Jalil Haddadin
1710132
Experiment 3: Spectrophotometer
Report 3
In this experiment, you will study the basic principles of spectrophotometry and serial dilution. A
spectrophotometer is a very powerful instrument used in both the chemical and biological
sciences, filtered to a precise wavelength (or very narrow range of wavelengths), through a
sample and onto a light meter. Some basic properties of the sample can be determined by the
amount and wavelengths of light absorbed by the sample.
Objectives
1. You should understand: the basic mechanics of the spectrophotometer
2. The basic principles of spectrophotometry including transmittance and absorbance.
3. The A max for a compound and how it is determined.
4. The application of Beer’s law to determine concentrations and extinction coefficients.
5. The use of spectrophotometry to identify compounds the use of standard curves in
analyzing data
Introduction:
A spectrophotometer is an instrument that measures the amount of light absorbed by a sample.
Spectrophotometer techniques are used to measure the concentration of solutes in solution by
measuring the amount of the light that is absorbed by the solution in a cuvette placed in the
spectrophotometer . Ultraviolet (UV) Spectrophotometer: Uses ultraviolet light of wave lengths
from 200 to 350 nm. Visible (VIS) Light Spectrophotometers: Uses visible light (white light) of
wave lengths from 350 nm to 700 nm.
1
The detector measures the amount of light being transmitted by the sample and reports that value
directly) transmittance %) or converts it to the amount light absorbed in absorbance units using
Beers Law.
Materials Required:
1. Spectrophotometer
2. Cuvette
3. Blank solution
Reagents:
1. Cobalt (II) chloride
2. Hexaaquacobalt (II) ion
3. Ferrocene
4. Crystal violet
5. Rose bengal
6. Coumarin
Procedure:
Determination of Molar Absorption Coefficient:
1. Select a blank cuvette and place it in the spectrophotometer. Close the lid.
2. Click on 0 ABS 100%T button, the instrument now reads 0.00000 A.
3. Choose a solution with known concentration and measure the absorbance between the
wavelengths 350 nm to 700 nm.
4. Record the wavelength at the maximum absorbance value.
5. Calculate the value of molar absorption coefficient , using the equation .
Determination of Unknown Concentration:
1. Set the wavelength to the value corresponding to maximum absorbance (recorded above).
2. Place the cuvette with same solution but at an unknown concentration.
3. Read the absorbance for this wavelength.
4. Calculate the concentration with the help of the equation, molarity
5. Enter the calculated concentration value in the given box. (Note : Should enter the value correct
to four decimal places)
6. Repeat the same procedure for a second solution.
2
Observations and Calculations:
Conc. Absorbance λmax
0.01 mM 0.8357 583 nm
0.005 mM 0.3327 583 nm
Unknown (C) 0.1774 583 nm
Concentration (C)of Solution = 0.01 mM
Absorbance (A) of known conc. solution = 0.8357 A
Cuvette length (L) = 1 cm
Waveleng that maximum absorbance value = 583 nm
Molar Absorption Coefficient, = 83570 M−1 cm−1
Absorbance of given solution = 0.1774 A
Therefore, concentration = 0.00212 mM
Result:
Concentration of the given solution =0.00212 mM
3
Points to Remember while Performing the Experiment in a Real Laboratory:
1. Always wear lab coat and gloves when you are in the lab. When you enter the lab, switch on the
exhaust fan and make sure that all the chemicals and reagents required for the experiment
are available. If they are not available, prepare the reagents using the components for reagent
preparation.
2. Make sure to clean all your working apparatus with chromic acid and distilled water and ensure
that all the apparatus are free from water droplets while performing the experiment.
3. Make sure to calibrate the electronic weigh balance before taking the measurements.
4. Ensure that the spectrophotometer is working properly.
5. Ensure that you are handling the cuvette with tissue paper .Never touch it with your hand.
6. Wipe the cuvette with tissue paper before placing the spectrophotometer.
7. Clean all glassware with soap and distilled water. Once the experiment is completed recap the
reagent bottles. Switch off the light and exhaust fan before leaving the lab.
8. Discard the used gloves in a waste bin.
Discussion:
We calculated the unknown concentration using beers law and it was logically correct
where the absorbance was even less than 0.005mM absorbance sot it is predicted to have a
lower concentration than 0.005 ml.
Conclusion:
we conclude that our experiment was correctly done depending on our results. And that
we can predict the concentration value from its absorbance where in this experiment we
could have predict that the concentration of the unkown sample equals 1/2 of the (0.005
mM) sample because its absorbance is also 1/2 of the (0.005 mM) absorbance.
