Environmental Engineering Reference
In-Depth Information
chemistry laboratory for the identification of biomolecules in complex samples,
including peptides, proteins, oligosaccharides, and oligonucleotides. The first
reports demonstrating successful MALDITof-MS biochemical analysis were
published in the late 1980s from the labs of Tanaka et al. [
8
] and Karas and
Hillenkamp [
9
].
13.2.9 Ultraviolet-Visible Spectroscopy (UV/VIS)
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis
or UV/Vis) refers to absorption spectroscopy in the ultraviolet-visible spectral
region. This means it uses light in the visible and adjacent (near-UV and near-
infrared (NIR)) ranges. The absorption in the visible range directly affects the
perceived color of the chemicals involved. In this region of the electromagnetic
spectrum, molecules undergo electronic transitions. This technique is complemen-
tary to fluorescence spectroscopy, in that fluorescence deals with transitions from
the excited state to the ground state, while absorption measures transitions from the
ground state to the excited state [
10
].
Molecules containing
-electrons or nonbonding electrons (n-electrons) can
absorb the energy in the form of ultraviolet or visible light to excite these electrons
to higher antibonding molecular orbitals [
11
]. The more easily excited the electrons
(i.e., lower energy gap between the HOMO and the LUMO), the longer the
wavelength of light it can absorb.
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantita-
tive determination of different analyses, such as transition metal ions, highly
conjugated organic compounds, and biological macromolecules. Spectroscopic
analysis is commonly carried out in solutions but solids and gases may also be
studied.
• Solutions of transition metal ions can be colored (i.e., absorb visible light)
because d electrons within the metal atoms can be excited from one electronic
state to another. The color of metal ion solutions is strongly affected by the
presence of other species, such as certain anions or ligands. For instance, the
color of a dilute solution of copper sulfate is a very light blue; adding ammonia
intensifies the color and changes the wavelength of maximum absorption (
ˀ
ʻ
max
).
• Organic compounds, especially those with a high degree of conjugation, also
absorb light in the UV or visible regions of the electromagnetic spectrum. The
solvents for these determinations are often water for water-soluble compounds,
or ethanol for organic-soluble compounds. (Organic solvents may have signif-
icant UV absorption; not all solvents are suitable for use in UV spectroscopy.
Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can
affect the absorption spectrum of an organic compound. Tyrosine, for example,
increases in absorption maxima and molar extinction coefficient when pH
increases from 6 to 13 or when solvent polarity decreases.
