Introduction In this experiment, students receive small volumes of unknown liquid which is a mixture — Organic Chemistry Chemistry Question

Introduction
In this experiment, students receive small volumes of unknown liquid which is a mixture of allura red (C₁₇H₁₂N₂O₈S₂Na₂, abbrev. AR) and bromocresol green (C₂₁H₁₄Br₄O₅S, abbrev. BCG) in their basic forms. An aliquot is placed on a small silica gel column, which students prepare by filling silica gel in a Pasteur pipet. The students separate the dyes by stepwise elution with the solvents listed in Table 1. The students choose two eluents that are expected to give the best separation of the dyes. The samples are then diluted to a known volume and quantified by visible spectrophotometry. The components of the entire experiment – column preparation, separation, calibration curves, and analysis of the unknown – can be undertaken easily if students know how to perform quantitative serial dilution, operate a spectro–photometer, and know how to select the analytical wavelengths for the dyes.

[VISUAL]

Chemicals and materials
* allura red, solid,
* bromocresol green, solid,
* hydrochloric acid , w(HCl) = 0.35,
* triethylamine,
* methanol,
* ethyl acetate,
* silica gel, solid,

Apparatus and glassware
* visible spectrophotometer,
* analytical balance,
* sample cell (10–mm pathlength),
* twelve 10 cm₃ volumetric flasks,
* two 50 cm₃ volumetric flasks,
* two 50 cm₃ beakers,
* three 2 cm₃ Pasteur pipets,
* one micropipette (0.1 cm₃),
* pipet filler.

Procedures:
I. Preparation of stock solutions of dyes
These dye solutions tend to decompose over extended periods of time, so they should be prepared just prior to the experiment.
(1) Bromocresol green. An accurately weighed sample (300 mg) is dissolved in ethyl acetate in a 50 cm₃ volumetric flask to which sufficient triethylamine is added to produce the purple basic form.
(2) Allura red. An accurately weighed sample (about 100 mg) is dissolved in about 30 cm₃ of methanol in a 50 cm₃ volumetric flask to which triethylamine is added dropwise until the dye is completely dissolved. Methanol is then added to the mark to dilute the mixture.
Note: Allura red should be pre–purified as follows: Dissolve the sodium salt of allura red in methanol and then filter the mixture. Add an excess of concentrated HCl to the filtrate obtained. After about 30 min the resulting crystals of the protonated form can be filtered off.

II. Preparation of standard solutions of dyes
Prepare five standard solutions for each dye by diluting the stock solution in the same solvents used for the preparation of the stock solutions in 10 cm₃ volumetric flasks. To the allura red standard solutions 3–4 drops of concentrated HCl are added to obtain the dye in its acidic form. The concentration of the standard solutions should be in the range of one tenth to nine tenths of the original concentration of the stock solution.

III. Preparation of calibration curves of dyes
Prepare a calibration curve for each dye using its absorbances at respective peak maximum vs. concentration in ppm.

IV. Preparation of a chromatographic column
Prepare a chromatographic column by placing a small plug of glass wool (or cotton) at the constriction of a 2 cm₃ Pasteur pipet. Silica gel (45/70 mesh) slurried in ethyl acetate is added to the pipet to produce 4–5 cm of packed gel in the column.

V. Column chromatography and quantification
(1) Transfer a 40 µdm₃ aliquot of unknown liquid to the column.
(2) Rinse the walls of the column with a few drops of an eluent selected from Table 1, pass the eluent through the column and collect the eluate in a 10 cm₃ volumetric flask containing 2–3 drops of triethylamine.
(3) Pass a second eluent through the column selected from Table 1, and collect the eluate in a separate 10 cm₃ volumetric flask containing 3 – 4 drops of concentrated HCl.
A small band of impurity may be left behind at the top of the column.
(4) Dilute the first fraction in its purple, basic form to volume with the first eluent.
(5) Dilute the second fraction in its acidic, red form to volume with the second eluent.
(6) Find the concentration of each sample, and thus the amount (mg) of each dye in the unknown from the calibration curve for each dye.

Table 34.1 Possible eluent systems
Eluent No. | Eluent Set I | Eluent Set II
1 | ethyl acetate | methanol
2 | methanol | ethyl acetate
3 | ethyl acetate–HCl a) (200 : 1 v/v) | methanol–HCl (200 : 1 v/v)
4 | ethyl acetate–TEA (200 : 1 v/v) | methanol–TEA (200 : 1 v/v)
5 | methanol–HCl (200 : 1 v/v) | ethyl acetate–HCl (200 : 1 v/v)
6 | methanol–TEA (200 : 1 v/v) | ethyl acetate–TEA (200 : 1 v/v)
a) concentrated hydrochloric acid.