Paper chromatography simultaneously

In each chromatographic technique, one of the four mechanisms predominates, but it should be emphasized that two or more may be involved simultaneously. Partition and adsorption frequently occur together and in paper chromatography, for example, ion-exchange and exclusion certainly play minor roles also. 

A better method has been described by Schwarz (S6). Washed red blood cells are lysed, precipitated with trichloroacetic acid below 0°C and the supernatant quickly neutralized. Speed and low temperatures are necessary to prevent hydrolysis of galactose-l-phosphate which is very sensitive to acid. Barium acetate and ethanol are added, and the precipitated barium salt of galactose-l-phosphate washed with 80% ethanol. The barium salt is then hydrolyzed by heating with dilute hydrochloric acid, acid and salts removed with mixed ion-exchange resins, and the galactose estimated by paper chromatography as described above. It is probably better to avoid the Amberlite MB-1 resin used by Schwarz and, instead, to use a weak base resin mixture, such as Amberlite MB-4. Recovery of added galactose-l-phosphate should be determined simultaneously. 

A similar ion-exchange resin method was used by Ling in 1955 (LI) for the examination of combined amino acids in urine. According to this procedure urine was desalted and simultaneously freed from amino acids by using Amberlite IR-112, H+-form resin. The effluent collected from the column was then fractionated on Amberlite IRA, OH--form resin, by successive elution with 0.16 N acetic acid, 0.08 N formic acid, 0.25 N formic acid, 0.08 N hydrochloric acid, and finally with 0.16 N formic acid. The solutions of all acids contained 10% of acetone. The collected fractions were hydrolyzed with hydrochloric acid and the liberated amino acids identified by means of paper chromatography. 

Paper Chromatography.—A ketonic solvent system has been recommended for the rapid determination of nucleotide-inorganic phosphate mixtures,221 and a procedure for the simultaneous determination of metabolites of various chemical classes has been developed.222... 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

One of the classical approaches of liquid chromatography, paper chromatography, was used for chiral resolution about 50 years ago but is not part of modem practice. In paper chromatography, the stationary phase is water bonded to cellulose (paper material), which is of course chiral and hence provides a chiral surface for the enantiomers. However, some workers used chiral mobile phase additives also in paper chromatography [73,74]. In 1951 some research groups independently [73,75-77] resolved the enantiomers of amino acids. Simultaneously, numerous interesting publications on chiral resolution by paper chromatography appeared [70]. 

Although bacterial nucleic acids and nucleoproteins have been recognized since the turn of the centuryonly the last decade has witnessed research on the low-molecular nucleotides and nucleosides in bacterial cells. Simultaneously, a variety of such substances from other materials (such as plants, yeasts, and animal tissues) have been isolated. Most of these substances are present in small amounts in the cell, and the introduction of refined techniques (such as ion-exchange chromatography, paper chromatography, and paper electrophoresis) were required for their isolation. The isotope technique has also contributed substantially to our knowledge of these substances. 

While Pasteur made the historical discovery, subsequent advances in the resolution of enantiomers by crystallization were based on empirical results. Several attempts to separate enantiomers using paper chromatography were met with unsystematic results. In 1952 Dalgliesh postulated that three points of simultaneous interaction between the enantiomeric analyte and the stationary phase are required for the separation of enantiomers [2]. 

The use of quantitative, paper chromatography is discussed in this Section, since one of the most important factors in its application to wood-cellulose analysis is that it permits a satisfactory determination of mannan. Reasonably reliable methods for the determination of xylan have been available for some time, but mannan determinations, for the reasons discussed above, have been less satisfactory. The fact that (on chromatograms) xylose, as well as other carbohydrates, can be determined simultaneously with mannose is an added attraction in the use of this technique. 

In the same year paper chromatography was first attempted by Benassi (B4) for the simultaneous analysis of 8 tryptophan metabolites (kyn-urenine, 3-hydroxykynurenine, kynurenic acid, xanthurenic acid, anthra-nilic acid, 3-hydroxyanthranilic acid, 2-aminoacetophenone, and 2-amino-3-hydroxyacetophenone), separated by means of a mixtiue of methanol, n-butanol, benzene, and water and revealed through the fluorescence in ultraviolet light of 3655 A. Each compound elicits a different fluorescent color (cf. Table 1). 

Bll. Bondy, P. K., and Upton, G. V., Simultaneous determination of cortisol and corticosterone in human plasma by quantitative paper chromatography. Proe. Soc, Exptl. Biol. Med. 94, 585-589 (1957). 

The apparatus used for continuous-flow paper electrophoresis can be used for separation with an elution rather than a continuous-flow method. The sample is spotted at the top of the paper and only the buffer solution is continuously added. If the electrophoresis is stopped before the sample components drip from the bottom of the paper, the technique combines the separating ability of descending paper chromatography (separation occurring vertically) with that of electrophoresis (separation occurring horizontally) to yield spots simultaneously developed in two dimensions. 

When the solvent has almost reached the top of the paper, the paper is removed and quickly dried. The paper is developed to locate the positions of colorless fractions by spraying with a suitable chemical, e.g. ninhydrin, or by exposure to ultraviolet radiation. The components are identified by comparing the distance they have traveled up the paper with standard solutions that have been run simultaneously, or by computing an J F value. A simplified version of paper chromatography uses a piece of filter paper. The sample is spotted at the center of the paper and solvent passed through it. Separation of the components of the mixture again takes place as the mobile phase spreads out on the paper. 

The most suitable method for the detection and identification of alcohols is the utilization of the reaction with 3,5-dinitrobenzoyl chloride the reaction has been very thoroughly studied and it can be used in combination with paper chromatography for simultaneous detection and identification. As they serve for identification purposes, the esters of 3,5-dinitrobenzoic acid were prepared from a maximum number of alcohols. Another advantage consists in the possibility of preparing these derivatives even when the alcohols are available only in the form of their aqueous solutions. 

A reliable method for the identification of the alcoholic component consists in the cleavage of the ester with hydriodic acid and conversion of the alkyl iodide formed either to S-alkylthiuronium 3,5-dinitrobenzoate or to an ester of 3,5-dinitrobenzoic acid. The procedure is described in detail on p. 201 simultaneously, the alcoholic component of the ester may be identified by paper chromatography. The advantage of this method consists in the small amount of substance (several milligrams) necessary for the analysis. 

Chromatographic methods, notably hplc, are available for the simultaneous deterrnination of ascorbic acid as weU as dehydroascorbic acid. Some of these methods result in the separation of ascorbic acid from its isomers, eg, erythorbic acid and oxidation products such as diketogulonic acid. Detection has been by fluorescence, uv absorption, or electrochemical methods (83—85). Polarographic methods have been used because of their accuracy and their ease of operation. Ion exclusion (86) and ion suppression (87) chromatography methods have recently been reported. Other methods for ascorbic acid deterrnination include enzymatic, spectroscopic, paper, thin layer, and gas chromatographic methods. ExceUent reviews of these methods have been pubHshed (73,88,89). 

---