AN ASSIGNMENT ON UV-

SPECTROMETER
Course Name: Pharmaceutical Analysis - ll
Course ode: BPM-321

Submitted to
MOHAMMAD ANWAR HOSSAIN
Lecturer
Dept. of Pharmacy
Manarat International University

Submitted by
MD. MUSTAFIZUR RAHMAN
ID: 0813BPM00379
Student
Dept. of Pharmacy
Manarat International University

Submission Date 25th May 2011

MANARAT INTERNATIONAL UNIVERSITY
Introduction:
Ultraviolet and visible spectrometers have been in general use for the last
35 years and over this period have become the most important analytical
instrument in the modern day laboratory. In many applications other
techniques could be employed but none rival UV-Visible spectrometry for
its simplicity, versatility, speed, accuracy and cost-effectiveness.

Ultraviolet (UV) spectroscopy is a method of determining which

wavelengths (colors) of visible light a sample absorbs or emits. There are
three main types of UV spectroscopy: transmittance, absorbance, and
emission.

Transmittance and absorbance spectroscopy are opposites of one another.

In transmittance spectroscopy, different wavelengths of monochromatic
light are shot at a sample, and the wavelengths that do not interact with the
sample are measured by a detector on the other side of the sample. In
absorbance spectroscopy, the light that is absorbed (that is to say, the light
that does interact with the sample) is measured using the same setup. If one
were to take a graph of transmittance and invert it, it would be an
absorbance.graph.

Emission is done in a slightly different way. Often, when molecules absorb

light, they are put into a higher energy level (in one way or another,
rotationally, vibrationally, electronically, etc.) which is less stable than their
ground state. When the molecule returns to its ground state, it releases
light of equal wavelength to that it absorbed. The prime difference between
the light that is emitted and the light that was absorbed is direction.
Emitted light can be released in any direction. Thus, in order to avoid
measuring light from the source, rather than only light that has been
emitted, detectors in emission spectroscopy are often put at a 90 degree
angle from the source light beam.

Principle:
A spectrophotometer consists of two instruments, namely a spectrometer
for producing light of any selected color (wavelength), and a photometer
for measuring the intensity of light. The instruments arc arranged so that
liquid in a cuvette can be placed between the spectrometer beam and the
photometer. The amount of light passing through the tube is measured by

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the photometer. The photometer delivers a signal to the display. The signal
changes as the amount of light absorbed by the liquid change.
Theory:
When monochromatic light (light of a specific wavelength) passes through
a solution (compound must be unsaturated and conjugated)

Fig : Acyclic butadiene

there is usually a quantitative relationship (Beer's law) between the solute

concentration and the intensity of the transmitted light, that is,

It=I010-kcl
where It sub I0 is the intensity of transmitted light using the pure solvent, I
is the intensity of the transmitted light when the colored compound is
added, c is concentration of the colored compound, I is the distance the
light passes through the solution, and k is a constant. If the light path I is a
constant, as is the case with a spectrophotometer, Beer's law may be
written,
It/ I0=10-kc

where k is a new constant and A is the absorbance of the solution. There is

a logarithmic relationship between Absorbance and the concentration of
the colored compound. Thus,

log It/ I0 =-kc

or, A=kc
( Absorbance A = log It/ I0 & Avoid negative symbol)

Aαc (k is constant)

The Absorbance is directly proportional to the concentration of the colored

compound. Most spectrophotometers have a scale that reads both in

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absorbance units, which is a logarithmic scale, and in % transmittance,
which is an arithmetic scale.

Instrumentation:
1. Diagram of a double-beam UV-Vis. spectrophotometer

Fig: Diagram of a double-beam UV-Vis. spectrophotometer

2. Description:

The light source

Need a light source which gives the entire visible spectrum plus the near
ultra-violet so that you are covering the range from about 200 nm to about
800 nm. (This extends slightly into the near infra-red as well.)

It can't get this range of wavelengths from a single lamp, and so a

combination of two is used - a deuterium lamp for the UV part of the
spectrum, and a tungsten / halogen lamp for the visible part.
The combined output of these two bulbs is focused on to a diffraction
grating.

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The diffraction grating and the slit:

It probably familiar with the way that a prism splits light into its
component colors. A diffraction grating does the same job, but more
efficiently.

The blue arrows show the way the various wavelengths of the light are sent
off in different directions. The slit only allows light of a very narrow range
of wavelengths through into the rest of the spectrometer.

By gradually rotating the diffraction grating, it can allow light from the
whole spectrum (a tiny part of the range at a time) through into the rest of
the instrument.

The rotating discs:

This is the clever bit! Each disc is made up of a number of different

segments. Those in the machine we are describing have three different
sections - other designs may have a different number.

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The light coming from the diffraction grating and slit will hit the rotating
disc and one of three things can happen.

1. If it hits the transparent section, it will go straight through and pass

through the cell containing the sample. It is then bounced by a mirror
onto a second rotating disc.

This disc is rotating such that when the light arrives from the first
disc, it meets the mirrored section of the second disc. That bounces it
onto the detector.

It is following the red path in the diagram:

2. If the original beam of light from the slit hits the mirrored section of
the first rotating disc, it is bounced down along the green path. After
the mirror, it passes through a reference cell (more about that later).

Finally the light gets to the second disc which is rotating in such a
way that it meets the transparent section. It goes straight through to
the detector.

3. If the light meets the first disc at the black section, it is blocked - and
for a very short while no light passes through the spectrometer. This
just allows the computer to make allowance for any current
generated by the detector in the absence of any light.

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The sample and reference cells:

These are small rectangular glass or quartz containers. They are often
designed so that the light beam travels a distance of 1 cm through the
contents.

The sample cell contains a solution of the substance you are testing -
usually very dilute. The solvent is chosen so that it doesn't absorb any
significant amount of light in the wavelength range we are interested in
(200 - 800 nm).

The reference cell just contains the pure solvent.

The detector and computer:

The detector converts the incoming light into a current. The higher the
current, the greater the intensity of the light.

For each wavelength of light passing through the spectrometer, the

intensity of the light passing through the reference cell is measured. This is
usually referred to as Io - that's I for Intensity.

The intensity of the light passing through the sample cell is also measured
for that wavelength - given the symbol, I.

If I is less than Io, then obviously the sample has absorbed some of the light.
A simple bit of math’s is then done in the computer to convert this into
something called the absorbance of the sample - given the symbol, A.

For reasons which will become clearer when we do a bit of theory on

another page, the relationship between A and the two intensities is given
by:

On most of the diagrams you will come across, the absorbance ranges from
0 to 1, but it can go higher than that.

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An absorbance of 0 at some wavelength means that no light of that
particular wavelength has been absorbed. The intensities of the sample and
reference beam are both the same, so the ratio Io/I is 1. Log10 of 1 is zero.

An absorbance of 1 happens when 90% of the light at that wavelength has

been absorbed - which means that the intensity is 10% of what it would
otherwise be.

In that case, Io/I is 100/I0 (=10) and log10 of 10 is 1.

The chart recorder

Chart recorders usually plot absorbance against wavelength. The output

might look like this:

This particular substance has what are known as absorbance peaks at 255
and 395 nm. How these arise and how they are interpreted are discussed
on another page.

Operation Procedure:
1. Make sure the unit is plugged in.
2. Turn on the unit (switch is on the back of the instrument on the right
side).
3. Flip up the display screen on the front of the instrument.
4. Instrument will go through a series of diagnostics. "Multi-CellOrg."
diagnostic will fail. Proceed to the next step.
5. Allow the UV source to warm up for a few minutes in order for the
lamp energy to stabilize.

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 6. Press the number 1 on the keypad to select "Photometric
Measurement".
7. Press the GOTO l, button. A cursor will appear at the bottom of the
display screen.
8. Enter the desired wavelength using the numeric keys. Press Enter.
The wavelength will appear in the centre of the display. (If the
wavelength is entered incorrectly, repeat steps 7-8.)
9. Press the Fl button to toggle between absorbance and o/o
transmittance modes. Abs will appears on the display.
10. Fill two plastic cuvette (for aqueous solutions) or quartz cuvette (for
organic solutions) about 50% filI with solvent. One is sample and one
is reference solution
11. Place those solution in the sample and reference compartment. The
cuvette will not go down that far. Not push down very hard on the
cuvette.
12. Make sure the sample compartment door is closed.
13. Press the AUTO ZERO button to "zero" the spectrophotometer.
14. The reading will appear in the centre of the display.
15. Turn off the unit (switch is on the back of the instrument on the
right side).
16. Flip down the display screen on the front of the instrument.

Trouble Shooting:
Possible Causes Action

Light path is blocked. Ensure that the light path is free and
clear of all obstructions

Cuvette or flow cell not installed Check and correct cell installation.
correctly.

Air bubble sticking on the quartz Use cell cleaning fluid to passivity
window of the flow cell. flow cell.

Source lens or spectrograph lenses Clean lenses.

dirty or fogged.

Shutter not functioning or partly Exchange shutter assembly.

blocking light.
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Spectrophotometer data acquisition Exchange SDA board.
(SDA) board may be defective.

Spectrophotometer lamp supply Exchange SLS board.

(SLS) board may be defective.
Possible Causes
Action

Main power supply (MPS) may be Exchange MPS.

defective.

Spectrograph electronics may be Exchange the optical unit.

defective.

One of the lamps may be turned off Switch on proper lamp.

when making measurements.
Measurements taken with the
deuterium lamp off exhibits excessive
noise in the UV wavelength range.
Measurements taken with the
tungsten lamp off exhibits excessive
noise in the visible wavelength range.

Noise in the UV wavelength range Exchange the lamp. Lifetime of the

may be caused by a weak or defective deuterium lamp may be influenced by
deuterium lamp. the number of ignitions.

Flow cells and cuvettes, which reduce Change cell to standard type. Glass
and/or distort the colimated light absorbs in the low UV region and
beam used in the spectrophotometer, causes high noise. Make sure that
can result in low lamp intensity at the your flow cells and cuvettes are made
spectrograph. from quartz

Solvent or buffer blocks light in a If information is required in such a

certain wavelength range. wavelength range, a solvent or buffer
that is transparent in that range

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 should be used.

Fingerprints on cuvettes or flow cells Clean your flow cells or cuvettes with
typically absorb light in the UV range a lens cleaning tissue.
of the spectrum..

Source lens or spectrograph lenses Clean lenses.

dirty or fogged.

Spectrophotometer lamp supply Exchange SLS board.

(SLS) board may be defective.

Remedies:
1. Operators must be wear hand gloves.
2. UV- Spectrometers power plug connect properly.
3. Set λmax properly for specific compound.
4. Cuvette tube must be cleaned with distilled water.
5. High concentrated liquid must not be used, e.g.: Semisolid,
suspension etc.
6. Taking absorbance from the display properly up to four digits after
decimal.
7. Work properly & sincerely .

Application:
1. Determination of various chemical compound purity test of various
active pharmaceutical ingredients (API)
2. Determination of amount and concentration of API in various
pharmaceutical dosage form
3. The Determination of Food Colors in Mixtures
4. Thermal Analysis of DNA
5. Kinetics Rate Law Investigations
6. Multi-Component Analysis of a Vitamin B Mixture
7. Automation of Good Manufacturing Practice Requirements
8. Automated Measurement of the Color of Red Wines

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 9. Fully Automated Water Nitrate Analysis and etc.

Conclusion:
Following the application we can say that UV-Spectrometer is one of the
most important analyzing and testing instrument. All pharmaceutical,
chemical industry and research institutes use it for their wide range of
testing and identifying.

Reference:
1. Khandpur RS. (2007) Hand Book of Biomedical Instrumentation 2 nd Ed,
Tata McGraw Hill.
2.Boyer, R. (2006) Biochemistry Laboratory, Modem Theory and
Techniques, Pearson Benjamin Cummings, p. 213 -25 I
3. http://www.en.wikipedia.org
4. Skoog, D, et al. "Fundamentals of Analytical Chemistry," 8ft Edition,
Thomson, 2004
5.http://www.chemguide.co.uk/analysis/uvvisible/spectrometer.html
6.http://wiki.answers.com/Q/What_is_UV_spectroscopy#ixzz1MZb172Es
7.http:// www.chem.agilent.com

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