AN INTRODUCTION TO SPECTROSCOPY
Spectroscopy is a powerful tool used to study biological molecules, including
proteins, lipids, carbohydrates, and nucleic acids. Absorbance spectroscopy
involves the study of the pattern of light absorption by substances. A beam of
monochromatic light (light of a specific single wavelength) is passed through a
solution (sample), and the amount of light transmitted is measure by a
detector (Fig 1). When UV or visible light (radiation) is passed through an
organic compound/molecule solution, it is either absorbed or transmitted
depending on the wavelength. Absorbance (A), also known as optical density
(OD), is the quantity of light absorbed by the solution and can be expressed
as;
𝑰𝟎
A = Log10( ) Where;
𝑰
I0 = intensity of incident light
I = intensity of transmitted light
Fig 1: Representation of a Basic UV/Vis setup.
https://orbitbiotech.com/molecular-analysis-using-uv-visible-spectroscopy-spectroscopy-uv-absorption-reflection-
spectra-electromagnetic-radiation/ (EDITED)
Molecules absorb and emit light based on the kinds of bonds within the
molecule. Thus spectroscopic approaches enable molecules to be identified,
including characterising changes occurring to their chemical structures. An
absorption spectrum is a graph that shows the amount of light absorbed by a
solution at different wavelengths. The wavelength at which most light is
absorbed is referred to as Lambdamax (λmax).
Beer-Lambert law establishes a linear relationship between absorbance and
the concentration of molecules absorbing light in the solution, given the path
length travelled by the light. Therefore, UV-Vis spectroscopy can also be used
to determine the light absorbing molecules' concentration for a fixed path
length. Beer-Lambert Law is expressed as;
A=ε•c•l Where; A = Absorbance
Ɛ = Molar absorption coefficient (M/cm)
C = molar concentration
l = length travelled by light (cm)
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METHOD
You are provided with;
• A stock solution (20 µL + 5 mL H2O) of food dye (concentration will be
provided during the lab practical).
• Water.
• Test tubes.
• Solution M (Food dye solution of unknown concentration).
• An answer sheet and graph paper to complete and submit at the end
of the lab.
PART A: Absorption Spectrum of Food dye.
1. Switch on the UVmini-1240 (Fig 2) and allow it to go through all initialising
stages. This will take a few minutes.
2. The photometric mode screen will eventually appear, displaying
previously set wavelength and measured absorbance. If you don't see the
photometric mode screen, press the RETURN button to get to the Mode
menu screen (Fig 3). Then set the spectrophotometer to photometric
mode by pressing 1 on the main menu.
3. For this exercise, we will measure Absorbance (ABS). The F1 button
allows you to switch between measuring the % transmittance (T%) and
absorbance (ABS). Ensure the data reading displayed is absorbance
(ABS); if not, press the F1 button to switch to ABS mode.
4. Set the wavelength by pressing the GOTO WL button. Enter the
wavelength (360 nm), then press the ENTER button.
5. Add 2 mL of your blanking solution to the cuvette. Insert the cuvette into
the receptacle (Fig 4) with the clear sides facing left and right and close
the lid. Make sure to wipe away any fingerprints/stains on the cuvette
before inserting it.
6. Press the AUTO ZERO button to blank the spectrophotometer at the set
wavelength. Then remove the blank from the receptacle.
7. Add 2 mL of stock solution to the cuvette and insert it into the receptacle,
close the lid and record the absorbance displayed on the screen in your
answer sheet.
8. Using your 2 mL stock solution, record the absorbance of the solution
from 360 − 740 nm at 20 nm intervals on your answer sheet. You do not
have to blank the spectrophotometer before each absorbance reading but
do so after every five readings.
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9. Plot an absorption spectrum of the food dye using your results and
determine the λmax of the solution.
PART B: Standard/Calibration curve of Food dye.
1. Prepare different concentrations of food dye from the stock solution
following the table below. Calculate the amount of water required and the
final food dye concentration for each tube. Record these in your answer
sheet.
2. Set the spectrophotometer wavelength to the λmax of the solution
determined in part A.9.
3. Blank the spectrophotometer and record the absorbance of tubes 1 – 5
and Solution M, recording your results in your answer sheet.
Tube Stock Water Total Concentration Absorbance
solution (mL) (mL) (mL)
1 1.0 5.0
2 0.8 5.0
3 0.6 5.0
4 0.4 5.0
5 0.2 5.0
M Solution M − −
4. Plot a standard curve of the food dye using your results.
5. Express the relationship of food dye concentration and absorbance as an
equation, i.e. y = mx + c
6. Determine the concentration of Solution M by;
a. Extrapolating the concentration from the standard curve (show
this on your standard curve).
b. Using your equation from part B.5 to calculate concentration.
7. Calculate the concentration of a food dye solution with an absorbance of
1.600.
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Fig 2: The on/off switch is located at the back of the spectrophotometer
Fig 3: Photometric mode and Mode menu screen
Fig 4: Receptacle where cuvette is inserted and examples of cuvettes.
