Prabhakar Singh- II_SEM-Paper V_COLORIMETER & SPECTROPHOTOMETER

1. TEJASVI NAVADHITAMASTU
“Let our (the teacher and the taught) learning be radiant”
Let our efforts at learning be luminous and filled with joy, and
endowed with the force of purpose
Paper V: Instrumentation and Analytical Techniques
PHOTOMETRY: COLORIMETER & SPECTROPHOTOMETER

2. PHOTOMETRY
COLORIMETER & SPECTROPHOTOMETER

3. ULTRAVIOLET AND VISIBLE LIGHT SPECTROSCOPY
Molecular (sub-)structures responsible for interaction with electromagnetic
radiation are called chromophores. In proteins, there are three types of
chromophores relevant for UV/Vis spectroscopy-
•peptide bonds (amide bond);
•certain amino acid side chains (mainly tryptophan and tyrosine)
•certain prosthetic groups and coenzymes (e.g. porphyrine groups such as in haem)

4. Energy scheme for molecular orbitals (not to scale). Arrows indicate possible electronic
transitions. The length of the arrows indicates the energy required to be put into the
system in order to enable the transition. Black arrows depict transitions possible with
energies from the UV/Vis spectrum for some biological molecules. The transitions shown
by grey arrows require higher energies (e.g. X-rays)

5. Light is electromagnetic radiation and can be described as a wave propagating
transversally in space and time. The electric (E) and magnetic (M) field vectors are
directed perpendicular to each other. For UV/Vis, circular dichroism and fluorescence
spectroscopy, the electric field vector is of most importance. For electron paramagnetic
and nuclear magnetic resonance, the emphasis is on the magnetic field vector.
ELECTROMAGNETIC RADIATION
Electromagnetic radiation is
composed of an electric and a
perpendicular magnetic vector,
each one oscillating in plane at right
angles to the direction of
propagation. The wavelength is the
spatial distance between two
consecutive peaks (one cycle) in the
sinusoidal waveform and is
measured in submultiples of metre,
usually in nanometres (nm). The
maximum length of the vector is
called the amplitude

6. ELECTROMAGNETIC SPECTRUM AND THEIR PROPERTIES
Various spectrometer and their working range in electromagnetic radiations

7. Photometry broadly deals with the study of the phenomenon of light absorption
by molecules in solution. The specificity of a compound to absorb light at a
particular wavelength (monochromatic light) is exploited in the laboratory for
quantitative measurements. Colorimeter and spectrophotometer are the
laboratory instruments used for this purpose. When a light at a particular
wavelength is passed through a solution (incident light), some amount of it is
absorbed and, therefore, the light that comes out (transmitted light) is diminished.
The nature of light absorption in a solution is governed by Beer-Lambert law.
BEER-LAMBERT LAW
Beer’s law states that the amount of transmitted light decreases exponentially
with an increase in the concentration of absorbing material (i.e. the amount of
light absorbed depends on the concentration of the absorbing molecules).
Lambert’s law states that the amount of transmitted light decreases exponentially
with increase in the thickness of the absorbing molecules (i.e. the amount of light
absorbed is dependent on the thickness of the medium).
COLORIMETER & SPECTROPHOTOMETER

8. By combining the two laws (Beer-Lambert law), the following derivation can
be obtaine-
I = I0
ɛcd
where I= Intensity of the transmitted light
I0 = Intensity of the incident light
H= Molar extinction coefficient
c = Concentration of the absorbing substance (moles/l or g/dl)
d = Thickness of medium through which light passes.
Transmittance (T)
When the thickness of the absorbing medium is kept constant (i.e. Lambert’s
law), the intensity of the transmitted light depends only on concentration of
the absorbing material. In other words, the Beer’s law is operative. The ratio
of transmitted light (I) to that of incident light (I0) is referred to as
Transmittance (T).

9. Absorbance (A) or optical density (OD)
Absorbance (A)or optical density (OD) is very commonly used in laboratories.
The relation between absorbance and transmittance is expressed by the
following equation.

10. Deviations from the Beer–Lambert law
According to the Beer–Lambert law, absorbance is linearly proportional to
the concentration of chromophores/ Produced colour. This might not be
the case any more in samples with high absorbance. Every
spectrophotometer has a certain amount of stray light, which is light
received at the detector but not anticipated in the spectral band isolated
by the monochromator.
In order to obtain reasonable signal-to-noise ratios, the intensity of light at
the chosen wavelength should be 10 times higher than the intensity of the
stray light (Istray). If the stray light gains in intensity, the effects measured at
the detector have nothing or little to do with chromophore concentration.
Secondly, molecular events might lead to deviations from the Beer–
Lambert law. For instance, chromophores might dimerise at high
concentrations and, as a result, might possess different spectroscopic
parameters.

11. The absorption spectrum of a chromophore is only partly determined by its chemical
structure. The environment also affects the observed spectrum, which mainly can be
described by three parameters:
•protonation/deprotonation (pH, RedOx);
•solvent polarity (dielectric constant of the solvent); and
•orientation effects.
Protonation/deprotonationarises either from changes in pH oroxidation/reduction
reactions, which makes chromophores pH- and RedOx-sensitive reporters. As a rule of
thumb the sample will displays a batho- and hyperchromic shift, if a titratable group
becomes charged.
Solvent polarity affects the difference between the ground and excited states. Generally,
when shifting to a less polar environment one observes a batho- and hyperchromic effect.
Conversely, a solvent with higher polarity elicits a hypso- and hypochromic effect.
Orientation effects, such as an increase in order of nucleic acids from single stranded to
double-stranded DNA, lead to different absorption behavior.
 A wavelength shift to higher values is called Red Shift or Bathochromic Effect.
 A shift to lower wavelengths is called Blue Shift or Hypsochromic Effect.
 An increase in absorption is called Hyperchromicity (more colour).
 A decrease in absorption is called Hypochromicity (less colour)

12. COLORIMETER
Colorimeter (or photoelectric colorimeter) is the instrument used for the
measurement of coloured substances. This instrument is operative in the visible range
(400-800 nm) of the electromagnetic spectrum of light.
The working of colorimeter is based on the principle of Beer-Lambert law.
The colorimeter, in general consists of light source, filter sample holder and detector
with display (meter or digital). A filament lamp usually serves as a light source. The
filters allow the passage of a small range of wave length as incident light. The sample
holder is a special glass cuvette with a fixed thickness.
The photoelectric selenium cells are the most common detectors used in colorimeter.
Diagrammatic representation of the components in a colorimeter.

13. SPECTROPHOTOMETER/ UV-Visible Spectrophotometer
The spectrophotometer primarily differs from colorimeter by covering the
ultraviolet region (200-400 nm) of the electromagnetic spectrum.
Further, the spectrophotometer is more sophisticated with several
additional devices that ultimately increase the sensitivity of its operation
several fold when compared to a colorimeter.
A precisely selected wavelength (say 234 nm or 610 nm) in both ultraviolet
and visible range can be used for measurements. In place of glass cuvettes
(in colorimeter), quartz cells are used in a spectrophotometer.
The spectrophotometer operation is also based on the Beer-Lambert law.

14. Instrumentation
 UV/Vis spectrophotometers are usually dual-beam spectrometers where the first
channel contains the sample and the second channel holds the control.
 The light source is a tungsten filament bulb for the visible part of the spectrum, and a
deuterium bulb for the UV region. Since the emitted light consists of many different
wavelengths, a monochromator, consisting of either a prism or a rotating metal grid
of high precision called grating, is placed between the light source and the sample.
 In a dual-beam instrument, the incoming light beam is split into two parts by a half
mirror. One beam passes through the sample, the other through a control (blank,
reference). This approach obviates any problems of variation in light intensity, as
both reference and sample would be affected equally.
 Wavelength selection can also be achieved by using coloured filters as
monochromators that absorb all but a certain limited range of wavelengths. This
limited range is called the bandwidth of the filter. Filter-based wavelength selection
is used in colorimetry, a method with moderate accuracy, but best suited for specific
colorimetric assays where only certain wavelengths are of interest. If wavelengths
are selected by prisms or gratings, the technique is called spectrophotometry

15. Optical arrangements in a dual-beam spectrophotometer. Either a prism or a grating
constitutes the monochromator of the instrument. Optical paths are shown as green lines.

16. Applications
The usual procedure for (colorimetric) assays is to prepare a set of standards and produce a
plot of concentration versus absorbance called calibration curve. This should be linear as long
as the Beer–Lambert law applies. Absorbances of unknowns are then measured and their
concentration interpolated from the linear region of the plot.
Qualitative and quantitative analysis
1. Qualitative analysis may be performed in the UV/Vis regions to identify certain
classes of compounds both in the pure state and in biological mixtures (e.g.
protein-bound).
2. The application of UV/Vis spectroscopy to further analytical purposes is used for
quantification of biological samples either directly or via colorimetric assays. In
many cases, proteins can be quantified directly using their intrinsic chromophores,
tyrosine and tryptophan.
3. The region from 500 to 300 nm provides valuable information about the presence
of any prosthetic groups or coenzymes.
4. Protein quantification can also be done by single wavelength measurements at
280 and 260 nm.