DIAGNOSTIC IN CLINICAL CHEMISTRY I

MKEB 2404

TITLE:
• INSTRUMENTATION (SPECTOPHOTOMETER)

AIM AND OBJECTIVES:

• Learn how to use a simple spectrophotometer.
• Learn how to evaluate a spectrophotometer.

PRINCIPLES:
1. Checking a spectrophotometer for stray light.
Stray light is a radiation at any wavelength other than the required wavelength.
The most common causes 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. There are two methods employed to check a spectrophotometer for
stray light.
• By using an aqueous solution of sodium nitrite, 5g/100ml. The percentage
transmission read at 340 nm is a direct measurement of the stray light of
the instrument. Ideally the value should be 1% or less.
• Spectro grade acetone read against water should show zero transmission
over the range of 250-320 nm. Readings above 2% indicate the presence of
stray light.
2. 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 filter is placed in the light path, and the wavelength control is set at the
wavelength at which maximal absorbance is expected.

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 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.
For narrow band spectrophotometers, a holmium oxide glass may be scanned
over the range of 220nm to 650 nm. Selected absorption peaks for this filter, suitable for
calibration purposes occur at 279.3nm, 287.6nm, 333.8 nm, 360.8nm, 4l8.5nm, 536.4nm,
and 637.5nm. With broad bandwidth instruments, a didymium filter may be used. This
filter has a sharp minimum percentage transmission at 530nm against air blank.
3. Determination of linearity of a spectrophotometer.
Either a didymium filters or nickel sulphate solution (NiS04.6H20) 12g/100ml in
0.05M H2SO4 can be used. For didymium filter, zero the spectrophotometer with air at
550nm. Read absorbance of filter and record value. Remove filter and set absorbance to
0.25. Insert didymium filter and record value.
Repeat by setting the absorbance to 0.5, 0.75, 1.00 and 1.25 calculate the change in
absorbance for each increment in setting. Linearity is satisfactory as long as the change
in absorbance remains constant and within specified limits. If we do not have either of
these, the linearity of a spectrophotometer can be checked by using potassium
dichromate solution.

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PROCEDURE:
1. Checking a spectrophotometer for stray light.
a) Prepare an aqueous solution of sodium nitrite, 5g/100ml.
b) Fill a 10 mm cuvette and set the wavelength of the spectrophotometer to
340nm.
c) Set the 100% transmission with a cuvette filled with distilled water.
d) Place the cuvette containing the sodium nitrite in the spectrophotometer
sample compartment.
e) The percentage transmission read at 340 nm is a direct measurement of the
stray light of the instrument. Ideally the value should be 1% or less.
2. Determination of wavelength accuracy of a spectrophotometer.
a) For narrow band spectrophotometers, a holmium oxide glass may be
scanned over the range of 220nm to 650 nm.
b) Selected absorption peaks for this filter, suitable for calibration purposes
occur at 279.3nm, 287.6nm, 333.8 nm, 360.8nm, 4l8.5nm, 536.4nm, and
637.5nm.
3. Determination of the linearity of a spectrophotometer.
a) Prepare a solution of 0.93g of potassium dichromate K2Cr207 in 100 ml of 0.05
M H2S04. Make up to IL with distilled water, shaking the flask all the time.
b) Calculate the precise mol by dividing the exact weight of dichromate used
with 294.2 (relative molecular mass of potassium dichromate).
Mol = 0.93g/294.2
= 0.00316
c) The concentration of the stock solution (K2Cr207)in this case would be
Cone = mv
= 0.00316 X I L
= 3.16X103 mol/L

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d) A series of dilutions of the stock solution as follows (to total 10 ml).
o 1 part of stock solution to 39 parts of distilled water.
o 3 part of stock solution to 37 parts of distilled water.
o 5 part of stock solution to 35 parts of distilled water.
o 7 part of stock solution to 33 parts of distilled water.
o 9 part of stock solution to 3-1 parts of distilled water.
e) The concentrations of all dilutions are recorded.
f) About 3 ml of the 1:39 dilution in a 10 mm cuvette.
g) The spectrophotometer is set to wavelength 325 nm and the
reference level of the instrument with a cuvette that containing
distilled water.
h) The cuvette containing the prepared dilution in the sample
compartment and absorbance is recorded.
i) Repeat steps 6 to 7 at wavelength increments of 10 nm up to
405nm and record absorbance at each wavelength setting.
j) Plot the results as absorbance against wavelength.
k) To determine more precisely the wavelength of maximum
absorbance repeat the measurement from 340nm to 360 nm at
increments of 5 nm.
l) From the graph note the wavelength of maximum absorbance
for this solution.
m) The wavelength is set of the spectrophotometer to the
wavelength noted in step I 1 and record both absorption and
transmission of all dilutions of the stock solution of potassium
dichromate prepared earlier.
n) On the same graph paper prepare two plots, one of absorbance
against concentration and one of transmission against
concentration.
o) Lastly the absorbance is plotted linear to about two absorbance
readings and transmission plot is exponential.

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RESULT:
1. Checking a spectrophotometer for stray light.
• The percentage of transmission: 340 nm: 0.2 % T
• Meaning: The spectrophotometers don’t have stray light.
2. Determination of wavelength accuracy of a spectrophotometer.
WAVELENGTH FROM EXPERIMENT
ACTUAL WAVELENGTH (NM)
(NM)
278.00 279.3
287.40 287.6
333.30 333.80
361.50 360.80
421.10 418.50
536.40 536.40
636.90 637.50

3. Determination of the linearity of a spectrophotometer.

• Result: : 3 ml (1:39 dilution)
WAVELENGTH (NM) ABSORBANCE
330 0.206
340 0.250
350 0.285
360 0.284
370 0.260
380 0.206
390 0.153
400 0.104
410 0.072

• Result: 3 ml (1:39 dilution) = 340 nm - 360 nm (+5 nm increment).

WAVELENGTH (NM) ABSORBANCE
340 0.251
345 0.273
350 0.283
355 0.288
360 0.282

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 • Result: Maximum absorbance: 0.288 (355 nm).
• Result : All dilutions (Stock solution : Potassium Dichromate ) = 355 nm
PERCENTAGE
NUMBER OF SAMPLE ABSORBANCE
TRANSMISSION (% T)
SAMPLE 1
0.285 51.88
(1:39)
SAMPLE 2
0.725 18.84
(3.37)
SAMPLE 3
0.636 X 2 = 1.272 5.35
(5: 35)
SAMPLE 4
0.598 X 3 = 1.794 1.61
(7:33)
SAMPLE 5
0.786 X 3 = 2.358 0.44
(9:31)

CALCULATIONS:
1. Concentration of all dilutions.
• 1 part of stock solution to 39 parts of distilled water.
M1V1 = M2V2
X 10-3 mol/L (0.1 L) = M2 (4.0 L)
M2 = 3.16 X 10-3 (0.1) / 4.0
= 7.9 X 10-5 Mol/L
• 3 part of stock solution to 37 parts of distilled water.
M1V1 = M2V2

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X 10-3 mol/L (0.3 L) = M2 (4.0 L)
M2 = 3.16 X 10-3 (0.3) / 4.0
= 2.37 X 10-4 Mol/L
• 5 part of stock solution to 35 parts of distilled water.
M1V1 = M2V2
3.16 X 10-3 mol/L (0.5 L) = M2 (4.0 L)
M2 = 3.16 X 10-3 (0.5) / 4.0
= 3.95 X 10-4 Mol/L
• 7 part of stock solution to 33 parts of distilled water.
M1V1 = M2V2
3.16 X 10-3 mol/L (0.7 L) = M2 (4.0 L)
M2 = 3.16 X 10-3 (0.7) / 4.0
= 5.53 X 10-4 Mol/L
• 9 part of stock solution to 3-1 parts of distilled water.
M1V1 = M2V2
3.16 X 10-3 mol/L (0.9 L) = M2 (4.0 L)
M2 = 3.16 X 10-3 (0.9) / 4.0
= 7.11 X 10-4 Mol/L

2. Calculation of percentage transmission of each sample.

• Sample 1 (1:39)
A = 2 – Log % T
0.285 = 2 – Log % T
Log % T = 1.715
% T = Antilog 1.715
= 51.88 %
• Sample 2 (3:37)
A = 2 – Log % T

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 0.725 = 2 – Log % T
Log % T = 1.275
% T = Antilog 1.275
= 18.84 %
• Sample 3 (5:35)
A = 2 – Log % T
1.272 = 2 – Log % T
Log % T = 0.728
% T = Antilog 0.728
= 5.35 %
• Sample 4 (7:33)
A = 2 – Log % T
1.794 = 2 – Log % T
Log % T = 0.206
% T = Antilog 0.206
= 1.61 %
• Sample 5 (9:31)
A = 2 – Log % T
2.358 = 2 – Log % T
Log % T = -0.358
% T = Antilog -0.358
= 0.44 %
DISCUSION:

A spectrophotometer is used to measure the light transmitted by a

solution to determine the concentration of the light-absorbing substance in the solution.
From the experiment A, show that the percentage transmission of
sodium nitrite, 5 g/100 ml at 340 nm give result 0.2 % transmission. This means, the
stray light are acceptable for the spectrophotometer used. As definition, stray light
refers to any wavelengths outside the band transmitted by the monochromator. If the
percentage transmission is more 1%, the stray light of the spectrophotometer are not

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suitable for reading any sample. This may happen because of dust, scratched cuvette,
or others thing that affects diffraction of the stray light of the spectrophotometer. As a
result, a error would be happen for each reading sample.
In the experiment B, the spectrophotometer is being tested for accuracy
of the wavelength. On this experiment, the result is slightly different with true value of
the wavelength of the spectrophotometer. This means, the accuracy of the
spectrophotometer with compare to the result that get is not very fully accurate.
For experiment C, the spectrophotometer is determined for the
linearity. As definition, the linearity is demonstrated when a change in concentration
results in a straight-line calibration curve. The precaution on this experiment is when
using the solution of potassium dichromate, this because of the carcinogen
characteristics. From the plotted graph, the linearity is quiet satisfactory, as shown that
the maximum wavelength is 355 nm. The graph is fulfilled with Beer’s Law where the
graph absorbance versus concentration and percentage transmission versus
concentration.

CONCLUSION:

1. Experiment A shown that the spectrophotometer have no stray light since the
percentage of transmission is 0.2%.
2. In the experiment B, the wavelength of the spectrophotometer is not fully
accurate because there is a slightly differences with true wavelength of the
spectrophotometer.
3. For experiment C, the optimum absorbance is 355 nm and it satisfactory since
the absorbance remains constant and within specified limits.