Spectrophotometer

23rd January 2023

9:00 am

Dr Hadi Al-Sagur
PhD in Medical Physics (United Kingdom)
Occupation: Lecturer

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 Second experiment
Apparatuses

1. Spectrophotometer Instrument
2. Sensitive balance
3. Cell (cuvette)
4. Potassium permanganate
5. Deionized water
6. Pipette
7. Tips

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Theory
Absorption of light
The intensity of incident light (I0) is greater than the intensity of emerging light (I)
when the light passes through a media. This leads to weakness in the intensity I comparing
to I0 because of the reflection in the limits surface between them, (I & I0) or because of
dispersion suffered by the beam because of matter in the media or direct absorbed by the
matter.

Absorption process is the principle factor and the amount of absorption light depends on:
1 - Intensity of incident ray.
2 - Absorption coefficient depends on the physical nature of the material.
3 – Path length for absorption medium.

Can express on this relationship by mathematics equation the following:

𝑑𝐼
= −𝐾𝐶𝑑𝑥 (1)
𝐼

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The blood absorbs the incident light that will lead to reduce the energy of transmitted light.

HEMOGLOBIN METER
λ = 540 nm

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Where:
I: intensity of light at certain wavelength i.e. the energy for the unit area and unit time.
dI: change of intensity of the light resulting from absorption in the layer of thickness
(dx) and concentration [C]. Distance x is measured through the cell in the direction of
the beam of light that is being absorbed. The concentration [C] is usually expressed in
(mol.L-1), K: is a factor depends on the physical nature of the material.

Since it is convenient to use logarithms to the base 10, the Beer- Lambert law is

used in the form:

I
Log
0
= e cl = A
…..(2)
I

A is Absorbance!
𝑰
= 𝒆− 𝜺𝒄𝒍 …..(3)
𝑰𝑶

𝐾
𝜀=
5
2.303
The ratio between I/I0 called Transmittance (T).
After this information we understood that spectrophotometry is an analytical tool in
laboratory.

Method Part
The preparation of Sample solution has been prepared, according to the following
relationship:

M x V x W
W
C =
…..(4)
1000

Where
C the matter concentration (mole/liter)
M w Molecular weight of the matter (g/mole)
V the volume of the solvent (ml)
W weight of the dissolved dye (g)

Spectrophotometer is an instrument that measures the amount of light absorbed by

substance

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Figure (1) : spectrophotometer instrument Figure (2) : Sensitive balance

Figure (3): Schematic diagram illustrate the single beam UV-Visible spectrophotometer
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 Figure (4): Electromagnetic Spectrum

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Procedure
1. By using the pipette pure a ( 4) ml of Deionized water.
2. Turn on the spectrophotometer.
3. Put the cell filled with solvent (blank which is DI water).
4. Set the spectrum at lower value of the wavelength (λ) range (400 nm)
5. Calibrating: when we put the blank we need to make sure that the absorbance A=0 which
means T=100.
6. Then we put the solution (DI water mixed with Potassium permanganate).
7. In the mode of A, we read the absorbance of the solution and write this reading in the table.
8. Increase the wavelength at which the spectrophotometer work by 10 nm and take a blank
reading. (Every time we calibrate the blank for very new wavelength).
9. Continue with increasing the wavelength 10 nm till 600 nm and record the reading each
time.
10. Press the (100 button) which will eliminate the effect of the blank absorbance in the
previous range on the coming readings of the sample.
11. Take a (0.01g) of Potassium permanganate which has a molecular weight (158.034 g/mol)
12. Mix DI water with Potassium permanganate to prepare 0.007 M of the concentration C
(molary) based on equation (4).

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13. Repeat the steps (6 – 9) for the cell filled with the sample.
14. Tabulate the results of the sample as the below table.
Wavelength (λ)
Absorbance
nm
400
The molecular weight of Potassium permanganate is: 410
420
KMnO4 = (39.1 x1)+(54.94x1)+(16x4) = 158.034 g/mole 430
440
450
460
470
158.034 x 4 ml x w 480
490
0.007 M =
500
1000 510
520
530
540
550
560
570
580
590
600

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Medical Application: ultraviolet and visible spectrophotometers represent an analytical
tool of great versatility and power

1. In clinical applications, it is used for determination of hemoglobin concentration.

The spectrophotometer that is used for this purpose called, HEMOGLOBIN METER

HEMOGLOBIN METER
λ = 540 nm

2. Determination of urea concentration in blood and urine

3. Spectrophotometric assay of creatinine in human serum sample
4. Play great role in research on vitamins and hormones.
5. Pharmaceutical analysis
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Extra info:

Figures explain the preperation differences between the

Deionised and Distilled water.
Deionised Water has been treated to remove all ions – typically,
that means all of the dissolved mineral salts.

Distilled water has been boiled so that it evaporates and then

re-condensed, leaving most impurities behind

Pipette, tips, and cuvette are as shown respectively

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