Barry L. Karger

Hydrophobic effects in reversed-phase liquid chromatography☆

Abstract The role of hydrophobic effects in revered-phase liquid chromatography is examined. The molecular weight or structural selectivity resulting from hydrophobic effects relative to dispersion forces is discussed, and it is shown that in general a selectivity up to twice as great is possible in the former case. Hydrophobic selectivity can be accounted for using a new topological index, the molecular connectivity, to represent solute surface area. Selectivity as a function of organic modifier type and composition is explored. By simultaneously varying both of the above parameters such that retention is normalized, it is shown that hydrophobic selectivity is approximately independent of organic modifiers. Some differences in separation as a function of organic solvent are, however, observed when mixtures containing solutes of different functional groups are employed. Finally, hydrophobic selectivity is roughly constant under time normalization conditions based on temperature-organic phase composition changes.

Use of complexing agents for selective separation in high-performance capillary electrophoresis. Chiral resolution via cyclodextrins incorporated within polyacrylamide gel columns

Abstract The incorporation of a complexing agent within a polyarcylamide gel column provides a general means of manipulating the selectivity of a capillary electrophoresis separation. As an example of this approach, chiral resolution of dansylated amino acids by high-performance capillary electrophoresis has been achieved by adding β-cyclodextrin within the gel matrix. A retention model has been developed which can be used for selectivity optimization. Various parameters, such as concentration of cyclodextrin, addition of methanol to the buffer, and temperature, have been examined in terms of their influence on retention and selectivity. From this study high-performance separations have been developed. Efficiencies as high as 100 000 plates in 15 cm have been achieved.

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.

Dansylation of amino acids for high-performance liquid chromatography analysis.

Abstract An amino acid dansylation procedure compatible with reversed-phase high-performance liquid chromatography is presented. The method provides high yield derivatization while the yield appears to be independent of the ratio of dansyl chloride to amino acid over a 1000-fold range. Studies of derivatization yields as a function of reaction time at ambient temperature and at elevated temperatures permit the proper choice of reaction conditions. A comparative study of the dansyl-liquid chromatography, gas chromatography, and amino acid analyzer methods of analysis for amino acids in complex bacteria broths demonstrates the advantageous use of the dansyl-liquid chromatography approach.

High-performance sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis of peptides and proteins

High-performance capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been developed for the separation and molecular weight (MW) determination of peptides and proteins. In this work, acrylamide was polymerized in fused-silica capillaries of 75 microns I.D. and 10 or 20 cm length. On-line UV detection was employed by placing the capillary in the optical path of the detector. Rapid separations of myoglobin fragments and of a mixture of standard proteins were demonstrated with efficiencies of the order of 40,000 plates for a column, 20 cm in length. Linear plots of log MW vs. mobility for the SDS protein complexes were observed. Variation in the percentage monomer composition (% T) of the polyacrylamide yielded the expected common intercept of log mobility vs. % T, confirming the size separation mechanism in the gel capillaries. The separation of the A and B chains of insulin with a 10-cm column in less than 10 min was also demonstrated.

On-column transient and coupled column isotachophoretic preconcentration of protein samples in capillary zone electrophoresis

Abstract Two strategies for the isotachophoretic preconcentration of samples were evaluated using a standard protein mixture as an example. In the first, on-column transient isotachophoretic migration permits the injection of relatively large volumes of sample into a commercial instrument. Depending on the composition, 30–50% of the column length can be filled with the sample while maintaining good resolution of the sample components. When proper electrolyte compositions are selected, the conventional single-column instrument can be used for an isotachophoretic sample preconcentration of 50-fold or more without any modification. In the second strategy, a coupled-column system was examined which, in principle, provides a higher degree of freedom in the selection of capillary zone electrophoretic running conditions and the possibility of injection of higher sample volumes. The gain in detection level is at least a factor of 1000 in the coupled-column arrangement; however, the instrumentation is more complicated.

The role of organic modifiers on polar group selectivity in reversed-phase liquid chromatography

Abstract The influence of the organic modifiers methanol (MeOH), acetonitrile (AN) and tetrahydrofuran (THF) on polar group selectivity in reversed-phase liquid chromatography has been studied. In order to elucidate polar group effects, we have first explored the influence of the surface coverage of bonded phase on selectivity. Using a series of synthesized n -octy bonded phases, we have been able to observe significant differences in group contribution with bonded phase coverage, the largest differences arising from MeOH H 2 O as mobile phase and the least from THF H 2 O as mobile phase. The importance of using phases that minimize accessible silanol groups in order to study the influence of the mobile phase has been emphasized. We have selected a high coverage n -octyl phase that is silanized for these studies. In order to examine polar group effects, we have normalized the methylene group increment in the MeOH H 2 O, AN H 2 O and TH H 2 O binary phases. As the hydrophobic selectivity is thus roughly normalized, meaningful relative polar group contributions are observed. Plots of log k ′ (THF H 2 O) vs . log k ′ (MeOH H 2 O) reveal particularly striking polar group differences. The practical usefulness of the plots is shown in the peak reversal of solute mixtures with the two mobile phases. Further studies reveal that polar group selectivity can be powerfully controlled using ternary phases of MeOH THF H 2 O. Thus, the choice of mobile phase can greatly influence separation in reversed-phase liquid chromatography.

High-performance capillary electrophoresis using open tubes and gels

SummaryThis paper overviews several aspects of high performance capillary electrophoresis (HPCE), a promising new method of analytical and micropreparative separation of biochemically important samples. The basic migration equations of electrophoresis are first presented and the benefit of high fields for rapid analysis and high performance emphasized. Since power is generated with high voltages, Joule heating results and this heat must be dissipated. The use of capillary columns is shown to be important in efficient heat removal and in minimizing the temperature differences within the column. The various factors influencing band broadening are next described, and it is shown how plate counts close to 106 can be achieved. Various results from our laboratory on open tube and gel columns are then presented to illustrate the potential of this method. Chiral resolution of dansylated amino acids using a chiral metal chelate micelle in open tube HPCE is shown. With the gel columns, the baseline separation of a 2-chain variant from methionine growth hormone (met-hGH) under non-denaturing conditions at fields close to 1000 V/cm is presented. Finally, the micropreparative purification of a 20-mer oligonucleotide using the gel column is described.

An expanded solubility parameter treatment for classification and use of chromatographic solvents and adsorbents : Parameters for dispersion, dipole and hydrogen bonding interactions

A basis for calculating specific solubility parameters for dispersion, δd, induction, δln, orientation, δo, and hydrogen bonding, δa and δb, is presented. These parameters provide a classification scheme of solvents and adsorbents that can be used to estimate selectivity in chromatography. The dispersion solubility parameter is determined via the Lorentz-Lorenz function and the refractive index of the pure compound. The orientation parameter is obtained from the dipole moment of the substance and the molar volume. The use of bond dipoles (i.e. effective dipole moment) is found to be necessary in describing solution interactions. The induction solubility parameter can be obtained from the orientation parameter and the molar volume. For hydrogen bonding, the δa δb product of alcohols obtained from the vaporization energy is correlated with the heat of hydrogen bonding. With the definition of equal values of δa and δb for alcohols, it is then possible to determine the parameters for other compounds via heats of hydrogen bonding. Evidence is presented of the general validity of the relationships derived.

High-performance capillary electrophoresis in the biological sciences

High-performance capillary electrophoresis (HPCE), the instrumental approach to electrophoresis, is a method undergoing rapid development at the present time. There is a high expectation that HPCE will become a widely applicable tool within the biochemical community. This review presents principles and instrumentation followed by typical applications of HPCE in the biological area. In the instrumentation area, particular attention is paid to current and future detectors, including laser-induced fluorescence and HPCE-mass spectrometry. In the applications section, both peptide or protein and oligonucleotide high-resolution separations are described for open-tube and polyacrylamide gel capillary columns. The use of isoelectric focusing and electrokinetic chromatography to manipulate separation is also presented. Future directions of methodology are suggested, and it is predicted that in the next few years HPCE will become a complementary tool to liquid chromatography and slab gel electrophoresis.