detectors. At about 350 nm most instruments require a change of the light source from
visible to UV light. This is achieved by mechanically moving mirrors that direct the
appropriate beam along the optical axis and divert the other. When scanning the
interval of 500–210 nm, this frequently gives rise to an offset of the spectrum at the
switchover point.
Since borosilicate glass and normal plastics absorb UV light, such cuvettes can only
be used for applications in the visible range of the spectrum (up to 350 nm). For UV
measurements, quartz cuvettes need to be used. However, disposable plastic cuvettes
have been developed that allow for measurements over the entire range of the UV/Vis
spectrum.12.2.4 Applications
The usual procedure for (colorimetric) assays is to prepare a set of standards and
produce a plot of concentration versus absorbance calledcalibration curve. This
should be linear as long as the Beer–Lambert law applies. Absorbances of unknowns
are then measured and their concentration interpolated from the linear region of
the plot. It is important that one never extrapolates beyond the region for which an
instrument has been calibrated as this potentially introduces enormous errors.
To obtain good spectra, the maximum absorbance should be approximately 0.5 which
corresponds to concentrations of about 50mM(assuminge= 10 000 dm^3 mol^1 cm^1 ).Qualitative and quantitative analysis
Qualitative analysis may be performed in the UV/Vis regions to identify certain classes
of compounds both in the pure state and in biological mixtures (e.g. protein-bound).
The application of UV/Vis spectroscopy to further analytical purposes is rather limited,
but possible for systems where appropriate features and parameters are known.
Most commonly, this type of spectroscopy is used for quantification of biological
samples either directly or via colorimetric assays. In many cases, proteins can be
quantified directly using their intrinsic chromophores, tyrosine and tryptophan.
Protein spectra are acquired by scanning from 500 to 210 nm. The characteristic
features in a protein spectrum are a band at 278/280 nm and another at 190 nm
(Fig. 12.6). The region from 500 to 300 nm provides valuable information about
the presence of any prosthetic groups or coenzymes. Protein quantification by single
wavelength measurements at 280 and 260 nm only should be avoided, as the presence
of larger aggregates (contaminations or protein aggregates) gives rise to considerable
Rayleigh scatterthat needs to be corrected for (Fig. 12.6).Difference spectra
The main advantage of difference spectroscopy is its capacity to detect small absorb-
ance changes in systems with high background absorbance. A difference spectrum is
obtained by subtracting one absorption spectrum from another. Difference spectra
can be obtained in two ways: either by subtraction of one absolute absorption
spectrum from another, or by placing one sample in the reference cuvette and another
in the test cuvette. The latter method requires usage of a dual-beam instrument,
the former method has become very popular due to most instruments being controlled490 Spectroscopic techniques: I Photometric techniques