Paper chromatography components

Paper chromatography in particular frequently enables the components of a mixture to be separated and identified when only 1-2 mg. of the mixture are available, the process being independent of the relative solubilities of the components. 

Paper chromatography (benzene-chloroform 1 1—formamide system) of representative chromatogram fractions indicates the presence of a small quantity of a more polar ultraviolet absorbing component that gives a negative blue tetrazolium test and a very polar component (no ultraviolet negative tetrazolium test). These materials have not been characterized. 

C. Isolation and purification of XK-62-2 100 g of the white powder obtained in the above step B are placed to form a thin, uniform layer on the upper part of a 5 cm0X 150 cm column packed with about 3 kg of silica gel advancely suspended in a solvent of chloroform, isopropanol and 17% aqueous ammonia (2 1 1 by volume). Thereafter, elution is carried out with the same solvent at a flow rate of about 250 ml/hour. The eluate is separated in 100 ml portions. The active fraction is subjected to paper chromatography to examine the components eluted. XK-62-2 is eluted in fraction Nos. 53-75 and gentamicin Cja is eluted in fraction Nos. 85-120. The fraction Nos. 53-75 are combined and concentrated under reduced pressure to sufficiently remove the solvent. The concentrate Is then dissolved in a small amount of water. After freeze-drying the solution, about 38 g of a purified preparate of XK-62-2 (free base) is obtained. The preparate has an activity of 950 units/mg. Likewise, fraction Nos. 85-120 are combined and concentrated under reduced pressure to sufficiently remove the solvent. The concentrate is then dissolved in a small amount of water. After freeze-drying the solution, about 50 g of a purified preparate of gentamicin Cja (free base) is obtained. 

FIGURE 1 Two stages in a paper chromatography separation of a mixture of two components. (a) Before separation f (b) after separation. The relative... 

A technique of chemical analysis in which the components of a liquid mixture are adsorbed in separate layers in a column of adsorbing material. Variations of the technique are paper chromatography and gas chromatography. 

High-voltage electrophoresis and subsequent paper chromatography of the fractions obtained made possible the isolation from the analyzed mixture of twenty-two components giving colored spots with ninhydrin and isatin. Among these, fourteen were identified as peptides and their amino acid composition established (Table 5). In the case of eight peptides, also N- and C-terminal amino acids were determined (Table 6). 

TLC is related to paper chromatography (PC) as both use a stationary phase and a liquid phase to move the sample [12]. A common example of PC is the separation of black ink into its individual colors. Because the individual molecules behave differently when exposed to a solvent such as water or isopropyl alcohol, they are retained on the paper at different intervals, creating a visible separation of the individual components. This helps to identify each component in a mixture. 

Quantitative analysis is also possible. The spot representing the component of interest can be cut (in the case of paper chromatography) or scraped from the surface (TLC), dissolved, and quantitated by some other technique, such as spectrophotometry. Alternatively, modern scanning densitometers, which utilize the measurement of the absorbance or reflectance of ultraviolet or visible light at the spot location, may be used to measure quantity. 

TLC has similar applications to paper chromatography. The stationary phase is a coating, such as silica gel, on a glass or plastic plate. Depending on the TLC plate used, components may be separated based on differences in molecular weight, charge, or polarity (see Chapter 11). TLC with a 70% isopropyl alcohol mobile phase and a silica gel plate is an effective substitute for paper chromatography separation of amino acids. Nucleotides may be separated on a special silica gel plate and a 20% ethanol (in water) mobile phase. 

Paper chromatography is a common way to separate various components of a mixture. The components of the mixture separate because different substances are selectively absorbed by paper due to differences in polarity. In this field or laboratory investigation, you will separate the various pigments found in leaves. You also will calculate the ratio called Rf for each of them. The ratio Rf compares the distance traveled by a substance, Ds, to the distance traveled by the solvent, Df. The ratio is written as Rf = Ds / Df. 

Thin-layer chromatography (TLC) is often used as a faster alternative to paper chromatography. Instead of paper, a thin layer of silica gel or alumina coated onto glass, metal or plastic is used. The water held on the silica gel or alumina is the stationary phase. The mobile phase is a suitable solvent or a mixture of solvents. The solvent flows through the stationary phase and carries the components of the mixture with it. Different components in the mixture travel across the stationary phase at different rates. 

A very efficient thin layer form of circular paper chromatography makes use of a circular glass disc coated with an adsorbent (silica, alumina or cellulose). The apparatus is called a Chromatotron (available from Harrison Research, USA). The disc is rotated by a motor, and the sample followed by the eluting solvent are allowed to drip onto a central position on the plate. As the plate rotates the solvent elutes the mixture, centrifugally, while separating the components in the form of circles radiating from the central point. When elution is complete the revolving circular plate is stopped and the circular bands are scraped off and extracted with a suitable solvent. 

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