clinical a concurrent process for the automatic...

Journal of Automatic Chemistry of Clinical Laboratory Automation, Vol. 8, No. 3 (July-September 1986), pp. 151-154

A concurrent process for the automaticpreparation of biological samples combinedwith high pressure liquid chromatographicanalysis

D. C. Turnell and J. D. H. CooperThe Department of Biochemistry, Coventry & Warwickshire Hospital, StoneyStanton Road, Coventry CV1 4FH, UK

Introduction

The objectives of sample preparation for high pressureliquid chromatography (HPLC) analysis are the elimina-tion of compounds that would otherwise progressivelyreduce the performance of the analytical column anddirectly interfere with the separation and quantitation ofanalytes. Approaches to achieve these objectives haveincluded liquid-liquid extraction [1 and 2], proteinprecipitation [3], ultrafiltration [4], dialysis [5] and theuse of sorbants, by either pre-column [6] or columnswitching and back-flushing techniques [7]. All theseoperate as batch processes with the exception of columnswitching methods and those employing robots.

The technique of automatic sequential trace enrichmentof dialysates (ASTED) has been shown to be aninexpensive and effective technique for deproteinizingserum samples prior to HPLC analysis: the traceenrichment cartridge can be regenerated for subsequentanalyses hundred oftimes [8 and 9]. However, it operatedin a purely sequential mode with the HPLC, such that thesecond sample was not processed until the first had beenanalysed and consequently half of the system was alwaysidle. This paper describes modifications to the basicASTED system that enable concurrent processing ofadjacent samples so that the second sample may beprepared whilst the first is being chromatographed.

Equipment and processes

HPLC unit

The HPLC equipment was loaned by Gilson Interna-tional, Villiers-Le-Bel, France, and comprised two 303/5SC HPLC pumps, a 620 Data Master and 502 ContactModule, all controlled by a 704 System Manager (version2.1) operating in an Apple IIe microcomputer.

Sample preparation unit

The sample preparation unit comprised a modifiedAuto-Analyser sampler II and dialyser block fitted with aCuprophan C-type membrane (molecular weight cut-off10000 Daltons, Technicon, Basingstoke, Hampshire,UK), a Minipuls and 303/5SC HPLC pump (GilsonInternational, Villiers-Le-Bel, France), a Rheodyne 7010

injection valve with a 12 V pneumatic controller (Ana-chem, Luton, Bedfordshire, UK), a Sinclair Spectrummicrocomputer and a purpose-built interface based on anZ80A parallel input/output controller (Radio Spares,London). The injection loop of the Rheodyne valve wasreplaced with a trace enrichment cartridge (TEC), whichhas been described previously [8]. The Auto-AnalyserSampler and the Minipuls were modifed so that theycould be controlled by the Spectrum microcomputer.

The hydraulic connections are shown in figure 1. TheMinipuls was used to aspirate the sample from the samplecup into the donor channel of the dialyser block.Dialysate in the recipient channel of the dialyser blockwas pumped through the TEC by the 303/5SC pump(TEC pump). The Rheodyne valve could switch the TECbetween the dialysate or HPLC solvent flow.

The operations of the sampler, Minipuls, injection valve,and 303/5SC pump are controlled by a BASIC programin the Spectrum microcomputer via the Z80A interface.The electrical connections between these components areschematically shown in figure 1.

Operation of the sample preparation unit

The operations required to prepare a single sample forinjection are shown in the timing diagram (figure 2) andare as follows:

(1) The sampler probe is moved from the washreservoir into the sample cup, the Minipuls energized,and sample is drawn into the donor channel of thedialyser block. Once the donor channel is filled, thesampler probe is returned to the wash reservoir and theMinipuls is switched off.

(2) With the sample held in the donor channel of thedialyser, the TEC pump is energized and dialysate isdrawn from the recipient channel of the dialyser andpassed through the TEC. The TEC retains the analytesin the dialysate which have diffused across the Cupro-phan membrane. The TEC pump is then switched off.

(3) The TEC is introduced into the solvent stream ofthe HPLC by rotating the Rheodyne valve to the injectposition and the retained analytes eluted from the TEConto the analytical column.

(4) With the Rehodyne valve in the inject position,both the Minipuls and TEC pump are energized topurge the sample and dialysate from the dialyser blockand associated tubing.

151

D. C. Turnell and J. D. H. Cooper Automatic preparation of biological samples combined with HPLC

HPLC Unit Sample Preparation Unit|,

Gilson 502|

Apple lie

HPLC pump

Print er

"-Data.master ’iIDetectr t"’lumn- ]"

Waste

--Spectrum

Interface

r---’---’t

SamplerSample

C pump . Dialyser

RecipientSolvent

WaSte

[ -M’inipuls

1 WaSte

Figure 1. Schematic diagram ofthe hydraulic ( ) and electrical ( ) connections of the sample preparation and HPLC units.

Out

Off

Off

Injection Inject

Figure 2. Timing diagram for the operation ofa single ASTEDcycle to prepare a single sample

(5) The Rheodyne valve is turned to the load positionand recipient solvent passed through the TEC by theTEC pump. Both the Minipuls and TEC pumps arethen switched off. In this way the retaining conditionson the TEC are regenerated by re-equilibrating withrecipient solvent in preparation for the next sample.

152

Configuration of the system

The system consisted of the HPLC and the samplepreparation units combined together (figure 1), so thatthe preparation and analysis of adjacent samples mayoperate concurrently. The flow diagram for this operationis shown in figure 3 and is shown schematically in figure4. Once the first sample has been prepared and injectedthe processes only interact at the moment of injection,such that the faster process will wait for the slower. Thisallows the sample preparation unit to treat the nextsample whilst the current sample is being chromato-graphed. The interactions between the two units duringthe injection sequence ensure that no samples are iost ifeither unit fails. Neither of the controlling programsneeds to refer to the cycle time of the other processsequence and the Apple IIe controls the number ofsamples analysed.

The time taken for dialysis and trace enrichment dependson the amount ofanalyte required for HPLC analysis, theflow rate of recipient solvent, and the retention volume ofthe analyte on the TEC under the conditions used.Although efficient removal of analyte from the sample isachieved using long dialysis times and fast flow rates of


SPECTRUM APPLE

Start Start

Wait for Apple

Take a Sampleand Prepare

Sample Rea.dyto Injecz

Wait for Apple

Start Apple I Start Integratin

Gradient in Progress

Inject (__ Next Sample

Purge

Regenerate TEC

No

Figure 3. Flow diagram ofthe algorithm to control the concurrentASTED system. The Spectrum microcomputer controls theASTED system and the Apple microcomputer controls the HPLC.

Sample I Sample 2...

recipient solvent, if the retention volume is exceededretained analyte on the TEC will be gradually eluted andlost. Also the time taken for sample preparation, purgingand regeneration should, ideally, be less than the timerequired to chromatograph the analyte. To optimize thesystem, the constant parameters, analyte retention vol-ume and analytical sensitivity required, should bedetermined first, followed by the variables recipientsolvent flow rate and dialysis time.

Discussion

ASTED utilizes two separation methods, dialysis andtrace enrichment, to selectively isolate analytes in a formsuitable for subsequent HPLC analysis. Although clas-sical dialysis is an efficient deproteinization technique,when applied to HPLC sample preparation it has thefollowing limitations: (1) dialysis rate is proportional tothe concentration gradient across the membrane; (2) atequilibrium the concentrations of the diffusable analytesacross the membrane are equal; (3) the time taken toreaizh equilibrium may be up to 10 h. Thus usual dialysistechniques can provide either a high yield of solute in alarge volume of solvent or a low yield of solute in a smallvolume of solvent. Serum dialysates have concentrationsof analytes that are usually not detectable by HPLCanalysis. When applying this technique to the sequentialpreparation of samples for HPLC, short dialysis timesmust be used which further reduces the yield ofanalytes.

The technique ofASTED overcomes this problem in twoways. Firstly, dialysis is performed by holding the samplestationary while the recipient solvent is moved continu-ously. This maintains a high concentration gradient

Sample 3 Sample 4

HPLC-

Sample I

In]tion

Sample 2

Sample 3

Injection

0Time

Figure 4. Schematic diagram of the concurrent operation of the ASTED and HPLC systems. A load sample; B dialysis and traceenrichment; C purge; D regeneration of the TEe.

153


Table 1. Comparison ofsomefeatures ofASTED with those ofatypical batch sample preparation technique.

ASTED Batch

Volatile solvents requiredGuard column requiredInternal standard requiredCan be regeneratedRunning costsFlexibility ofwork scheduleEfficiency ofanalytical controlStafftime required

No YesNo YesNo YesYes NoLow HighHigh LowGood PoorLow High

across the Cuprophan membrane, which results in a

higher analyte yield in a shorter time than is possiblewhen both donor and recipient streams are held static[10]. Secondly, the dilute analytes in the dialysate areretailed on the trace enrichment cartridge so that theyare available in a concentrated form for the HPLCinjection. Due to the small physical size of the enrichmentcartridge analytes have a very low retention volume in theanalytical solvent and therefore are eluted in a smallvolume of solvent even when the TEC and analyticalcolumn are packed with the same material.

When compared with batch procedures for pre-treatingsamples, ASTED has many advantages (table 1). More-over, when ASTED is operated concurrently with HPLC,it offers the added benefits of optimal use of equipmentwith rapid analysis of samples. With the exception of thefirst specimen, sample preparation occurs during thechromatographic separation of the previous sample(figure 4). Since the system is controlled according to thealgorithm shown in figure 3, the operator does not have toconsider the co-ordination of both processes. By the timethe third sample is taken the system will have optimizedits operation such that the speed of analysis is limited bythe slower of the two processes (figure 4). Thus it is easyto interchange varioUs ASTED and HPLC procedures ofdifferent lengths.

The use of sequential overlapping processes for samplepreparation and HPLC theoretically provide higheranalytical rates with less operator involvement thancomparable batch preparation methods [11]. Althoughthe individual steps of a batch process are simple to linktogether the resulting analytical system is difficult tocontrol efficiently. Ifthere is a failure in part ofthe samplepreparation process, it is usually not detected until the

HPLC results are obtained. At this stage much time andmany samples would have been lost. In contrast, a failurewithin a sequential sample preparation process is detec-ted almost immediately allowing appropriate action to betaken quickly with the loss of only the sample beingtreated at that time. This also permits a more flexiblework schedule enabling rapid analysis of urgentspecimens or repeat analyses.

At present there is no sample preparation method thatachieves both total protection of the analytical columnperformance and elimination of interfering chromato-graphic components. Elimination of high molecularweight components using dialysis maintains the perfor-mance of the analytical column whilst trace enrichmentpresents the diffusible analytes in a form suitable forHPLC analysis. ASTED provides an effective samplepreparation method for HPLC that is rapid, inexpensiveand simple to operate.

Acknowledgements

We wish to thank Gilson International, Villiers-Le-Bel,France for the loan ofthe HPLC apparatus and AnachemLtd, Luton, Bedfordshire, UK for its maintenance.

References

1. ADAMS, R. F., S(2HMIDT, G.J. and VANDEMARK, F. L.,Journalof Chromatography, 145 (1978), 275.

2. TURNELL, D. C., TRrvoR, S. C. and CooPeR, J. D. H.,Annals of Clinical Biochemistry, 2(I (1983), 37.

3. BLANCXARD, J., Journal of Chromatography, 226 1981 ), 455.4. GR.N, D. J. and PRMAN, R. L., Clinical Chemistry, 26

(1980), 796.5. NORDMEV.R, F. R. and HANSON, L. D., Analytical Chemistry,

54 (1982), 2605.6. NARASXMHACHARI, N.,Journal ofChromatography, 225 (1981),

189.7. A’FFL, A., ALFREDSON, T. V. and MAJORS, R. E., Journal of

Chromatography, 206 1981 ), 43.8. CooPeR, J. D. H. and TURNLt., D. C., Journal ofAutomatic

Chemistry, 7 (1985), 181.9. CooPER, J. D. H. and Tu.N.a, D. C., Journal of Chromato-

graphy, in press.10. TURNELL, D. C. and COOPER, J. D. H., Journal ofAutomatic

Chemistry, 7 (1985), 177.11. ZN1, F. H., International Laboratory, March (1985), 50.

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