This document describes an experiment to determine the calibration curve of cobalt nitrate using a spectrophotometer. Key steps include preparing cobalt standard solutions of known concentrations, measuring their absorbance, and using the results to construct a calibration curve relating absorbance to concentration. The calibration curve will then allow determination of the concentration of an unknown cobalt solution based on its measured absorbance. The document provides background on spectrophotometry and Beer's Law, which states that absorbance is directly proportional to concentration and path length.

1. INSTRUMENTAL ANALYSIS
LAB.
Mr Haydar A.M.S
BSc, MSc
Faculty of Science
Chemistry Department
Email: Haydar.kovly@gmail.com

2. Exp. 3 Determination the Calibration Curve of
Cobalt Nitrate by Spectrophotometer
Objectives
In this experiment, you will
• Prepare a set of Co+2 standard solutions of known concentrations,
• Measure the A or %T of the known solutions and construct a calibration
curve, and
• Measure the A or %T for unknown solution and determine its concentration
from the calibration curve.

3. Ultraviolet/Visible Molecular Absorption
Spectrometry
The instrument used to measure the amount of light absorbed by the sample is a
spectrophotometer.

4. Components of a Spectrophotometer
4

5. Wavelength Selectors
5

6. Light source
Grating
Rotating the grating
changes the wavelength going
through the sample
slits
slits
Sample
filter
Phototube
Wavelength Selectors
When blank is the sample
Io is determined
otherwise I is measured
Separates white light
into various colors
detects light &
measures intensity
- white light of constant intensity
6

7. 7

8. 8
Sample Containers

9. Range

12. Transmittance (T) is defined as the amount of light passing through the sample solution (I)
divided by the amount of incident radiation (Io).
T = I/ Io
•Transmittance is often expressed as a percentage or
%T = I/Io × 100%
The Absorbance (A) of a medium is defined by the equation:
A = log Io/I = -log T

13. As the radiation is absorbed in the sample, the total
intensity of radiation is reduced as it travels through
the sample. This results in a non-linear relationship
between transmittance and concentration
Absorbance (A) is based on the amount of light
absorbed by the solution and is defined as the log
of the inverse of the transmittance.
A = log
(1/T)
10
Within limits, the relationship between
absorbance and concentration is linear.

14. The blank
• The blank contains all substances except the analyte.
• Is used to set the absorbance to zero:
A blank = 0
• This removes any absorption of light due to these substances and the cell.
• All measured absorbance is due to analyte.
Analyte is a substance undergoing analysis.

15. Beer’s law
Beer’s law states that the amount of light absorbed by a solution (colored) is directly
proportional to the path length (b) through the medium and the concentration (c) of the
absorbing species,
A (absorbance) = log Io/I = a b c
• Where (a) is the proportionality constant called the absorptivity
• b is the path length of the sample i.e. the inside cross section of the sample cuvette
(cm).
• When the concentration is expressed in (mol/L), the absorptivity is called the molar
absorptivity and is given the symbol (є)
A = є b c (units of є = L.mole-1.cm-1)

16. Determination the calibration curve of
cobalt nitrate by spectrophotometer

17. Procedure
1. Obtain TWO matched cuvettes, the first cuvette is for distilled water and the second one is
for the cobalt solution.
2. Prepare the following solutions
From stock solution (0.2M)
Co(NO3)2.6H2O
(M)
Absorbance
(A)
0.02
0.04
0.06
0.08
0
Part A Calibration Curve

18. Procedure Continued
3. Set the wavelength dial 505 nm
4. Measure the absorbance of the blank (water).
5. Measure the absorbance of the cobalt solutions and record the absorbance
6. Empty and rinse the cuvettes thoroughly with water.
7. plot the absorbance versus concentration
8. Measure LOD, LOQ and sensitivity

19. Procedure Continued
Part B Determination the absorbance of the unknown
1. Measure the absorbance of the unknown concentration
2. use a calibration curve that you plot it in part A and determine the concentration
of the unknown solution.

20. 20
Absorbance
1.00
0.60
0.20
0.30 0.40 0.50
0.80
0.40
0.10 0.20
Concentration in mol / Litre
0
0