SPECTROPHOTOMETRIC MEASUREMENT OF ABSORBANCE

INTRODUCTION

In 1760, Johann Lambert formulated the law of light absorption along with August Beer which relates the absorption to the composition of the material that it passes through. When a beam of light goes perpendicularly into a solution, some of the photons are absorbed by the contents of the solution. This is expressed by two concepts, transmittance and absorption.

where

: molar absorption, l : the length of the path that the beam travels inside the solution and c : concentration of the solution. Thus, theres a linear correlation between the concentration and absorptioni.

While were calculating the absorption of a solute in a solution, we also need to consider the absorption of the solvent itself. This problem is solved by taking transmittance as the ratio between the light intensity through the sample and the light intensity through the solvent aloneii. In spectrophotometers, machines designed to do the calculations above for different wavelengths and solutions, blank calculations consisting only of the solvent are either done beforehand or at the same time with the real sample to be examined.

AIM

The reason we conducted this experiment was that we need to be familiar to spectrophotometry, a critical technique in biological research which allows us to determine the concentration of a specific substance in a solution.

MATERIALS

Lab equipments used: Spectrophotometer Cuvettes Micropipette & disposable tips Plastic container tubes

Chemicals used Distilled water Bromophenol blue A BPB solution of unknown molarity

METHOD

Our experiment consisted of two sub-experiments. i. Determination of the wavelength of maximum absorbance by BPB a. We first calibrated the spectrophotometer with pure distilled water, to prevent the machine from miscalculating due to the absorbance of water in the BPB solution. i. We filled one of the cuvettes with distilled water, and the other with a stock solution of 0.02mg/ml BPB. ii. The cuvette with distilled water was placed inside the machine, paying attention to the placement of the straight and the curved sides of the cuvette. iii. The calibration reading was performed by the machine automatically. b. We started the absorbance readings on the 0.02mg/ml BPB stock solution. i. We performed 9 readings manually in 25 nm intervals from 475 nm to 675 nm and recorded the outcomes.

ii.

Construction of a standard graph to determine the relationship between absorbance and concentration: a. We prepared 4 additional diluted samples (of 0.0025, 0.0050, 0.1, 0.015 mg/ml) using the provided stock solution of 0.02mg/ml BPB, by adding water on small amounts of the solution itself (see Appendix 1.1). b. At the highest absorbance wavelength determined in Step I, we performed readings of all samples and the unknown sample provided to us. c. The data obtained was recorded and interpreted.

RESULTS Our two experiments served to provide different types of results, one aiming to determine the maximum absorbance wavelength for BPB and the other using this data to correlate absorbance to concentration, and help us determine the composition of the unknown sample given to us. Experiment 1: Determination of the wavelength of maximum absorbance by BPB During this experiment, we did 9 measurements using the stock solution of 0.02mg/ml BPB:

Wavelength (nm) 475 500 525 550 575 600 625 650 675

Absorption 0,081 0,165 0,321 0,589 0,99 1,029 0,159 0,026 0,014

Absorption 1.2 1 0.8 0.6 0.4 0.2 0 0

Absorption

200

400

600

800

Figure 1.1.a: Data table consisting of the absorption measurements at different wavelengths on the stock solution.

Figure 1.1.b: A preliminary graph generated from the data tables.

As we can interpret from the data, the maximum absorption wavelength is a value around 600 nm. We took this value as 625 nm for the rest of the experiment. Experiment 2: Construction of a standard graph to determine the relationship between absorbance and concentration: This experiment consisted of readings at 625 nm wavelength of different concentrations of BPB. The data obtained is as follows:

Concentration of sample (mg/ml) 0,0025 0,005 0,01 0,015 0,02 Unknown

Absorption 0,157 0,219 0,549 0,769 1,042 0,983

Figure 1.2.a: A data table showing the results obtained from the different solutions at 625 nm.

Absorption 1.2 1 0.8 0.6 0.4 0.2 0 0 0.005 Linear (Absorption)

Absorption
y = 51.827x + 0.003

0.01

0.015

0.02

0.025

Figure 1.2.b: A graph showing the linear correlation between the concentration and absorption. The line generated by the Excel software is an approximation derived from the given data.

Using the approximation from the data provided in Figure 1.2.b, we calculated the concentration of the unknown sample as ~0,0189mg/ml (See Appendix 2.1)

DISCUSSION

i ii