BioPHYSICAL chemistry

a reference sample cell that contains everything that is in the sample to
be measured, except for the molecules being measured. For example, if
you are measuring the absorbance of myoglobin in a phosphate buffer,
the reference would be an identical cell with the phosphate buffer but no
myoglobin. The advantage of using two beams is that the contribution
of everything but the molecule of interest can be subtracted, yielding the
signal of only that molecule.
Typically, the sample will be in a cuvette with a pathlength of 1 cm, so
usually εis provided with units of mol−^1 cm−^1. The absorption spectrum
of a biological sample will consist of a number of bands spread through
the spectral region, as seen for bacteriochlorophyll in bacterial reaction
centers (Figure 14.5). Many different types of molecular system have
widely spaced electronic orbitals and therefore absorb in
the ultraviolet region of the spectrum, including aromatic
rings of proteins, the bases of nucleic acids, and many small
aromatic molecules. Molecules with large conjugated
systems, including bacteriochlorophylls (Figure 14.5),
carotenoids, and hemes, have transitions corresponding
to the visible and near-infrared regions as predicted by
the particle-in-a-box model (Chapter 13). Remember that
the color that you see is what the molecule does not absorb.
For example, a flower is red when it absorbs blue light
and reflects red light.
In some cases, the absorption spectrum may contain
more than one contribution. This may occur if a reaction
is occurring in the sample. Alternatively, if a protein has
a metal cofactor that can be in different redox states, it

Figure 14.4A sketch of a simple version of a double-beam ultraviolet–
visible absorbance spectrophotometer with a sample compartment (S), a
reference compartment (R), and photodetector (PD).

CHAPTER 14 OPTICAL SPECTROSCOPY 295

D 2 lamp

Tungsten bulb

R

S

PD

A to D

converter

A to D

converter

Print

PD

Monochromator

700 800 900 1000

Wavelength (nm)

Figure 14.5

The absorbance

spectrum of a

bacterial reaction

center (see

Chapter 20).