9780521516358book.pdf

Alternatively, one can record the control spectrum first and use this as internal reference

for the sample spectrum. The latter approach has become very popular as many spectro-

meters in the laboratories are computer-controlled, and baseline correction can be carried

out using the software by simply subtracting the control from the sample spectrum.

The light source is a tungsten filament bulb for the visible part of the spectrum, and

a deuterium bulb for the UV region. Since the emitted light consists of many different

wavelengths, amonochromator, consisting of either a prism or a rotating metal grid

of high precision calledgrating, is placed between the light source and the sample.

Wavelength selection can also be achieved by using coloured filters as monochromators

that absorb all but a certain limited range of wavelengths. This limited range is called

thebandwidthof the filter. Filter-based wavelength selection is used incolorimetry,

a method with moderate accuracy, but best suited for specific colorimetric assays

where only certain wavelengths are of interest. If wavelengths are selected by prisms

or gratings, the technique is calledspectrophotometry(Fig. 12.6).

Example 1ESTIMATION OF MOLAR EXTINCTION COEFFICIENTS

In order to determine the concentration of a solution of the peptide MAMVSEFLKQ

AWFIENEEQE YVQTVKSSKG GPGSAVSPYP TFNPSS in water, the molar

absorption coefficient needs to be estimated.

The molar extinction coefficienteis a characteristic parameter of a molecule and

varies with the wavelength of incident light. Because of useful applications of the

law of Beer–Lambert, the value ofeneeds be known for a lot of molecules being

used in biochemical experiments.

Very frequently in biochemical research, the molar extinction coefficient of

proteins is estimated using incrementaleivalues for each absorbing protein residue

(chromophore). Summation over all residues yields a reasonable estimation for the

extinction coefficient. The simplest increment system is based on values of Gill and

von Hippel.^1 The determination of protein concentration using this formula only

requires an absorption value atl¼280 nm. Incrementseiare used to calculate a molar

extinction coefficient at 280 nm for the entire protein or peptide by summation over

all relevant residues in the protein:

Residue Gill and von Hippelei(280 nm) in dm^3 mol^1 cm^1

Cys 2 120

Trp 5690

Tyr 1280

For the peptide above, one obtainse¼(1 5690 þ 2 1280) dm^3 mol^1 cm^1 ¼

8250 dm^3 mol^1 cm^1

(^1) Gill, S. C. and von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino
acid sequence data.Analytical Biochemistry, 182 , 319–326. Erratum:Analytical Biochemistry(1990),
189 , 283.
488 Spectroscopic techniques: I Photometric techniques