Spot elution technique

The importance of TLC methods as assay methods considering the several difficulties experienced during its use, has decreased considerably, and only few official methods can be found in the pharmacopoeias usually with a spot elution technique for quantitation. 

A general approach to quantitation in the pharmaceutical analysis using TLC may be made on the basis of methods used forevaluation. Two basic approaches can be distinguished direct methods, in which the separated spots are evaluated in situ on the plate, and indirect methods, in which quantitative measurements are carried out after elution of the spots on the chromatoplate. Although the importance of direct methods has increased considerably, the spot elution technique is also being used (e.g. some assay methods in USP Pharmacopoeia). 

Table 3 Advantageous and Disadvantageous Properties on Spot Elution Technique and In Situ Densitometry...

The spot elution technique is a simple procedure in the quantitative TLC of steroids. After elution from the plate, the separated steroids can be quantified by colorimetric, fluorimetric, gas chromatographic, mass spectrometric or radiochemical methods. By adding the high efficiency and selectivity of TLC to the sensitivity of the method chosen for quantitation, many difficult analytical problems can be solved in small laboratories equipped with basic TLC equipment and a spectrophotometer or fluorimeter. Besides its advantages, the use of the spot elution technique also has some problems. These are as follows ... 

As a result of the above factors and because of the development of other highly selective chromatographic methods including in situ TLC techniques, the importance of spot elution techniques in steroid analysis is decreasing and will significantly decline in the future. 

In one metiiod of direct extiaction, tiie adsorbent is scraped from oimd the spot, the glass adjacent to the spot is carefully cleaned, and tiie adsorbent is eluted in situ directiy on to a wall of potassium bromide built around the tip of the spot. The potassiiun bromide is then pressed into a disk. This technique is only suitable for well-resolved spots. Elution of the spot sideways will reduce contamination from compoimds tiiat are not as well resolved. The recovery of material from chromato-... 

Quantitative evaluation of thin-layer chromatograms can be performed by direct, in situ visual, and indirect elution techniques. Visual evaluation involves comparison of the sizes and intensities of color or fluorescence between sample and standard zones spotted, developed, and detected on the same layer. The series of standards is chosen to have concentrations or weights that bracket those of the sample zones. After matching a sample with its closest standard, accuracy and precision are improved by respotting a more restricted series of bracketing standards with a separate sample spot between each of two standard zones. Accuracy no greater than 5-10% is possible for trained personnel using visual evaluation. The determination of myco-toxins in food samples is an example of a practical application of visual comparison of fluorescent zones. 

After carrying out visualization of invisible spots by any of these procedures, comparision of the distance travelled by each component of the test mixture with the distance travelled by known substances can be made, and the unknown substances are identified on the basis of the Rf values thus measured. Once this distance and hence the position of the spot of each componant of the mixture has been determined, the corresponding areas of the adsorbent can be separated without subjecting these to the aforesaid spot-location techniques and each componant eluted separately. The simplest method of elution consists in scraping off the appropriate area of the thin-layer with the tip of a spatula, followed by extracting the substance from this powder with a solvent. 

Quantitative evaluation of thin-layer chromatograms can be performed by direct, in situ visual and indirect elution techniques. Visual evaluation involves comparison of the sizes and intensities of color or fluorescence between sample and standard zones spotted, developed, and detected on the same layer. The series of standards is chosen to have... 

Removal of Spots from the Plate and Elution Technique... 

INFORMATION. The TLC analysis is carried out using Eastman Kodak silica gel-polyethylene terephthalate plates with a fluorescent indicator. Activate the plates at an oven temperature of 100 °Cfor 30 min and then place them in a desiccator to cool until needed. After spotting, elute the plates using methylene chloride as the solvent. Visualize the spots with a UV lamp. The course of the reaction is followed by removing small samples (2-3 drops) of solution from the hot test tube at set time intervals with a Pasteur pipet and placing them in separate -dram vials. See Technique 6Afor the method of TLC analysis and the determination of Rf values. Approximate Rf values trans = 0.72 cis = 0.64. 

Figure 2 Transfer and elution technique for concentration of the material from a TLC spot into a glass capillary tube for introduction into the source of the mass spectrometer. Adapted from (26).

Most pharmacopoeial techniques for steroid analysis use the color reaction with ethanolic (or methanolic) sulfuric acid for visualization of the separated spots. Evaluation under UV-wavelength of 254 nm is also of great importance. The technique of spot elution is only used in USP. In USP the same visualization methods are used for both active ingredients and formulations, as well as for... 

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