Spectrophotometry is a technique that uses the measurement of light absorption to determine the concentration of chemical substances. It operates based on Beer's Law, which states that absorbance is directly proportional to concentration. The methodology involves using a spectrophotometer to measure the intensity of light passing through reference and sample solutions. Applications include concentration measurement, detection of impurities, structure elucidation, and more. Spectrophotometry is a widely used analytical technique in clinical chemistry.

1. SPECTROPHOTOMETRY
Dr. Ofonmbuk Umoh
IN CLINICAL CHEMISTRY

2. OUTLINE
• Introduction
• Operating Principle
• Methodology
• Instruments
• Applications
• Conclusion
• References

3. INTRODUCTION
• Photometry is defined as the measurement of light;
• Spectrophotometry is defined as the measurement of the
intensity of light at selected wavelengths.
• Spectrophotometric analysis is a widely used method of
quantitative and qualitative analysis in Clinical Chemistry.
• Spectrophotometer measures the intensity of the light entering
a sample and the light exiting a sample and compares the two
intensities.

4. …introduction contd
It does this by:
- diffracting the light beam into a spectrum of wavelengths
- Directing it unto an object
- receiving the light reflected or returned from the object
- detecting the intensities with a charge-coupled device
- displaying the results on the detector and then the display device

5. • Spectrophotometer was invented in 1940 by Arnold Beckman and
Howard H Cary at National Technologies Laboratories
• This discovery streamlined and simplified chemical analysis, by
allowing researchers to perform a 99% accurate biological assessment
of a substance within minutes as opposed to the weeks required
previously for results with only 25% accuracy.
…introduction contd

6. …introduction contd

7. Spectrophotometry operates on the principles of Beer-Lambert’s Law. Beer’s Law
states that the Concentration of a substance is directly proportional to the amount of
light absorbed, or inversely proportional to the logarithm of the transmitted light.
The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance
and concentration of an analyte. The general Beer-Lambert law is usually written as:
A = a b c
where A is the measured absorbance, a is the wavelength(λ)-dependent absorptivity
coefficient, b is the light’s path length in cm, and c is the analyte concentration in
moles per litre.
Beer’s Law
OPERATING PRINCIPLE
of Spectrophotometry

8. The linearity of the Beer-Lambert law is limited by chemical and instrumental
factors. Causes of nonlinearity include
• High Concentration: deviations in absorptivity coefficients at high
concentrations (>0.01M) due to electrostatic interactions between
molecules in close proximity
• Scattering of light due to particulates in the sample
• Fluoresecence or phosphorescence of the sample
• Changes in refractive index at high analyte concentration
• Shifts in chemical equilibria as a function of concentration
• Non-monochromatic radiation, deviations can be minimized by using a
relatively flat part of the absorption spectrum such as the maximum of an
absorption band
• Stray light
Limitations of the Beer-Lambert’s Law

9. …operating principle
• The intensity of transmitted light passing through a solution
containing an absorbing substance (chromogen) is decreased by the
absorbed fraction. This fraction is detected, measured and used to
relate the light transmitted or absorbed to the concentration of the
analyte in question.
• If an incident light beam with intensity Io, passes through a square cell
containing a solution of a compound that absorbs light of a certain
wavelength, λ, given that the intensity of the transmitted light beam
is Is, then transmittance (T) of light is defined as: T = Is/Io. Percentage
Transmittance %T = Is/Io. X 100
• …and the amount of light absorbed is expressed as Absorbance, and
related to Transmittance by the equation A = -log T

10. • A portion of the incident light, however,
may be reflected by the surface of the cell
or may be absorbed by the cell wall or
solvent.
• To focus attention on the compound of
interest, elimination of these factors is
necessary. This is achieved using a reference
cell identical to the sample cell, except that
the compound of interest is omitted from
the solvent in the reference cell.
• The transmittance (T) through this
reference cell is IR divided by IO; and the
transmittance for the compound in solution
then is defined as Is divided by IR.
…operating principle

11. …operating principle
• In practice, the light beam is blocked and the detector signal set to
zero transmittance, then a reference cell is inserted and the detector
signal adjusted to an arbitrary scale reading of 100 (corresponding to
100% transmittance), followed by the cell containing the sample to be
measured, and the percentage transmittance reading is made on the
sample.
• Modern absorption instruments can usually display the data as either
transmittance, %-transmittance, or absorbance. An unknown
concentration of an analyte can be determined by measuring the
amount of light that a sample absorbs and applying Beer's law. If the
absorptivity coefficient is not known, the unknown concentration can
be determined using a working curve of absorbance versus
concentration derived from standards.

12. METHODOLOGY
• Turn the switch at ‘ON’ and “Warm up” the instrument for about 15 min.
• Zero adjust to read infinite absorbance (Zero % transmittance). With no
cuvette in the chamber, a shutter cuts off all light from passing thru the
cuvette chamber.
• Adjust wavelength control to read the wavelength at which the test is
desired.
• Blank, Control & Standard adjustments: fill the blank cuvette with the
solvent used to dissolve specimen. Polish to clean, insert into the cuvette
chamber, aligning mark to front of light source. Close chamber cover.
• Sample 1 & 2: Then, fill the cuvette with the desired specimen, insert into
the cuvette chamber, aligning mark to front of light source. Close chamber
cover.

13. INSTRUMENTATION

15. …instruments
• Light source: provide a sufficient source of light which is
suitable for marking a measurement.
• The light source typically yields a high output of polychromatic
light over a wide range of the spectrum.
Types of light source
INCANDESCENT LAMPS: Tungsten light, Hydrogen light, Deuterium light
LASER light – extremely intense, focused & nearly non-divergent beam
of monochromatic light

16. Light spectrum

17. …instruments
• Monochromator: Accepts polychromatic input light from a
lamp and puts out monochromatic light.
• Monochromator consists of these parts:
I.Entrance slit
II.Collimating lens or mirror
III.Dispersion element
IV.Focusing lens or mirror
V. Exit slit
9

18. …instruments
10
• Dispersion devices: A special plate with
hundreds of parallel grooved lines.
• The grooved lines act to separate the white light
into the visible light spectrum.
The more lines
the smaller
the wavelength
resolution.

19. …instruments
• Focusing devices: Combinations of lenses, slits, and mirrors.
• relay and focus light through the instrument.
11

20. …instruments
• Cuvettes: (also cuvet) designed to hold samples for
spectroscopic experiments. made of Plastic, glass or silica
• should be as clear as possible, without impurities that might
affect a spectroscopic reading.

21. …instruments
• Detectors: Convert radiant energy (photons) into an electrical
signal.
The photocell and phototube are the simplest photodetectors, producing
current proportional to the intensity of the light striking them.

22. …instruments
• Display devices: The data from a detector are displayed by a
readout device, such as an analog meter, a light beam reflected on a
scale, or a digital display, or LCD .
• The output can also be transmitted to a computer or printer.
14

23. Single & Double Beam Spectrophotometer

24. • SINGLE BEAM;
Advantage include: low cost, high throughput and hence high sensitivity
because of optical system is simple.
Disadvantage include: An appreciable amount of time elapses between taking
the reference and making the sample measurement such that there can be
problems with drift.
• DOUBLE BEAM: this is designed to eliminate drift by measuring blank and
sample virtual simultaneously.
Advantage: High stability because reference and sample are measured
virtually at the same moment in time.
Disadvantages include: Higher cost, lower sensitivity as throughput of light is
poorer because of the optical complexity.

25. APPLICATION of Spectrophotometry
• Spectrophotometry has a wide range of application in Clinical
Chemistry

26. …applications
1. Concentration measurement
–
–
Prepare samples
Make series of standard solutions of known concentrations

27. − Set spectrophotometer to the λ of maximum light
absorption
− Measure the absorption of the unknown, and
from the standard plot, read the related
concentration.
…applications

28. 17
2. Detection of Impurities
•UV absorption spectroscopy is one of the
best methods for determination of impurities in
organic molecules.
Additional peaks can be
observed due to impurities
in the sample and it can be
compared with that of
standard raw material.
…applications

29. 3. Structure elucidation of organic compounds.
•From the location of peaks and combination of
peaks UV spectroscopy elucidate structure of
organic molecules:
othe presence or absence of unsaturation
…applications

30. 4. Chemical kinetics
•Kinetics of reaction can also be studied using UV
spectroscopy. The UV radiation is passed through
the reaction cell and the absorbance changes can
be observed.
…applications

31. 5. Detection of Functional Groups
•Absence of a band at particular wavelength
is regarded as an evidence for absence of
particular group
…applications

32. 6. Molecular weight determination
•Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable
derivatives of these compounds.
•For example, if we want to determine
the molecular weight of amine then it is converted in to
amine picrate.
…applications

33. CONCLUSION
• Spectrophotometric analysis is a widely used method of quantitative
and qualitative analysis in Clinical Chemistry. It is a veritable
diagnostic technique for rapid turnover of analytes

34. THANK YOU
FOR
LISTENING

35. REFERENCES
1. Tietz Textbook of Clinical Chemistry & Molecular Medicine 6th Edition
2014
2 Clinical Chemistry – Principles, Techniques, Correlations; Bishops; 7th
Edition 2015
3. Fundamentals of UV-visible spectroscopy, Tony Owen, 1996
4. UNIT: Spectrophotometry, Clinical Chemistry Lab Manual