Advances in Liquid chromatography (LC) is one of the most commonly performed separation methods because of its applicability to a wide array of compounds. Since the emergence of modern LC in the late 1960s, advances in column materials and improvements in column design have changed the face of this versatile method. LC continues to evolve. Separation processes are being applied to new areas such as biotechnology. Advances in column materials are making oncedifficult separations commonplace. Understanding of basic chromatographic mechanisms is allowing optimal chromatographic conditions to be determined more quickly than ever before. One of the most popular forums for discussion and presentation of recent advances in this field has been the International Symposium on Column
FOCUS Liquid Chromatography. This symposium, which has alternated between Europe and the United States, has brought together an increasing number of chromatographers from around the world. The twelfth such meeting, known as HPLC '88, took place June 19-24 in Washington, DC. Chaired by Georges Guiochon of the University of Tennessee and Oak Ridge National Laboratory, HPLC '88 was a huge success; more than 1300 scientists and 115 exhibitors attended. Recent advances in LC discussed at the meeting's plenary session included fluorescence-based automated DNA sequencing, the use of reversed-phase high-performance liquid chromatography (HPLC) for characterizing recombinant proteins, NMR imaging of the chromatographic process, the increasing role of cyclodex-
trins in separations, and electrically driven LC in packed capillary columns. Fluorescence-based automated DNA sequencing was discussed by J. M. Prober of Du Pont. DNA sequencing, said Prober, is the cornerstone analytical technique of modern molecular biology. Although manual sequencing methods, which have been available for about a decade, are extremely elegant, they can be time consuming, labor intensive, and subject to errors in data transcription and interpretation. To solve some of these problems, Prober and his colleagues have developed an automated DNA sequencing system that closely integrates chemistry, instrumentation, and molecular biology. The Du Pont system is based on the Sanger method of sequencing, said Prober, which determines the sequence of DNA by using an enzymatic process to relate the sequence information to fragment length. The fragments are then placed on an electrophoretic gel. In traditional manual sequencing, the fragments are radioactive, but in Du Pont's method, the radioactivity is replaced with a set of fluorescence tags. The system detects the fluorescencetagged fragments and then determines whether the final base in the fragment is an adenine, thymine, cytosine, or guanine. The sequencer can detect as little as 10" 18 mol of DNA fragments. Four fluorescent dyes are used, continued Prober—one for each of the four DNA bases. In designing the system, Prober and his colleagues needed to determine which dyes to use and where to attach them to the fragments. They were able to engineer four succinyl fluoresceins, but they had difficulty getting them on the DNA sequencing fragments. To obtain certain performance characteristics, they wanted to have the enzyme place the fluorescence tag on the fragments as part of the termination process rather than on the ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988 · 1045 A
FOCUS primer, as is usually done. Although there are trade-offs in deciding wheth er to use the primer or the terminator approach, Prober and his colleagues believed that the terminator approach was best. (For a discussion of automat ed sequencing using the primer ap proach, see Lloyd Smith's REPORT in the March 15, 1988, issue.) The usefulness of the system isn't tied just to sequencing. Prober men tioned that the DNA analysis system can also be used for other applications in molecular biology, including DNA mapping and fingerprinting. W. S. Hancock of Genentech dis cussed the use and limitations of reversed-phase HPLC for the analysis of both recombinant proteins and their corresponding tryptic digests. The two approaches can yield complementary information on the same sample. Han cock went on to explain the application of these two approaches to characteriz ing two recombinant DNA-derived proteins: human growth hormone and tissue plasminogen activator. According to Hancock, the retention of polypeptides on reversed-phase col umns has been related to the surface hydrophobicity of the sample. Also, only certain regions of a given protein molecule interact with the stationary phase. LC can thus provide useful in formation about the intact protein. Tryptic mapping techniques, which involve the separation of relatively small peptides, can provide definitive information on the entire sequence of the molecule. This is useful in charac terizing a molecule for the first time and also for monitoring consistency of the manufacturing process. However, warned Hancock, the digestion of a protein with a proteolytic enzyme can introduce ambiguities in the tryptic map because of side reactions that oc cur during the digestion process. In ad dition, information on the threedimensional structure of the protein is destroyed upon digestion. In the case of human growth hormone, the tryptic map can be used to determine if the disulfide bonds have been formed be tween the correct residues. Hancock then focused on the chem istry of the intact polypeptide sample. Chromatography of the intact mole cule does not lead to side reactions, and sensitivity is better than with the tryp tic map. However, a substitution may not be detected because it may be shielded by the three-dimensional structure of the protein. The question then becomes, "How can you predict if a given substitution can be detected?" Hancock used growth hormones as an example, tak-
Separation processes are being applied to new areas such as biotechnology, φ β
ing one sample that has an additional ^-terminal methionine and one that corresponds to pituitary growth hor mone without the extra residue. Reso lution depends on the chromatograph ic conditions. With harsh chromato graphic conditions, which more strongly denature the protein, the reso lution of the two growth hormones is lost. This is consistent with what is known about the structure of the mole cule in that the N-terminus is believed to be near the surface. Extreme denaturation either folds away the N-terminus and loses it, or additional hydro phobic residues are exposed from the interior and "swamp out" the differ ence. He concluded that when separat ing similar variants, if the difference is on the surface, one should minimize denaturation. If the difference is in the interior, however, one should attempt to denature the proteins to expose the difference. Ernst Bayer next discussed how NMR imaging can be used to study liquid chromatographic separations. NMR methods, said Bayer, can also be used to study the chemical structure of column materials. Bayer described how NMR imaging has been applied to study and monitor solute and solvent movement in the LC column. He then showed a movie of how NMR imaging can be used to visu alize flow profiles, packing density, po rosity, and the diffusion process. The method of NMR imaging is largely based on differences in the relaxation times Τ ι, which can be measured bycertain pulse sequences, and then com posed to a picture. The relaxation times Τι and To correlate with the mo bility of solvents and solutes.
1046 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988
NMR relaxation times show large differences in various kinds of silica and can be correlated with chromato graphic properties, said Bayer. The dif fusion of solvents and solutes in poly mer beads can also be measured. • The reasons behind the increasing use of cyclodextrins in separations were discussed by Daniel Armstrong of the University of Missouri-Rolla. Ac cording to Armstrong, the ability of cy clodextrins to form inclusion complex es has been widely known for a long time, but the use of cyclodextrins in analytical separations didn't become popular until recently. There has been an exponential growth in the use of cy clodextrins in the last few years, said Armstrong. The structure and complexing abili ties of cyclodextrins must be known if one is to fully use them in separations. Cyclodextrins are natural macrocyclic polymers of glucose that contain from 6 to 12 D-(+)glucopyranose units that are bonded via «-(1, 4) linkages. The most common types of cyclodextrin, abbreviated as α-CD, β-CD, and γ-CD, contain six, seven, and eight glucose units, respectively. The cyclodextrin molecule has certain features that make it useful for separations. The main feature is that the molecule is chiral and can form inclusion complex es with a variety of compounds because of its hydrophobic interior. In all chiral columns, Armstrong ex plained, the separation ability varies depending on the compounds being separated. The advantage of the cyclo dextrin column is that it can be used to separate geometric and structural iso mers as well as enantiomers. This wide application range is attributable to three factors: cyclodextrins separate these compounds very well, cyclodex trins are relatively inexpensive, and the column is stable compared with other chiral columns. Armstrong described two approach es whereby cyclodextrins can be used in separations: as a mobile-phase additive and as a bonded stationary phase. As a mobile-phase additive, cyclodextrins have been used successfully to separate structural and geometric isomers. The use of cyclodextrins as a bonded sta tionary phase is a more recent advance ment and is particularly amenable for separating enantiomers. Racemic mix tures of drugs have also been separated by using this approach. Cyclodextrins have also been used in areas other than separations, Arm strong added. Myron Bender at North western University has used cyclodex trins for catalysis as models for active sites of membranes. Armstrong and his
FOCUS colleagues have successfully demon strated the application of cyclodextrins as membranes and are continuing their work in this area. The next speaker, J. H. Knox, dis cussed the use of electrically driven LC in packed capillary columns. Knox said that an electrical potential gradient can be used to induce the flow of eluent through the medium. The characteris tics of electroosmotic flow suggest that higher efficiencies may be obtained than those observed with pumped flow. He cautioned that because of ohmic heating, narrow-bore columns must be used, preferably with on-column detec tion. They have investigated the use of an electric field to propel eluent through narrow-bore capillaries packed with conventional LC materi als. Eluted solutes have been detected by on-column fluorescence. Knox stated that their experiments with particles of varying size show no sign of a significant decrease in elec troosmotic flow velocity upon reduc tion of particle size. Thus, he conclud ed, electrical double-layer overlap is not a problem unless the particle diam eter is reduced below 1 μτα. Enhanced efficiencies can be ob tained with electroosmotic flow com pared with pressure-driven flow, con tinued Knox, provided that a suitable concentration of electrolyte is present. Reduced plate heights as low as 0.7 have been achieved under ideal condi tions for 5-μτα spherical silica with electrically driven flow, compared with 2.0-2.5 for the same column using hy drostatic pressure. He stated that plate-height velocity curves show evi dence of a reduced contribution from the structure-dependent van Deemter Α-term, as expected from the flat ve locity profile of electroosmotic flow. Similar reductions in plate height are observed for all particle sizes when using electroosmotic flow. Under prac tical operating conditions, efficiencies of 500,000 plates per meter can be ob tained for 1.5-μπι particles at a linear velocity of 2 mm s _1 . Knox concluded that the prospects are excellent for ob taining very high-efficiency separa tions with short analysis times by using electroosmotic flow and submicrondiameter particles. This year's symposium provided chromatographers from around the world an opportunity to expand their knowledge of LC. HPLC '89, to be held in Stockholm, Sweden, will provide yet another opportunity. Inquiries may be directed to the chairman, D. Westerlund, of Uppsala University Biomedi cal Center, Uppsala, Sweden. Keith B. Belton
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