by computers which allows easy processing and handling of data. From a purist’s
point of view, the direct measurement of the difference spectrum in a dual-beam
instrument is the preferred method, since it reduces the introduction of inconsistencies
between samples and thus the error of the measurement. Figure 12.7 shows the two
absolute spectra of ubiquinone and ubiquinol, the oxidised and reduced species of
the same molecular skeleton, as well as the difference spectrum.Example 2DETERMINATION OF CONCENTRATIONS
Question (1) The concentration of an aqueous solution of a protein is to be determined
assuming:
(i) knowledge of the molar extinction coefficiente
(ii) molar extinction coefficenteis not known.
(2) What is the concentration of an aqueous solution of a DNA sample?
Answer (1) (i) The protein concentration of a pure sample can be determined by using the
Beer–Lambert law. The absorbance at 280 nm is determined from a protein
spectrum, and the molar extinction coefficient at this wavelength needs to be
experimentally determined or estimated:¼AM
"d
wherer* is the mass concentration in mg cm^3 andMthe molecular mass of
the assayed species in g mol^1.
(ii) Alternatively, an empirical formula known as the Warburg–Christian
formula can be used without knowledge of the value of the molar extinction
coefficient:
¼ð 1 : 52 A 280 0 : 75 A 260 Þmg cm^3
Other commonly used applications to determine the concentration of
protein in a sample make use of colorimetric assays that are based on
chemicals (folin, biuret, bicinchoninic acid or Coomassie Brilliant Blue)
binding to protein groups. Concentration determination in these cases
requires a calibration curve measured with a protein standard, usually
bovine serum albumin.
(2) As we have seen above, the genetic bases have absorption bands in the UV/Vis
region. Thus, the concentration of a DNA sample can be determined
spectroscopically. Assuming that a pair of nucleotides has a molecular mass of
M= 660 g mol^1 , the absorbanceAof a solution with double-stranded DNA at
260 nm can be converted to mass concentrationr* by:¼ 50 mgcm^3 A 260The ratioA 260 /A 280 is an indicator for the purity of the DNA solution and should
be in the range 1.8–2.0.491 12.2 Ultraviolet and visible light spectroscopy