Spectrophotometric Analysis of a Mixture

Resolving a two-component mixture using simultaneous equations

Colorimetry Advanced (University) 75 min ~$12.00

Objective

Determine the individual concentrations of two dyes in a mixed solution using absorbance measurements at two wavelengths and simultaneous equations.

Background

When a solution contains two absorbing species with overlapping spectra, a single wavelength measurement cannot determine both concentrations. However, by measuring absorbance at two wavelengths (ideally the λmax of each component) and applying the additive property of absorbance (Beer-Lambert), a system of two simultaneous equations can be solved. This technique is the basis of multicomponent analysis in spectrophotometry.

Safety Warnings

  • Indicator dyes stain clothing
  • Handle cuvettes by frosted sides only to avoid fingerprints
  • Clean spills immediately

Required PPE

goggles lab_coat

Materials

  • Methyl orange solution (0.01%) (100 mL)
    Component A
  • Bromothymol blue solution (0.01%) (100 mL)
    Component B
  • Unknown mixture of both dyes (50 mL)
    Prepared by instructor
  • Distilled water (200 mL)

Equipment

UV-Vis spectrophotometer Cuvettes (matched pair) Volumetric flasks (50 mL, 6 total) Pipettes Calculator

Procedure

1

Scan pure methyl orange from 350–700 nm. Record λmax (approximately 464 nm in acidic pH).

8 min
2

Scan pure bromothymol blue from 350–700 nm. Record λmax (approximately 615 nm at neutral pH).

7 min
3

Prepare 3 standard concentrations of each pure dye. Measure absorbance of each at both λmax values (464 and 615 nm).

15 min
4

Calculate the molar absorptivity (ε) for each dye at each wavelength from the standard curves: ε(A,464), ε(A,615), ε(B,464), ε(B,615).

15 min
5

Measure the absorbance of the unknown mixture at both wavelengths.

5 min
6

Set up the simultaneous equations: A(464) = ε(A,464)·C(A) + ε(B,464)·C(B), A(615) = ε(A,615)·C(A) + ε(B,615)·C(B). Solve for C(A) and C(B).

15 min
7

Verify your results by comparing the calculated total absorbance at several wavelengths with the measured mixture spectrum.

10 min

Expected Results

The simultaneous equations yield positive concentrations for both dyes. The reconstructed spectrum (sum of individual contributions) should closely match the measured mixture spectrum, validating the Beer-Lambert additivity principle.

Cleanup

Pour dilute dye solutions down the drain with running water. Rinse all cuvettes and volumetric flasks thoroughly.

Details

Category
Colorimetry
Difficulty
Advanced (University)
Duration
75 min
Est. Cost
$12.00
Steps
7
Materials
4

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Frequently Asked Questions

What is the objective of Spectrophotometric Analysis of a Mixture?
Determine the individual concentrations of two dyes in a mixed solution using absorbance measurements at two wavelengths and simultaneous equations.
How difficult is Spectrophotometric Analysis of a Mixture?
This experiment is rated as Advanced (University). It takes approximately 75 minutes to complete.
What safety precautions are needed for Spectrophotometric Analysis of a Mixture?
Key safety precautions include: Indicator dyes stain clothing; Handle cuvettes by frosted sides only to avoid fingerprints; Clean spills immediately.
What materials are needed for Spectrophotometric Analysis of a Mixture?
The main materials required are: Methyl orange solution (0.01%), Bromothymol blue solution (0.01%), Unknown mixture of both dyes, Distilled water.
What results should I expect from Spectrophotometric Analysis of a Mixture?
The simultaneous equations yield positive concentrations for both dyes. The reconstructed spectrum (sum of individual contributions) should closely match the measured mixture spectrum, validating the Beer-Lambert additivity principle.