Evaluation Without Derivatization

When the solvent residues have been removed from the TLC plate, this is the moment to take the first look at it to find out whether the work done so far has given the desired result. 

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It would have been possible to combine Chapter 5 Evaluation without Derivatization with the present Chapter 7 to form a single Chapter Evaluation , as the earlier Chapter is mainly of general application. However, evaluation before derivatization in the course of the TLC process needed to be clearly emphasized. As the use of a TLC scarmer was described in detail in Section 5.2, it would be a good idea to reread this Section at this point in the book. 

This measurement has already been mentioned in Section 5.2.3 Direct Optical Evaluation below 400 nm , as the evaluation is performed without derivatization. However, the importance of this example hes in the quantitative results of the measmements of trimethoprim and sulfamethoxazole (both being pharmaceutically active), so that it seems more appropriate to discuss it here. 

The traditional way to evaluate the hotness in peppers or hot sauces is through a taste panel, measured by the Scoville scale, through a series of comparative evaluations of diluted samples. The taste panel method is subjective, costly, and time consuming. Capsaicins can be analyzed directly (without derivatization) by HPLC. The method is precise and rapid (20 minutes), and requires only a drop of filtered hot sauce. Figure 7.8 shows the... 

Recently, CE coupled with Ru(bpy)3 ECL for highly sensitive detection of metformin hydrochloride (MH) derivatized with acetaldehyde is reported. The precolumn derivatization of MH with acetaldehyde was performed in phosphate buffer solution (0.3 mol L pH 7.5) at room temperature for 120 min. The factors affecting this method of analysis, e.g., acetaldehyde concentration, buffer pH, electrokinetic voltage and injection time were examined and under optimization of these conditions, the MH ECL detection sensitivity was more than 120 times that without derivatization. The detection of 0.3 ng mL with S/N = 3 was attained. The proposed method is simple, economical, and sensitive and is used for the determination of MH in urine [42]. A method combining CE with Ru(bpy)3 " ECL detection that can be applied to amine-containing clinical species was developed, and the performance of CE-ECL as a quantitative method for the determination of sulpiride in human plasma or urine was evaluated [43]. 

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)]. 

Bechtel utilized a strong polar stationary phase (HI-EFF 8B) 0.75 % on Chromosorb G HP for the same purpose and succeeded in determining the main opium alkaloids in opium extracts without any derivatization. The relative standard deviation for the individual alkaloids lay between 0.6 t and 8.8 %, and for morphine at 2.3 %. An internal standard was used for the evaluation. On a packed column with a 1 1 mixture of OV-17 and SE-30 (3 % on Varaport 30... 

In the open (off-line) TLC process all fractions are stored on the plate. Their optical properties are measured by densitometry. Such densitometric evaluation, including the recording of in situ spectra can be repeated without the need to repeat chromatography. Densitometric re-evaluation may be combined with postchromatographic derivatization. The decision to measure with different parameters, or to interpose a derivatization step can be made upon reviewing first results, and without the necessity to repeat chromatography. 

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