Experiment 2: Method/Instrumentation

Principles of UV Spectroscopy:

Absorption spectroscopy relates the amount and type of radiant energy absorbed by a
material to its structure, concentration and identity. Electromagnetic radiation is
usually viewed as a stream of discrete packets of energy called photons. According to
quantum mechanics, atoms and molecules can occupy a limited series of energy states
that correspond to distinct energy levels. To be absorbed, the energy of the incoming
radiation must exactly match the difference between two of the substance energy
levels. Since atoms and molecules have mostly unique energy configurations (electron
configurations, vibrational/rotational modes etc.), detection by absorption
spectroscopy can be made specific. Furthermore, since absorption is proportional to
the concentration of sample analyte, spectroscopy can be used to quantify the amount
of material present in an unknown.

UV-Vis spectroscopy is based on the selective absorption of electromagnetic radiation

in the 180-780 nm wavelength range. UV-Vis radiation has sufficient energy to cause
transitions in bonding electrons (as opposed to atomic innershell or valence electrons)
and thus, is correlated best with the behavior of bonds and functional groups in the
analyte. Functional groups having electrons with relatively low excitation energies
absorb strongly in the UV-Vis region, and are called chromophores.

Absorption in the UV-Vis range is due to electrons participating directly in bond

formation or to unshared, outer electrons that are localized about electronegative
atoms such at oxygen, the halogens, sulfur and nitrogen (8). Either type of electron
can be promoted to a higher energy molecular orbital. Molecular orbitals result from
the overlap of atomic electron orbitals during bond formation between two atoms. The
valence electrons of each atom are arranged in molecular orbitals in a way that
minimizes repulsive coulombic forces between neighboring electrons, and between
the positively charged nuclei. When two atomic orbitals combine they form both a
low energy bonding molecular orbital, and a high energy antibonding molecular
orbital (designated by an *). Moreover, s and p atomic orbitals overlap in different
ways. The head-on overlap of two s or two p atomic orbitals is called a sigma bond
and the parallel overlap of two p atomic orbitals is called a pi bond. Single bonds are
usually sigma bonds, where as double bonds contain one sigma bond and one pi bond.
Thus, the net number of molecular orbitals for a double bond are sigma, sigma*, pi,
and pi*. Each of these molecular orbitals represents a different energy level as shown
in Figure 1 (8).
FIGURE 1

Each atom in a organic molecule in its ground state (low energy state) will contain
bonding electrons in sigma and pi molecular orbitals, and outer, nonbonding unshared
electrons (designated by n). The energy relationship between electrons in bonding,
antibonding and nonbonding orbitals is: bonding <="" antibonding="" where="" a=""
bonding="" orbital="" has="" the="" least="" energy,="" and="" is="" most=""
stable.="" there="" are="" four="" possible="" transitions="" for="" configuration=""
discussed:="" sigma="">sigma*, n --> sigma*, n -->pi* and pi --> pi*. Transitions
among these orbitals are brought upon by radiation with energy equaling exactly the
energy difference between the specific orbitals.

Transitions to sigma* orbitals (sigma --> sigma*, n --> sigma*) requires high energy
radiation of wavelengths less than 200 nm which usually is not encompassed in a UV-
Vis spectral analysis. Vacuum UV or X-ray radiation is necessary to cause sigma -->
sigma* transitions. Molecules such as water and methanol absorb maximally at 167-
184 nm due to n --> sigma* transitions.

Transitions to a pi* orbital requires the presence of an unsaturated functional group

(chromophore) to supply the pi* orbitals. Radiation in the 200-700 nm range brings
about these transitions making molecules with chromophores convenient for analysis
using a UV-Vis spectrophotometer. Furthermore, n electrons are very sensitive to the
stabilizing effect of polar solvents making the solvent another factor in identification
and interpretation. Outer nonbonding electrons can form extensive, stabilizing
hydrogen bonds with water and alcohols, whereas inner p electrons are unaffected by
solvent choice (8).

Organic molecules with conjugated double bonds, carbonyl groups, carboxyl groups,
and nitro groups are the best absorbers in the UV-Vis range. Each functional group has
a wavelength associated with an absorption maximum that can be used for qualitative
identification in an unknown sample. The maxima are strongly affected by substituant
groups. For instance, aromatic hydrocarbons have a distinct absorption band at around
280 nm due to a pi --> pi* transition.

Beer's Law:

UV spectroscopy can be used to quantify the amount of an absorbing material present.

The absorbance A of a solution is defined as:

(1) A = -log10T = log10(Po/P)

Where T, the transmittance, is the fraction of the incident radiation that is transmitted
by the solution, P0 is the power of the incoming radiation and P is the power of the
attenuated radiation that has passed through the sample. Absorption is proportional to
the path length b through a sample solution and the concentration c of an absorbing
species according to Beer's Law.

(2) A = ebc

The constant of proportionality in Beer's Law, e, is the molar absorptivity, and is

expressed in units L mol-1 cm-1 when c is expressed in molarity and b in cm. To
avoid reflection and scattering error by the sample holding cell and solvent, the power
of the radiation beam transmitted by the solution is compared to the power of a beam
transmitted by an identical cell containing only the solvent. Thus, Beer's Law is
usually written as:

(3) A = log10(Psolvent / Psolution)

Beer's Law can be derived by considering that the fraction of the incoming beam
absorbed is proportional to the probability of capture by the analyte in solution. The
probability of capture is proportional to the surface area of absorbing particles which
can then be related to the number, and concentration of particles. Using Beer's Law,
the absorbance of a sample can be related to its concentration. Absorbances are
additive, and Beer's Law can be used to determine the concentrations of a mixture of
species, as long as they are not interacting. Beer's Law is limited in that it applies only
to dilute solutions. In concentrated solutions (>0.01 M) particles interact altering the
analyst's ability to absorb a certain wavelength of radiation which causes non linear
deviations from Beer's Law. Changes in the refractive index of the solution, and
chemical reaction will also cause error in applying Beer's Law to a sample. Finally,
Beer's Law is only valid for monochromatic radiation. Thus, the most accurate result
will be obtained by using a source able to produce intense radiation at a single
wavelength.

UV-Visible Spectrophotometers:

Spectrophotometers are made up of stable source of radiant energy, a transparent

sample container, a device for isolating specific wavelength, a radiation detector
which converts transmitted radiation to a usable signal, and a signal processor and
readout. Each of these components will be discussed in reference to the Cary 1-E UV-
Vis Spectrophotometer used in this experiment.

Students measure sample spectra on a Cary UV-Vis Spectrophotometer

Sources
A radiation source for spectroscopy must generate a beam with sufficient power,
wavelength range and stability for detectable and reproducible results. Many UV-Vis
spectrophotometers such as the Cary 1-E, use a deuterium lamp for the UV range and
switch to a tungsten filament lamp at 350 nm for the visible range. The electrical
excitation of deuterium at low pressure results in a continuous spectrum of emitted
radiation from 160 nm to the beginning of the visible (375 nm). An arc is formed
between a heated, oxide-coated filament and a metal electrode. When about 40 Volts
is applied to the heated filament, a direct current is produced resulting in an intense
ball of radiation.

At around 350 nm the Cary 1-E switches its radiation source to a tungsten filament.
The radiant energy emitted from a heated tungsten filament approaches that of a black
body and thus, is temperature dependent. The operating temperature is usually 2870 K
which results in radiation in the 350-2500 nm range. The lower limit is due to
absorption at 350 nm by the glass container housing the filament.
The problem of source stability can be resolved by using a duel beam system where
radiation is directed at a sample and a solvent reference at the same time. Fluctuations
in the source will be canceled. The Cary 1-E utilizes a duel beam system.

Sample containers
Sample containers (cells or cuvettes) must be constructed of a material that is
transparent to radiation in the wavelength range of interest. Containers of quartz or
fused silica are necessary in the UV range, and can be used into 700-3000 nm infra-
red region. Glass and plastic can be used in the visible region as well. To minimize
reflection cuvettes should have windows that are perfectly normal to the direction of
the beam. Quartz cuvettes with a 1 cm path length were used with the Cary 1-E.

Wavelength Selector, Radiation Detector and Signal Processor

A schematic of the Cary 1-E inner workings is shown in Figure 2. Table 1 summarizes
the components of the Cary 1-E.

FIGURE 2
TABLE 1

Number Label Description

D2/QI
1 Radiation Source
lamp
A colored glass filter wheel that isolates a rough region of wavelengths
2 Grating 1
around the desired wavelength.
Entrance Isolates a thin beam of radiation coming from the filter wheel, limits the
3
slit range of incidence angles.
A holographic grating that disperses the radiation allowing a very precise
4 Grating 2 selection of wavelengths. The grating is 30 x 35 mm in size and can move
between wavelengths at 3000 nm/minute.
Radiation from the holographic grating is reflected by a mirror to the exit
slit. The desired wavelength is selected by rotating the grating relative to
5 Exit slit
the exit slit. Radiation of undesired wavelengths is absorbed by black matte
walls surrounding the grating.
Radiation from the exit slit is directed by mirrors at the first chopper. The
chopper consists of three sections as shown in Figure 4 and rotates at 30 Hz.
The chopper has a cut-out section as shown, which allows radiation to pass
through the chopper to a mirror which directs it to the sample cell. The next
6 Chopper 1
section on the chopper is a mirrored which reflects the beam to another
mirror, and then to the reference solvent cell. Light hitting the black section
is absorbed. Thus, the beam hits the sample and reference almost
instantaneously.
Sample The beam is focused on the center of the sample compartment to allow
7
position maximum light throughput and reduce noise.
The second chopper ensures that the sample beam and reference beam hit
the phototube detector at the same position and angle since the detector is
sensitive to these quantities. The mirrored section reflects the sample beam
8 Chopper 2 to another mirror, and then to the phototube detector. The chopper then
rotates to the cut-out section which allows the reference beam through to
the detector. The solid section of the chopper absorbs all radiation and
blocks the beam from hitting the phototube detector.
The phototube detector first corrects for any residual signal in the detector
by zeroing itself during the moment when the black section of the choppers
block the light beam. Next the sample and reference beams are sequentially
9 Phototube measured. The transmission is determined by:
T = (sample - dark signal) / (reference - dark signal)
The transmission signal is sent to a computer where it is converted to
absorbance. The result is show on the CRT.
Interferences:

The most obvious source of error in correlating UV-Vis absorption with DOC is from
nonabsorbing organic material present in the sample. Simple sugars, aliphatic acids,
alcohols and amino acids, such as glycine, do not absorb in the UV range which
results in an underestimation of the DOC (4). However, these compounds are those
most likely to be consumed by microbes as food, and are not expected to be present in
natural water samples from rivers and lakes at high concentrations.

Other possible sources of interference with the desired the UV-Vis spectra can come
from particulate matter, nitrate/nitrite, chlorine, and/or ozone in the sample. Nitrate, a
nutrient ubiquitous to natural water, absorbs at 313 nm due to a n-->pi* transition.
Nitrite also absorbs at 280 nm and 360 nm, but is not usually found in natural water or
treated waste water. The nitrate used to lower the pH of our samples may have
interfered with the absorption spectra by giving a higher absorbance than for sample
alone.

The presence of oxidants such as chlorine or ozone in the samples can cause a
reduction in the absorbance at 254 nm without a corresponding reduction in DOC.
Small amounts of these compounds can partially oxidize some of the NOM to CO2. In
the process, less aromatic, unabsorbing by-products are formed. For example, the by-
products of chlorine addition to natural water can result in di- and tri- chloroacetic
acid (5). It also has been shown in laboratory experiments that ozone addition to
fulvic acid solutions causes a reduction in absorbance at 254 nm (5).

Particulate matter can affect the UV-Vis absorbance by scattering incoming radiation.
Scattering by suspended material in the sample increases with particle size with
maxim scattering, for a given weight of sample, occurring at a particle size equal to
the wavelength of incoming radiation. Therefore, UV radiation is more sensitive to
turbidity as it is scattered by smaller particles (<500 nm diameter) than is radiation of
longer wavelength. Although the natural water sample in this study was unfiltered,
little particulate organic carbon was present, and no particulate material was visible.
Dobbs et. al found that unfiltered, clear samples of natural water were adequate to
preform error-free concentration studies with UV-Vis absorbance (4).

Calibration Procedure:

A standard curve relating A254 to the concentration of KHP in ppm C was plotted as
shown in Figure 3. A typical regression analysis was performed on the data resulting
in the linear curve fit and equations as shown. The relevant parameters are displayed
in the RESULTS section. All errors are reported at the 95% confidence level.

FIGURE 3