Automation

Figure 1 A fully automated HPLC system including an autoinjector, a pump, a detector and a collector controlled by centralizedsoftware.

CHROMATOGRAPHY

Automation

E. VeH rette, Gilson, Villiers-le-Bel, FranceCopyright^ 2000 Academic Press

The last 20 years have seen an important increase inpublished papers on chromatography, reporting fullyautomated systems or complete online techniques.This shows a strong interest in automation inchromatography to obtain faster, safer, more cost-effective and convenient analytical procedures,that satisfy regulatory compliance and good laborat-ory practice directives. Automation in chromatogra-phy takes place in the more general laboratory auto-mation context, which is the application of comput-ing, robotics, electronics and mechanical engineeringtechnologies to laboratory problems for enhancedthroughput, reproducibility and traceability. This hasbeen made possible as a result of considerable im-provements in equipment and techniques, fromautosamplers, sample processors and reliable eluentdelivery devices, to universal or more speciRc de-tectors, collectors, single point control software ableto collect and handle accurate data (Figure 1) and,last but not least, laboratory information manage-ment systems.

The chromatographic techniques whose automa-tion is principally discussed in this article are gaschromatography (GC), high performance liquid

chromatography (HPLC) and supercritical Suidchromatography (SFC). Other chromatographicmethods such as planar chromatography can also beautomated, but full automation is more difRcultto achieve.

One of the most important aspects of automation isthe introduction of interfaces between instrumentsand computers, and centralized user-friendly soft-ware for system control and data handling. The mainqualities required for interfaces, designed to collectanalog signals and digitize them to reSect detectoroutput accurately, are good sampling rates and highresolution. Other important aspects of automationare directly related to the evolution of individualinstruments, and the possibility of coupling them on-line. This article is not an exhaustive description ofautomation in chromatography, but highlights theprincipal trends in this Reld. It emphasizes samplepreparation and application in chromatographic sys-tems, multidimensionality and fully automated solu-tions widely used today such as high throughputscreening and preparative chromatography.

Autosamplers

Not far behind computerized data acquisition andanalysis, autosamplers are one of the biggest factorsin automation, allowing long unattended series ofanalyses, therefore releasing the operator for othertasks. Autosamplers also give considerable improve-ment in reproducibility. Autoinjectors for HPLC, GC

II /CHROMATOGRAPHY /Automation 343

and SFC are generally of Cartesian displacement or ofcarousel types.

High Performance Liquid Chromatography

HPLC is considered to be the major chromatographictechnique available today for involatile or heat-sensi-tive substances. Sample injection is commonly per-formed using a switching valve via an external orinternal sampling loop. Sample containers range fromsimple vials with a wide range of volumes to dif-ferent types of microtitre plates (96 and 384 wells),with the option of temperature control. Using 384-well microtitre plates, some modern instruments canstore and handle over 4600 samples unattended.Many autosamplers have three injection modes, total,partial and centred loop Rlling, depending on thesample volume limitations. In recent years the needfor faster analysis has been a major consideration;therefore, the shortest time required between twoinjections (injector cycle time) appears to be an im-portant criterion for autoinjectors, and may be lessthan 20 s in the fastest. Other important criteria areinjection precision and the level of carryover, withtypical values below 0.3% (relative standard devi-ation) and 0.02% respectively for the best instru-ments.

Gas Chromatography

Because of its extremely high separation efRci-ency, speed of analysis and wide range of sensitivedetectors, GC and particularly capillary GC is anappropriate procedure for analysing many com-pounds in a great variety of applications, providedthese compounds are volatile, thermally stable andwith reasonable polarities to avoid problems encoun-tered with derivatization procedures. However, untilrecently its major critical point was the injection oflarge volumes, especially when traces of organic com-pounds have to be analysed, and the suitability of thesample solvent. The injector inSuences accuracy, pre-cision, resolution and analyte recovery. The use ofnew automatic injection devices has provided themeans to enhance the limit of detection signiRcantly.Five modes of operation (split, split-less, direct, coldon-column injection, and programmed temperaturevaporizer) allow maximum injector Sexibility.Among these, the programmed temperature vaporizer(PTV) is of major utility for the automated injectionof large sample volumes (Figure 2). This technique isbased on the selective elimination of the solvent,while simultaneously trapping the components with amuch lower volatility. Liquid samples are introducedinto a large capacity, sorbent-packed liner insertedin the injector head. By temperature-programming

the liner, the solvent is vented through the splitexit while analytes are retained. The split valve isthen closed and the injector is rapidly heated totransfer the analytes to the GC column. With sucha technique, injection volumes in split-less capillaryGC can be increased up to 1 mL and this proceduretherefore brings enormous improvements in limits ofdetection.

Static or dynamic headspace sampling, is anotherinteresting way of injecting volatile compoundsfrom complex matrices into a GC. In this process,the vapour in equilibrium above a sample held atconstant temperature in a sealed vial is drawnand analysed. Headspace GC is used to analysea wide range of compounds in solid and liquidcomplex matrices, and fully automated devices areavailable today with computer-activated heating andpneumatic systems.

Sample Preparation

For a long time, sample preparation has been con-sidered to be the bottleneck of the analysis. However,the advances of automation in this area have untilrecently been distinctly more modest compared withother areas. The move towards automation in samplepreparation occurred for several reasons, includinghigher productivity, fewer and more skilled laborat-ory personnel, better analytical results and less haz-ardous conditions. Basically, there are two almostcomplementary types of equipment, anthropomor-phic arms and Cartesian liquid handlers, used accord-ing to the nature and quantity of samples to be pro-cessed and the preparation complexity (Figure 3).Anthropomorphic arms are capable of grabbing ob-jects and moving them from one place to anotherwith high accuracy. They need a set of dedicatedstations to carry out different operations such asgrinding, homogenizing, drying, weighing, etc. Theprincipal preparation steps used with Cartesian(XYZ) samplers are dilution, addition of reagents orinternal standards and mixing. With additionalequipment they can also perform temperature con-trol, evaporation or Rltration.

Solid-phase Extraction (SPE)

SPE has become a technique of choice for sampleclean-up and trace enrichment in the Relds of phar-maceutical, clinical, food and environmental analysis.Indeed, it offers better versatility and selectivitythan other sample preparation techniques. Comparedwith liquid}liquid extraction, it is quicker, it requiresmuch less solvent, it prevents possible formation ofemulsions, sample volumes can be smaller, and it is

344 II /CHROMATOGRAPHY /Automation

Figure 2 Schematic representation of a PTV injector and temperature profile of injection.

much more amenable to automation. The US Envir-onmental Protection Agency has adopted more than20 SPE methods as alternatives to liquid}liquid ex-traction. SPE formats range from traditional dispos-able extraction columns and membrane discs to 96-well plates and SPE pipette tip conRgurations. Today,a wide range of sorbents is available to increase selec-tivity and/or simplify the SPE procedure, from silica-to polymer-based phases, including speciality phaseswith mixed-mode functionality.

An experimental SPE procedure typically followsfour steps } conditioning, loading, washing and elut-ing. With ofSine systems using disposable col-umns, liquids go through the extraction columns

either by aspiration (vacuum manifolds), or by posi-tive air pressure; the latter can be completely auto-mated. Figure 4 shows typical SPE automation, basedon the mobility of a dedicated rack consisting of threeparts. After elution and a Rnal mixing step, the eluatecan be automatically introduced into the chromato-graph via an injection valve for analysis.

Fully automated ofSine SPE brings an easytransfer of manual methods, which results in a quicktransposition of hundreds of existing protocols withlittle additional development. Since the introductionof the large volume GC injection with a PTV injectorinterface, automated SPE has been extended tonumerous GC applications. Some SPE instruments


Figure 3 Diagram of estimated automation requirements according to sample number and preparation complexity.

propose a Sexible process to develop new methodssuch as the possibility for multicollection, multi-mo-dal SPE or column drying, and more recently thepossibility of using 96-well SPE microplates witha multiple probe equipment.

In online conRgurations, an extraction cartridge isinserted as part of the chromatographic equipment,and is directly connected to the high pressure streamof the mobile phase. Such automated SPE equipmentcan use either the same cartridge for multiple extrac-tions, or carry out automatic cartridge exchange.Online SPE processes are also known for pre-columnconcentration techniques, and may involve columnswitching or coupled-column procedures. The onlineapproach provides high throughput and preserves thesample integrity, but the technique is still dependenton particular applications and the reproducibility ofpacking materials.

Solid-phase Microextraction (SPME)

SPME, introduced by Pawliszyn in the early 1990s, isa sample preparation and introduction method inwhich analytes migrate from a liquid, headspace orair sample on to a polymer (principally polydimethyl-siloxane or polyacrylate) that is coated on a fusedsilica rod. The Rbre is then displaced into a thermal orsolvent desorption interface for analysis respectivelyby GC or HPLC. All these steps can be fully auto-mated using autosamplers speciRcally adapted toSPME, such as the recent in-tube SPME device forHPLC. This technique, which is used repeatedly, pro-vides fast, easy-to-use and inexpensive sampling, andeliminates organic solvents. Initially applied to theanalysis of relatively volatile environmental pollu-tants in simple matrices, it now allows sampling ofa broader range of analytes, including drugs in morecomplex media, with improved analytical perform-ance.

Valve Switching Devices for Sample Preparation

Valve switching devices allow variable combinationsof columns, mobile phases and/or detectors, whichcontribute to the overall system selectivity. Whenlarge numbers of substances are present in complexsamples like biological Suids, and the compounds ofinterest are at very low concentrations, such devicesare used sequentially to carry out sample clean-upand trace enrichment. Different set-ups are avail-able depending on the type of application, permittingthe transformation of ofSine multi-step methodsto single-step procedures. The simplest conRgurationuses one pre-column between the sample injector anda six-port valve which rotates after venting the un-wanted components to waste (straight-Sush mode).A more powerful process can be achieved by operat-ing in backSush mode, but necessitates additionalequipment. In recent years, switching devices haveregained interest due to the introduction of the bi-modal restricted access sorbents. Designed for thedeproteinization of biological samples, restricted ac-cess sorbents are characterized by a hydrophilic ex-ternal surface incorporating reversed-phase coatedpores. Interfering proteins are excluded from thepores by hydrophilic and size exclusion interactions,and low molecular analytes like drugs and metab-olites are extracted and enriched on the bonded phasein the sorbent pores. This new type of separationmedium is best used in the form of an extractioncartridge Rtted to the six-port valve of a samplinginjector, before analysis with a more conventionalcolumn.

A schematic diagram of a fully automated set-upincluding an auto-injector with two switching valvesis shown on Figure 5, where a preliminary onlineRltration step is performed to extend the lifetime ofthe cartridge when analysing crude samples. After


Figure 4 Automated solid-phase extraction based on ASPECTM (Gilson) technology. (A) The four basic steps: conditioning (C),loading (L), washing (W) and eluting (E). (B1) Conditioning, loading and washing over the drain container. (B2) Eluting into the collectiontubes.

injection the Rltration device is regenerated with suit-able solvents in backSush mode. Membrane-basedsample clean-up such as dialysis, electrodialysisand ultraRltration is another efRcient and innova-tive way of discardingmacromolecular andmicropar-ticulate matters before analysis. These devices canbe automated and associated online with a chro-matographic system, mostly HPLC, using switchingvalves.

Figure 6 shows a commercially available systemautomatically performing liquid transfer steps likeaddition of internal standards or derivatization, dialysisand trace enrichment from crude samples, for the onlineroutine analysis of a large number of samples. Thisinstrument consists of a XYZ autosampler, equippedwith a dual-syringe pump, a Sat-bed dialyser insertinga porous membrane and two six-port valves. After eachanalysis, the system is completely regenerated.


Figure 5 Online filtration and clean-up of biological fluid samples using valve-switching devices. AC, Analytical column; D, detector;F, filtration cartridge; IP, injection port; MB, mobile phase; RA, restricted-access sorbent cartridge; W, waste.

Figure 6 Online dialysis and trace enrichment of liquid samples using ASTEDTM (Gilson) technology. AC, Analytical column, DB,dialyser block; DC, donor channel; DE, dilutor eluent; EC, enrichment cartridge; IP, injection port; MP, mobile phase; R, reagent rack;RC, recipient channel; RE, recipient eluent; RR, result rack; SP, dual-syringe pump; SR, sample rack; T, tray; W, waste.

Supercritical Fluid Extraction (SFE)

Supercritical Suids have physical and chemical prop-erties that make them particularly useful for extrac-tion of analytes from solid and liquid matrices. Abovetheir critical temperature, they can be compressed toincrease their density and therefore their solvatingpower; moreover, they keep gas-like viscosity anddiffusivity, and they can easily be evaporated bylowering the pressure. Because of these properties,SFE has received much attention as an alternative to

Soxhlet extraction. Automated SFE can be incorpor-ated online with various analytical techniques such asHPLC, GC or SFC.

Multidimensionality

In the analysis of complex samples, a single analyticalcolumn combined with a selective detector does notalways provide the required sensitivity and selectiv-ity. Automated multidimensional chromatographyhas emerged as a convenient answer to these


problems. Multidimensional separation techniquesconstitute a powerful class of methods in which twoor more independent steps are linked together.Roughly, multidimensionality includes two majorbranches: hyphenation and coupled-column tech-niques. A hyphenated instrument can be described asthe combination of two (or more) instruments auto-mated as a single integrated unit via a hardware inter-face whose function is to reconcile the output limita-tions of one instrument and the input limitations of theother.

Couplings of Chromatographs andSpectral Technique Devices

In recent years, numerous publications have de-scribed coupled techniques associating chromato-graphic instruments like HPLC, GC or SFC withsophisticated detectors or spectrometric devices, suchas mass spectrometers (MS), MS coupled with diodearray detector (DAD), MS-MS, Fourier transforminfrared and nuclear magnetic resonance. However,for these online couplings, complete automation isgenerally required, and they are only feasible if suit-able interfaces are available. Among these combina-tions, GC-MS and HPLC-MS are the most widelyused in laboratories.

Gas Chromatography+Mass Spectrometry MS dataprovide the qualitative information for identiRcationand characterization of sample components that islacking in most other GC detectors. The availabilityof highly inert, durable and selective fused-silica col-umns and the ease of coupling GC systems withrelatively low cost mass spectrometers make GC-MSa method of choice for numerous analyses, and thisautomated combination is now a routine analyticaltechnique in many laboratories. The main restrictionis that the analytes must be sufRciently volatileand thermostable for transfer to the vapour phasewithout decomposition, or that they can be de-rivatized to provide these properties.

High performance liquid chromatography+massspectrometry Of all LC detectors, MS is a verypowerful conRrmatory tool for screening applica-tions, and is widely used for impurity identiRcation orquantiRcation of drugs and metabolites in biologicalSuids. Nevertheless, no method of interfacing HPLCand MS exists that can be universally applied to alltypes of analyses. However, considerable progresshas been made recently in the development of softionization techniques such as matrix-assisted laserdesorption ionization, and also in sample introduc-

tion devices like particle beam, electrospray/ion sprayor atmospheric pressure chemical ionization inter-faces. When nonvolatile buffers like phosphatesor ion-pairing agents are used, removal of these addi-tives can be automatically performed, changing thephase system with additional equipment, usingcoupled-column and valve-switching techniques.

Couplings of Chromatographic Systems

These techniques are versatile and powerful, and arewell suited to the separation of multicomponent mix-tures. They include zone-cutting methods, in whichfractions from one chromatographic column are usu-ally transferred via a loop installed on a switchingvalve, to one (or more) secondary column(s) in seriesfor additional separation(s), or to another chromato-graphic system. In LC-LC, a special area of interest ofcolumn-switching is the resolution of enantiomericdrugs in biological Suids, by coupling chiral systemsto conventional reversed-phase columns.Other usefulassociations combine size exclusion or ionchromatography with reversed-phase chromatogra-phy. Parallel settings using switching valves are alsopossible for column and mobile-phase selection. Suchsystems have been reported for fully automated, rapidand easy method development to analyse differ-ent types of compounds, including enantiomers.

LC-GC is a very convenient combination whichassociates the selectivity of LC with the high efR-ciency capillary GC. Moreover, GC possesses manysensitive and speciRc detectors. Such a coupled tech-nique enables the analysis of samples with complexmatrices, and considerably prolongs the GC columnlifetime. The Rrst set-up was described by Majors in1980; since then important improvements in develop-ment have been achieved. With this transfer tech-nique, speciRc attention must be paid to interfaces.Several interfaces are available to couple the twosystems online, such as on-column, loop-type andPTV interfaces which permit the transfer of largevolume samples into the gas chromatograph. Inser-tion of a short trapping column between HPLC andGC also enables direct coupling from reversed-phasesystems.

Fully Automated Dedicated Systems

The production of chemical compounds at the labor-atory or plant scale requires fully automated andreliable separation techniques. This is true from dis-covery, through process control, to the Rnal stage ofpuriRcation and quality control of a new compoundor drug. Numerous complete solutions exist today,


Figure 7 Automated analytical/preparative supercritical fluid chromatography on packed columns, with a solvent-less injectionmode, based on Series SF3TM (Gilson) technology. AC, Analytical column; AS, dual-valve autosampler; DE, detector; EC, enrichmentcartridge; IG, inert gas; IP, injection port; MM, mixing module; OV, oven; PC, preparative column; PR, pressure regulator (a, upstreampressure and pulse damper; b, heat exchanger; c, downstream pressure regulator); P1, liquid carbon dioxide pump; P2, modifier pump;P3, collection solvent pump; RV, pressure relief valve; SM, switching valve module; SP, dual-syringe pump; W, waste.

such as online systems available in biotechnology,which involve automated process monitoring for thedetermination of small organic molecules, and inter-active controls of the physicochemical and biochemi-cal growth conditions in culture media. In manyRelds, including combinatorial chemistry, preparativeseparations are certainly widely used and require themost complete online solutions.

Preparative Chromatography

The objective of preparative chromatography is toobtain sufRcient quantities of compounds at therequired purity, with the highest throughput and atthe lowest price. Material to be puriRed can be simpleor more complex, such as fermentation broths andnatural products. In such instances, fully automatedpreparative HPLC on a laboratory scale (milligramsto grams) is one of the best separation techniques forthis objective, and also because of its Sexibility toaccommodate frequent application changes and vari-able quantity ranges.

The requirements of a preparative HPLC systemdiffer according to the quantity of product to becollected, and the nature and number of samples to bepuriRed.However it should provide reliable and com-plete automation for unattended operations witherror-handling methods, and total traceability of allevents, including the collected fractions. Fractioncollectors are key instruments in preparative HPLC,and should be selected according to their collectionfacilities and fractionation software. Devices existthat can perform peak collection with automatic

tracking of the baseline drift. With such instruments,collection is initiated if the detector signal is aboveuser-set parameters in relation to threshold level andpeak width. The graphic sample tracking which isfeasible with some recent centralized software is alsoan important feature in preparative chromatography.Indeed, it can provide useful information to the useron sample identiRcation, collection positions andassociated chromatograms.

Since the introduction of independent program-ming of mobile-phase pressure, composition, andSow rate, SFC with packed columns has receivedconsiderable interest for several reasons, including itssuitability and exceptional performances in prepara-tive works. Consequently, this technique may becomea serious competitor to preparative HPLC in the nearfuture. Figure 7 shows a completely automated sys-tem for analytical and preparative SFC. A versatiledual-valve autosampler of large capacity is used toperform both injection and collection. A solvent-lessinjection mode and a column-switching procedure arerealized by two extra six-port valves. Such a systemperforms analytical development and switches easilyand quickly to preparative chromatography for linearscale-up, and back to analytical for the purity checkof the collected fractions. All these steps are auto-matically monitored by centralized software.

Analysis and Puri\cation in CombinatorialChemistry

Combinatorial chemistry appeared a few years ago inthe pharmaceutical and biotechnological industries as


a novel approach to Rnding new drugs, and hasundergone outstanding development since then. It isdeRned by a variety of automated high throughputsynthesis techniques, to produce large numbers ofsmall organic molecules, for screening these com-pounds against several biological targets for drugdiscovery. Combinatorial chemistry includes threebasic steps } synthesis, candidate screening andlibrary product puriRcation } but chromatographyis principally involved in the last two.

In drug candidate screening, library product veriR-cation can be performed with various analytical tech-niques. Among these, gradient reversed-phase HPLCwith UV detection is widely used. Such a system,Rtted with an autoinjector which accommodatesmicrotitre plates, provides a robust and highly auto-mated approach. Fast HPLC methods (generally ofless than 5 min) generate up to 300 analyses per dayper system and are required to produce libraries ofproducts with widely differing polarity. Thesegeneric methods use short C18 columns with sharpslope gradients from acidiRed water to acidiRed or-ganic solvents, high Sow rates and UV detection set at220 or 254 nm. Automated HPLC-MS, HPLC-DAD-MS and HPLC-MS-MS have also become importantroutine tools for the combinatorial chemist, becausethey provide structural information to identify thecompounds which have been synthesized.

Automated puriRcation to an extremely high levelwith sample quantities of up to 100 mg can beachieved with a preparative HPLC system equippedwith an autoinjector and a fraction collector. Thetechnique uses the same packing material and mobilephases as those used in analytical separation. Fast andeasy clean-up can also be performed using SPE car-tridges or microplates. Samples may therefore be pro-cessed in parallel with an automated device for opti-mum throughput. And last but not least, preparativeSFC, although with limited applications so far, alsoprovides a high productivity approach to puriRcation,and in the near future should acquire a much biggerplace in combinatorial chemistry.

Future Trends

Automation in chromatography has been a majorpreoccupation in recent years, mostly for pharma-ceutical and clinical laboratories, and this will cer-tainly be more intensive in the future, with concomi-tant developments in other Relds like chemical,agrochemical, food or environmental analysis. Themain goal is to provide more analyses giving themaximum and the most accurate information in theshortest time, at the lowest cost. This is the currentsituation in pharmaceutical and biomedical research

for high throughput screening in combinatorial chem-istry, genomics or proteomics. Future trends in auto-mation will surely follow the evolution of instru-ments, including mixed-mode techniques like capil-lary electrochromatography, more efRcient inter-faces, and more versatile and user-friendly software.They will also follow the progress in chromato-graphic media like restricted-access sorbents or ad-vanced stationary phases with sub 3 m particles forLC, and newmaterials for sample preparation such as96-well SPE extraction plates.

Miniaturization in chromatography, includingautomated micro- and nano-techniques, is certainlyan important step towards this goal, and a goodexample is given by the fast separation of complexmixtures with miniature gas chromatographs, whichcan now be performed in the Reld. Moreover, inaddition to allowing the analysis of very limited vol-ume samples with good sensitivity, and to reducemobile-phase consumption, miniaturization tendsto reduce work space, robot displacements andliquid transfers and consequently overall analysistime. The evidence of the release on the marketof 384-well SPE extraction plates will obviouslyconRrm this trend. Down-sizing even further, the useof 1536-well microtitre plates automated by sampleprocessors equipped with distribution heads and chipformat technology will certainly be expanded in thevery near future. This goal involves reRnement of thetechnique to carry out continuous separations withsub-second time resolution and attomole detectionlimits.

See also: II /Chromatography: Liquid Chromatography -Gas Chromatography. Chromatography: Gas: HistoricalDevelopment; Detectors: Mass Spectrometry.Chromatography: Liquid: Detectors: Mass Spectro-metry; Instrumentation; Large-Scale Liquid Chromatogra-phy. Multidimentional Chromatography. Electrophoresis:Capillary Electrophoresis. Extraction: Solid-Phase ex-traction; Solid-Phase Microextraction; Supercritical FluidExtraction.

Further Reading

Anton K and Berger C (eds) (1998) Supercritical FluidChromatography with Packed Columns } Techniquesand Applications. Chromatographic Sciences series.New York: Marcel Dekker.

Grob K (1995) Review: development of the transfer tech-niques for on-line high-performance liquid chromatog-raphy } capillary gas chromatography. Journal ofChromatography A 703: 265.

Hills D (ed.) (1998) Current Trends and Developments inSample Preparation. LC GC International. Chester, UK:Advanstar Communications.


Niessen WMA and Tinke AP (1995) Review: liquidchromatography}mass spectrometry. General principlesand instrumentation. Journal of ChromatographyA 703: 37.

Poole CF and Poole SK (1991) Chromatography Today.Amsterdam: Elsevier.

Prinzis S, Fraudeau C, Hutchinson G and Paulus M (1997)Gilson Guide to SPE Automation. Villiers-le-Bel,France: Gilson.

Stevenson D and Wilson ID (eds) (1994) Sample Prepara-tion for Biomedical and Environmental Analysis. NewYork: Plenum Press.

Convective Transport in Chromatographic Media

A. E. Rodrigues, LSRE Faculty of Engineering,University of Porto, Porto, PortugalCopyright^ 2000 Academic Press

New liquid chromatographic media have been de-veloped in the last decade containing large pores forconvective mass transport and smaller diffusivepores to provide adsorption capacity. These per-meable packings are at the heart of perfusionchromatography, patented in 1991, leading to bettercolumn efRciency and speed of separation withapplications in the rapid analysis of biological macro-molecules and preparative scale puriRcation of pro-teins. This improved column efRciency is basedon the concept of augmented diffusivity by con-vection, available from parallel development in thechemical reaction engineering area.

The analysis of column performance requires anextended van Deemter equation for the HETP (heightequivalent to a theoretical plate) as a function of thesuperRcial velocity. A methodology for obtainingbasic data for design of perfusive chromatography issuggested involving elution chromatography of pro-teins in nonretained and frontal chromatographyexperiments.

Permeable Chromatographic Media

Packing materials in bead form can be groupedinto four classes: homogeneous cross-linkedpolysaccharides (e.g. agarose), macroporous poly-mers based on synthetic polymers (e.g. POROS par-ticles used in perfusive chromatography with largepores of 600}800 nm and diffusive pores of50}100 nm), tentacular adsorbents allowing fasterinteraction between proteins to be separated andfunctional groups and materials based on the conceptof soft gel in a rigid shell combining the goodcapacity of soft gels with the rigidity of compositematerials.

In conventional packings, solutes are carried tosites on the particle surface by bulk convective Sow ofthe mobile phase through the column and then dif-fuse to binding surfaces inside the particle. The intra-particle diffusion process can be quite slow forlarge molecules (proteins, peptides). In order to maxi-mize capacity and resolution, interaction with asmany sites as possible is required; however, this willbe more difRcult at high Sow rates and thereforetrade-offs between speed, resolution and capa-city are needed. In permeable, Sow-through particlesused in perfusive chromatography the Sow rate canbe increased one order of magnitude compared withconventional packings because of the short diffu-sion path length inside the microspheres.

Newmaterials aim to achieve higher sorption capa-city and better sorption kinetics. Some developmentsconsider new geometries: Rbres, membranes or discs(cellulose, polymethacrylate) or continuous beds(rods, monoliths). In continuous rods of acrylamide-acrylate polymers, Sow pores are of 3}4 m diameterand in methacrylate-styrene polymers large pores of0.5}2 m and even 20 m are obtained with micro-spheres of less than 0.5 m. Similar developments oncontinuous silica rods are taking place. In continuousbed technology, channels are of 3}5 m with micro-spheres of 0.5}1 m, whilst in bead form, packingsare typically of 3}50 m with pore size of 0.1 m.Also superagarose beads of 300}500 m have beenprepared with superpores of 30 m as well as 3 mmthick membranes. Table 1 reports examples of con-vective chromatographic media.

The Concept of AugmentedDiffusivity by Convection

The design of chromatographicmedia aims to elimin-ate or reduce the mass transfer resistance inside par-ticles by coating a nonporous support with activespecies, decreasing the particle size or increasing par-

352 II /CHROMATOGRAPHY /Convective Transport in Chromatographic Media

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