Fluorescent derivatives HPTLC

Many derivatization techniques, based on chemical reactions or even heat and chemical reactions, can be applied to TLC or HPTLC to specifically determine some compounds or groups of compounds this is specific to TLC techniques. For instance, exposure to fluorescamine in acetone wiU convert sulfonamide antibiotics into fluorescent derivatives which can be visualized under UV radiation [8]. Other techniques are reported and, as suggested earher, the reader should refer to the latest edition of the Official Methods of Analysis pubhshed by the Association of Official Analytical Chemists (Arlington VA, U.S.A.) for complete details about aU the available techniques for the routine analysis of drugs. 

The quantitative analysis of thiamine hydrochloride (vitamin Bi), using HPTLC on sUica gel plates with two different mobile phases, was elaborated. After TLC separation, vitamin Bi was derivatized by the use of tert-butyl hypochlorite or potassium hexacyanoferrate (ni)-sodium hydroxide as reagents. The tert-butyl hypochlorite reagent formed yeUow-fluorescing derivatives... 

TLC is a sensitive technique, and it is important to think small with respect to sample size. The highest resolution will always be obtained with the smallest sample commensurate with the ability to visualize the analyte. This is also one of the prerequisites for reproducibility, especially if densitometry is to be used for quantitation (see Chap. 13.8). Zones that are too concentrated re.sult in integrated curve areas that behave in a nonlinear way on regression plots. Depending on the nature of the analyte, sample. sizes as small as picogram amounts are not uncommon, especially in high-performance thin layer chromatography (HPTLC). This is especially true in the analysis of fluorescent substances. Thus, for ultimate sensitivity it is sometimes desirable that a fluorescent derivative of the analyte be prepared. 

Klaus et al. (1991) presented a TLC method for the simultaneous analysis of uric acid, creatine, creatinine, and uric acid together with glucose in the urine and serum of patients with diabetes and other carbohydrate anomalies. The method requires no special sample preparation, and separations are done on amino-modified HPTLC precoated plates. Detection of all substances is achieved by simply heating the plates to give stable fluorescent derivatives. 

Fluorescent derivatives of prostaglandins were recently separated and determined by OPLC on HPTLC plates using ethyl acetate-diethyl ether-benzene-dioxane-hexane (45 12 5 8 30, v/v) mixture as eluent (88). The linearity of calibration graphs were good in the range 1-100 ng. This range was satisfactory for the determination because the detection limit of analytes were 10-40 pg. 

False-negative results arising from disturbance of normal peak ratios of the analyte ions may be overcome by a variety of means including reinjection of the same derivative on another column, application of an alternative derivatization technique, and/or performing a second analysis with a different method. Thus, the identity, for example, of an illegal hormone residue in a suspect sample has been given by a series of different events including two values for the ratio to the front in two-dimensional HPTLC, a characteristic fluorescence after sulfuric... 

Vitamin Bi, vitamin B2, and nicotinic acid, all of which frequently occur together in foods, were separated by TLC and fluorimetrically determined by using a commercially available fiber optic-based instrument. A fluorescent tracer (fluoresceinamine, isomer II) was used to label the nicotinic acid. Vitamin B1 was converted to fluorescent thiochrome by oxidizing with potassium ferricyanide solution in aqueous sodium hydroxide. These vitamins were separated by HPTLC on silica gel using methanol-water (70 30 vol/vol) as mobile phase. Under these conditions, the Rf values of the vitamin Bi, vitamin B2, and nicotinic acid derivatives were 0.73, 0.86, and 0.91, respectively. 

Junker-Burcheit, A. Jork, H. Prechromatographic in-situ derivatization of fatty acids in the picomole range. Part 1 HPTLC of fluorescent monodansylpiperazine and mono-dansylcadaverine derivatives. Fresenius Z. Anal. Chem. 1988, 331 (3 ), 387-393. 

Lai et al. (1994) reported a simple technique to determine free porphyrins in urine and feces for the diagnoses of porphyria based on C-18 HPTLC with detection of fluorescent zones under long-wavelength UV light. Kus (1996) compared the TLC behavior of several crown ether derivatives of tetraphenylporph-yrin on silica gel, alumina, and cellulose layers with different organic mobile phases. 

The simultaneous detection of benzoic, citric, and lactic acid in soft drinks (168), benzoic acid in textile fibers (169), in Chinese traditional medicine from Daemonorops draco (170), in foods (171-173), and by fluorimetric detection and quantification (173a) have been effected. Alkyl 4-hydroxybenzoates have been separated by circular HPTLC (174), on silufol (preferred visualization, iodine vapor) (175), by reflectance spectrophotometry at 272 nm on sil G HR UV plates (176), and by remission photometry (177). Hydroxybenzoic acids in grape leaves, berries, and wine have been separated on micropolyamide layers (178), and preservatives in wine (179) on polyamide layers containing a fluorescent pigment. Silanized silica gel H (F254) has been employed for the quantitative analysis of alkyl 4-hydroxybenzoates by off the plate differential spectrophotometry (180). Salicylic acid and its derivatives have been analyzed by a variety of TLC procedures (181-190), Sources of error have been discussed (191) and manipulative problems examined (192). 

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