Locating reagents

Spraying of locating reagents should always be performed in a fume cupboard. 

If the compound shows no convenient intrinsic feature, it may be possible to develop a detectable characteristic by chemical modification and, particularly in thin-layer chromatography and electrophoresis, many locating reagents which are specific for various groups of compounds are available. 

Table 9.5 Examples of locating reagents suitable for the TLC of carbohydrates... 

The nature of the amino acids is an important factor in the choice of a solvent and different solvents will permit better resolution of acidic, basic or neutral components (Table 10.7). In general, increasing the proportion of water in the solvent will increase all RF values and the introduction of small amounts of ammonia will increase the RF of the basic amino acids. Some solvents contain noxious chemicals, e.g. phenol, and this may restrict their routine use. The chemical composition may also limit the range of locating reagents which can be satisfactorily applied. For example, sulphanilic acid reagent cannot be used with phenolic solvents. 

Other reagents that are more specific for particular amino acids have been described (Table 10.8) and their use significantly assists in the identification process. The different locating reagents may be applied either to separate chromatograms or as part of a multi-dip sequence, when they should be used in the recommended order to prevent interference of one reagent by another. Many reagents are hazardous and must be handled in accordance with approved safety procedures. 

The use of dansyl derivatives is not recommended for routine analysis of free amino acids but is very suitable in the identification of an unknown amino acid that has been selectively extracted from the original sample and is present in small quantities. Both kinds of derivative can be easily separated by chromatography or electrophesis and no locating reagent is required for either because the DNP derivatives are themselves yellow in colour and the dansyl derivatives are fluorescent. 

Location reagents Applications of TLC analysis Qualitative identity tests Limit tests HPTLC... 

There is a huge number of location reagents available and these reagents range from those which are fairly specific for a particular type of analyte to those which will detect many different compounds. 

TLC is often used by BP monographs as part of a number of identity tests performed on pure substances. For extra confirmation of identity, more than one solvent system may be used and also different types of spray reagent may be used. Some examples of identity checks based on TLC have been mentioned earlier. Table 13.4 lists a few of the compounds which have their identity checked by TLC and a variety of location reagents and mobile phases are used to illustrate the fact that there is much less uniformity about TLC methodology than there is in the case of HPLC or GLC methodology. 

Location reagent acidified iodoplatinate solution oversprayed the plate, giving a positive color, with Rf value of 70. 

Stevens, P.J., Thin-layer chromatography of steroids specificity of two location reagents, J. Chromatogr., 14, 269, 1964 Chem. Abs., 61, 2491b, 1964. 

Suitable location reagents for detecting organophosphorus-, organochloro- and carbamate insecticides on TLC plates are given below. Details of their composition and the treatment of the plate necessary with each reagent are also given. 

Location Reagents. Examine the plate under ultraviolet light. 

Akematively, System TB (p. 167) or System TC (p. 167) may be used. For these systems, plates impregnated with potassium hydroxide are used. Location Reagents. Examine the plate under ulftaviolet light. Cover columns 3 to 8 with a glass plate and spray... 

Location Reagents. Spray the plate with the following reagents in the sequence shown, drying die plate under warm air after each application. 

Location Reagents. Spray file plate developed m System TA, and one of file plates developed in System TE, with acidified iodoplatinate solution. Lightly spray the remaining plate developed in System TE with 9M sulphuric acid phenothiazine drugs and tiieir metabolites give pink, red, or blue spots. Overspray with acidified iodoplatinate solution the presence of the acid deepens the blue colour produced when morphine reacts with iodoplatinate solution, and this increases the sensitivity of detection of this compound. 

Location Reagents. Spray the plate with hydrochloric acid and heat in an oven at 100° for 10 minutes in order to hydrolyse any thiazide present... 

Remove the plates and dry under a hot-air blower. Location Reagents. Danthron is visible as a yellow spot on the first plate, or as an orange spot on the second plate. Examine the plates under ultraviolet light at 254 nm and 366 nm. All the substances absorb at 254 nm danthron and rhein give an orMige fluoresence at 366 nm. 

Suitable locating reagents are listed under the TLC systems described at the end of this chapter, and details of their composition are given in the Reagent Appendix. The reagents described under Colour Tests (p. 128) may also be used as TLC location reagents. 

Systems TA, TB, or TC, previously described, may be used together with the associated location reagents. 

Location Reagents. The reagents given for Systems TD, TE, and TF can be used as well as those given below. 

Systems TD, TE, or TF, previously described, may be used. System TF gives the best separations. Location Reagents. The reagents given for Systems TD, TE, and TF can be used as well as that given below. 

Location Reagents. For Systems TL and TM. Naphthoquinone sulphonate solution. Spray the plate with the reagent, then spray with a 10% v/v solution of hydrochloric acid and heat at 110° for 20 minutes. 

Location Reagents. For Systems TN and TO. Acidified iodoplatinate solution, Violet, blue-violet, grey-violet, or brown-violet spots on a pink background are given by quaternary ammonium compounds. 

Plates. Kieselguhr, 250 pm thick, impregnated with a mixture of acetone propylene glycol (9 1). Mobile Phase. Cyclohexane toluene (1 1). Location Reagents. For Systems TP, TQ, TR, and TS. 

Mobile Phase. Chloroform methanol (70 30). Location Reagents. For Systems TF, TT, TU, and TV. 

Location Reagent. Allow the plate to dry in air, heat at 110° for 2 hours, allow to cool, spray with... 

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