample ispulled or pushed through a fixed loop or aspecial case in which the injection needleforms part of the loop. Although otherdesigns and hybrids exist, I will discuss onlythe three basic autosampler variations here.

Pull-to-Fill AutosamplersThe pull-to-fill autosamplers get their namebecause the sample is drawn into thesample loop by syringe suction. This typeof autosampler was popular in the earlydays of autosampler development becauseof its simplicity and reliability, but it hasdropped from favour with the advent ofthe other two designs.

Figure 3(a) illustrates the pull-to-filldesign. In this example, a syringe isattached to the waste port and a needlewith connecting tubing is attached to thesample port. The needle is inserted in asample vial and the syringe is drawn backuntil the loop is filled with sample. Therotation of the valve results in the injectionof the loop contents. This autosamplerdesign is quite simple, often with a needlethat moves in just one axis (up and down)

with a sample tray rotating until the desired vial is below the needle. Onepopular variation is even simpler — it usesa tight-sealing vial cap that is presseddown into the vial to displace sample intothe loop rather than using a syringe at theother end.

If simplicity is the strong point of thepull-to-fill autosampler, sample waste is itsweak point. As Figure 3(a) shows, samplemust fill the needle and all the connectingtubing before it reaches the sample loop.This sample, sometimes as much as10–100 µL, is wasted. This waste may ormay not be important, depending on theavailable volume of sample.

Because of the simple design, most ofthese autosamplers use a rotating sampletray with a fixed sequence and apredetermined number of injections pervial. The flushing mechanism may require awash vial between each sample vial or mayuse a separate wash station.

Push-to-Fill AutosamplersA more common autosampler design usesthe push-to-fill technique. This technique isvery similar to manual injection. Thesyringe moves to the sample vial, pulls upthe desired sample volume, moves it to theinjection port, and delivers the sample tothe loop, as shown in Figure 3(b). Typically,users need much less extra sample withthis design than for the pull-to-fill design.Syringe filling and dispensing is under thecontrol of a stepping motor, which canprovide very precise and accurate sampledelivery. As a result, an imprecision of lessthan 0.5% is common in both filled- andpartial-loop modes.

Usually, push-to-fill autosamplers addfeatures to the basic pull-to-fill designs,such as random vial access, more effectiveflushing and programmable samplevolumes. The tray layout may be round ormay use an x,y,z axis arm that moves tofind the desired vial from a rack of vials ora 96-well plate. The valve canaccommodate various loop sizes, although100–500 µL volumes are the mostcommon. Some older autosamplers used a5 mL or even a 10 mL sample loop thatcould not be changed.

Integral-Loop AutosamplersIn recent years, the integral-loopautosampler has become popular. Thestrong point of this design is that nosample is wasted, and that can be veryimportant for trace analysis when samplevolume is limited. The action of theintegral-loop autosampler is morecomplicated, as illustrated in Figure 4.

Figure 2: Schematics of (a) filled- and (b) partial-loop autosampler injection modes.

(a) (b)

Sample

Waste

Column

Pump

Sample

Waste

Column

Pump

Figure 3: Schematics of (a) pull-to-fill and (b) push-to-fill autosampler designs in the fill mode.Arrows show direction of sample flow during the fill cycle.

(a)

Column

Pump

(b)

Column

PumpWaste

Low-pressureneedle

seal

V

LC•GC Europe - May 20014 LC troubleshooting

Figure 4(a) shows the valve in the flushposition, in which wash solvent is pulled,pushed, or both through the loop,depending on the system design. Note thatthe needle is fitted firmly into a high-pressure seal. The needle is moved to theautosampler vial and sample is pulled intothe loop (Figure 4(b)). Finally, the needle isinserted into the high-pressure seal againand the rotor is moved to the inject position,as shown in Figure 4(c). Because the sample

is contained only in the swept portion of theloop, 100% of the sample is injected. Themaximum sample volume is dictated by theloop size, which is difficult, if not impossible,to change. Typically 100 µL is the maximuminjection volume for these autosamplers,whereas the other two designs canincorporate any loop size desired.

One potential weak point of the integral-loop injector is the high-pressureseal. The push-to-fill autosampler designuses a low-pressure seal that is generallytrouble-free. Because of problems with thehigh-pressure seals, some manufacturersincorporate an additional valve instead of ahigh-pressure seal to perform a similarfunction, but this change adds cost andcomplexity. The vial access, injectionvolume flexibility (up to as much as theloop volume), and programmability areequivalent to the push-to-fill designs.

Trade-offsSome of the strong and weak points ofeach autosampler are listed above. Table 1compares some aspects of the threeautosampler designs.

The pull-to-fill design has the simplestmechanical design, whereas the integral-loop autosampler tends to be the mostcomplex. This feature often translates tothe reliability or number of problems thatcrop up during routine operation.

The pull-to-fill autosamplers may have noindependent flushing procedure, so flushingthe needle and transfer tubing may requireplacing a wash vial between each sample.An alternative would be to allow the nextsample to provide the flush, but it wouldrequire large sample volumes. The othertwo autosamplers can use an independentwash solvent and they generally allowmultiple flushes between samples.

The mechanical simplicity of the pull-to-fill design often means that the samplesare placed in a tray that indexes one

position at a time, so the vials must beloaded in the injection order. It may alsorequire that the same number of injectionsbe performed for each vial. The other twodesigns usually allow random access tovials, a variable number of injections, andeven a variable injection volume for eachvial. Programming is simplest if the vials areinjected in order, but the flexibility ofrandom access can be a useful feature,particularly if one or more samples orstandards must be injected at intervalsduring the run sequence.

Because all the sample is contained inthe swept needle-loop tubing, the integral-loop autosampler wastes little or nosample. If analysts use microvials or vialinserts, nearly all the sample in the vial canbe drawn into the loop and injected. Thisfeature can be attractive in trace analysesin which sample volume is limited. Push-to-fill syringes generally waste little sample,but a small volume of sample is usually leftin the syringe and connecting passagesand is wasted.

The push-to-fill design generally providesthe most flexibility in terms of injectionvolume. Small volumes of as little as 1–2 µLcan be injected if the syringe mechanism isdriven precisely. By fitting a large loop on theinjector, chromatographers can inject nearlyany large volume. The integral-loop designalso allows programmed injection volumesas large as the loop volume, but the loop isnot changed easily, so the upper end of theinjection volume range is more restrictedthan it is for the push-to-fill models.

The high-pressure needle seal is one of the weak points of the integral-loopautosamplers, and users should take careto ensure that the seal is in goodcondition. The low-pressure seal of thepush-to-fill injector is rarely a problem, andit can be adjusted easily by tightening afitting in most models. No needle seal isused in the pull-to-fill autosampler.

Table 1: Comparison of Autosampler Features.*

Features Pull-to-Fill Push-to-Fill Integral Loop

Mechanical simplicity � 0 �

Ease of flushing � � �

Random access � � �

Conserves sample � 0 �

Variable injection volume � � �

Needle seal problems � 0 �

Syringe precision required � � �

Impact on dwell volume � 0 or � 0

* � is a positive trait, � is a negative trait, 0 is neither a positive nor a negative trait and � is theautosampler’s strongest trait.

Figure 4: Schematic of an integral-loopautosampler in its (a) needle- and loop-flushing, (b) loop-filling and (c) injection positions. Arrows show direction of sampleor flush solvent flow.

(a)

ColumnWaste

High-pressureneedle seal

Pump

(c)

WasteColumn

Pump

V

(b)

ColumnWaste

Pump

V

V

“The push-to-filldesign generallyprovides the mostflexibility in terms ofinjection volume.”

5LC•GC Europe - May 2001 LC troubleshooting

Both the integral-loop and the push-to-fill models rely on the syringe to determinethe injection volume in most instances.Therefore, the syringe control must beprecise and accurate for good performance.Fortunately, the mechanical controls inthese units are very good, and they shouldprovide an imprecision of less than 0.5% ifthe autosampler is operating properly.

The dwell volume of an LC system is thevolume from the point at which themobile-phase components are mixed tothe head of the column. This volumeaccounts for an unintentional isocratic holdbuilt into every gradient run. Large dwellvolumes can result in methods that arehard to transfer and have longer-than-desired gradient run times. Dwell volumehas no practical effect on isocraticseparations. Dwell volumes for common LCequipment range from 0.2 mL to 5 mL ormore, depending on the equipmentdesign. The dwell volume comprises thesolvent mixing chamber and all theconnecting tubing between the mixer andthe column. For low-pressure mixingsystems, the dwell volume includes thepump volume.

A final important portion of the dwellvolume is the autosampler loop volume,because the loop in the inject position ispart of the volume between the mixer andthe column. As long as the loop’s volume isno larger than approximately 100 µL, it isgenerally an unimportant contributor tothe dwell volume. However, it is easy toconceive unintentional dwell volumechanges when a loop is changed. Forexample, if the system had a 300 µL dwellvolume with a 50 µL loop, then the dwellvolume would change to 1250 µL if a 1 mL loop were substituted. This changecould affect the appearance of thegradient chromatogram, especially ifanalytes were eluted within the first fewminutes of the run or if the separationused a narrow-bore column. Analystsshould consider the effect on the dwellvolume and the implications for theseparation when they change a loop.

ConclusionsI have described three basic autosamplerdesigns. Some commercial models may use a combination of these designs, so they may not fall clearly into one categoryor another. Each design has its appropriateuses and, in general, will performsatisfactorily if used within its capabilities. In the second instalment of this series, I’ll look at some common problems that users may encounter with routineautosampler use.

“LC Troubleshooting” editor John W. Dolan ispresident of LC Resources Inc., of WalnutCreek, California, USA, and a member of theEditorial Advisory Board of LC•GC Europe.Direct correspondence about this column to“LC Troubleshooting,” LC•GC Europe,Advanstar House, Park West, Sealand Road,Chester CH1 4RN, UK,e-mail: dhills@advanstar.com

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