trends in analytical chemistry, vol . 1, no. 8, 1982

MEETING REPORT

A report on the InternationalSymposium on the HPLC of Pro-teins and Peptides held in Wash-ington, D.C ., U .S.A ., on 16-17

November 1981 .

The analysis of biochemical sub-stances has been central to the historyof liquid chromatography ever since itsdevelopment by Tswett and its renais-sance in the separation of carotenoidsat the hands of Lederer . It is thereforenot surprising that the InternationalSymposium on the HPLC of Proteinsand Peptides was filled with enthusias-tic practitioners, who included expertsin the fields of separation science aswell as protein and peptide chemists,keen to discuss the latest results andprospects in this young and rapidlygrowing area of high performanceliquid chromatography (HPLC) .

A plenary lecture and six technicalpapers constituted each of the six

ON

HPLC of proteins and peptides

formal sessions . An additional 94papers were presented in two postersessions that were separately sched-uled . The first day ended with roundtable discussion groups on retentionmechanisms, column technology,instrumentation and isolation andsemi-preparative techniques .

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For the most part, the first day of thesymposium was given over to discus-sion of the chromatographic tech-niques of value . to protein chemistry,while the second day was dedicated toapplications of those techniques . Thekeynote lecturer, Csaba Horvath fromYale, discussed some of the features of

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chromatography particularly signifi-cant for macromolecules . Ourexperience of classical protein chro-matography reaches back 40 years andincludes ion-exchange, size exclu-sion and affinity chromatography .However, the advantages conferred bythat experience and the efficiency ofthe supports developed are lost if anisocratic separation is not performedin the optimal flow velocity range . Thehigh molecular weight of macromole-cules means that they are usuallychromatographed at much reducedvelocities, with correspondingly greatplate heights and band spreading -which is further contributed to by slowreversible interconversion of mole-cular forms . The pore diameter of thesupports used should be in the order of50 nm (to satisfy a rule of thumb (fromGLC) that pore diameter be eighttimes particle diameter to avoid micro-pore effects) .Because of the high molecular

weight of proteins their chromato-graphic retention factors are large andselectivities between different speciesare marked. This effect is clearlymanifested, for example, in the selecti-vities observed between hemoglobinvariants which differ in only one aminoacid . The wide range in retentionfactors and selectivities mandates theuse of gradient elution in practicalprotein separations employing ion-exchange and reversed-phase chro-matography .

Plenary lecturesThe first plenary lecturer, Klaus

Unger of the University of Mainz,discussed the synthesis and propertiesof non-interactive, i .e . size exclusion,phases . Even nominally non-interactive phases can exhibit hydro-phobic and ionic adsorptions whichcloud the interpretation of chromato-grams. The standardization of col-umns to allow the use of plots of log(molecular weight) versus KD, forexample, for identification purposes, ismade difficult by adsorption, the widerange in the aspect ratio of solutes, anduncertainties in the internal volumesof columns . The main advantages ofsize exclusion systems are that scale-up and interpretation are both mucheasier than in interactive systems .Moreover, the packings can be heavilyloaded. On the other hand, peak

capacity is limited and optimization ofseparation is tedious due to the weakinteractions .

In the second plenary lecture, FredRegnier of Purdue University pointedout that in cases where proteins haveseveral points of attachment to ion-exchange supports, gradient elutionbecomes necessary . In this situation,the column length is almost irrelevantto the separation . Regnier also showedthat packings with relatively smallnumbers of exchange sites can havegreater loading capacity for proteinsthan those with more exchange sites, ifthe pore diameter of the more heavilyloaded packing is too small to allowaccess to macromolecules . Propertiesof recently developed non-swellingsulfopropyl, diethylaminoethyl andcarboxymethyl phases were describedin the subsequent technical session .

Stanley Stein of Hoffman LaRochcdiscussed the reversed-phase chro-matography of proteins and peptides,with emphasis on opioid peptides,their precursors and interferons . Thesystems he described used n-propanolfor gradient development and formicacid/pyridine or acetic acid/pyridinebuffers with detection by post-columnderivatization . Reports in the tech-nical sessions pursued this theme -gradient elution with acetonitrile orn-propanol was used predominantlywith aqueous trifluoroacetic acid ortriethylamine phosphate in the mobilephase . The existence of different selec-tivities on octyl-, octadecyl-, cyano-propyl- and diphenylsilica columnswas also noted in this plenary lecture .

Technical sessionsThe technical sessions covered a

range of topics which included the useof Gilbert theory in the evaluation ofsize exclusion chromatograms of asso-ciating systems, analysis of the activa-tion products from prothrombin andthe separation of tryptic fragments andvarious enzymes . Ion exchange wasused for the separation of hemoglobinvariants and the quantitation of fetalhemoglobin, as well as for the separa-tion of adenylosuccinate synthetaseand hexokinase isozymes .Reversed-phase chromatography

was used for purification of chymo-trypsin and trypsin . This, and otherreports, showed that it is a powerfultool for demonstrating heterogeneity

trends in analytical chemistry, vol . 1, no. 8, 1982

in commercially available protein pre-parations . Several reports showed thatretention time in a gradient is relatedto peptide structure and can be used topredict retention .

The second day was prefaced with alecture by M . T. W. Hearn on HPLCin polypeptide structural studies . Thedependence of retention on the pH andcomposition of the mobile phase(including, when present, ion-pairingagents) was discussed . The greatresolving power of reversed-phasechromatography was noted in anexample in which ca. 300 peaks wereidentifiable under conditions whereonly nine were resolved in size-exclusion chromatography undercomparable conditions . The technicalpapers that followed described howthese properties can be exploited in theuse of two columns, size exclusion andreversed-phase, to isolate peptidesafter proteolysis or CNBr treatment ofproteins . The genetic and evolutionaryimplications of the sequences of thepurified peptides were frequently dis-cussed . The characterization of anisozyme population by use of thereversed-phase chromatogram of thetryptic peptides as a fingerprint wasdemonstrated .

The final sessions focussed on theseparation of biologically active pep-tides and on the use of novel detectionsystems and separation methods . Theseparation techniques coveredincluded chromatofocussing and fieldflow fractionation . Post-column reac-tors, immobilized enzyme systems andmass spectrometry detection were dis-cussed .

Most of the papers presented at thissymposium will be included in aforthcoming special issue of Analy-tical Biochemistry . The organizers,M. T . W . Hearn, Tim Wehr and FredRegnier did a superb job, both withregard to the scientific program, andthe physical arrangements . The parti-cipants are indebted to them for astimulating meeting that may wellprove to be seminal in this importantarea of separation science .

WAYNE MELANDER

Dr Melander is in the Department of ChemicalEngineering, Mason Laboratory, Yale Univer-sity, P.O. Box 2159, New Haven, CT 06520,U.S.A .