Postchromatographic Derivatization

It is an inherent advantage of Thin-Layer Chromatography that fractions remain stored on the plate and can be derivatized after chromatography. By derivatization substances that do not respond to visible or UV light can be rendered detectable. In many cases, substances or classes of substances can be identified by specific reagents.

Reasons for choosing derivatization as a step in TLC include:

Changing non-absorbing substances into detectable derivatives
Improving the detectability (lowering detection limits)
Detecting all sample components
Selectively detecting certain substances
Inducing fluorescence.

From a technical point of view only one principal decision must be made: how to transfer the reagent to the plate? Derivatization can be achieved with gas, by liquid spraying or dipping (immersion). In any case the reagent needs to be homogenously transferred to the chromatogram.

By immersing a TLC plate into the derivatizing reagent a very homogenous reagent transfer can be achieved. Dipping and withdrawing has to be performed smoothly in order to avoid tidemarks. Using the Chromatogramm Immersion Device the reproducibility of the derivatization step can be significantly improved compared to spraying. Furthermore, no fumes are generated during this derivatization technique and the exposure to hazardous chemicals is limited.

If the reagent is suitable, dipping should be preferred over spraying.

However, the fact is that spraying is most widely used for reagent transfer onto the TLC plate because it is simple and quick. No expensive equipment is necessary and only small volumes of reagent are needed. In addition spraying is very flexible and indispensable when reagents have to be applied in sequence. Also during method development, when searching for the most suitable reagent, spraying is more frequently mentioned.

Spraying on the other side generates substantial amounts of obnoxious and hazardous fumes, which must be carefully removed using e.g. the TLC Spray Cabinet.

During spraying, particularly for quantitative evaluation, it must be ensured that a homogenous fine spray mist is generated. Reproducibility of derivatization by spraying is greatly dependent on the skill of the operator. Most chemical reactions used in derivatization require heating for completion. The two principal heating devices are ovens and plate heaters. Ovens have two major shortcomings: One, fumes from derivatization agents can be corrosive and two, cross contamination may become an issue.

Derivatization of capsaicin with dichloroquinone chloroimide reagent/
ammonia by spraying (1 g/L, left side) and by dipping (0.25 g/L, right side)

It is therefore advantageous to use a TLC Plate Heater designed to homogenously heat the TLC plate to the selected temperature.If visualization is not required, derivatization may not be advantageous. For example, progesterone has a chromophor and absorbs UV light at 254 nm, it can thus be analyzed without derivatization. If necessary, the substance can be visualized by derivatization with sulfuric acid. The comparison of the standard deviation for the densitometric evaluation of derivatized and non-derivatized progesterone is given below. The best results are obtained without derivatization, derivatization by spraying yields the least reproducible results across the plate.

Evaluation of progesterone

	No Derivatization	Derivatization by immersion	Derivatization by spraying
Densitometry	Abs. 254 nm	Abs. 536 nm	Abs. 536 nm
Progesterone, CV of 16 samples, 6 mm bands	1.1%	2.4%	2.9%

CAMAG offers a complete range of derivatization here.