As a continuation of a previous paper on the retention behavior of recombinant human growth hormone (rhGH) in reversed phase chromatography at pH 6.5 (Oroszlan, P., et al. Anal. Chem. 1992, 64, 1623-1631) the effect of 1-propanol (1-PrOH) and acetonitrile on the conformation of rhGH at this pH has been investigated by circular dichroism (CD), second-derivative UV spectroscopy, fluorescence anisotropy, fluorescence quenching, and fluorescence lifetime measurements. Addition of 1-PrOH up to a concentration of 10% (v/v) does not cause any significant changes in protein structure. However, above this concentration, a transition from the native to a new state is observed; the transition is completed above 30% (v/v) of 1-PrOH, the composition for completion being dependent on temperature. This change in structure correlates with retention changes observed in reversed phase chromatography. The new rhGH conformation retains much of the alpha-helicity and possesses a slightly expanded hydrodynamic radius relative to native rhGH. Second-derivative UV spectroscopy suggests that the hydrogen bond between Trp 86 and Asp 169, spanning two alpha-helices, remains intact. On the other hand, the near-UV CD intensity changes from positive to negative in the Trp region of the spectrum, signaling an alteration in the Trp environment. In addition, fluorescence quenching measurements with trichloroethanol reveal greater accessibility to solvent of the Trp residue after the conformational transition has occurred. From the results, it is concluded that a molten globule state (compact state retaining much of the secondary structure of the native state but with a disrupted tertiary structure) is produced with the addition of > 30% (v/v) 1-PrOH.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.

Partition coefficients of phenol, salicylic acid, and several environmentally important chloro- and nitrophenols in a supercritical CO2-water system were measured using direct cocurrent extraction of aqueous solutions of the individual solutes with CO2. Partitioning data on the nitrophenols and salicylic acid were obtained for the first time. To bypass the troublesome and error-prone analysis of the CO2-rich phase, the present method employed only the solute concentrations in the aqueous phase before and after extraction to determine the partition coefficient. Unlike most previous engineering studies of phenol partitioning in a CO2-water system, the concentrations of phenolic solutes approached infinite dilution in both phases. This makes the results relevant to analytical-scale SFE of environmental water samples with CO2. Because of effective infinite dilution of the solutes, the partition coefficients provide a direct measure of relative CO2-philicity/hydrophilicity of the individual phenols. Compared to the octanol-water partition coefficients of substituted phenols, the CO2-water partition coefficients are more sensitive to substitution in the position neighboring the hydroxyl group.

Abstract. The influence of coiling on column efficiency in capillary electrophoresis (CE) has been examined. In open tubular columns of 75-pm i.d., no significant effect of column coiling on efficiency was observed, even at plate counts of 2 x lo6 m-l. The small column internal diameter and radial diffusion in free solution was able to minimize the potential deleterious effect of coiling. With gel-filled columns of very high efficiency (-lo7 plates m-l), the influence of coiling on column efficiency was, however, significant, e.g., a factor of 3 or more loss in plate count per coil. For relatively rigid gels (e.g., 9 % T linear polyacrylamide with 7 M urea), this observed loss of column efficiency was attributed mainly to the inability of the polymer network to permit diffusional relaxation of the coiling effects. A significant influence of coiling on column efficiency was also found with medium concentration polymer networks (e.g., 3-6 % T linear polyacrylamide with no urea). The polymer network structure was assumed to change under the influence of mechanical stress caused by coiling, resulting in anisotropy across the column. Furthermore, the changes in structure and in efficiency were found to be time dependent and, in general, reversible. The effect appears to be caused by the shear force imposed on a polymer network structure, since changes are only observed when the column shape is altered after the polymer has been loaded into the capillary.

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A criterion is presented for the evaluation of the polarity of GC stationary phases, based on the concept of reluctance of a polar substance to accept a nonpolar one. An additivity of the partial molar excess Gibbs free energies of the individual groups in the molecule is assumed, which makes it possible to define the partial molar excess Gibbs free energy of a methylene group by: AG£(CH2) = -RT dln(VgP°)/d/7 where R, T, Vg, P°, and n stand for the gas constant, column temperature, specific retention volume of solute on the given phase, saturated vapor pressure of pure solute at the column temperature, and the number of methylene groups in the molecule of solute, respectively. The AG£(CH2) value appears to be a universal criterion of the polarity of sorbents.

A theoretical analysis of the effects of the background detector response on the net response to a substance was performed. The effects were investigated experimentally with the use of two stationary phases of different volatilities, employing the flame ionization detector. The change in relative response factors caused by bleeding of the stationary phase and the systematic errors incidental to the use of a volatile stationary phase in quantitative PTGC are demonstrated, ANY GAS CHROMATOGRAPHIC DETECTOR, operated under usual conditions in a conventional set-up, produces a certain background response. Despite attempts to compensate for this response, it usually enhances the noise level and should therefore be kept as low as possible. There are some exceptional cases in which the response to the presence of the component eluted (net response) is given by a decrease in the background response [e.g. the electron capture detector ( I ) ] or in which the positive response is proportional to the background response (2, 3) [e.g., the alkali flame ionization detector (4, 5)]. These special cases obviously require a compromise in the setting of the detector sensitivity to attain optimum performance.There is still another problem associated with the background detector response. AS shown below, in spite of various methods of compensating for the background response, it may cause a decrease in the net response. In this

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