R il2pR I

A number of possible reaction pathways can then occur. Table 1 lists some of the common reactions that take place on the chromatographic layer with iodine. Iodine also possesses fluorescence quenching properties, hence, chromatographic zones that contain iodine will appear as dark zones on a silica gel layer containing a fluorescent indicator when viewed under UV light at 254 nm.

In the case of both reversible and non-reversible reactions with iodine vapour, the chromatographic zones can be 'fixed' by further treatment with a 0.5-1.0% aqueous starch solution. The well-known deep blue iodine-starch complex is formed, which has good stability. As the reaction is very sensitive it is important to make sure that little iodine remains in the background, otherwise the whole background will be coloured blue!

The detection limits are usually a few micrograms of substance per chromatographic zone, but there are some cases where the detection is lower still (e.g. 200 ng glucose). Iodine may also be applied as a solution and is usually prepared in an organic solvent, such as petroleum spirit, acetone, methanol, chloroform or diethyl ether. A suitable dipping solution would be 250 mg iodine in 100 mL petroleum spirit. Such solutions have the advantage that, in some cases, the iodine is enriched to a greater extent in the chromatographic zones when dissolved in a lipophilic environment than a hydrophobic one. Hence, the sensitivity can be improved.

Ammonia vapour Ammonia vapour has a number of uses in chromatographic zone detection:

1. When pH indicator solutions are used to detect aliphatic and aromatic carboxylic acids and fatty acids, ammonia vapour can help to intensify the colours against a contrasting background (often a reversible reaction).

Table 1 Examples of iodine reactions on the TLC layer with a common range of organic substances



Polycyclic aromatic hydrocarbons, indole and quinoline derivatives

Quinine alkaloids, barbiturates and calciferol Opiates, brucine, ketazone and trimethazone

Thiols and thioethers

Formation of oxidation products

Addition of iodine to the double bonds

Iodine addition to the tertiary nitrogen for the opiates Addition reaction with the -OCH3 group of the brucine Ring-opening reaction in the case of the ketazone and the trimethazone

Oxidation of sulfur and addition across the double bond in the thiazole ring

Alkaloids, phenthiazines and sulfonamides

Complex formation

2. There are a number of instances where residual chemical detection reagent gives a background colour. Often exposure to ammonia vapour has the apparent effect of bleaching it from the layer. (An example of this is in the use of molybdophos-phoric acid reagent, where the background yellow colour is removed.) This effect increases sensitivity of detection.

3. Stabilization of some reactions on the layer is also possible with ammonia vapour. An instance of this is the blue colour of tryptamine after reaction with Gibb's reagent.

Nitric acid vapour Most aromatic compounds can be nitrated with the fumes from fuming nitric acid. The reaction works best if the developed chrom-atogram is heated to about 160°C for 10 min and introduced whilst still hot into a chamber containing the nitric acid vapour. Generally the chromato-graphic zones are rendered yellow or brown in colour. They also absorb in UV light at 270 nm. Some substances such as sugars, xanthine derivatives, testosterone and ephedrine fluoresce yellow or blue when excited by long wavelength UV light after such treatment.

Group specific reagents Many reagents give specific reactions with certain organic chemical groups and are called group-specific reagents. In most cases, the reaction mechanism is clearly understood. As a general rule these reagents are very sensitive. It is these reagents that make up the major part of the detection reagent lists that are readily available in a large number of TLC publications. The relative merits of a few of the major group-specific reagents are discussed below.

Oxidation/reduction reactions Often the most frequently used visualization techniques, oxidation/reduction reactions are group-specific depending on the particular reagent used. Some examples of oxidation reactions used in TLC are as follows: Emerson reagent (4-aminoantipyrine-potassium hexacyanofer-rate [III]) for detection of arylamines and phenols; phosphomolybdic acid reagent for lipids and some sterioids; chlorine-o-toluidine reagent for vitamin B2, B6 and triazines; chloramine T reagent for steroids and purine derivatives; and chlorine-potassium iodide-starch reagent for amino, imino and amido groups, and triazine herbicides. Examples of reduction reactions include: tin(II) chloride-4-dimethyl-aminobenzaldehyde reagent for the detection of aromatic nitrophenols; blue tetrazolium reagent for corticosteroids, Tillman's reagent (2,6-dichloro-phenolindophenol) for organic acids including vit amin C; and silver nitrate-sodium hydroxide reagent for reducing sugars and sugar alcohols.

Hydrazone formation The reagent mainly employed for hydrazone formation is 2,4-dinitrophenyl-hydrazine in acidic solution, which provides a specific reagent for carbonyl compounds (aldehydes, ketones and carbohydrates). Yellow or orange-yellow hy-drazones, or osazones in the case of carbohydrates, are formed on the chromatogram. Ascorbic acid and dehydroascorbic acid also respond to this reagent. The sensitivity limit is of the order of 10 ng per chromatographic zone.

Dansylation Dansyl chloride and other derivatives are used to produce fluorescent dansyl derivatives of amino acids, primary and secondary amines, fatty acids and phenols. The dansylation of fatty acids is indirect as the acid amides must be formed first. This conversion is readily achieved with the reagent di-cyclohexylcarbodiimide. In the next step, dansyl cadaverine or dansyl piperidine is used to form fluorescent derivatives of the amides. The detection limit is 1-2 ng fatty acids.

Diazotization Azo dyes are strongly coloured and can be produced on the TLC layer by reduction to primary aryl amines, diazotization and coupling with phenols. Conversely, phenols can be detected by reaction with sulfanilic acid in the presence of sodium nitrite (Pauly's reagent). The coloured zones formed by such reactions are often stable for a period of months.

A few named reagents exist that rely upon a diazotization reaction to detect specific groups of compounds. Two well-known ones are the Bratton-Marshall reagent and Pauly's reagent. The Bratton-Marshall reagent consists of two spray solutions: the first, is sodium nitrite in acid to effect the diazotiz-ation; and the second is a mainly ethanolic solution of N-(1-naphthyl)ethylenediamine dihydrochloride. This reagent is used specifically to visualize primary aromatic amines, sulfonamides and urea, and carba-mate herbicides. Pauly's reagent is used to visualize phenols, amines, some carboxylic acids and imidazole derivatives.

Metal complexes The cations of a variety of transition metals are electron acceptors and are therefore capable of forming complexes with colourless organic compounds that are electron donors. Coloured complexes are the result caused by electron movement within the orbitals of the central metal ion. The most important of these metal ions that form chelates are Cu2 +, Fe3 + and Co2 +, which have particular affinity for compounds that contain oxygen and nitrogen. Examples of this type of chemistry are the biuret reaction with proteins (resulting in the formation of a reddish-violet complex) and the reaction of the Cu2 + ion with aromatic ethanolamines (to form blue-coloured chelates). In addition there is the formation of reddish-violet colours of phenolic compounds with the Fe3 + ion.

Schiffs base reaction The Schiff's base reaction is a group-specific reaction for aldehydes. The reaction usually occurs under basic conditions with aromatic amines to form a Schiff's base. Aniline is normally used to form a coloured anil or Schiff's base with an aldehyde. Carbohydrates can be visualized with 4-aminobenzoic acid with the formation of coloured and fluorescent Schiff's bases. A similar reaction mechanism occurs with 2-aminobiphenyl for aldehyde detection. One of the most sensitive reagents for reducing sugar visualization, the aniline phthalate reagent, is also a Schiff's base reaction. The limit of sensitivity is 10 |g per chromatographic zone.

Other reactions There are a number of less well-used reactions such as halogenation with bromine or chlorine vapour, esterification of alcohols, hydrolysis reactions, and the formation of charge transfer complexes. Many other popular reagents do not fit into the above categories, yet they do constitute a major part of visualization reagent lists. For some of these, the reaction mechanism has not been fully elucidated. Table 2 lists a selection of visualization reagents together with the classes of compounds visualized.

Sequencing reactions If it is known that particular functional groups may be present in the separated chromatographic zones, then reactions can be ex ploited more specifically, not necessarily to give direct identification, but to increase the evidence of the presence or absence of particular analytes. Here, specific reagent sequences can be used to give a wealth of evidence visually. Sequencing reactions are particularly useful where a number of differing functional group compounds are present on a chrom-atogram. An example illustrating the use of four well-known detection reagents is shown in Figure 1.

Where excess of one reagent has been used that may then interfere with the next reagent in the sequence, washing or 'destaining' steps will be necessary. Rinsing troughs in the form of dipping chambers can be used. Such sequential reactions are always carried out either to prepare a substance for a colour reaction that will follow later or to increase the amount of information that is obtained by exploiting a combination of different independent reactions. Therefore, information is provided that could not be obtained using a single reagent step.

Pre-chromatographic derivatization to enhance detection Group-specific reagents can prove very useful not only in detection but also in determining to some extent the choice of mobile phase for the development of the separation. If we are looking to determine the concentration of a particular compound, or similar compounds with the same functional group in a complex mixture, then prechromatographic derivatization with a group-specific reagent is an effective way of accomplishing this. The derivatization reaction is normally carried out before application of the sample to the chromatographic layer. Lengthy extraction procedures on sample preparation columns to clean up the sample before chromatography are not necessary. Such derivatizations are simple 'test tube' reactions, which usually are not time consuming.

Table 2 Popular visualization reagents for TLC

Visualization reagent

Compoundgroups visualized

Anisaldehyde-sulfuric acid

Terpenes, steroids, glycosides


Fatty acids, triglycerides

Copper(II) acetate-phosphoric acid

Lipids, prostaglandins

Diphenylboric acid-2-aminoethyl ester

Flavonoids, carbohydrates

Dragendorff reagent

Alkaloids and other nitrogen-containing compounds

Ehrlich's reagent

Amines, indoles

Folin and Ciocalteu reagent


Gibb's reagent

Phenols, indoles, thiols, barbiturates


Amino acids, peptides, amines, amino-sugars

Pinacriptol yellow

Alkyl and aryl sulfonic acids

Potassium hexaiodoplatinate

Alkaloids, nitrogen compounds, thiols

Thymol-sulfuric acid

Sugars, sugar alcohols

Vanillin-sulfuric acid

Essential oils, steroids



Amino acids, primary amines (violet, pink, yellow and brown zones)

Iron(lll) chloride

Phenothiazines, dibenzazepines (brown and blue zones)

Dragendorff reagent

Alkaloids (yellow and orange zones)

Potassium hexaiodoplatinate

Secondary and tertiary amines (brown, green and violet)

Figure 1 A typical visualization sequence for a TLC chromatogram. The sequence enables the identification of a number of different groups of nitrogen function organic compounds.

An example of such an analytical procedure is the determination of vitamin C in fruit juices. Although it is possible to apply the fruit juices directly to the TLC/HPTLC layer, develop the plate in a suitable solvent mixture, and then use a detection reagent to locate the vitamin C, a more precise and better resolution of the vitamin C can be achieved by derivatizing the sample first with 2,4-dinitrophenylhydrazine to form hydrazones with the keto groups (>C = O) present. The sample applied to the plate is then observable by its yellow colour. Using an appropriate solvent mixture for development, the yellow chromatographic zones can be visually observed as they separate and become resolved from each other.

An even more elegant and simple way to achieve prechromatographic derivatization is to carry out the reaction on the layer. At the point where the sample is to be applied on the chromatographic layer, the de-rivatization reagents can also be applied. Either the sample can be applied to the layer first, followed by the derivatization reagent shortly afterwards, or it can be done in the reverse order. The advantage of applying the derivatization reagent first is that a complete track across the width of the adsorbent layer can be applied, resulting in complete reaction with the sample when it is dosed, usually as bands, on top of the derivatization reagent. After appropriate drying and reaction time, development of the chromatogram can proceed using a solvent mixture that takes into consideration the polarity of the newly formed compounds.

It is also possible to carry out such 'functional chromatography' within the framework of 2-dimen-sional separations. The first ascending development is carried out in the usual way with underivatized sample. Before the second development, the separation track is subjected to treatment with a detection reagent specific to the functional group present in the substances (e.g. acids can be esterified, alcohols can be oxidized to ketones or aldehydes, carbonyls can be reduced to alcohols, carbonyls can form hydrazones or semicarbazones). The second development then follows in the usual way at 90° to the first.

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