Before highly efficient methods like chromatography or genetic engineering revolutionalized the eluc — Physical Chemistry — Thermodynamics Chemistry Question
Rhodanilic acid or its salts used in protein structure analysis
Before highly efficient methods like chromatography or genetic engineering revolutionalized the elucidation of protein structure the analysis of protein hydrolyzates was very complicated. Several reagents were developed to selectively precipitate individual amino acids from mixtures often containing 20 or more different compounds. One such widely used reagent was rhodanilic acid or its salts, which contain the rhodanilic complex ion ([Cr(SCN)4(PhNH2)2] –). This problem demonstrates the use of this reagent.
Procedure
Chemicals and reagents
* KCr(SO4)2 · 12 H2O, solid
* KSCN, solid
* Aniline
* L-Alanine, solid
* L-Phenylalanine, solid
* L-Proline, solid
* L-Glutamic acid, solid
* Methanol, abs.
* Ninhydrin, 0.5 % (in acetone) (Ninhydrin is a selective reagent for amino acids. Dip the developed and dried plates in a 0.5% acetone solution of ninhydrin, dry the plates and heat them for a short time with a heat-gun. Contact of the reagent with skin should be avoided since it produces a rather long-lasting purple discoloration. Use forceps!)
* n-butanol
* NH3, conc. aqueous solution
* Acetic acid, 100 %
* Acetic acid, 50 %, aqueous solution
* HCl, aqueous solution (c = 0.25 mol dm–3)
Preparation of ammonium rhodanilate
In a 100 cm3 flask mix 5 g hydrated chromium(III) potassium sulfate, 5.8 g potassium thiocyanate and 5 cm3 water and heat it in a 80 ˚C water bath for 10 minutes. Under the hood add 5 cm3 aniline and continue heating for an additional 60 minutes. Dilute the product with 50 cm3 water and add 10 cm3 glacial acetic acid. Keep the mixture in an ice-water bath for 10 minutes with occasional scratching using a glass rod. Filter off the purple precipitate on a sintered glass filter and wash it with water.
In a flask dissolve the product in 20 cm3 methanol. Filter out any insoluble impurities. To the solution add 10 cm3 of concentrated ammonia solution and 50 cm3 water. Collect the precipitate with filtration, wash it with water and dry on an open Petri dish.
The reaction of ammonium rhodanilate with amino acids
Dissolve 0.35 g proline in 15 cm3 aqueous HCl solution (0.25 mol dm–3) in a beaker. In a separate beaker dissolve 1.5 g ammonium rhodanilate in 20 cm3 methanol. Mix the two solutions. Filter the precipitate on a glass filter. Wash with three 10 cm3 portions of distilled water. Dry the product in an open Petri dish.
TLC experiments
Dissolve approximately 10 mg samples of alanine, proline, phenylalanine and glutamic acid (separately) in 1 cm3 of water. Additionally, make a sample mixing the four standard solutions. In a separate test tube mix 0.1 cm3 samples of these solutions with 0.1 cm3 ammonium rhodanilate solution (5 % in methanol). Filter the solutions on paper using a small funnel.
Analyze the solutions by TLC on silica plates. Find an appropriate eluent by mixing 50% acetic acid and n-butanol. Visualise the spots with ninhydrin. Summarize your results. Explain your findings!
Model Answer
The students should find an eluent that is capable of separating all the amino acids (e.g. n-butanol : 50% acetic acid = 2:1).
The plates show that unreacted amino acids are present in the filtrates with the exception of proline. Thus, the reagent selectively removes proline from the mixture. A precipitate is formed in every test tube, as the reagent itself precipitates.
Note that proline produces a yellow discoloration with ninhydrin, while the other amino acids give purplish-blue spots. The mechanism of the chromophore generation is rather complex and is not needed to do the problem.