Thin-Layer Chromatography

32 2 Physical Methods of Detection
100
90
80
70
60
50
40
30
I
I?
II
I?
11
li
li
100 •
90-
80-
70-
60-
50-
40-
30-
\ I
220 240 260 280 300 320 nm 220 240 260 280 300 320 nm
Fig. 23: Reflectance spectra (o—o—) of 3 |ig testosterone (A) and 3 p.g ^*-androstendione-
(3,17) (B) taken up on a silica gel layer compared with the absorption spectra determined in
methanohc solution (•— —)
Fig. 24: Schematic representation of the recording of an absorbance scan. — M = measuring
slit
Indirect Determination of Absorption (Fluorescence Quenching)
If the substance under investigation absorbs with wavelengths between 250 and
300 nm, it should be checked whether it is possible to employ chromatographic
layers containing luminescence indicators. For the inorganic indicators (Sec. 2.2.2)
also absorb in this range and emit, for example, yellow-green long-wavelength
radiation. Hence, it is the radiation that has not been absorbed by the substance
plus the fluorescence/phosphorescence radiation emitted by the indicator that
arrives at the detector. The signal produced is, therefore, a composite signal of
these two radiation types.
When working with a deuterium lamp the radiation energy is so low that the
luminescence radiation only makes up a few percent of the total radiation. This
can be easily checked in the majority of scanners by setting the total radiation (e.g.
X = 260 nm) to 100% reflectance and then inserting a cut-off filter in the beam.
This filter absorbs the short-wavelength radiation before it enters the detector
(Fig. 22B). The energy that remains comes from the emission of the indicator or
is produced by stray light. The remaining signal is almost always small. Hence,
when a deuterium lamp is employed absorption determinations are only falsified
to a small extent. This falsification is also reduced by the fact that at the site of a
zone the absorbing substance also reduces the emission. The absorbing substances
absorb energy in the excitation range of the luminescent indicator and, hence, less
light is available for the stimulation of luminescence.
However, the optical train illustrated in Figure 22B allows the determination of
fluorescence quenching. The "interfering effect" described above now becomes the
major effect and determines the result obtained. For this purpose the deuterium
lamp is replaced by a mercury vapor lamp, whose short-wavelength emission line
(k = 254 nm) excites the luminescence indicator in the layer. Since the radiation
intensity is now much greater than was the case for the deuterium lamp, the
fluorescence emitted by the indicator is also much more intense and is, thus, readily
measured.
The emission of the indicator is reduced in places where there are substance zones
that absorb at A = 254 nm present in the chromatogram. This produces dark zones
(Fig 4A), whose intensity (or rather lack of it) is dependent on the amount of
substance applied. If the plate background is set to 100% emission the phosphorescence
is reduced appropriately in the region of the substance zones. When the
chromatogram is scanned peaks are produced, whose position with respect to the
start can be used to calculate Rf values and whose area or height can be used to
construct calibration curves as a function of the amount applied (Fig. 25).