Principles of Spectrophotometric Methods

UV-Vis
Basic principles

Spectrophotometry: any procedure that uses light to measure

concentration of an analyte.

Properties of light: described in terms of both particles and

waves.
Light waves consist of perpendicular, oscillating electric and
magnetic fields.

Electomagnetic radiation

 = c
Wavelength, ; frequency, ; speed of light, c

Interactions between atoms and molecules and the electric

and magnetic field vectors are possible
 With regard to energy, it is more convenient to think of light
as particles called photons. Each photon carries the energy, E,
which is given by:

E = h

Where h is Plank’s konstant (= 6.626 x 10-34 J.S)

Changes in atomic or molecular energies are associated with absorption,
emission and scattering of photons

UV-Vis spectrophotometry: uses ultra violet or visible light,

corresponding to electron transition in molecules or atoms.

Fig 18-2. Molecular processes occurring at absorption of electromagnetic

radiation of different wavelength.

There are several other important techniques using electromagnetic radiation with wavelengths from
radio- to X-ray range (NMR, NIR, FTIR, XAFS, etc), although these will not be covered here.
The colour of a solution is the complement
of the colour of the light that it absorbs. The
colour we perceive depends not only on the
wavelength of light, but on its irradiance
(intensity).
Molecular orbital diagram of formaldehyde
Possible Electronic States Resulting
from an n → * transition a) excited
singlet state, S1 and b) excited triple
state, T1
What happens to the energy absorbed by the
molecule?

When a molecule absorbs a photon, the energy of the

molecule increases. We say that the molecule is promoted to
an excited state. If a molecule emits a photon, the energy of
the molecule is lowered. The lowest energy state of a
molecule is called the ground state.

Combined Electronic, Vibrational, and Rotational Transitions

When a molecule absorbs light having sufficient energy to cause an
electronic transition, vibrational and rotational transitions (changes
in the vibrational and rotational states) occur as well.
3 principally different ways to measure the concentration of chemical
substances by light:

Absorption (by ground-state molecules or atoms)

Emission (by nonradiational excited molecules or atoms, i.e. analytes

excited by heat or chemical reactions)

Luminescence (by radiational excited-state molecules or atoms)

Fluorescence
Phosphorescence
 The first process after absorption is vibrational
relaxation to the lowest vibrational level.
 In this radiationless transition (R1 in figure 17-
15) vibrational energy is transferred to other
molecules (ex. solvent) through collisions, not
by emission of a photon.
 As an effect part of the energy of the absorbed
photon is converted into heat that is spread
through the entire medium.
 The relative rates of internal conversion (IC),
intersystem crossing (ISC), fluorescence, and
phosphorescence depend on the molecule, the
solvent, and conditions such as temperature and
pressure.
 The energy of phosphorescence is less than the
energy of fluorescence i.e., phosphorescence
comes at longer wavelengths than fluorescence.
 Schematic diagram of a Single Beam Spectrophotometer for
Absorbance measurements

P
Transmittance T
P0
defined as the fraction of light passing through the sample cuvette,
often expressed as %.

P 
Absorbance A  log 0    log T
P
absorbance is often used since it is linearly proportional to concentration (see Box
17-1, page 397).

Beer´s law A    bc
Where  is the molar absorptivity (M-1cm-1), b the path length (cm) and c the
concentration (M)

Beer’s law (Beer’s model) is an approximation with limited validity. The

model requires several conditions to be fulfilled, e.g.

• Monochromatic light
• Dilute solutions
Although UV-Vis uses electron transitions for measurements,
very broad absorption spectra are obtained due to vibration and
rotation band in molecules.

Limited resolution of UV-Vis spectrophotometers makes these

transitions unresolved. In addition the orbitals/energy levels are
shifted by interactions with solvent and matrix compounds.

This gives poor selectivity for UV-Vis spectrophotometry,

especially in the UV-range.

Often analytes are reacted with compounds to form coloured

complexes, absorbing light in the visible region, prior to
measurements, e.g. Fe2+ (below), NO2- / NO3-, PO43-.

Fig 18-7. Visible absorption spectra for of the complex (ferrozine)3Fe(II).

Luminescence measurements

Most commonly excited molecules return to ground state by

radiationless relaxation, e.g. via vibration and rotation transition,
converting the excess energy to heat.

However, emission of light (photons) also occurs, which is used in

fluorescence and phosphorescence measurements. These processes are
inherently more sensitive as the emitted light is measured against a dark
background (in contrast to absorbance measurements).

Fig 18-19. Schematic diagram of luminescence spectrophotometer.

The relationship

I   k P0 c
is valid for certain conditions and especially at low analyte
concentrations. At high concentrations so called “self-absorption” of
emitted light causes problems. k is a constant including, among other
things, the observation volume and the probability of transition at .
Applications of Spectrophotometry
UV-Vis spectrophotometry is not only important for quantitative chemical
analysis, but also a useful tool for fundamental studies in inorganic-, physical-
and biochemistry.
Chapter 19 describes several examples of this, and also the analysis of mixtures
of several analytes with overlapping spectra.

Flow Injection Analysis (FIA)

Fig 19-9. Schematic diagram of FIA systems.

UV-Vis is a common detector in FIA-systems, although FIA can be used with any
continuously measuring detector. The analysis of NO2- / NO3- in environmental control
laboratories is one example where FIA with UV-Vis detection is used. FIA allows
automation and fast analysis.

Immunoassays

Fig 19-12 and 13. Schematic diagrams ofenzyme-linked immunosorbent assay.

Highly selective, fast and simple analytical techniques.

Pregnancy test is one example where immunoassays are used.
Instrumentation spectrophotometers

Single-beam spectrophotometer for absorbance measurements (Fig 18-4)

Double-beam spectrophotometer (Fig 20-1)

Interior of double beam spectrophotometer (Fig 20-2).

Light sources
Deuterium arc lamp

Tungsten filament lamps

Fig 20-3. Emission spectra of deuterium and tungsten lamps.

Monochromators
Monochromators are used to select which wavelength interval to use for
measurements.

A grating, consisting of closely spaced grooves, is used to disperse light of

different wavelength (in simple ectrometers prisms or filters are used instead).

Concave mirrors are used to collimate (make parallel) the light beams before
hitting the grating and, after wavelength dispersion, to focus each wavelength
at a different point in the focal plane.

Only the wavelength interval that passes through the exit slit is used for
measurements.

By rotating the grating different wavelengths are projected on the exit slit.
Fig 20-5.Czerny-Turner grating monochromator.

The wavelength resolution of the monochromator depends on the dispersion

capability of the grating, focal length and slit width.

The refraction grating is ruled with a series of closely spaced parallel groves
with distance d.

Constructive interference occurs when the difference in length of the two

paths is an integran multiple of the wavelength of light.

n = a + b whre n is the diffraction order

Grating equation: n = d(sin + sin)

where d is the distance between adjucent groves.
Resolution of grating:

/ = nN
Where  is wavelength, n is the diffraction order, and N is the
number of prooves of the grating that are illuminate.

The more grooves in a grating, the better the resolution

between closely spaced wavelengths:

Ex. if we need to resolve lines that are 0.05 nm apart

at a wavelength of 500 nm, the required resolution is
/ = 500 nm/0.05 nm = 104.

Dispersion: a measure of the ability to separate

wavelengths differing by  through the difference in
angle, .

Dispersion of grating:

 / = n/dcos
where  is the angular dispersion and n is the difraction
order
The effect of changing slit width
The slit width is normally variable on monochromator instruments and can be changed
to change the resolution of the spectrometer to suit different applications.
Unfortunately, decreasing the slit width does not only increase resolution, but does also
decrease the light throughput of the monochromator. This deteriorates the signal-to-noise
ratio and thereby detection limit.

The wider the exit slit (in figure 20-5) the wider the band of wavelengths selected by the
monochromator.

A wide slit increases the energy reaching the detector and gives a high signal-to-noise
ratio, leading to good precision in measuring absorbance.

A monochromator bandwidth that is 1/5 as wide as the absorption peak generally gives
acceptably small distortion of the peak shape.
 Detectors
Photomultiplier tube
Emits electrons from a photosensitive, negatively charged surface (the cathode)
when struck by visible light or ultraviolet radiation.

Electrons emitted from the photosensitive surface strike a second surface, called
dynode, which is positive with respect to the photosensitive emitter.

This process is repeated several times, so more than 10 6 electrons are finally
collected for each photon striking the first surface.
As a result extremely low light intensities are translated into measurable electric
signals.

Fig 20-12. Photomultiplier detector

The detector response depends on the wavelength of the incident photons

Photodiode Array

Fig 20-14. Photodiode array spectrometer

In a diode array spectrophotometer all wavelengths are recorded

simultaneously, allowing rapid acquisition of the spectrum

Each diode receives a different band of wavelengths, and all

wavelengths are measured simultaneously

Resolution depends on how closely spaced the diodes are and how
much dispersion is produced by the polychromator

Photodiode array instruments have almost no moving parts

Stray light

Light, of wavelengths outside the bandwidth expected from the

monochromator, that reaches the detector.

 Stray light coming through the monochromator from the

light source arises from
o diffraction into unwanted orders and angles
o unintended scattering from optical components and
walls
 Stray light coming from outside the instrument, if the
sample compartment is not perfectly sealed.

Practical aspects

Selection of spectral band pass:

The spectral bandwidth of emitted light should typically be set to ≤

1/5 of the half-width of the absorbance peak for quantitative
measurements and typically ≤ 1/10 for determinations of . This
will be further illustrated in your laboratory work (see also Fig 20-
8).

• Keep spectrometer/cuvet house closed to external light

• Prevent dust from entering spectrometer and cuvets
• Avoid fingerprints on cuvet!!!!!!
• Insert the cuvet reproducible in the spectrometer and
never rotate it 180 between measurements

Exercises and problems- Spectrophotometry

Harris 8th edition or (7th edition) 17-19 (18-19), 17-24 (18-22), 17-25 (18-23), 19-1 (20-1), 19-4 (20-4),
19-5 (20-5), 19-6 (20-6), 19-11 (20-10), 19-12a (20-11)