Handout 7

07. Column Chromatography
Important concepts
Heterogeneous phase equilibrium, adsorption chromatography, stationary phase and mobile phase, eluents,
Beer Lambert’s law. Absorption photometry in visible range, absorbance vs. concentration calibrating curve.
Objective:
In this experiment you will use column chromatography for the separation of two dyes (Sudan III and
Erioglaucine) from their common solution. After separation you will determine the dye concentrations by
measuring the absorbance of the solutions at appropriate wavelengths.
Background:
All the various types of chromatography are based on the fact that different components of a multicomponent
gas or liquid system are differently distributed between a phase attached to a porous solid carrier and a fluid
phase (gas or liquid) flowing through that solid.
According to the underlying physico-chemical principle we can distinguish between adsorption, partition,
and ion-exchange chromatography.
According to the physical state of the fluid phase (mobile phase) we have liquid chromatography (LC) and
gas chromatography (GC).
The method that is used here is a simple version of liquid chromatography and the underlying physicochemical principle is adsorption.
In the present experiment you will make use of the fact that adsorption of the following substances is different
on Al2O3:
water(solvent component) > Erioglaucine (dye component) >
> ethanol (solvent component) > Sudan III (dye component).
Figure 1. Dye molecules are bounded to active sites of
solid (alumina) at the interface.
Apparatus
Figure 2. The chromatographic apparatus
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Procedure
1. Assemble the apparatus shown in Fig. 2.
2. Prepare the chromatography column. Place the Witt platelet in the glass tube and put an even layer of
5 mm of cotton over it. Wet the cotton layer with 96% ethanol, and press against the bottom of the glass tube
with a glass rod. Measure in a 25 ml beaker 10 ml of alumina (appr. 2 spools) and 7 ml 96% ethanol, and mix
them well with a glass rod. Add more 96% ethanol if needed to get a suspension like pancake batter. Fill the
whole amount of suspension into the tube. Fill a ≈1 cm thick layer of 96% ethanol over the column.
Handling of vacuum apparatus.
Turning on the water aspirator: The vacuum control stopcock and the vacuum inlet on the filter flask are
opened. Open the tap of water aspirator completely, and close gently the vacuum control stopcock (on
the second filter flask for pressure adjusting).
Turning off the water aspirator: Having finished the suction open the vacuum control stopcock first, and after
that close the tap of water aspirator.
Turn on the water aspirator. Keep ethanol on the top of the column and compact it for a minute. Turn off the
water aspirator, the column is ready for use.
Make sure that there always remains some liquid layer of at least 0.5 cm (this time 96% ethanol) over the
settled Al2O3 column otherwise it will dry out or crack (even if you cannot see that happen) and you will have
to prepare a new one.
Separating the dyes.
3. Pipette 1 ml from your assigned unknown solution over the column. Start suction.
4. Stop suction when the red ring originating from Sudan III left the column and the cotton layer. Disassemble
the apparatus. Check if the solution is opalescent (some alumina can get into the filter flask) or not. If it is
opalescent you have to filter it using a glass filter before you wash it quantitatively into a 25 ml volumetric
flask. Fill the volumetric flask up to the ring mark with 96% ethanol.
5. Fit the column again onto a clean, dry filter flask and continue experiment by feeding the column with 30%
ethanol. Start suction. Stop suction when the blue ring originating from Erioglaucine left the column and the
cotton layer. Disassemble the apparatus. Check if the solution is opalescent or not. If it is opalescent you have
to filter it using a glass filter before you wash it quantitatively into a 25 ml volumetric flask. Fill the volumetric
up to the ring mark with 30% ethanol.
Be careful in filling a proper amount of solvent. Use as maximum volume only 15 ml for the separation process
and 7 – 10 ml for washing the content of filter flask.
Photometry
The spectrum of Sudan III. and Erioglaucine, i.e. the absorbance vs. wavelength function are taken by
Metertech 880 single beam spectrophotometer. Before working, the photometer has to be warmed up and
ready to measure.
We have to measure the spectra of separated dye solutions and the spectrum of the 25 times diluted original
unknown solution (ask the technician for the solution).
When components are separated well the sum spectra of the separated dyes should be identical to the one got
from the technician.
For the sake of the highest signal to noise ratio in detecting absorbances we determine the wavelength
belonging to the highest absorbance, λmax for the separated dies.
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Settings
From Function menu item chose Spectrum. In the appearing active Instrument window set parameters
available in Table 1.
Table 1 Settings
Parameter
Start wavelength / nm
Stop wavelength / nm
Measure mode
Low value (ABS)
High value (ABS)
Scan speed / nm/min
Value
400
700
ABS
0
3.000
normal
Reference reading
Use a plastic cuvette of 1 cm optical pathlength either for 96% ethanol (Sudan III.) or 30% ethanol
(Erioglaucine). Fill the reference solution to cover the aperture of optical system i.e. all the incoming photons
transported through the liquid in the cuvette (fill it up to ¾ of total height). The outer active surfaces of a
cuvette must be stainless, so clean them. Place the cuvette into cuvette holder, put a teflon lid on cuvette and
close the door of cuvette compartment. Click on knob
for running base-line
Measurement
Fill the dye containing liquid in a 1 cm cuvette. Place the cleaned cuvette into cuvette holder, put a teflon lid
on cuvette, close the door of cuvette compartment. Click on knob
for running sample spectrum. When
spectrum is taken use command File\data export to Excel and chose a file name according to the sample. (The
file name is noted in Lab report also).
Save after each spectrum taken, if not the data are lost. The data files can be found in folder
„C:\Program files\Metertech\SP880\Mate”.
Calculation
The absorbance values can be read and processed from the saved Excel file.
The linear calibrating functions of dyes are given below. Note, absorbances and intercepts are dimensionless,
therefore the term c·l·ε should also have no dimension.
ASudan III = 0.00365 + 915.58706 · (100 cm2 / g) · cSudan III
(at 509 nm)
AErioglaucine = –0.01178 + 1014.52947 · (100 cm2 / g) · cErioglaucine
(at 626 nm)
The maximum wavelength of dyes observed can differ by 1 – 2 nm, the error arising from this can be neglected
to that of the error of separation.
From equations given, you can calculate the concentrations of unknown, by taking into account the 25 times
dilution.
Graphs, results, data to be reported:
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the λmax values of dyes
the absorbances read at the given wavelengths
the calculated concentrations in units g/100 cm3 for the original unknown solution in three significant
figures
the spectrum of the unknown sample after separation and the spectrum of the 25 times diluted original
solution without separation (ask the technician for the solution). All three spectra should be in the same
figure.
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Sample questions and answers for the entrance quiz.
These questions are devoted for practicing and preparing to labs at home. Please note that these are samples only and
the question of the entrance quiz are not restricted to these ones!
MLT Q and A:
Q1. Describe the concentration determination process of separated dye components.
A1. The separated dye components are diluted to known volume and their absorbance are measured by
photometer. From absorbance - dye concentration calibration line the concentration of separated dyes can be
read.
Q2. Give the general law for the concentration dependence of the absorbance!
A2. At a single wavelength mostly at λmax of a spectral band the Beer Lambert’s law can be written as
I
A(λ ) = log 0 = ε (λ ) ⋅ c ⋅ l
I
where A(λ ) is the absorbance, ε (λ ) is the molar absorbance, c is the concentration of absorbing species and
l is the optical path length of the solution. ε (λ ) is a material coefficient which depends on the wavelength. I0
and I are the incoming and outgoing intensity of radiation.
Q3. What are the stationary phase and mobile phase used in this experiment?
A3. The stationary phase: Al2O3, and mobile phase: water – ethanol mixture.
Q4. What eluent mixtures are applied?
A4. Eluent mixtures are: 96% ethanol comes first, 30% ethanol is the next.
Q5. What are the components of slurry (the column material), and how do you prepare the column?
A5. A slurry is prepared by mixing the first eluent, 96% ethanol with the powder of stationary phase and then
carefully poured into the column. Care must be taken to avoid air bubbles. The solution of the dye mixture is
pipetted on top of the stationary phase.
Q6. What is the strongest adsorbate of the four components?
A6. The surface of Al2O3 is hydrophilic and water adsorbs the most on.
Q7. What is the adsorption order develops on alumina in the four component liquid system?
A7. H2O > Erioglaucine > Ethanol > Sudan III. The most polar molecule water adsorbs to polar alumina the
strongest way.
Q8. Describe the separation process of dye mixture.
A8. At first eluent 96% ethanol extracts Sudan III. Erioglaucine and water are strongly adsorbed to alumina
and they remain on the top of the column. Sudan III, the red component on active sites is replaced by ethanol
and water along the length of the column.
In the second stage eluent 30% ethanol washes Erioglaucine from column, because of its increased water
content. Water is capable to desorb blue component Erioglaucin.
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