1. [SALUM MKATA B.PHARM 3]
2014
1
DATE: May 28, 2014.
ULTRA-VIOLET VISIBLE SPECTROSCOPY PRACTICAL REPORT:
Aim: To apply the Beer-Lambert relationship to an aqueous solution containing
an absorbing substance and thus determine its respective concentrations.
INTRODUCTION AND THEORY:
Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-
Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the
ultraviolet-visible spectral region (about 190-820nm).The Ultraviolet-visible
absorption based on molecules containing π-electrons or non-bonding electrons (n-
electrons) which can absorb the energy in the form of ultraviolet or visible light to
excite these electrons to higher anti-bonding molecular orbitals. The more easily
excited the electrons (i.e. lower energy gap between the HOMO and the LUMO), the
longer the wavelength of light it can absorb.
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative
and qualitative determination of different analytes, such as transition metal ions,
highly conjugated organic compounds, and biological macromolecules.
Spectroscopic analysis is commonly carried out in solutions but solids and gases may
also be studied
The ultraviolet region (about 400-190) is particularly important for the qualitative
and quantitative determination of many Organic compounds, especially those with a
high degree of conjugation, also absorb light in the UV or visible regions of the
electromagnetic spectrum. The solvents for these determinations are often water for
water-soluble compounds, or ethanol for organic-soluble compounds. While in the
visible region (about 400-820nm), spectrophotometry methods are widely used for
the quantitative determination of many trace substances, especially inorganic species.
The basic principle of quantitative absorption spectroscopy lies in comparing the
extent of absorption of a sample solution with that of a set of standards under
radiation of a selected wavelength through the application of Beer-Lambert law.
The Beer-Lambert law states that the absorbance of a solution is directly
proportional to the concentration of the absorbing species in the solution and the path
length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine

2. [SALUM MKATA B.PHARM 3]
2014
2
the concentration of the absorber in a solution. It is necessary to know how quickly
the absorbance changes with concentration.
Equation:
A =
Where:
A= absorbance
P= power of transmitted
PO =power of incident
ϵ= extinction coefficient (mol-1
dm3
cm-1
)
b= path length of cell (cm)
c= concentration of absorbing species.
Note: In many cases, the sample compound does not absorb radiation appreciably in
the wavelength provided, it’s then necessary to form an absorbing substance by
reacting a compound in question with other reagents.
What a special about Ultraviolet-visible spectrometer:
The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis
spectrophotometer. It measures the intensity of light passing through a sample ( ),
and compares it to the intensity of light before it passes through the sample ( ). The
ratio is called the transmittance, and is usually expressed as a percentage (%T).
The absorbance, , is based on the transmittance:
The UV-visible spectrophotometer can also be configured to measure reflectance. In
this case, the spectrophotometer measures the intensity of light reflected from a
sample ( ), and compares it to the intensity of light reflected from a reference

3. [SALUM MKATA B.PHARM 3]
2014
3
material ( ) (such as a white tile). The ratio is called the reflectance, and is
usually expressed as a percentage (%R).
The basic parts of a spectrophotometer are; a light source, a holder for the sample,
a diffraction grating in a monochromator or a prism to separate the different
wavelengths of light, and a detector. The radiation source is often a Tungsten
filament (300-2500 nm), a deuterium arc lamp, which is continuous over the
ultraviolet region (190-400 nm), Xenon arc lamp, which is continuous from 160-
2,000 nm; or more recently, light emitting diodes (LED) for the visible wavelengths.
The detector is typically a photomultiplier tube, a photodiode, a photodiode array or a
charge-coupled device (CCD).
Single photodiode detectors and photomultiplier tubes are used with scanning
monochromators, which filter the light so that only light of a single wavelength
reaches the detector at one time. The scanning monochromator moves the diffraction
grating to "step-through" each wavelength so that its intensity may be measured as a
function of wavelength. Fixed monochromators are used with CCDs and photodiode
arrays. As both of these devices consist of many detectors grouped into one or two
dimensional arrays, they are able to collect light of different wavelengths on different
pixels or groups of pixels simultaneously.
Figure.1

4. [SALUM MKATA B.PHARM 3]
2014
4
APPARATUS AND CHEMICAL USED
Apparatus:
1) Spectrophotometer
2) Cuvettes
3) Measuring flask (10mls.)
4)1mls and 10mls capacity pipettes
5) Pipette filler
6) Storage bottle Figure.2 spectrophotometer.
7) Digital mass balance.
8) Test tube rack
9) Beaker.
10) Stopper
11) Stop watch
Figure.3 Digital mass balance
Chemical/ Reagents:
1) Salicylic acid (0.1%)
2) Acetate buffer (0.05M)
3) Distil water

5. [SALUM MKATA B.PHARM 3]
2014
5
PROCEDURES:
1) We turn on the spectrophotometer and allow it to warm up for at least 20 min.
Then we determine the absorption spectrum using the standard acetate buffer.
2) We select one of the cuvettes for the blank solution (in this case acetate buffer)
and we do not interchange it with the other cuvettes also we did not handle the
lower portion of cuvettes through which the light passes.
3) We always rinse the cuvettes with several portions of solution by using acetate
buffer before taking a measurement.
4) Then we wipe the outside of cuvettes with tissue paper.
5) Then we inserted the cuvette into the cell holder with the index line facing us
to avoiding scratching.
6) Then we turn the wavelength control knob to 265nm with blank solution
calibrate the spectrophotometer.
A. Preparation of the solution:
1) We prepared a stock solution of 0.1% salicylic acid by: accurately measuring
100mg of salicylic acid by digital mass balance then we dissolved the obtained
mass in 100ml of acetate buffer in beaker.
2) From that stock solution above we prepared serial dilution containing 0.05%,
0.025%, 0.01%, 0.005%, 0.0025, and 0.001%.(Note all percentages are in
weight by volume ).
We apply dilution law of same substance as:
C1 V1=C2V2
Where:
M1=concentration of concentrated (stock) solution.
V1= volume of concentrated (stock) solution.

6. [SALUM MKATA B.PHARM 3]
2014
6
M2= concentration of diluted solution.
V2= volume of diluted solution.
Note: V2 will be equal to 10ml because we have to diluting the stock solution by
adding sufficient amount of acetate buffer to make a solution of 10ml in measuring
flask. Also C1 and C2 known then V1 obtained by following formula
3) Then we labeled test tube from 1 up to 6. After obtained required volume of
stock solution to be taken from beaker containing stock solution of salicylic
acid. Then we add sufficient amount of acetate buffer to make 10ml solution
results summarized in following table.
TABLE.1;
S/N 1 2 3 4 5 6
Concentration
(%w/v)
0.05 0.025 0.01 0.005 0.0025 0.001
Volume of
stock solution
need(ml)
5.00 2.50 1.00 0.50 0.25 0.10
Volume of
diluted
solution(ml)
10 10 10 10 10 10
4) Then we prepared (standard blank) in which 20ml of acetate buffer is
substituted for the sample.
B. Determination of absorbance
1) We set blank solution in the cuvette and calibrate at wavelength at wavelength
control of 296 nm.
2) Then we inserted cuvette containing the sample- then we read and recorded the
absorbance for all solutions in above section A.
3) Then we determine the absorbance of unknown sample.

7. [SALUM MKATA B.PHARM 3]
2014
7
Results obtained as follows
NOTE: since we have to represents concentration in MOLARITY then:
Concentration in Molarity= Concentration in g/dm3
Molar mass in g/mol
Concentration in g/dm3
:
Since
Y g in 1000 cm3
X g in 100 cm3
Since X is mass of sample in above table then mass Y which is in 1000cm3
can be obtain.
But molar mass of salicylic acid= 138g/mol.
Then we change those concentrations in w/v% in Table 1 above into Molarity
Then we obtain Table below.
TABLE .2;
TRIAL CONCENTRATION(MOL/LITRE) ABSORBANCE
1 7.24 x 10-5
0.007
2 1.81x 10-4
0.003
3 3.62x 10-4
0.007
4 7.24x10-4
0.011
5 1.81x10-3
0.015
6 3.62x10-3
0.018
Unknown Z 0.042

8. [SALUM MKATA B.PHARM 3]
2014
8
DISCUSSION:
We plot the graph of Absorbance vs. concentration in Excel sheet as shown below:
ANSWERS TO GIVEN QUESTIONS AND SOURCES OF ERROR:
1) Yes, it can, if an appropriate mathematical model can be fitted to the data
obtained from standards.
For example the older versions of the Sedex Evaporative Light-Scattering
Detector (ELSD) (image below) gave quadratic response to most analytes, so
one had to use a quadratic fit for the standards, and calculating the
concentration of unknowns required solution of by the quadratic formula.
Figure.4
y = 3.7129x + 0.006
R² = 0.8294
0
0.005
0.01
0.015
0.02
0.025
0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004
Absorbance
concentration (mol/litre)
ABSORBANCE VS. CONCENTRATION (MOLE/LITRE)

9. [SALUM MKATA B.PHARM 3]
2014
9
In NIR spectroscopy, non-linear fit methods such as Principal Component
Analysis and Multiple Linear Regression are often used because the sample
response may not strictly obey Beer's law, especially for reflectance spectra.
2) There no any added advantage of using choosing a wavelength at
absorbance plateau other than the maximum since:
It really depends on what is the largest source of error. Taking the readings at
the peak maximum is best at low absorbances because it gives the best signal-
to-noise ratio, which improves the precision of measurement. If the dominant
source of noise is photon noise, the precision of absorbance measurement is
theoretically best when the absorbance is near 1.0. So if the peak absorbance is
below 1.0, then using the peak wavelength is best, but if the peak absorbance is
well above 1.0, you might be better off using another wavelength where the
absorbance is closer to 1. Another issue is calibration curve non-linearity,
which can result in curve-fitting errors.
The non-linearity caused by polychromatic light is minimized if you take
readings at either a peak maximum or a minimum, because the absorbance
change with wavelength is the smallest at those wavelengths. On the other
hand, using the maximum increases the calibration curve non-linearity caused
by stray light.
But, Very high absorbances cause two problems:
 The precision of measurement is poor because the transmitted
intensity is so low, and the calibration curve linearity is poor due to
stray light. The effect of stray light can be reduced by taking the
readings at a wavelength where the absorbance is lower or by using
a non-linear calibration curve fitting technique. Finally,
 If spectral interferences are a problem, the best measurement
wavelength may be the one that minimizes the relative contribution
of spectral interferences (which may or may not be the peak
maximum). In any case, don't forget: whatever wavelength you use,
you have to use the exact same wavelength for all the standards and
samples.

10. [SALUM MKATA B.PHARM 3]
2014
10
3) Cuvettes may be circular, square or rectangular (the latter being uncommon),
and must be constructed of a material that will transmit both the incident and
emitted light. Square cuvettes or cells will be found to be most precise since
the parameters of path length and parallelism are easier to maintain
during manufacture. However, round cuvettes are suitable for many
more routine applications and have the advantage of being less expensive.
Figure 5.Examples of sample cells for UV/Vis spectroscopy. From left to right
(with path lengths in parentheses): rectangular plastic cuvette (10.0 mm),
rectangular quartz cuvette (5.000 mm), rectangular quartz cuvette (1.000 mm),
cylindrical quartz cuvette (10.00 mm), cylindrical quartz cuvette (100.0 mm).
4) The Beer-Lambert Law will not be obeyed if
 The photons of light striking the detector do not all have an equal chance
of absorption by the sample. This can happen if they have different
absorption coefficients, different path lengths through the sample, or if
they encounter different concentrations of sample molecules. Also
 If anything else is present in the sample that absorbs light or causes
light scattering, the measured absorbance will not be zero when the
analyte's concentration is zero, contrary to Beer's Law.
 If the absorber undergoes any type of chemical reaction or equilibrium
that varies as a function of concentration, Beer's Law will not be obeyed
with respect to the overall or total concentration, because the

11. [SALUM MKATA B.PHARM 3]
2014
11
concentration of the actual absorbing molecule is not proportional to the
overall concentration of the solution. The "c" in Beer's Law refers to the
concentration of just the absorber, not to the total concentration of all the
compounds reacting with or in equilibrium with the absorber. Even if
Beer's Law holds exactly for each individual compound, the total
absorbance of the mixture will not follow Beer's Law with respect to the
total concentration if the proportion of each compound changes with
concentration (unless by chance the absorptivity of all those compounds
happens to be exactly the same).
 Deviations in absorptivity coefficients at high concentrations (> 0.01M)
due to electrostatic interactions. Changes in refractive index at high
analyte concentration.
 Fluorescence or phosphorescence of the sample and Non-
monochromatic radiation.
5) From the graph we obtain the equation below:
Y = 3.7129X + 0.006
Hence the unknown concentration can be obtained since:
Y=value from y-axis i.e. the absorbance
X=value from x-axis i.e. the concentration in Mol/litre
But absorbance (Y value) of unknown sample is obtain which is equal to 0.042
Then we can find the value of unknown concentration!
Take
0.042=3.7129X+ 0.006
Then
0.042-0.006=3.7129X
0.036=3.7129X
Then divide by 3.7129 both side to obtain X=9.696x 10-3
There fore
Unknown concentration in mol/litre of solution is equal to 9.696x 10-3

12. [SALUM MKATA B.PHARM 3]
2014
12
6) The molar extinction coefficient of SA
From below equation
A
Where:
A= absorbance
ϵ= extinction coefficient (mol-1
dm3
cm-1
)
b= path length of cell (cm)
c= concentration of absorbing species.
Take
A1 =0.007 and A2=0.003
Also
C1= 3.62x 10-4
and C2=1.81x 10-4
b =1cm
A1=ϵ1c1b and A2=ϵ2C2b
0.007= ϵ1 x 3.62x 10-4
x1cm …………………………………….eqn.1
0.003= ϵ2x1.81x 10-4
x1cm…………………………………………eqn.2
Then
ϵ1=19.33
ϵ2=16.57
Then molar absorbivity of SA Will be 19.33+16.57=35.9
35.9/2=17.95moll-1
cm-1

13. [SALUM MKATA B.PHARM 3]
2014
13
Source of errors:
1) Stray Light: A problem when working at limits of a spectrometer’s range.
2) Cells and Solvents: Everything else except the sample should be as
transparent as possible.
3) Sample Preparation: If two samples are prepared so that one carries along a
greater concentration of insoluble particulates, then additional scattering will
lead to an apparent greater absorption.
4) Slit Width Affects Absorbance Measurements: If a significant variation in
absorptivity occurs over the spectral bandwidth admitted by the slit, a non-
linear variation (non-Beer’s Law) with concentration will be observed. This
arises because the spectrometer measures the average transmissivity over the
spectral bandwidth, but transmissivity and concentration are not linearly
related. Keep slit width large to increase S/N ratio, but must keep it small
enough to maintain a linear relationship with concentration changes. This
effect is minimized if the absorptivity changes slowly with wavelength. Select
a wavelength near a peak maximum. Use a slit width to provide a bandwidth
about 1/10 the spectral feature width.
5) Wavelength error: In liquids, the extinction coefficient usually changes
slowly with wavelength. A peak of the absorbance curve (a wavelength where
the absorbance reaches a maximum) is where the rate of change in absorbance
with wavelength is smallest. Measurements are usually made at a peak to
minimize errors produced by errors in wavelength in the instrument, that is
errors due to having a different extinction coefficient than assumed.
CONCLUSION, ACKNOWLEDGEMENT AND REFERENCES:
CONCLUSION:
From above we can conclude that UV/Vis spectroscopy is best method
which routinely used in analytical chemistry for the quantitative
determination of different analytes, such as transition metal ions, highly
conjugated organic compounds, and biological macromolecules.

14. [SALUM MKATA B.PHARM 3]
2014
14
Spectroscopic analysis is commonly carried out in solutions but solids and
gases may also be studied.
A UV/Vis spectrophotometer may be used as a detector for HPLC. The
presence of an analyte gives a response assumed to be proportional to the
concentration. For accurate results, the instrument's response to the analyte
in the unknown should be compared with the response to a standard; this is
very similar to the use of calibration curves. The response (e.g., peak
height) for a particular concentration is known as the response factor.
ACKNWOLEDGEMENT:
1. TO MR. EDSON-LAB. TECHNICIAN
2. TO Dr. E. KAALE.
3. TO Dr. SEMPOMBE-HEAD OF DEPARTMENT OF MEDCINAL CHEMISTRY
4. TO MY FELLOW GROUP MEMBER AND THE REST OF CLASS.
REFERENCES:
1) http://en.wikipedia.org/wiki/Molar_extinction_coefficients
2) http://www.thestudentroom.co.uk/showthread.php?t=1424595
3) https://answers.yahoo.com/question/index?qid=20130721231123AABslPR
4) http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_Wit
h_a_Biological_Emphasis/Chapter__4%3A_Structure_Determination_I/S
ection_4.3%3A_Ultraviolet_and_visible_spectroscopy
5) http://en.wikipedia.org/wiki/Cuvette
6) Practical protocol by Department of Medicinal chemistry-School of
Pharmacy, MUHAS.