Unlike visual evaluation of a chromatograms before derivatization, which can only give qualitative or semiquantitative results, direct optical evaluation using instruments enables quantitative results to be obtained. For this, a traditional TLC scanner, diode- array scanner or video equipment, either alone or in combination with a flat-bed scan- ner, is used. Quantitative evaluation with these instruments is described in more detail in Sections 7.2–7.4. However, the limits of this book would be exceeded if we gave a detailed description ofallthe commercially available equipment that can be used to quantify substances on TLC plates. Training in the use of TLC scanners can be ob- tained in company seminars (e.g. CAMAG) and detailed instructions are provided by the manufacturer when the equipment is purchased.
5.2.1 Principle of Operation of a Traditional TLC Scanner
Quantitative analyses are obtained by densitometry. The TLC scanner used for this is linked to a personal computer (PC) and is controlled by an evaluation program. The PC performs the calculation of the result, supports the protocol, and provides an ar- chive of all the parameters of the equipment and evaluation program, the raw data and the numerical and graphical results.
The measurement consists of a direct photometric evaluation. The TLC plate is scanned lane by lane by monochromatic light of the appropriate wavelength. The mea- suring slit is adjusted to give a light beam whose dimensions suit the size of the spot.
Spots obtained by pointwise application are scanned over their whole diameter. With bandwise application, which is preferable for quantitative evaluation, an aliquot of 50–75 % is taken from the homogeneous central part of the band.
The light that is diffusely reflected (re-emitted) by a blank part of the TLC plate is measured by a photomultiplier (PM) and is set equal to 100 % (0 % absorption). When the absorbing substances are scanned, the absorption increases with increasing amount of substance. A plot of the absorption signal against the migration distance gives the so-called absorption-position curve (representation of the chromatogram).
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5.2.2 Direct Optical Evaluation Above 400 nm
When TLC scanners are used for absorption measurements in the 400–800 nm range, illumination is by tungsten filament lamp. To determine the optimum wavelength for measuring a substance (the absorption maximum), the absorption spectrum is mea- sured at the centre of the spot obtained by chromatography. This spectrum can then be kept in a library of spectra and later used for identifications of other samples.
Dyes that absorb in the visible range, e.g. those found in the analysis of writing inks, can be quantified.
5.2.3 Direct Optical Evaluation Below 400 nm
A deuterium lamp is used for absorption measurements in the 190–400 nm range. Here also the optimum wavelength for measurements is determined by obtaining an absorp- tion spectrum. At an excitation wavelength of 254 nm, UV-active substances can be di- rectly measured on TLC plates. Alternatively, it is possible to use a high-pressure mer- cury vapor lamp, which gives an extremely intense line at 254 nm (see also Section 5.2.4). Examples of measurements at an excitation wavelength of 254 nm using a high- pressure mercury vapor lamp can be found in the literature [79]. The chromatograms of the sulfonamides in Section 7.2.2.3 were measured with deuterium lamps using an irradiation wavelength of 285 nm.
5.2.4 Direct Optical Evaluation With 365-nm UV Light (Fluorescence Measurement)
Substances that themselves fluoresce (self-fluorescence) are determined by measuring this fluorescence. A high-pressure mercury vapor lamp, which is usually used as the light source, emits a line spectrum in the range 254–578 nm. The fluorescence light of longer wavelength emitted by the substance on the TLC plate after it has absorbed the light at the selected excitation wavelength is recorded by the PM. To prevent the re- flected light which is also emitted from the TLC plate at the excitation wavelength from reaching the PM, this is eliminated by a cut-off filter or narrow-band filter. For fluorescence measurements in general it is standard practice to use an excitation wave- length of 366 nm combined with a cut-off filter which is not transparent to light of a wavelength less than 400 nm. As with absorption measurements, the fluorescence in- tensity is plotted against the migration distance, giving a fluorescence-position curve [80].
The chromatograms of greater celandine shown in Section 10.2.3 “Effect of Light”
are examples of fluorescence measurements without previous derivatization.
117 5.2 Direct Optical Evaluation Using Instruments
Practical Tipsfor working with traditional TLC scanners:
Determination of the optimal measurement wavelength
With older equipment which cannot be used to obtain spectra, the optimal measu- rement wavelengths are determined as follows: If it is known, for example, that the maximum is at a wavelength less than 300 nm, the chromatogram lane containing the substance to be determined is scanned several times using a scheme such as the following:
(a) From 220 to 300 nm in 10-nm steps (9 ×) Result: Maximum is in the medium range (b) From 250 to 270 nm in 5-nm steps (5 ×)
Result: Maximum is between 260 and 270 nm (c) From 260 to 270 nm in 1-nm steps (11 ×)
Result: Maximum is at 265 nm
Measurement with incomplete separation of the substance
If two substances on a chromatogram lane are not separated at the base line, but only one of the two substances is needed for the analytical result, the spectra of both substances are obtained and the measurement wavelength is selected such that there is a minimum for the substance not used. In the ideal case, however, the sub- stance to be determined has a maximum or at least gives a satisfactory signal.
Measurement with multiple wavelength scan (MWLS)
The use of MWLS gives additional information enabling compounds to be identi- fied or at least classified as members of a group of substances. An example of the use of MWLS is the analysis of drinking water for pesticides by AMD using Bur- ger’s method [81]. Here, the chromatograms are measured successively at the wave- lengths 190, 200, 220, 240, 260, 280 and 300 nm. The measurement curves of the chromatogram at the various wavelengths are all printed together, i.e. superim- posed, using a multicolor plotter to enable them to be visually evaluated (Fig. 75).
In UV multidetection, each substance is thus characterized by its chromatographic migration distance and its behavior.
Multiple wavelength measurements are also performed in the analysis of natural substances, which have complex compositions, e.g. in the determination of flavo- noids after AMD separation. Here, absorption measurements are performed at wavelengths of 235, 286, 300 and 314 nm [82].
Figure 75:see Photograph Section.
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Advantages of fluorescence measurement over absorption measurement:
– The limits of detection are lower by about 1–3 orders of magnitude.
– The calibration curves are linear over a greater range of concentrations.
– The selectivity of the determination is usually greater [80].
Intensification and stabilization of a fluorescence signal
The intensity and the stability of a fluorescent spot can be increased by Triton X- 100 in chloroform or paraffin inn-hexane. For example, in the determination of tes- tosterone, the TLC plates are dipped twice for 1 s in a solution of Triton X-100 in chloroform (1 + 4). Between the two dipping operations, the plate is air dried for 30 min in the absence of light until the chloroform has completely evaporated. This causes an increase in the spot intensity by a factor of 2.5 [83].
In the determination of polycyclic aromatic hydrocarbons (PAHs), the separation is performed either on caffeine-impregnated HPTLC silica gel 60 plates, on HPTLC- RP-18 plates with a concentration zone or on precoated HPTLC silica gel 60 plates with a concentration zone (Merck Item Nos. 1.15086, 1.15037, 1.13728). Subsequent dipping in paraffin/n-hexane (1:4) gives not only stabilization of the PAHs on the plate but also an increase in the fluorescence intensity by a factor of 4–5 [83a].
Sensitive samplesare often destroyed when subjected to measurement by fluores- cence. This sensitivity should be borne in mind when performing a validation and, if appropriate, it should be stated in the testing procedures that the chromatogram should be measured only once. Our own investigations into the determination of dried extract of nettle root showed that a second determination of scopoletine on the same chromatogram lane gave a value that had decreased by ca. 5 %.
Replacement of lamps
If the sensitivity of a measurement decreases, e.g. because of loss of intensity, the lamps should be replaced. Here, the operating instructions should be followed strictly. It is advisable to practice the changing of the lamps, which are usually pre- set, on delivery of a TLC scanner.
Measurement at 190 nm and 200 nm in a nitrogen atmosphere
To obtain reliable results at wavelengths of 190 nm and 200 nm, it is strongly recom- mended to purge the monochromator housing with nitrogen (ca. 0.5 L/min). This is because short-wave UV light causes oxygen to be converted into ozone, the shorter the wavelength the greater being the extent of conversion. Consequently, local
“clouds” of ozone escape intermittently through the ventilation holes. Ozone ab- sorbs in the short-wave UV range with a maximum at 254 nm. Purging with nitro- gen prevents ozone formation and hence prevents fluctuations in the intensity of the short-wave UV light [84].
119 5.2 Direct Optical Evaluation Using Instruments