2 Methods of determining the concentration of a compound in solution
Using beer-lambert’s law directly
If coefficient of absorptivity is known, and spectrophotometer gives the absorbance of the analyte, the concentration can be calculated directly by using the Beer’s equation;
Absorbance = 𝜀Cl
Calibration curve/GRAPH
This method is based on the absorbance measurement of a set of standard solutions for which the Beer’s law must be valid.
Then a graph of light absorbance against concentration is plotted.
The concentration of unknown sample can be extrapolate or calculated from the calibration curve if its absorbance is known.
Calibration factor
The calibration factor is calculated by dividing the concentration with the appropriate absorbance to obtain a set of numbers, from which you can calculate a mean value - the calibration factor.
The absorbance of unknown sample is multiplied by the calibration factor to get the concentration.
Standard sample
The absorbance of unknown sample (c2) and the standard sample (c1) are determined simultaneously.
After measuring the absorbance of both samples we can calculate the concentration of unknown sample according to equation: C1:A1=C2:A2
This relationship allows us to rearrange the equation to solve for the concentration of the unknown sample (C2) as follows:
This method is widely used in spectrophotometry, ensuring accurate results when determining concentrations in various solutions.
EXPERIMENT 1 (A) ABSORPTION OF COLORED SOLUTION
INTRODUCTION:
Colored solutions absorb certain wavelengths while allowing other wavelengths to pass through.
AIM:
To determine the wavelength of maximum absorbance of a given coloured solution using the spectrophotometer.
PROCEDURE/PROTOCOL:
Rinse cuvettes with water before use.
Fill one spectrophotometer cuvette with distilled water (3/4 full) to act as a blank.
Fill another cuvette with an amount of a colored solution.
Keep both cuvettes clean on the outside, and hold them near the top, away from the region through which a light beam passes.
Set the spectrophotometer to a recommended wavelength.
Measure the absorbance at 20 nm intervals. (Do not forget to “blank” or “zero” with the blank at each wavelength)
Record the absorbance.
Complete the table below for the colored solution.
Plot the absorption spectrum. (absorbance versus wavelength)
Find the wavelength of maximum absorbance for the coloured solution from the graph.
What colour of light corresponds to the wavelength.
What is the reason for maximal absorption at that wavelength.
POST LAB QUESTIONS
Differentiate between colorimetry and spectrophotometry
Outline the procedure of operating the spectrophotometer
Explain the term “Blanking” as used in specrophotometry?