62

3.2  Basic UV-VIS-IR Absorption, Emission, and Elastic Light Scattering Methods

Conventional FTIR is limited by a combination of factors including an inevitable trade-​off

between acquisition time and SNR and the fact that there is no spatial localization informa­

tion. However, recent developments have utilized multiple intense, collimated IR beams

from synchrotron radiation (see Chapter 5). This approach permits spatially extended detec­

tion of IR absorption signals across a sample allowing diffraction-​limited time-​resolved high-​

resolution chemical imaging, which has been applied to tissue and cellular samples (Nasse

et al., 2011).

Many biomolecules that contain chemical bonds that absorb in the IR will also have a

strong Raman signal. The Raman effect is one of the inelastic scattering of an excitation

photon by a molecule, resulting in either a small increase or decrease in the wavelength of the

scattered light. There is a rough mutual exclusion principle, in that strong absorption bands

in the IR correspond to relatively weak bands in a Raman spectrum, and vice versa. Raman

spectroscopy is a powerful biophysical tool for generating molecular signatures, discussed

fully in Chapter 4.

Long UV light (~200–​400 nm) is also a useful spectrophotometric probe especially for

determining proteins and nucleic acid content in a sample. Peptide bonds absorb most

strongly at ~280 nm wavelength, whereas nucleic acids such as RNA and DNA have a peak

absorption wavelength of more like ~260 nm. It is common therefore to use the rate of absorp­

tion at these two wavelengths as a metric for protein and/​or nucleic acid concentration. For

example, a ratio of 260/​280 absorbance of ~1.8 is often deemed as “pure” by biochemists for

DNA, whereas a ratio of ~2.0 is deemed “pure” for RNA. If this ratio is significantly lower, it

often indicates the presence of protein (or potentially other contaminants such as phenol that

absorb strongly at or near 280 nm). With suitable calibration, however, the 260 and 280 nm

absorption values can be used to determine the concentrations of nucleic acids and proteins

in the absence of sample contaminants.

In basic spectrophotometers, the transmitted light intensity from the sample is ampli­

fied and measured by a photodetector, typically a photodiode. More expensive machines will

include a second reference beam using an identical reference cuvette with the same solvent

(generally water, with some chemicals to stabilize the pH) but no sample, which can be used as

a baseline against which to reference the sample readings. This method finds utility in meas­

uring sample density containing relatively large biological particulates (e.g., cells in suspension,

to determine the so-​called growth stage) to much smaller ones, such as molecules in solution.

To characterize attenuation, if we assume that the rate absorption of light parallel to the

direction of propagation, say z, in an incrementally small slice through the sample is propor­

tional to the total amount of material in that thin slice multiplied by the incident light inten­

sity I(z), then it is trivial to show for a homogeneous tissue:

(3.3)

I z

I

Cz

( ) = ( )

−

(

)

0 exp

(

σ λ

)

This is called the “Beer–​Lambert law,” a very simple model that follows from the assumption

that the drop in light intensity upon propagating through a narrow slice of sample is

TABLE 3.1  Common Covalent Bonds in Biological Matter, Which

Absorb Infrared Electromagnetic Radiation, with

Associated Typical Absorption Ranges Indicated

Peak IR Absorption Range (cm⁻¹)

Bond in Biomolecule

730–​770

C—​H

1180–​1200

C—​O—​C

1250–​1340

C—​N

1500–​1600

C=​C

1700–​1750

C=​O

2500–​2700 (and other peaks)

O—​H

3300–​3500 (and other peaks)

N—​H