Overpressure derivatization

Overpressure derivatization (OPD) is a relatively new method in which an absorbent polymeric pad prewetted with detection reagent is pressed onto the TLC plate. OPD was found to give more reproducible results when compared to spraying of ninhydrin reagent in the quantitative analysis of tryptophan on a cellulose layer using densitometric scanning. Use of OPD for a variety of other detection reagents and application to the analysis of a multivitamin preparation was described by Postaire et al. (1990). 

Postaire, E., Sarbach, C., Delvordre, P., and Regnault, C. (1990). A new method of derivat-ization in planar chromatography overpressure derivatization. J. Planar Chromatogr.—Mod. TLC 3 247-250. 

New derivatization techniques to replace spraying and to improve reproducibility, i.e., overpressured derivatization, dipping (32)... 

Panthenol and pantothenic acid have been identified and quantified in pharmaceutical preparations by extraction with ethanol or benzyl alcohol, separation by TLC on silica gel and the chromatogram developed in propan-2-ol-water (85 15). Spots were measured by spectrodensitometry (13). The overpressure derivatization technique has also been applied following separation of calcium pantothenate from other hydrophilic vitamins on silica gel HPTLC layers developed in butan-l-ol-pyridine-water (50 35 15) (11). 

An overpressured layer chromatographic procedure, with photodensitometric detection for the simultaneous determination of water-soluble vitamins in multivitamin pharmaceutical preparations, was developed and evaluated. HPTLC on silica gel plates with 1-butanol-pyridine-water (50 35 15 vol/vol) as mobile phase was used. The quantitation was carried out without derivatization [vitamin B2 (Rf value 0.30), vitamin Bg (Rf value 0.64), folic acid (Rf value 0.37), nicotinamide (Rf value 0.80), and vitamin C (Rf value 1.02)] or after spraying ninhydrin reagent [calcium panthothenate (Rf value 0.72)] or 4-demethylaminocin-namaldehyde [vitamin B12 (Rf value 1.84) and biotin (Rf value 0)]. 

Chromatographic analysis of folate compounds including methotrexate and other antifolates has been reviewed by McCormack and Newman (32). Process impurities in the reduced folate compound leucovorin calcium have been monitored using a TLC method with fluorescence detection (33). An overpressure layer HPTLC procedure has been used to improve the separation of folic acid from other water-soluble vitamins with good recovery and resolution. The method uses silica gel layers developed in butan-l-ol-pyridine-water (50 35 15) at a rate of 0.25 ml/min for baseline separation. Quantitation was achieved without derivatization (11). 

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