Analytical Techniques in Biotechnology:

Lab Component
Name: Saumya S
Reg. no.: 15BBT0054
Date:
Lab 6:
Part 1-Solvatochromism
Aim:
To observe the phenomenon of solvatochromism using crystal violet
and methyl red dyes on various solvents.
Principle:
Solvatochromism is defined as the ability of a chemical substance to
change its color by changing the solvent polarity. The Solvatochromic
effect or solvatochromic shift refers to a strong dependence of
absorption and emission spectra with the solvent polarity. Since
polarities of the ground and excited state of a chromophore are
different, a change in the solvent polarity will lead to different
stabilization of the ground and excited states, and thus, a change in
the energy gap between these electronic states. Consequently,
variations in the position, intensity, and shape of the absorption
spectra can be direct measures of the specific interactions between the
solute and solvent molecules.
Solvent effects are sum of the bulk solvent properties like
polarity,
dielectric
property,
dispersive-induction-polarization
interactions, viscosities and/or specific solute-solvent interactions. In
the absence of inter-molecular hydrogen bond, the spectral shifts are
mainly sensitive to the solvent polarity. Solvent-solute interactions in
the cases of non-polar solvents like saturated hydrocarbons are
negligible. Therefore, the absorption spectrum of a solute molecule in
these nonpolar solvents shows fine spectral details similar to that in
pure gaseous state.

Apparatus
UV Sperctrophotometer
Methyl orange dye
Crystal violet dye
Ethanol
Chloroform
Acetone
Distilled water
Ependoff tubes
Micro-pipette
Procedure
Take 4 ependoff tubes and add 990 micro litres of water, chloroform,
ethanol and acetone in each one respectively.
Make another set of such tubes.
To the 1st set add 10 micro litres of crystal violet dye to each of the 4 to
make up to 1mL
To the 2nd set add 10 micro litres of methyl orange dye to each of the 4
to make upto 1mL.
Shake well.
Observe the colour variations.
Now switch on the UV Spectrophotometer and wait for 30 minutes to
initialize the instrument.
Now load the samples one by one in the cuvette and measure the
absorbance in the range of 800 nm to 200 nm.
Save the readings.
Observations and Results

Experiment done results attached as excel

sheet.

Figure 1 Solvents with crystal violet dye

Figure 2 Solvents with Methyl Orange dye

Discussion
The solavtochromism is observed much more clearly in the set
containing methyl orange as the dye. This shows that methyl orange is
s much better dye for detection of this phenomenon.
The differences in the set containing crystal violet dye is very subtle
but can be observed much clearly when spectrophotometry is done.
Precautions
Wear lab coats and gloves while dealing with chemicals.
Most of the solvents are volatile in nature, so, use carefully.

Always use a reference cuvette for precise results.

Analytical Techniques in Biotechnology:

Lab Component

Name: SAUMYA S
Reg. no.: 15BBT0054
Date:

Lab 6:
Part 2-Absorption Spectroscopy

AIM:

To study the effect of solvents on the UV-visible absorption spectra of a

molecule.
PRINCIPLE:
Solvent effects are sum of the bulk solvent properties like polarity,
dielectric property, dispersive-induction-polarization interactions, viscosities
and/or specific solute-solvent interactions. In the absence of inter-molecular
hydrogen bond, the spectral shifts are mainly sensitive to the solvent
polarity. Solvent-solute interactions in the cases of non-polar solvents like
saturated hydrocarbons are negligible. Therefore, the absorption spectrum of
a solute molecule in these nonpolar solvents shows fine spectral details
similar to that in pure gaseous state.
We know that the electronic transitions modify the charge distribution of
the absorbing molecule. Therefore, depending on the solvent polarity and
kind of transition, solvent-solute interactions vary which in turn determine
the energies of the ground and excited states of the light absorbing
molecule. This affects both the peak position (max) and absorptivity () of the
absorbing molecule. The absorptivity, a characteristic of the absorbing
substance, is a useful quantity that gives us idea about the transition
probabilities in the molecules and the effective light capture area (the crosssection for light absorption) of the species. If the chromophore involved in
the transition is more polar in its ground state than in its excited state, then
the ground state is more stabilized than the excited state by a more polar
solvent due to solvation.
Many molecules absorb ultraviolet or visible light. The absorbance of a
solution increases as attenuation of the beam increases. Absorbance is
directly proportional to the path length, b, and the concentration, c, of the
absorbing species. Beer's Law states that
A = bc, where is a constant of proportionality, called the absorbtivity.
Different molecules absorb radiation of different wavelengths. An
absorption spectrum will show a number of absorption bands corresponding
to structural groups within the molecule. For example, the absorption that is
observed in the UV region for the carbonyl group in acetone is of the same
wavelength as the absorption from the carbonyl group in diethyl ketone.

Here absorptions of coumarin138 have been studied in a number of

polar and nonpolar solvents, namely cyclohexane, dioxane, acetonitrile,
ethanol and ethylene glycol, to demonstrate the solvent effect.

Molecular structure of coumarin 138

PROCEDURE:
1. Prepare five 1 10-5 M coumarin-138 solutions in solvents:
cyclohexane, dioxane, acetonitrile, ethyl alcohol and ethylene glycol.
Such dilute solutions can be prepared via dilution from 1 10-4 M stock
solutions in respective solvents.
2. The absorption measurements with all the solutions are carried out one
after another as follows.
3. To take a particular solution, click on the appropriate solvent on the
solvent selection bar and then click on the volumetric flask containing
the solution.
4. Click on the quartz cuvette (path length, 1 cm 1 cm) to take it to the
instrument table.
5. Click on the glass Pasteur pipette to collect about 3 mL of the
experimental solution which will be transferred into the quartz cuvette.
6. Click on the pipette to draw the solution into it.
7. Click on the pipette to take it out of the volumetric flask.

8. Click on the pipette again to transfer the solution into the cuvette. To
start the absorption spectral scan, click on the pop-up Start
Absorption Measurement.
9. Turn on the spectrophotometer clicking on the power button. In real
operation, it takes approx. 30 min for initialization of the instrument.
10.
Open the lid of the sample chamber of the spectrophotometer by
clicking on the lid for placing the sample in the cell-holder. Click on the
cuvette to place it in the sample holder. One has to use pure solvent as
the sample blank or reference in this measurement. Here a double
beam spectrophotometer is shown.
11.

Close the lid of the sample chamber by clicking on it.

12.
Open the measurement set-up screen by clicking on the
absorption measurement icon on the computer monitor.
13.
On the screen, enter the wavelength range. Start: 430 nm End:
275 nm. In real operation, the wavelength range of incident light for
the sample is chosen and the wavelength scan is run via the
accompanied computer software. One can run the scan in absorbance
(A) or transmittance (%T) mode.
14.
Click on the green Start button on the measurement set-up
screen to run the wavelength scan. Observe the wavelength scan.
15.
Click on Close button when spectral scan is complete. In real
operation, the scan data are stored in the computer. The instrument
stores data and therefore asks for the Sample File name. One enters a
file name to save the data.
16.
To take the cuvette out of the sample chamber, first click on the
sample chamber lid to open it and then on the cuvette. Click on the lid
of the sample chamber to close it.
17.

Click on Reset button to start over the measurements.

18.
Repeat the Absorption measurements with all the solvents by
clicking on the solvent selection bar first and then on the volumetric
flask containing the solution.
19.

Collect all data by clicking on the Data tab.

20.

Examine UV-visible absorption spectra with different solvents.

21.
Find out the maximum absorption wavelengths and
corresponding absorbance values for all the solvents and tabulate
them along with the solvent dielectric constants and refractive indices
values and note down the inferences.
OBSERVATION:
Cyclohexane:

Dioxane:

Acetonitrile:

Ethanol:

Ethylene Glycol:

Table 1: Solvent Properties

Observation Table:
Solvent
Absorbance

max

(approx.)

Cyclohexane

347.5

0.9

Dioxane

330

0.75

Acetonitrile

362.5

0.88

Ethanol

362.5

0.7

Ethylene Glycol

376

0.84