Thin-layer chromatography reproducibility

STATIONARY PHASES USED FOR THIN-LAYER CHROMATOGRAPHY. REPRODUCED WITH PERMISSION FROM [4. 1... 

IDENTIFICATION OF CEPHALOSPORINS USING THIN-LAYER CHROMATOGRAPHY REPRODUCED WITH PERMISSION FROM [ 126]... 

Figure 1. Action pattern of Bacillus circulans F-2 amylase. Culture supernatant was applied to PAGE and stained for amylase activity with starch—iodine method. Action patterns of groups I and II toward soluble starch were studied thin-layer chromatography. (Reproduced with permission from ref. 7. Cbpyright 1989 Elsevier.)...

Principles and Characteristics High-performance thin-layer chromatography (HPTLC), also known as planar chromatography, is an analytical technique with separation power and reproducibility superior to conventional TLC, which was first used in 1938 [7] and modified in 1958 [8]. HPTLC is based on the use of precoated TLC plates with small particle sizes (3-5 xm) and precise instruments for each step of the chromatographic process. 

Thin-layer chromatography does not provide quantitative information of the highest precision and accuracy. Linear relationships between the mass of a substance and the logarithm or square-root of the spot area can sometimes be established under very closely controlled conditions. The optical absorbance of a spot determined by reflectance measurements can be similarly related to mass, or the substances can be scraped from the plate and dissolved in a suitable solvent for a spectrometric determination. The main difficulties with area and density measurements lie in defining the boundaries of spots and controlling chromogenic reactions in a reproducible manner. Relative precision can be as good as 1-2% but is more usually 5-10%. 

This is considered to be largely a qualitative technique. Difficulties that arise in obtaining reproducible quantitative data are similar to those encountered in thin-layer chromatography. In addition, adsorption characteristics of dyes on macromolecules are so variable that only semiquantitative comparisons can be made. These are, however, still very useful... 

Thin layer chromatography is reproducible, relatively easy to perform, quick, and inexpensive. The resolution of TLC is greater than classical liquid column chromatography, although usually it is still not possible to resolve individual components from a complex mixture. It is, however, able to separate compound classes (e.g., aliphatic from aromatic) and its main use is,... 

The precipitate was filtered off, washed with ether, and dried under vacuum to yield 4.7 g of dry product (A-poly-2). Thin layer chromatography on silica gel using dichloromethane/methanol (93 7) showed only a trace of free monomer. This activated copolymer was soluble in water, THF, CH2CI2 and DMF. It was reproducibly prepared in good quantity and stored in the solid state for months, protected from moisture, without loss of activity. 

Measurements of the common physical constants such as boiling point or refractive index are not sufficiently sensitive to determine the trace amounts of impurities in question. Besides the common spectroscopic methods, techniques like gas chromatography (GC), high-pressure liquid chromatography (HPLC), or thin-layer chromatography (TLC) are useful. The surest criterion for the absence of interfering foreign compounds lies in the polymerization itself the purification is repeated until test polymerizations on the course of the reaction under standard conditions are reproducible (conversion-time curve, viscosity number of the polymers). 

Planar chromatography, also known as Thin Layer Chromatography (TLC), is a technique related to HPLC but with its own specificity. Although these two techniques are different experimentally, the principle of separation and the nature of the phases are the same. Due to the reproducibility of the films and concentration measurements. TLC is now a quantitative method of analysis that can be conducted on actual instruments. The development of automatic applicators and densitometers has lead to nano-TLC, a simple to use technique with a high capacity. 

Prinzler, H.W., Tauchmann, H., and Tzcharnke, C., Thin-layer chromatographic separation of organic sulfoxides and dinitrothioethers some observations on reproducibility and structural influence. II. Separation of sulfoxide mixtures by one- and two-dimensional thin layer chromatography, J. Chromatogr., 29, 151, 1967 Chem Abs., 67, 96615m, 1967. 

For a few tissue samples, the matrix deposition can be performed manually. A matrix solution is sprayed onto the tissue section with a hand-held thin layer chromatography (TLC) sprayer or an artist airbrush. The reproducibility of manual matrix deposition is an issue. When the manual sprayer is used, the MALDI target plate with the tissue section is held vertically about 15-25 cm from the sprayer nozzle. It is recommended to spray multiple coats of matrix across the tissue section and each coating cycle consists of passing the sprayer two to hve times across the tissue section and allowing the tissue to dry for about 1-5 min. This process is usually repeated between 10 and 20 cycles. 

There are two popular LC techniques in which the stationary bed is supported on a planar surface rather than in a column paper chromatography (PC) and thin-layer chromatography (TLC). PC preceded TLC by some 10 to 15 years, and a large number of excellent separations were devised for it. But beginning about 1956, it was found that TLC could also be used for most of these separations and that it was faster, more reproducible, more versatile, and more convenient. As a result, most laboratories have abandoned the use of PC with its large cumbersome glass chambers. Those who have not, continue to use PC because they feel that the original PC methods are superior or because of the lower cost of PC. 

Activate a silica-gel thin-layer chromatography (TLC) plate by heating for 5 min in a 100°C oven. This procedure drives off excess moisture from the plate so that the TLC separation is more reproducible. 

As a rule, chemical methods used in the examination of writing materials require initial preparation of a sample for study. Paper chromatography, thin-layer chromatography and capillary electrophoresis are experimental techniques often applied. These methods lead primarily to separation of the dyes contained in the ink under examination and to the discrimination of ink samples. The techniques are simple to use, require a small amount of sample for examination, are selective and give reproducible results. Their basic disadvantage, however, is the necessity to isolate the ink from the substrate (e.g. paper) on which the examined document has been prepared. Solvent extraction of the ink often leads to partial damage of the document. 

The significance of paper and thin-layer chromatography lies in the possibility of pre-separating single elements by simple means and also in the separation of element mixtures from different materials in connection with detection and determination methods. Disadvantages for quantitative trace analysis are reproducibility problems... 

Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc. Analysis of Food Colors by Thin-Layer Chromatography/Scanning Densitometry 3... 

Solute identification means qualitative analysis. Various methods are used today to identify a separated substance on a thin-layer chromatographic plate. Of all chromatographic methods, thin-layer chromatography (TLC) provides a unique simultaneous separation of up to 70 samples on the same plate therefore the reproducibility of the experimental conditions is not an issue because the experimental conditions are the same for all samples. This, together with the advantage of separating compounds with very different polarity and the possibility of using different detection methods for the same spot or for adjacent spots on the same plate, is the power of TLC. 

The 2-D TLC was successfully applied to the separation of amino acids as early as the beginning of thin-layer chromatography. Separation efficiency is, by far, best with chloroform-methanol-17% ammonium hydroxide (40 40 20, v/v), n-butanol-glacial acetic acid-water (80 20 20, v/v) in combination with phenol-water (75 25, g/g). A novel 2-D TLC method has been elaborated and found suitable for the chromatographic identification of 52 amino acids. This method is based on three 2-D TLC developments on cellulose (CMN 300 50 p) using the same solvent system 1 for the first dimension and three different systems (11-IV) of suitable properties for the second dimension. System 1 n-butanol-acetone -diethylamine-water (10 10 2 5, v/v) system 11 2-propanol-formic acid-water (40 2 10, v/v) system 111 iec-butanol-methyl ethyl ketone-dicyclohexylamine-water (10 10 2 5, v/v) and system IV phenol-water (75 25, g/g) (h- 7.5 mg Na-cyanide) with 3% ammonia. With this technique, all amino acids can be differentiated and characterized by their fixed positions and also by some color reactions. Moreover, the relative merits of cellulose and silica gel are discussed in relation to separation efficiency, reproducibility, and detection sensitivity. Two-dimensional TLC separation of a performic acid oxidized mixture of 20 protein amino acids plus p-alanine and y-amino-n-butyric acid was performed in the first direction with chloroform-methanol-ammonia (17%) (40 40 20, v/v) and in the second direction with phenol-water (75 25, g/g). Detection was performed via ninhydrin reagent spray. 

Thin-layer chromatography was not taken seriously as a quantitative method of analysis for a long time. The comparatively simple techniques could in fact be reproduced astonishingly well but were very labour-intensive. It was common practice to scrape the separated spots off the plate together with the adsorbent layer, then to elute and to measure the extracted substances in solution. An improvement on this was the direct elution from the plate. Several elution heads are fastened above the separated spots, solvents are pumped through and the extract is collected in spectrophotometer cells. Thus, several spots can be eluted simultaneously 1. ... 

Geiss, F., Schlitt, H., Klose, A. Reproducibility in thin-layer chromatography Influence of humidity, chamber form, and chamber atmosphere, Zeitschrift jilr Anal. Chem., 1965,... 

Complementary to paper chromatography, but used more frequently because of the wide availabihty of stationary phases, is thin layer chromatography (TLC). It is simple, fast, reproducible and can achieve high resolution. It is usually performed on a square plate or on strips. A variation of this type of planar chromatography is carried out on a rotating circular plate using an instrument called a Chromatotron. 

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