High-resolution chromatography of proteins in short columns and adsorptive membranes.

Short chromatographic columns prepared from stacks of microporous adsorptive membranes are promising for preparative-scale fractionation of even rather closely related proteins, but careful selection of operating conditions is needed for success. It has been shown that existing devices exhibit very low internal diffusional resistance, and that resolution is almost totally independent of percolation velocity. Total column length is short, however, and the number of plates exhibited under isocratic low-loading conditions is small, on the order of 100. Simulations using a large survey of protein thermodynamic data show that one can frequently obtain excellent protein separations in these short columns by using the sensitivity of protein adsorption equilibria to eluting solvent composition. In fact, some proteins can be separated in a single stage utilizing this 'on-off' behavior and properly selected solvent gradients. Solely on-off, or differential elution, behavior cannot often be depended upon under the mild conditions need for preparative operations. Carefully programmed gradient elution can frequently produce acceptable purification by maximizing both differential elution and differential migration, resulting in protein separations in columns of less than fifty plates. Means for doing this are described for some simple situations, and criteria are provided for selecting modulator gradient schedules.

Body composition by hydrostatic weighing at total lung capacity and residual volume.

Body density and percent fat were determined by hydro-static weighing (HW) at residual volume (RV), total lung capacity measured on land (TLCL), and total lung capacity measured in water (TLCW) in 50 male and 50 female subjects. Residual volume was measured on land by using the helium dilution method. Vital capacity was measured both on land and with the subject submerged to the neck in water. Total lung capacity was reduced during water submersion by 6.7 and 5.1% in males and females, respectively. Body density was 1.0588 +/- 0.0215, 1.0581 +/- 0.0207, and 1.0634 +/- 0.0214 for males, and 1.0246 +/- 0.0219, 1.0242 +/- 0.0233, and 1.0276 +/- 0.0238 for females at RV, TLCW, and TLCL, respectively. Percent fat was 17.7 +/- 9.7, 18.0 +/- 9.3, and 15.7 +/- 9.5 for males, and 33.4 +/- 10.3, 33.5 +/- 10.8, and 32.0 +/- 11.0 for females at RV, TLCW, and TLCL, respectively. Body density and percent fat were similar when measured by HW at RV and by HW at TLCW. Body density and percent fat measured by HW at TLCL were different than when measured by HW at RV (P less than 0.001). The subjects, using a modified 10-point Borg Scale, rated the HW procedure easier to perform at TLC than at RV (P less than 0.001). The results of this study indicate that measurement of body density and percent fat by HW at TLCW and HW at RV are similar, but if measured by HW at TLCL, body density is overestimated and percent fat is underestimated.