Guide Thin-Layer Chromatography: Reagents and Detection Methods

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Document information Title:. Journal of Chromatography. Table of contents — Volume , Issue 1 Show all volumes and issues Tables of content are generated automatically and are based on records of articles contained that are available in the TIB-Portal index. Normal-phase high-performance liquid chromatography with highly purified porous silica microspheres.

Influence of polymer morphology on the ability of imprinted network polymers to resolve enantiomers. Retention indices of phenols for internal standards in reversed-phase high-performance liquid chromatography. Application to retention prediction and selectivities of mobile phases and packing materials.

Part 10: TLC - Visualization of Spots (Thin Layer Chromatography)

Chromatographic analysis of the reaction between thiosalicylic acid and selenious acid in methanol. Salting-out solvent extraction for pre-concentration of benzalkonium chloride prior to high-performance liquid chromatography. Chromatographic investigations of oligomeric a,w-dihydroxy polyethers by reversed-phase high-performance liquid chromatography and evaporative light scattering and UV detection.

Comparison of different methods for the prediction of retention times in programmed-temperature gas chromatography. Theoretical aspects of chiral separation in capillary electrophoresis.

  • Preparation of TLC spray reagents..
  • Thin-Layer Chromatography of Lipids.
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Application to b-blockers. Chiral separations by complexation with proteins in capillary zone electrophoresis. Determination of gold I and silver I cyanide in ores by capillary zone electrophoresis. Estimation of inter-detector lag in multi-detection gel permeation chromatography. Multichannel coulometric detection coupled with liquid chromatography for determination of phenolic esters in honey. Analysis of phenolic and flavonoid compounds in juice beverages using high-performance liquid chromatography with coulometric array detection.

Stability of furosine during ion-exchange chromatography in comparison with reversed-phase high-performance liquid chromatography. High-performance liquid chromatographic determination of mexiletine in film-coated tablets using a new polymeric stationary phase. Mapping of derivatised biogenic amines by two-dimensional thin-layer chromatography. A comparative study. Electrophoretic determination of stability constants of Zn II - and Cd II -nitrilotriacetate-penicillamine mixed complexes.

Similar titles. Siouffi, A. British Library Online Contents Burns, D.

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Thorburn Elsevier Jork, H. Online Contents Elsevier Page navigation Document information Table of contents Similar titles. Commercial HP-TLC plates were utilized and they were first given a double development for part of the way in a polar solvent to separate the phospholipids, and then for the full length of the plate with a less polar mobile phase in order to resolve each of the simple lipids. The total elution time was only 30 minutes, in spite of the triple development. Although this methodology has been employed mainly in the medical field, I can envisage many similar applications in industrial laboratories.

HP-TLC plates have proved of particular value to research workers who deal with complex glycosphingolipids, such as gangliosides, as the extra resolving power is invaluable. A further advantage in this instance is that analysis times can be reduced from to hours [6], and that it is possible to use immunological techniques for detection purposes. Of course, it is possible to isolate the complex lipids from TLC plates and then re-chromatograph them with more polar solvents and with silica gel without added binder to isolate each of the individual phospholipid classes, say, with comparable resolution to HPLC.

It should also be noted that we can be sure that we see every lipid in the sample using this technique, but with HPLC it is always possible that some components may be strongly adsorbed and not eluted from the column. Figure 2 illustrates the nature of the separation for leaf lipids from Arabidopsis thaliana , a plant species much used in molecular biology. Each of the main individual phospholipids and glycolipids is clearly resolved.

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Preparation of TLC spray reagents.

Figure 2. Schematic representation of two-dimensional TLC separation of complex lipids from A. In this instance, plant phospholipids and glycolipids are separated by first developing the plate in chloroform-methanol-water After allowing sufficient time for drying, the plate is developed, at right angles to the first development, in chloroform-methanol-acetic acid-water , by volume.

We feel that this is less hazardous than the charring reagents containing concentrated sulphuric acid that are often used for universal detection. Alternatively, as an aid to identification, plates can be treated with a variety of specific reagents for different lipid types. Ninhydrin reagent shows up lipids containing amino groups, so it is particularly useful for verification of phosphatidylethanolamine and phosphatidylserine.

For nondestructive detection of individual polar lipids separated micropreparatively as above, we have found primulin 0. Lipids are observed under UV light, and give very clear spots even with light spraying with the primulin reagent. The components are extracted from the silica with polar solvent mixtures, prior to analysis by other means. My colleagues and I have been in the forefront of developing methods for separating complex phospholipids and glycolipids from plant leaf tissue by HPLC methods, and we have used these with thousands of samples for screening purposes.

However, when we require a precise detailed analysis of a sample, we go to TLC. This is tedious as it requires that we carry out a separation on the 5 mg scale, locate the spots with the spray reagent, isolate the lipids with an internal standard added and then transesterify for gas chromatographic analysis [1]. The important point is that we can get higher accuracy as well as much more information than by HPLC.

I can remember when silver ion TLC was viewed by industrial analysts as a rather esoteric technique, but it has come to be one of the standard methods for the determination of cocoa butter equivalents in confectionery fats, for example. Whether the HPLC or high-temperature gas chromatography methods will supplant it remains to be seen. Silver ion chromatography methods are reviewed in detail elsewhere on this web site. Reversed-phase TLC has been relatively little used for the analysis of triacylglycerols, mainly because it has been considered a messy technique and because the separated components are not easily visualised.

Methods are available which overcome many of these difficulties, and excellent separations of triacylglycerols have been reported [7]. The recommended stationary phase is a silanized kieselguhr, with a mobile phase consisting of acetonitrile-acetone-water mixtures. Charring densitometry is preferred for detection and quantification. However, I have little doubt that in this instance HPLC techniques are much better when the necessary equipment is available.

The disadvantages of various detectors in HPLC have been debated ad nauseam in recent years, and there is a review of the topic on this website here..

Detection Methods for Thin Layer Chromatography

What of quantification in TLC? The generally accepted method for multiple samples involves spraying with a corrosive reagent usually containing concentrated sulphuric acid , charring at high temperature for a set period, and finally densitometry of the carbon produced. Any experienced analyst coming fresh to lipid methodology would probably look askance at such a procedure. Yet it can be made to work, if great care is taken to standardise the charring conditions and to calibrate for each of the lipid classes with authentic standards, i. Modern scanning densitometers can give excellent results in skilled hands.

However, there is considerable scope for inexperienced analysts to compound the errors. In research applications with relatively few samples, I prefer to transesterify the separated lipids in the presence of an internal standard and use gas chromatography of the methyl ester derivatives of the fatty acids for quantification purposes [1].

What of those of us that have ready access to HPLC? I mentioned earlier that I had resurrected TLC in my own laboratory but with commercial pre-coated plates for the sake of a cleaner environment.

Visualizing agents for Thin layer chromatography

TLC has been missed most often when I have had a single sample whose purity or identity needed checking. It is often tedious, time-consuming and costly in terms of solvents to do this by HPLC. In the analysis of complex lipids, it is possible to use specific spray reagents to detect particular functional groups in lipids separated by TLC. I recall listening to a lecture many years ago in which the speaker described a chromatography-tandem mass spectrometry system in which phosphatidylcholine was eventually identified by the presence of a specific ion relating to the choline moiety; the same could have been done with a simple TLC spray.

We lack simple colorimetric methods for the identification of components separated by HPLC. What counts is the quality of the end result and not how it is obtained, and in skilled hands TLC offers considerable versatility and precision in lipid analysis with relatively low capital costs.

Of course, there are many parts of the world where HPLC is not available to analysts. If scientists in these countries can obtain equally good results by TLC, they should be encouraged to do so. Suffice it to say that traditional TLC is not dead, but alive and well, and likely to be around for some time to come. A recent review cited earlier makes this point very clearly [4]. This article has been updated appreciably from two earlier papers now amalgamated by the author that first appeared in Lipid Technology Christie, W.

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Has thin-layer chromatography had its day? Lipid Technology , 2 , ; Christie, W. Thin-layer chromatography-revisited. Lipid Technology , 11 , by kind permission of P. Some Practical Examples With TLC in the adsorption mode silica gel , the principal application in lipid analysis is for the separation of different lipid classes from animal and plant tissues. Quantification The disadvantages of various detectors in HPLC have been debated ad nauseam in recent years, and there is a review of the topic on this website here..