Thin-Layer Chromatography

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Fig. 9: HP-UVIS cabinet for UV inspection without a dark room (DESAGA).
mainly employed (although the absorption maxima of the indicators lie at k =
260 nm or 280 nm [37]) together with the long-wavelength double line at A = 365/
366 nm. While low pressure lamps deliver short-wavelength light almost exclusively,
the proportion of long-wavelength UV radiation is much higher in the case
of high pressure lamps. But these have the disadvantage that they require a starting
up time of 2 to 5 minutes. They also operate relatively hot and can only be reignited
after they have cooled down.
Note: The lamp can crack if the hot bulb comes into contact with a cold TLC
plate (protective housing!).
These restrictions do not apply to the less intense fluorescent tubes installed in the
UVIS or MinUVIS (Fig. 6) or Universal UV lamps (Fig. 7). Black glass surrounds
or screens serve as filters. Unfortunately account is often not taken of the fact
that the transparency for short-wavelength UV light decreases appreciably with
increasing duration of irradiation (Fig. 8). So it is advisable to change the filters
of lamps intended for short-wavelength radiation at regular intervals. They can
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in the long-wavelength region. This is particularly true if color
ate taken for documentation purposes.
Qjx&mA cop compact instruments, , where it is pb possible to switch from "daylight" dyg to
la^tW Short-wavelength UV light, are frequently offered for the examination of
thill-layer chromatograms (Fig. 9). These are often fitted with a camera holder.
CiMtkm: When working with UV light protective goggles should always be worn
in order to avoid damage to the eyes.
2.2.3 Photometric Measurement of Absorption
2.23.1 Apparatus
Photomultipliers are appreciably more sensitive sensors than the eye in their response
to line or continuum sources. Monochromators are fitted to the light beam
in order to be able to operate as substance-specifically as possible [5]. Additional
filter combinations (monochromatic and cut-off filters) are needed for the measurement
of fluorescence. Appropriate instruments are not only suitable for the qualitative
detection of separated substances (scanning absorption or fluorescence
along the chromatogram) but also for characterization of the substance (recording
of spectra in addition to hRf) and for quantitative determinations.
Monochromators
Today's commercially available chromatogram spectrometers usually employ diffraction
gratings for monochromation. These possess the following advantages
over prism monochromators which are still employed in the SCHOEFFEL doublebeam
spectrodensitometer SD 3000 and in the ZEISS chromatogram spectrometer:
• The wavelength scale is approximately linear; this also means
• that the wavelength scan is also linear making automation easier using appropriate
stepping motors;
• dispersion is almost constant and not wavelength-dependent, and
• the light transmission above k = 270 nm is higher than is the case for prism
monochromators.
However, the usable spectral region is limited by the waiiekaath-dependent efficiency
of the gratings.