Al-Quds University

Faculty of Health Professions

Department of Medical Laboratory Sciences

Clinical Laboratory Instrumentation

Lab (2): Selection of an Appropriate Wavelength for a Substance

Section: 1
Instructor: Ibrahim Ghannam
Students’ Names: Roaa Khalil 22111893
Saja Alkhatib 22111544
Shahd Abu Snineh 22111938
Date: 4/3/2023
Introduction

A single beam UV-Visible spectrophotometer was used in this lab, as it is

a quantitative technique used to measure how much a chemical
substance absorbs light. This is done by measuring the intensity of light
that passes through a sample with respect to the intensity of light that
passes through a reference sample. The composition of the reference,
with the exception of the absorbent to be measured, must be blank
identical to the composition of the titrated or unknown solution. So,
when measured with the device it gives a 100% transmittance, and 0
absorbance.
The absorbance and transmittance of two substances (KMnO4 and
K2Cr2O7) were measured using the device, and the wavelength at which
they perform maximum absorbance was obtained. This wavelength is
said to be the most appropriate to use for spectrophotometric analysis
for that substance. Then a mixture of both substances with unknown
concentrations, was measured with the device, to determine these
concentrations.
Results
For the KMnO4:
Wavelength (nm) Absorbance Transmittance
330 0.21 61.70%
340 0.201 62.90%
350 0.199 63.30%
360 0.183 65.70%
370 0.155 70%
380 0.128 74.50%
390 0.106 78.30%
400 0.09 81.30%
410 0.08 83.10%
420 0.074 84.30%
430 0.069 85.30%
440 0.068 85.50%
450 0.07 85.20%
460 0.074 84.20%
470 0.09 81.30%
480 0.108 78%
490 0.135 73.20%
500 0.168 68%
510 0.191 64.40%
520 0.221 60.10%
530 0.227 59.20%
540 0.216 60.80%
550 0.215 60.90%
560 0.141 72.30%
570 0.132 73.80%
580 0.075 84.10%
590 0.041 91%
600 0.033 92.60%
Curve (1): Absorbance and Wavelength for KMnO4
0.25

0.2
Absorbance

0.15

0.1

0.05

0
330 350 370 390 410 430 450 470 490 510 530 550 570 590 610 630
Wavelength (nm)

Curve (2): Transmittance and Wavelength for KMnO4

100.00%

90.00%

80.00%

70.00%
Transmittance (%)

60.00%

50.00%

40.00%

30.00%

20.00%

10.00%

0.00%
330 350 370 390 410 430 450 470 490 510 530 550 570 590 610 630
Wavelength (nm)
For the K2Cr2O7:

Wavelength (nm) Absorbance Transmittance

330 0.592 25.60%
340 0.765 17.20%
350 0.835 14.60%
360 0.787 16.30%
370 0.665 21.60%
380 0.506 31.20%
390 0.341 45.50%
400 0.212 61.30%
410 0.14 72.50%
420 0.121 75.70%
430 0.118 70.30%
440 0.113 77%
450 0.104 78.80%
460 0.088 81.70%
470 0.069 85.20%
480 0.051 89%
490 0.035 92.30%
500 0.022 95.10%
510 0.012 97.20%
520 0.006 98.60%
530 0.003 99.40%
540 0.001 99.80%
550 0 100%
560 0 100%
570 0 100%
580 0 100%
600 0 100%
600 0 100%
Curve (3): Absorbance and Wavelength for K2Cr2O7
0.9

0.8

0.7

0.6
Absorbance

0.5

0.4

0.3

0.2

0.1

0
330 350 370 390 410 430 450 470 490 510 530 550 570 590 610 630
Wavelength (nm)

Curve (4): Transmittance and Wavelength for K2Cr2O7

100.00%

90.00%

80.00%

70.00%
Transmittance (%)

60.00%

50.00%

40.00%

30.00%

20.00%

10.00%

0.00%
330 350 370 390 410 430 450 470 490 510 530 550 570 590 610 630
Wavelength (nm)
Calculations for the Unknown

Wavelength Absorbance ε of Absorbance ε of Absorbance of

(nm) of KMnO4 permanganate of K2Cr2O7 Dichromate unknown
(C = 0.1 m M) (C = 0.25 m
M)
Λ max for 0.244 2.44 0.005 0.02 0.072
kMnO4 =
525 nm
Λ max for 0.199 1.99 0.835 3.34 0.946
K2Cr2O7 =
350 nm

According to Beer’s law, the absorbance of a substance equals the

absorptivity of that substance at a specific wavelength multiplied by the
pathlength and the concentration of the substance (A = ε*b*c). And for
solutions containing two or more substances, the absorbance will be the
sum of the absorbance of the individual components in the mixture at a
specific wavelength. So, as we have a mixture containing both substances
of potassium permanganate and potassium dichromate in unknown
concentrations, we can use the following equation to determine the
concentrations:
 () = A1()+ A2()
 ()=A1 () + A2()
The pathlength is constant for the cuvettes (1cm), so we can say that:
 (1) = () c1 + () c2
A () = (2) c1 + (2) c2
When we do the calculation to find the concentrations, we will have
these two equations:
0.072 = (2.44*C1) + (0.02*C2) …………. (1)
0.946 = (1.99*C1) + (3.34*C2) …………. (2)
0.0946−( 3.34∗𝐶2)
Form the second equation, we can say that C1 = , which
1.99
also means that C1 = 0.4753 – (1.6783*C2) …………. (3)
By using the third equation in the first one we can find C2:
0.072 = 2.44 (0.4753 – (1.6783*C2)) + (0.02*C2)
0.072 = 1.159732 – (4.095*C2) + (0.02*C2)
-1.0877 = -4.075*C2
So, C2 = 0.266 m M, and C1 = 0.4753 – (1.6783*0.266) = 0.027 m M.
That means the unknown we had contained potassium permanganate
(KMnO4) in concentration 0.027 m M, and potassium dichromate in
concentration 0.266 m M.