Determination of Absorption Spectra

Group 6
Salvador
San Andres
Sanchez
Santos
Say
Learning Objectives:

1. Demonstrate the principles of spectrophotometry

2. Plot the absorption spectra of bromphenol blue (BPB) and methyl

orange (MO)

3. Identify the point of maximal absorption of BPB and MO

Definition and Principles
 Spectrophotometry – a study
of how much a chemical substance
absorbs light by measuring the
intensity of light as a beam passes
through sample solution.

Spectrophotometer
Light
Light is a form of electromagnetic
radiation,
transmitted via wavelengths,
measured in nanometers (nm)

a. Light can be reflected by the substance

b. It can be absorbed by the substance
c. It can be absorbed at certain wavelengths
and the remainder will be transmitted or
reflected
 Electromagnetic Spectrum
the plot of a sample’s absorbance of light at various wavelengths
Underlying Principles
Transmittance: ratio of the amount of light transmitted to the
amount of light that initially fell on the surface;

%T= I0 x 100

Absorbance: the negative logarithm of the transmittance

A = 2 – log%T
 REMEMBER!!!

Absorbance and transmittance

bear an

Inverse relationship
Absorbance & Transmittance

Computation
▪ First, the intensity of light (I0) passing through a blank is measured.
The blank is a solution that is identical to the sample solution
except that the blank does not contain the solute that absorbs
light. This measurement is necessary, because the cell itself scatters
some of the light.

%T= I0 x 100 and A = 2 – log%T

Absorbance & Transmittance
Computation
▪ Second, the intensity of light (I) passing through the sample
solution is measured.
▪ Third, the experimental data is used to calculate two
quantities: the transmittance (T) and the absorbance (A).

%T= I0 x 100 and A = 2 – log%T

Discussion on Experiment on Beer-
Lambert’s Law
Learning Objectives:

1. To discuss the Beer-Lambert’s Law

2. To demonstrate the Beer-Lambert’s Law using the experimental set-

up of bromphenol blue and methyl orange
Beer-Lambert Law

deals with the relationship between

absorbance and concentration, and between
absorbance and path length

▪ The concentration of a
substance is directly
proportional to the amount of
radiant energy absorbed
Beer-Lambert Law
Absorbance ∝ path length (l) • concentration
A= a•b•c = 2-log%T
Where:
a or ε = proportionality constant, the molar extinction
coefficient or molar absorptivity
b = path length of light traveled (cm)
c = concentration of absorbing molecules in mol/L
The relationship between absorbance and transmittance is illustrated in
the following diagram:

If all the light passes through a solution without any absorption,

then absorbance is zero, and percent transmittance is 100%.
If all the light is absorbed, then percent transmittance alpha is
zero, and absorption is infinite.
Flow of Experiment
Flow Chart of the Experiment
Determination of Absorption Spectrum
(Methyl Orange, Bromphenol Blue)

Take Readings of Optical Density of Diluted Sample

Set Spectrometer at 100 Transmission

Read test tube at different wavelength

Plot wavelength versus optical density

Draw a smooth curve through the points

Indicate a point of max absorption

Results
 Results of the
Absorbance of Methyl Orange at Different Wavelengths

Absorbance of Methyl Orange at Different Wavelengths

Wavelength: Group 2: Group 4: Group Group Group

6: 8: 10:

340 0.899 0.908 0.905 0.906 0.896

405 1.798 1.885 1.888 1.879 1.876

450 1.633 1.630 1.632 1.631 1.635

505 2.492 2.495 2.485 2.492 2.472

550 1.474 1.463 1.465 1.466 1.461

600 0.012 0.015 0.019 0.025 0.021

 Result of the
Absorbance of Bromphenol Blue at Different Wavelengths
Absorbance of Bromphenol Blue at Different Wavelengths

Wavelengt Group 1: Group 3: Group Group

h: 5: 7: Group
9:

340 1.402 1.355 1.295 1.119 1.218

405 2.056 2.048 2.040 2.046 2.038

450 1.662 1.647 1.637 1.628 1.538

505 2.060 2.017 2.020 2.038 2.011

550 2.476 2.209 2.418 2.420 2.416

600 2.916 2.921 2.925 2.939 2.941

 Theoretical Results vs Actual Results

Analyte Theoretical Result Actual Result

Methyl Orange 450nm 505nm

Bromphenol Blue 580nm 600nm

Absorbance of MO 460 nm

Test tube: Dilution Absorbance

(MO:Water)

1 1:4 1.612

2 2:3 1.540
Absorbance

3 3:2 1.631

4 4:1 1.649

5 Pure MO 1.675

Test tube and Dilution

Absorbance of BPB at 580 nm

Test tube: Dilution Absorbance

(BPB:Water)

1 1:4 3.021

2 2:3 3.090
Absorbance

3 3:2 3.050

4 4:1 2.992

5 Pure BPB 2.925

Test Tube and Dilution

Common Errors in Spectrometry

1. Error found in any dilution procedure is in the thoroughness of mixing the

solution
a. it is extremely important to mix the solutions thoroughly
before taking aliquots.
Common Errors in Spectrometry

2. Error involving the reading of the photometric values.

a. may be observed by glancing at the scale of optical density

that certain parts of the scale re-read with greater ease than
other. (Calibration)
Common Errors in Spectrometry

3. Improper fit of tubings

a. may produce “wiggling”, successive readings in the tube may

not be alike.
b. Scattering of light produced by the tubes may not be
perfectly clean.
Clinical Application
Clinical Application and Other Applications
HDN (Hemolytic Disease of Newborn)

RBC RBC ↑Unconjugated Jaundice ↑kernicterus

lysis bilirubin

Treatment / management

Measurement: Reading:
Spectrophotometry Zone 1 - “mildly affected”
Maximum absorbance Zone 2 - “moderately affected”
at 540nm Zone 3 - “ severely affected
SUMMARY