DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404
Instrumentation: spectrophotometer

Objective:
 To learn how to use a simple spectrophotometer
 To learn how to evaluate a spectrophotometer

Principles:
• Checking a spectrophotometer for stray light
Stray light is a radiation at any wavelength other than the required
wavelength. The most common cause of stray light are reflections of light
from scratches on optical surfaces or dust particles anywhere in the light
path and higher order spectra produced by diffraction gratings. The
major effect is absorbance error, especially in the high absorbance range.

• The two methods employed on checking a spectrophotometer for a stray

light is:

1. Using an aqueous solution of sodium nitrite, 5g/100ml. The

percentage transmission read at 340nm is a direct measurement of
the stray light of the instrument. Ideally the value should be 1% or
less
2. Spectro grade acetone read against water, should show zero
transmission over the range of 250-320nm. Reading above 2%
indicate the presence of stray light

Procedure:
1. An aqueous solution of sodium nitrite 5g/100ml was prepared
2. A 10mm cuvette was filled with sodium nitrite and the wavelength of the
spectrophotometer was set at 340nm
3. The 100% transmission was set with a cuvette filled with distilled water
4. The cuvette containing the sodium nitrite was placed in the
spectrophotometer sample compartment
5. The percentage of transmission read at 340nm was a direct measurement
of the stray light of the instrument. Ideally the value should be 1% or less

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Results:
From this experiment, we get the absorbance is 1.862 with transmissions
1.41%. It means that this spectrophotometer have a stray light

Discussion:
Stray light is light that scattered, separated, or happening accidentally or
randomly. If this situation occurs in spectrophotometer, it would cause an
error in reading the result from the spectrophotometer. The most common
cause of stray light are reflections of light from scratches on optical
surfaces or dust particles anywhere in the light path and higher order
spectra produced by diffraction gratings. To make sure that the
spectrophotometer is ideal to use, the value of transmission (%) read at
340nm should be 1% or less

Conclusion:
From this experiment we can conclude that the spectrophotometer that
was used have a stray light because the reading of transmission is 1.41%.
Normally, the reading of transmission should be 1% or less.

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Principles:
• Determination of wavelength accuracy of a spectrophotometer
Wavelength accuracy means that the wavelength indicated on the control
dial is the actual wavelength of light passed by the monochromator. It is
most commonly checked using standard absorbing solutions or filters
with absorbance of known wavelength. Didymium or holmium oxide in
glass is stable and is frequently used as filters. The filters is placed in light
path, and the wavelength control is set at the wavelength at which
maximal absorbance is expected. The wavelength control is then rotated in
either direction to locate the actual wavelength that has maximal
absorbance. If these two wavelengths do not match, the optics must be
adjusted to correctly calibrate the monochromator.

Procedure:
1. For narrow band spectrophotometers, a holmium oxide glass may be
scanned over the range of 220nm to 650nm
2. Selected absorption peaks for this filter, suitable for calibration purposes
occur at 279.3nm, 287.6nm, 333.8nm, 360.8nm, 418.5nm, 536,4nm, and
637.5nm

Results:
The result for this experiment was shown at below.

Discussion:
In experiment to determine the wavelength accuracy of the
spectrophotometer, some difference occurs between the results from the
spectrophotometer and suitable wavelength that was provided. The table
below showed the wavelength that the peaks occur and the actual
wavelength that was provided.
Wavelength from experiment (nm) Actual wavelength (nm)
278.3 279.3
287.7 287.6
334.4 333.8
362.0 360.8
421.4 418.5
536.7 536.4
637.2 637.5

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Conclusion:
From this experiment we can conclude that the wavelength accuracy of
the spectrophotometer is not fully accurate because there still some error
or difference between actual wavelength where the peaks occur and the
wavelength from the experiment.

Principle:
A. Determination of linearity of a spectrophotometer
For determination of linearity of a spectrophotometer, we can use either a
didymium filter or nickel sulphate solution (NiSO4.6H2O) 12g/100ml in
0.05M H2SO4 can be used
However for this experiment we use potassium dichromate solution

Procedure:
1. A solution of 0.93g of potassium dichromate K2Cr2O7 was prepared in 100
ml of 0.05 M H2SO4. Make up to 1L with distilled water. The flask was
shaked all the time
2. The precise mol was calculated by dividing the exact weight of
dichromate used with 294.2 (relative molecular mass of potassium
dichromate)
Mol = 0.93g / 294.2
= 0.00316
3. The concentration of the stock solution (K2Cr2O7) in this case would be:
Concentration = mv
= 0.00316 X 1L
= 3.16 X 10-3 mol/L
4. A series of dilutions of the stock solutions was made as follow (to total
volume of 10ml)
1 part of stock solution to 39 parts of distilled water
3 part of stock solution to 37 parts of distilled water
5 part of stock solution to 35 parts of distilled water
7 part of stock solution to 33 parts of distilled water
9 part of stock solution to 31 parts of distilled water
5. The concentration of all dilution was calculated and recorded
6. 1 ml of the 1:39 dilution was placed approximately in a 10mm cuvette
7. The spectrophotometer wavelength was set to 325 nm and the reference
level of the instrument with cuvette containing distilled water was set.
8. The cuvette containing the prepared dilution was placed in the sample
compartment. The absorbance was recorded
9. Steps 6 to 7 was repeated at wavelength increments of 10nm up to 405nm
and the absorbance at each wavelength settings was recorded
10. The results was plotted as absorbance against wavelength

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 11. The measurement from 340nm to 360 nm at increments of 5nm was
repeated in order to determine more precisely the wavelength of
maximum absorbance
12. The wavelength of maximum absorbance for this solution was notted
from the graph
13. The wavelength of the spectrophotometer was set to the wavelength
noted in step 11 and both absorption and transmission was recorded of all
dilutions of the stock solution of potassium dichromate prepared earlier
14. Two plots was prepared on the same graph paper, one of absorbance
against concentration and one of transmission against concentration
15. Note the absorbance plot is linear to about two absorbance readings and
transmission plot is exponential

Results and discussion:

For experiment involve 1ml of the 1:39 dilution:
Wavelength (nm) Absorption Transmission (%)
325 0.031 93.2
335 0.129 74.3
345 0.170 67.6
355 0.140 72.2
365 0.127 74.6
375 0.093 80.8
385 0.081 82.9
395 0.060 87.1
405 0.018 95.9

From this result, we get the wavelength at the maximum absorbance is

345nm with absorbance is 0.170 and transmission 67.6%. So for the next
step we take 345nm as the maximal absorbance at optimum wavelength.

NO 335nm 340nm 345nm 350nm 355nm

A T% A T% A T% A T% A T%
1) 0.124 75.2 0.126 74.8 0.152 70.4 0.148 71.1 0.135 73.3
1:39
2) 0.546 28.4 0.635 23.2 0.668 21.5 0.667 21.5 0.571 26.8
3:37
3) 0.847 14.2 0.933 11.7 1.065 8.6 1.015 9.7 0.415 38.5
5:35
4) 1.055 8.8 1.201 6.3 1.245 5.7 1.217 6.1 1.052 8.9
7:33
5) 1.268 5.4 1.320 4.8 1.344 4.5 1.293 5.1 1.096 8.0
9:31

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The table below shows the results from calculation of concentration of all
dilutions:
Series of dilutions Concentrations
1:39 Use M1V1 = M2V2
Stock solutions: 250 µl
Distilled water: 9750 µl
(M1V1) / V2= M2
(0.00316 X 250 µl) / 10,000 µl = 7.9 X 10-5 mol/L
3:37 Use M1V1 = M2V2
Stock solutions: 750 µl
Distilled water: 9250 µl
(M1V1) / V2= M2
(0.00316 X 750 µl) / 10,000 µl = 2.37 X 10-4 mol/L
5:35 Use M1V1 = M2V2
Stock solutions: 1250 µl
Distilled water: 8750 µl
(M1V1) / V2= M2
(0.00316 X 1250 µl) / 10,000 µl = 3.95 X 10-4 mol/L
7:33 Use M1V1 = M2V2
Stock solutions: 1750 µl
Distilled water: 8250 µl
(M1V1) / V2= M2
(0.00316 X 1750 µl) / 10,000 µl = 5.53 X 10-4 mol/L
9:31 Use M1V1 = M2V2
Stock solutions: 2250 µl
Distilled water: 7750 µl
(M1V1) / V2= M2
(0.00316 X 2250 µl) / 10,000 µl = 7.11 X 10-4 mol/L

From this experiment the optimum wavelength that produce maximum

peak is 345nm. The graph absorbance (A) against concentration (X 10-4
mol/L) produces a linear graph. However the graph transmission (%T)
against concentration (X 10-4 mol/L) produces a curve or exponential
graph.

Conclusion:
From this experiment we can conclude that the optimum wavelength that
produce maximum peak is 345nm. We can see that absorbance increase
with the increasing of the concentration (linear graph) and transmission
reduces with the increasing of the concentration (curve or exponential
graph)

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