Detection (Visualization) of Zones and Documentation of Chromatograms
After drying the developed plate, compound zones are detected on the layer by their natural color, natural fluorescence under
366-nm UV light, quenching of fluorescence under 254-nm UV light on an "F" layer containing a phosphor (termed a UV indicator),
or as colored, UV-absorbing, or fluorescent zones after reaction with an appropriate chemical reagent (postchromatographic
derivatization). Chromogenic and fluorogenic reagents are applied by spraying or dipping a layer or exposing it to reagent
vapors (for example, iodine). Chemical detection reagents can be nondestructive universal (iodine), destructive universal
(for example, charring with sulfuric acid), or selective (for example, ninhydrin for amino compounds). Most chemical derivatization
reactions require heating of the plate for completion. A forced air oven (with a solid glass or metal plate onto which the
TLC plate is placed for even heating) or a Camag plate heater can be used.
Effect directed analysis (EDA) is becoming more important in food, pharmaceutical, and phytochemical analyses. In EDA, there
is no attempt to analyze all compounds in a sample, but instead the focus is on just the relevant (for example, harmful or
bioactive) compounds detected by a bioassay coupled to TLC. These compounds can be the target analyte as well as unknown metabolites,
side products, process contaminants, adulteration products, and residues. Bioassays used with TLC include microbiological
assays (bioautography), bacterial assays (the Camag BioLuminizer bioluminescence detector utilizing Vibrio fischeri), and biochemical detections using enzymes, including immunoassays. TLC–EDA was reviewed by Morlock and Schwack (20).
If the chromatogram and its visualization produce visible results, it is possible to simply photocopy in black and white or
color, or scan the TLC plate to document it. However, a commercial system for photography is needed if documentation of a
combination of colored, fluorescent, and quenched zones is required. Camag, Desaga, and Analtech all offer photodocumentation
Figure 2: TLC–MS interface: (a) front of instrument and (b) schematic of extraction section. (Courtesy of Camag USA.)
Identification of Zones
Separated zones are initially identified based on the correspondence of R
f values and detection characteristics between sample and standard zones on the same plate, but must be independently confirmed
by other evidence, such as off-line and on-line coupling of TLC with various spectrometric methods. Scanning densitometers
have a spectral mode that allows measurement of UV–vis spectra of zones directly on the plate and comparison with standards.
In addition, peak purity can be examined by carrying out spectral studies at the peak start, middle, and end.
One of the most important and active areas of current research is hyphenation of TLC with MS for identification or structure
elucidation, which was reviewed by Morlock and Schwack (20,21). Many TLC–MS coupling approaches were covered in these reviews,
but the elution head-based technique is the most interesting because of the commercial availability since mid-2009 of the
Camag TLC–MS interface. Details of its use are available from Camag. The reviews by Morlock and Schwack also cover hyphenation
of TLC with UV–vis, fluorescence, Fourier transform infrared (FT-IR), and surface-enhanced Raman spectrometry in various combinations
to characterize samples as completely as possible.