Hugh J. McDonald

Some fundamental aspects of centrifugally accelerated paper chromatography

Abstract A study has been made of some of the fundamental factors involved in obtaining good separations and reproducible chromatograms, with the technique of contrifugally accelerated paper chromatography. A study was also made of variables which might influence R F values. Mixtures of bromphenol blue, methyl orange and methyl red were fractionated as well as solutions of lencine, methionine and glycine. For the separation of the dyes, Whatman No. 1 filter paper was used with a veronal buffer solution of ionic strength 0.05 and having a pH of 8.7. For this system, it was found that the R F values of the dyes were essentially unaffected by changes in rotational speed of the rotor from 300 to 925 r.p.m. It was found that the rate of addition of solvent was important in developing satisfactory chromatograms. A flow rate of 1.15 ml/min was found to be satisfactory at all rotational speeds above 250 r.p.m. The shape of the developed chromatogram was elliptical rather than circular, for all four types of paper which were studied. For Whatman No.1, the major axis of the ellipse was parallel to the machine direction of the paper. The R F values for the dye components were essentially the same for the following papers: Whatman No. 1, Eaton-Dikeman No. 613, and Cremer-Tiselius-Munktells. The amino-acid mixture was fractionated using Whatman-3MM paper and a solvent system of butanol-glacial acetic acid and water, in the volume ratio 40:10:10, respectively. The disk was rotated at 975 r.p.m. With a solvent flow rate of 1.2 to 2.1 ml/min, sharply defined zones were obtained. The time of development was 15 min for a flow rate of 1.2 ml/min and 7 min for a flow rate of 2.1 ml/min. The R F values obtained were 0.85 for lencine, 0.56 for methionine and 0.20 for glycine.

Paper chromatography in a centrifugal field

I n Fig. 1 s ieh t m a n Muster , die t ro tz sorgfSldger Glfihb e h a n d l u n g der P robe u m eine Vert iefuf lg e n t s t a n d e n sind. Es h a n d e l t s ich u m keimffSrmige Gebilde, die sich, d e m Weehse l der S p a n n n n g s r i c h t u n g folgend, k re i s f6rmig u m die ra t ion of at l eas t four ions in a m i x t u r e w h e n t he vo l t age is m a i n t a i n e d a t t50 V a n d t he c u r r e n t is passed for 5 hours . One drop (0.0t ml.) of a q u a r t e r n a r y m i x t u r e of so lu t ions of t h e ions of Pd ( I I ) , P t ( IV) , I r ( IV), R h ( I I I ) , R u ( I I I ) , Os(IV) a n d Au (III) con t a in ing on ly m i c r o g r a m a m o u n t s of each ion is suf f ic ient for s a t i s f ac to ry separa t ion . Sequences of s epa ra t i ons are in t h e order g iven below.

Ionography: Some aspects of ion migration on paper in an electric field

Abstract The relationships that exist between the mobility of amino acids migrating on paper under the influence of an electric field, and the pH of the solution, the ionic strength, the time and the voltage across the paper are presented. The rate of migration bears a linear relation to the potential gradient and to the time. The isoelectric points of histidine and glutamic acid have been determined to be 7.6 and 3.1, respectively.

Fractionation and characterization of the lipide stain, Sudan black B.

Abstract Sudan Black B has been separated chromatographically into ten component fractions, and the spectral characteristics of the isolated fractions are given. All the fractions have been shown to stain lipide material. A technique for determining the relative staining ability of the fractions, which utilized paper chromatographic methods, has been devised. The relative staining ability of the different components has been determined by means of the technique which was developed.

Electrophoresis of lipoproteins using pre-stained serum

Abstract A simple method for the paper electrophoretic determination of serum lipoproteins, pre-stained with a saturated solution of AcSBB in propylene glycol, is described. Pre-staining is carried out by adding 1 volume of the dye solution to 10 volumes of serum. Electrophoresis is performed on Macherey and Nagel No. 2214ff filter paper in veronal buffer, pH 8.6, ionic strength of 0.05. A potential gradient of 8 V/cm is used for a period of 2 h. The conditions for pre-staining are discussed and the influence of light and other factors are taken into consideration. The method is quite simple, inexpensive, and time-saving, showing a reproducibility of results which is within ± 3.0% for each individual fraction.

Polyvinylpyrrolidone: The Electromigration Characteristics of the Blood Plasma Expander

Before the metabolic role of the blood plasma expander, polyvinylpyrrolidone (PVP) can be studied by the technic of ionography, some fundamental ionographic characteristics of the polymer, per se, must be determined. This study is concerned with the influence of such variable factors as time, potential gradient, temperature, the pH of the buffer and its ionic strength on the mobility of the migrant when under the influence of an electrical field, and an attempt has been made to correlate them. This work, then, may serve as a basis for later investigations to be made on the combining power of PVP for certain physiologic products with which it comes into intimate contact when used as a plasma expander.

The separation of human serum lipoproteins by conventional descending and centrifugally accelerated paper chromatography

Abstract Employing the technique of chromatography on paper prewetted with a buffer solution (Veronal-oxalate-citrate or phosphate), two distinct lipoprotein fractions were resolved from human serum. The developing solution consisted of a mixture of the buffer solution with isopropyl alcohol or ethanol. Since the necessity of prewetting the paper negates the possibility of reporting the chromatographic properties in terms of Rf values, Rb values were introduced. They represent the ratios of the distances moved by a given substance under study divided by the distance moved by a reference substance, bromophenol blue. The correlation between the lipoprotein fractions obtained by the paper chromatographic technique and those obtained from ultracentrifugal methods was established. On the chromatograms of the lipoproteins, the zone which moved farthest from the origin corresponded to the high-density lipoproteins, the α-lipoproteins. The α-lipoproteins as prepared by ultracentrifugal procedures separated into at least two well-defined spots. Resolution of these two α-lipoproteins by the paper chromatography of serum was not found. The spot closest to the origin corresponded to the low-density lipoproteins. The limited studies on the effect of alterations in the composition of the developing solvent indicated that resolution of the lipoproteins is probably due in a large part to their lipid composition. Prestaining of the serum gave essentially the same type of chromatographic results as those obtained by the poststaining technique. The paper chromatographic separation of serum lipoproteins was compared with the ionographic method. The ratio of β-lipoproteins to α-lipoproteins as determined by paper chromatography agreed well with the results obtained by ionography. Centrifugally accelerated paper chromatography was adapted to the separation of serum lipoproteins. The paper was prewetted with Veronal, Veronal-oxalate-citrate, or phosphate buffer solution and developed with mixtures of these buffers with isopropyl or ethyl alcohol. The radial type of development on the whole circular sheet caused the migrant to “fan out”, yielding developed spots of an arc-type shape, which made ordinary densitometric scanning difficult. By cutting “spokes” into the paper sheet, the fanning out of the migrant zones as they moved radially outward from the point of origin was circumvented. The technique was found to be rapid and practical, and yielded chromatograms, especially of serum prestained for lipoproteins, of high quality, which were readily scannable.

A practical experimental method for converting electrophoretic mobilities in paper-stabilized media to free solution values : The isoelectric points of human serum lipoproteins☆

A practical experimental method for the conversion of ionographic mobilities of substances such as proteins and lipoproteins to free solution values has been presented. It is simple in concept, easily carried out from an experimental stand-point, theoretically sound and results in free solution values which agree very well with those determined directly. For the results to be valid, the ionographic mobility of both the migrant and the electro-osmotic indicator must remain constant throughout the course of the experiment. Four determinations are necessary for each set of experimental conditions. These include the experimental determination of the ionographic mobility and RD, a modified RF, of the migrant and of the electro-osmotic indicator. The measurements must be made under identical conditions of wetness and horizontality of the paper. The method has been used to determine the free solution mobilities for bovine plasma albumin, human serum β-lipoproteins, egg albumin and β-lactoglobulin. The values obtained were in close agreement with those determined experimentally using classical free solution electrophoresis. The isoelectric points of human serum α- and β-lipoproteins have also been determined and found to be in close agreement with previously reported values.

Effect of in Vivo Administration of Various Oral Hypoglycemic Agents on Hepatic Protein Synthesis

Summary The effects of the in vivo injection of two oral hypoglycemic agents (tolbutamide and phenethylbiguanide) upon the in vitro incorporation of leucine-14C into hepatic protein were measured. The acute injection of a large amount of either agent impaired the ability of the animals liver to synthesize protein. This impairment was seen to occur at the level of the cell sap and was not associated with the microsomes. The daily injection of smaller amounts of these compounds resulted in no significant effect on protein metabolism. The data appear to indicate that these agents may, under certain circumstances, have a deleterious effect on the protein balance of the organism.

The in vitro Effect of Two Oral Hypoglycemic Agents on Hepatic Protein Synthesis.

Summary Leucine-C14 was utilized to study the affects of 2 oral hypoglycemic agents (tol-butamide and phenethylbiguanide) on protein metabolism. Both compounds were seen to inhibit C14O2 production and incorporation of the amino acid into protein by rat liver ho-mogenate. Differences in the mode of inhibition are noted and discussed. Neither compound appears to be dependent on the presence of glucose for its in vitro affects on protein metabolism.