Paper chromatography techniques

The technique of paper chromatography has revolutionized biochemistry where difficult analyses with vanishingly small sample volumes are legion. The control of purity of pharmaceuticals, the detection of adulterants and contaminants in foods and drinks, the study of ripening and fermentation, the detection of drugs and dopes in emimals and humans, the analyses of cosmetics, and to top it all, the analyses of the reaction mixtures in biochemical labs are all performed routinely with paper chromatography technique. 

For the more advanced student, we have extended the section on Quantitative Semi-micro Analysis, and we have included a section dealing with Special Techniques in Separation and Purification, namely Adsorption Chromatography, Paper Chromatography, and Ion- Exchange Processes. 

High Pressure Liquid Chromatography. This modem version of the classical column chromatography technique is also used successfully for separation and quantitative analysis of dyes. It is generally faster than thin-layer or paper chromatography however, it requires considerably more expensive equipment. Visible and uv photometers or spectrophotometers are used to quantify the amounts of substances present. 

The detection of microgram quantities of pyrethrins, cinerins, keto alcohols, and chrysanthemum acids by paper chromatography and by application of these techniques to a study of possible metabolites enabled certain tentative conclusions that imply hydrolysis in insects of a large portion of the radioactive pyrethrins and synergists to corresponding keto alcohols and chrysanthemum acids. 

The identification of the target colorant is done directly by UV-Vis spectrometry (if the colorant is unique) or by a previous separation from a mixture. The most available techniques are planar chromatography (paper chromatography and thin layer chromatography) and, especially, high performance liquid chromatography... 

Spectrophotometric resolution for the discrimination of individual colorant molecules found in mixtures is lower than that of chromatographic techniques such as TLC or HPTLC and even low-cost paper chromatography. More expensive but more accurate determinations may be made by RP-HPLC, IP-HPLC with UV-Vis, and diode array detection. ... 

Thin-layer chromatography (TLC) is a type of liquid chromatography in which the stationary pease is in the form of a thin layer on a flat surface rather than packed into a tube (column). It is a member of a family cf techniques that include some types of electrophoresis and paper chromatography, more generally referred to as planar chromatccraphy. Since we will not discuss electrophoresis in this section, and since TLC has virtually superseded paper chromategr pby in most analytical... 

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. 

The bearing which these discoveries have had on the elucidation of the structure of ribopolynucleotides will be discussed later. It is important to stress here, however, that, for most purposes, the older methods of preparing nucleotides have been superseded by procedures which yield separate isomers of each. Of the techniques mentioned above, paper chromatography iB mainly of analytical value, and is the most convenient method for the qualitative detection of isomeric adenylic acids. The only disadvantage of this method is that the isomers are not completely separable from muscle adenylic acid. The presence of the latter, however, can be readily detected by hydrolyzing it to adenosine by means of the specific 5-nucleotidase present in snake venoms,66 or by deamination by a specific enzyme... 

Other techniques that have been used in the examination of hydrocarbons in seawater resulting from oil spills include gel permeation chromatography [88], mass spectrometetry [85], turbidimetry [89,90], and paper chromatography [91]. 

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. 

The column chromatography technique using Dowex 50 ion-exchange resin, introduced in 1951 (M2) and improved in 1954 (M3) by Moore and Stein, first made possible the precise quantitative analysis of amino acids liberated in the course of acid hydrolysis of urine. Similar results were also obtained by Muting in 1954 (M4), who used paper chromatography methods. In this procedure amino acids were quantitatively determined after staining on the paper and elution of the resulting spots. 

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. 

The efficiency of any chromatographic technique depends upon the number of sequential separations or equilibria that take place, which in the case of paper chromatography are due to the large number of compartments of cellulose-bound water. The test solutes are carried up the paper dissolved in the mobile phase and encounter successive compartments of water. At each one, rapid partition between the two phases occurs leaving the mobile phase to carry up the residual solute to the next water compartment and another partitioning effect. The solute, which is dissolved in the water and hence not carried up the paper, is now presented with fresh solvent rising up the paper and again is redistributed between the two phases. 

Paper chromatography has been used successfully for many years and is still a useful tool despite the fact that thin-layer techniques, especially with readily available commercially prepared plastic or foil-backed plates, offer advantages of speed, resolution and easier handling. Larger volumes of sample can be applied to paper, permitting the subsequent elution of a particular amino... 

The anthocyanin profile of the flowers of Vanda (Orchidaceae) was investigated with a similar technique. Flowers (2 kg) were extracted with 101 of methanol-acetic acid-water (9 l 10,v/v) at ambient temperature for 24 h. The extract was purified by column chromatography, paper chromatography, TLC and preparative RP-HPLC. Analytical HPLC was carried out in an ODS column (250 X 4.6 mm, i.d.) at 40°C. Gradient conditions were from 40 per cent to 85 per cent B in 30 min (solvent A 1.5 per cent H3P04 in water solvent B 1.5 per cent H3P04, 20 per cent acetic acid and 25 per cent ACN in water). The flow rate was 1 ml/min and analytes were detected at 530 nm. The chemical structures of acylated anthocyanins present in the flowers are compiled in Table 2.90. The relative concentrations of anthocyanins in the flower extracts are listed in Table 2.91. It can be concluded from the results that the complex separation and identification methods (TLC, HPLC, UV-vis and II NMR spectroscopy, FAB-MS) allow the separation, quantitative determination and identification of anthocyanins in orchid flowers [262],... 

Many types of substances can be separated and analyzed using this technique. In this experiment, you will use paper chromatography to separate the dyes in water-soluble black ink. 

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