Ion exchange separates amino acids.

related topics .First, the experimental purposelearn the operation method and basic principle of separating
amino acids
with cation exchange resin column.II, Experimental PrincipleIon Exchange Layering
(ICE) is a liquid-solid phase layering method for analyzing and preparing sample mixtures, based on cation or yin of the substance under test Static binding between ions and corresponding ion exchange agents, i.e. according to the differences between the
acid acid and alkali
of the substance, polarity and other differences, through the principle of adsorption and desorption between ions, the electrolyte solution is separated. It is one of the effective means of separating biological molecules of very similar nature from complex mixture systems. Because different charged substances have different affinity for ion exchange agents, by changing the ion strength and pH of the sequential fluid, control this affinity, these substances can be exhumed from the column in order of affinity size.Amino acids are gender electrolytes, the net charge on the
molecule
depends on the epectric point of the amino acid and the pH of the solution, the molecular structure of various amino acids is different, at the same pH the nature of the charge (positive, negative) and how many different, and the affinity of the ion exchange resin is different, so according to the affinity force from small to large order to be washed off, to achieve the effect of separation.III, instruments and
reagents(i) experimental equipment(1) glass
-layer column(2)
test tube(1) 3) Pipe(4) constant pressure wash-out bottle(5) part collector(6) water bath pot(7)
hydrometer(8) Electric furnace(ii) materials and reagents(1) styrene sodium sulfonate resin (100 to 200 eyes)(2) 2mol /L hydrochloric acid solution (3)2mol/L sodium hydroxide solution (4) standard amino acid solution: Tianmen dongine and lysine are rationed to 2 mg / 0.1mol/L hydrochloric acid solution of mL.(5) mixed amino acid solution: the above-mentioned Tianmen dongline and lysine solution in 1:4 ratio.(6) sodium citric acid-sodium hydroxide-hydrochloric acid solution (pH5.8, sodium ion concentration 0.45mol/L): take citric acid 14.25 grams, sodium hydroxide 9.30 grams dissolved in water after mixing, add 5.25 ml of hydrochloric acid, fixed capacity to 500 ml;(7) color agent: 2 g hydrate tritone dissolved in 95% ethanol, add water to 100 ml.4, the operation step gathering(1) layer column preparation: the strong acidic cation exchange resin with sodium hydroxide treatment into Na-type wash to neutral, stirring for 1 hour after loading into the triad column, so that it naturally sinks to a certain height, balanced with buffer.(2) Balance: Rinse the balanced ion exchange column with pH5.8 citric acid buffer, adjust the flow rate, collect the secuffed liquid to 4 times the bed volume to sample.(3) sample separation: the liquid surface slowly put close to the surface of the column, by the top of the column carefully added amino acid mixture 0.25 to 0.5 ml, at the same time began to collect fluid outflow. Immediately add 0.5 ml of citric acid buffer when the sample solution bends the moon face close to the top of the resin. Add an extra 0.5 ml of citric acid buffer when the buffer bends the moon face close to the top of the resin. Repeat so twice, then carefully inject the citric acid buffer with a dropper (do not stir the bed surface). Collect the semen with a test tube and 1 ml per tube until no amino acids flow out.(4) identification of amino acids: add 1 ml of hydrated tritone colorant to each tube collection solution and mix well,
heat
15 minutes in a boiling water bath, cold to room temperature to determine the light density value. The escape curve is drawn with the number of tubes collected as horizontal coordinates and the absorbance value A as the ordinate. (For the pure solution of the two known amino acids as samples, according to the above methods and conditions, the resulting escape curve and mixed amino acids of the escape curve control, you can determine what amino acids the two peaks are.) ) , thinking question 1 What is the ion exchange of amino acids? Which ion exchanger is used in this experiment?.2. Ion exchange resin with buffer balance, why rinse with buffer? Why use pH5.8 citric acid buffer in the experiment? Can I use buffers with other pH values? If so, how much pH should I use? And explain the principle..3.tricetone chromatin is in response to what group of amino acids when coloring with amino acids? What are the reaction conditions? Why should the boiling water bath be heated when the color is colored? What is the color-showing principle?. 4. Can the two amino acids isolated in this experiment be separated by anion exchange resin, and if anion resin is used, what are the conditions and results? Please explain the principle. . 5. In addition to amino acids, which substances are suitable for separation by ion exchange column layering? Why?
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