Thin-Layer Chromatography Evaluation |
Methods for Assessing TLC Results
TLC Evaluation without Derivatization
Direct visual assessment of TLC plates is still common practice, and recommended by pharmacopoeias. Evaluation can be performed in daylight, or through the use of short- or long-wave UV light.
Caution: Always wear dark glasses when working with UV lamps!
Visual Evaluation in Daylight
After separation, spots produced by colored compounds can be detected by the naked eye. This simple technique requires no instruments, and can be performed using pre-coated TLC plates without fluorescence indicators.
Detection with 254 nm UV Light
When exposed to short-wave UV light of 254 nm, UV-active compounds will undergo fluorescence quenching and appear as dark spots on a bright background. This method requires TLC plates with fluorescence indicators as well as UV lamps, and should be preferably performed in a darkroom.
Detection with 366 nm UV Light
Conversely, compounds that absorb 366 nm UV light will appear as bright spots on a dark background. This detection method requires TLC plates with fluorescence indicators, and a UV lamp with an excitation wavelength of 366 nm. A darkroom is also recommended.
TLC Evaluation with Derivatization
Evaluation of TLC plates after derivatization can be performed in daylight, in light of a wavelength greater than 400 nm, or by using UV light of 254 or 366 nm.
Important: Some derivatives may undergo rapid change or loss of color. Hence, chromatograms should be immediately assessed after derivatization.
Visual Qualitative Evaluation
Visual assessment of TLC plates is only acceptable for identity testing. In compliance with GxP protocols, records of photographic evidence or evaluation by a second analyst are also necessary.
Visual Semi-Quantitative Evaluation
For limit value tests, pharmacopoeias also require semi-quantitative evaluation of the TLC separation. In addition, photographic documentation or assessment by a second person is necessary to meet GxP guidelines.
Qualitative Evaluation with a TLC Scanner
For qualitative evaluation of samples with complex compositions, a “fingerprint” method is usually performed using a TLC scanner to create a spectrum for each spot on the plate. Results are then compared to standard spectra to identify the substances in the sample.
Quantitative Evaluation with a TLC Scanner
TLC scanners are the optimal choice for precise quantitative TLC evaluation. To ensure correct use of your TLC scanner, we recommend contacting the manufacturer for a product demonstration.
Tip: Practical advice for working with TLC scanners
Detection and Characterization by TLC-MS
TLC-MS coupling allows on-line evaluation of chromatograms using mass spectrometry (MS). This relatively new technique enables highly accurate identification of individual substances in complex mixtures based on the size and characteristics of their molecules. MS-grade TLC plates are recommended for this evaluation method.