C30F ANALYTICAL CHEMISTRY LABSCRIPT

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Paper chromatography of selected cations

Basic Theory of Experiment

Paper chromatography is a technique of separation and identification of chemical substances, effected by solvents diffusing through sheets or strips of filter paper, past a spot or streek containing a mixture of components. Differential rates of migration of components of a mixture along the paper arises from the different affinities of the components for the solvent (mobile phase) and the paper (adsorbent or stationary phase). The resulting pattern obtained is known as a paper chromatogram.

Either the ascending or descending technique (the terms underlined refer to the direction of solvent flow) may be used for the development of a paper chromatogram. Descending chromatography is generally faster than the ascending method, but at the expense of a slight increase in complexity of technique. The ratio of the distance traveled by the solvent front to that traveled by a spot from the point of application (origin) is known as the R_f value, which is dependent on the type of paper, solvent and other conditions of development used. Note that R_f values provides tentative, not confirmatory identification.

When the substances being chromatographed are colorless, faintly colored or very low in concentration, various techniques may be used to make the spots clearly visible. In this experiment, visualization will be effected by spraying the chromatograms with reagents that react with the components sought to produce colored complexes. Fluorescent reagents may also be used to provide a fluorescent background when viewed under ultraviolet (u.v.) light, with spots being visible as dark areas where fluorescence quenching occurs.

The sample provided in this experiment contains only cations for Groups IIIA and IIIB, as well as phosphate ions which must be removed before the two groups can be effectively separated. A strongly cationic ion exchanger will be provided to remove the phosphorus interference. After separation into the respective groups, the cations will be separated by paper chromatography.

Preparation of equipment and Materials

Prepare (a) developing solvent - mix in the following proportions :
Acetone:86 ml
Conc. HCl: 6 ml
Distilled water: 8 ml

Prepare (b) visualizing agent:
Dissolve: 0.25 g Oxime (8-hydroxyquinoline) in a mixture of 20 ml ethanol (95%) and 40 ml distilled water.

Sample Preparation

If sample is a solid, place approximately 0.1g of each sample in a crucible and add 0.5ml of conc. HCl. Digest sample on hot water bath in fume hood. When sample has completely dissolved, boil the xs acid off, using a flame if necessary. Transfer the sample to a semi-micro test tube with a few drops of distilled water.

If sample is a liquid, place 0.1g (~ 4 drops) into a semi-micro test tube.

To each sample add 0.5ml conc. ammonia solution to ppt. the gp IIIA cations, and mix the solution thoroughly on a vortex mixer. Allow to settle partially and add another drop of ammonia solution. If pptn. still occurs, add a further 0.25ml of ammonia solution and mix thoroughly. Centrifuge the mixture for two minutes and carefully decant the supernatant into a clean test tube.

To the supernatant, add 1ml thioacetamide solution to ppt. gp IIIB cations, and mix well. Centrifuge and decant off and discard the supernatant.

To each ppt. add 1ml of distilled water, mix thoroughly and centrifuge. Discard washings and repeat procedure once more.

Dissolve each ppt. in a minimum of conc. HCl, heating on a boiling water bath if necessary.
These solutions will be chromatographed.

Sample Application

Place a sheet of chromatographic paper (Whatman #1, 19 x 22cm) on a clean glass sheet. Samples should be placed about 1.5cm from one of the 22 cm edges. Beginning at about 1.5 cm from one of the 19 cm edges, mark points about 2 cm apart with a clean pointed glass rod (Fe contamination avoided) parallel to a 19 cm edge. Decide and note the order in which samples will be applied to the paper. Prepare a sheet for each group of cations. Very thin capillaries are required for placing a sample onto the paper. Ensure that capillaries to be used are broken or scored square so as to facilitate solution transfer from capillary to paper.

Reference solutions of the following metals (as chlorides) are provided in 1 mg/ml concentrations:

Group IIIA : Al⁺³, Cr⁺³, Fe⁺³

Group IIIB : Co⁺², Ni⁺², Mn⁺², Zn⁺²

Place a capillary into each solution and allow to fill. Touch the end of the capillary onto a marked point on the paper and allow a spot no greater than 3 mm to form before removing the capillary. Further applications may be made by drying the applied drop and re-spotting on the same point. In this case, the high standard solution concentrations used enables a single 5 ul spot to suffice. For sample solutions, a 10 ul per spot should suffice. Place a different sample on each point marked and allow the spots to dry before developing the chromatogram.

Development of the chromatogram

Place the sheet of paper, sample end downwards in the solvent, ensuring that the spots are not covered by the solvent. Using the capillary clips and rods, suspend the paper vertically and replace tank cover. Allow to develop until the solvent front has moved at least 10 cm past the points of application. Note the time taken for this development. Remove the paper from the tank, quickly mark the position of the solvent front and suspend vertically in an oven to dry.

Visualization

Fill the reservoir of spray bottle with with the prepared reagent, and reassemble. Suspend the dried chromatogram vertically in a fume hood and lower the window so that excess spray will be vented away from the operator.

Spray the chromatogram from a distance of 6 to 12 inches with oxime, moving horizontally across the paper in overlapping bands, but taking care not to saturate the paper with liquid.

Hold the damp paper at opposite edges and move each area slowly over a 250 ml beaker containing concentrated ammonia solution until yellow, green or brown spots are clearly visible. Dry the chromatogram in an oven on a glass plate at about 50° and then observe under ultraviolet fight, using protective lenses, and outline each spot with a pencil. Note the colors of each spot and background under normal and u.v. light.
Now spray the chromatogram with rubeanic acid, dry at 50° and observe under normal and u.v. light respectively.

Rf values

Measure the distance of the center of each spot from its origin, as well as the distance from each origin to the solvent front.
Calculate the R_f values of the standard reference samples and the unknown components.
Tabulate the R_f values with respective normal and u.v. color.

(a) Assuming that similarity in R_f values of sample and reference indicates the presence of a given component, what cations were present in your sample?

(b) Assuming that the volume of each applied spot was 5ug, estimate the limit of detection of the method for each metal.

(c) What advantages does this method have, if any, over the usual qualitative (semi-micro) analysis procedures for cations?

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