Ion exchange layering separates amino acids

the purpose requires(1) Learn the basic principles of using ion exchange resin
of amino acids
of amino acids.(2) master the basic operating techniques of ion exchange column layering.the experimental principleion exchange layering method is mainly based on the differences in the properties of the material and choose different ion exchanger separation method. The structure of various amino acid molecules is different, and the affinity with the ion exchange resin is different at the same pH, so it can be exhumed according to the order of affinity from small to large, to achieve the effect of separation.Reagents
and equipment 1, reagents and materialssodium styrene sulfonate resin (strong acid 1×8,100-200 eyes);Standard amino acid solutions: Winterline, lysine and histamine are all made into 2mg/mL of 0.1mol/L hydrochloric acid solution.Mixing amino acid solution: Mix 3 standard amino acid solutions in a ratio of 1:2.5:10.Sodium citric acid-sodium hydroxide-hydrochloric acid buffer (pH5.8, sodium ion concentration 0.45mol/L): Take citric acid (C6O7H8? H2O) 14.25g, sodium hydroxide 9.30g and strong hydrochloric acid 5.25mL dissolved in a small amount of water, fixed capacity to 500mL, refrigerator storage.Coloring agent: 2g hydratedin 75mL glycol monomethane ether, add water to 100mL.Second,
20cm × 1cm
; constant pressure wash bottle; part collector;
The 10th photonometer
.Operation method, resin treatmentthe dry strong acid resin soaked overnight with distilled water, so that it fully swelled. Soak for 1 hour with 2mol/L hydrochloric acid, 4 times the volume, pour out the liquid and wash to neutral. It is treated with 2mol/L sodium hydroxide, which is the same. Finally, soak with the buffer you want to use.

, the column
Take a 1cm diameter and a length of 10-12cm. Position the column vertically on the iron frame. From the top of the above-mentioned treated resin suspension, close the column outlet, after the resin subsides, release the excess solution, and then add some resin, to the resin sink to a height of 8 to 10cm can be.
Third, the exhumation of amino acids


the balance exchange column with pH5.8 citric acid buffer. The regulated flow rate is 0.5mL/min, which can be sampled when the outflow reaches 4 times the size of the bed. Carefully add the amino acid mixture 0.25-0.5mL from the top of the column and start collecting the fluid. When the sample fluid bends the ion surface close to the top of the resin, immediately add 0.5mL citric acid buffer to rinse and add the sample. Add a further 0.5mL buffer when the buffer bends the edging surface close to the top of the resin. Repeat twice, then carefully inject the citric acid buffer with a dropper (do not stir the bed surface) and attach the column to the washed-out bottle and part of the collector. Started with
test tube
collected semen, each tube collected 1mL, a total of 60-80 tubes.


4. Identification of amino acids added 1mL hydrate to each tube collection liquid tritone colorant and mixed well, accurately
heated
15min in boiling water bath and then cooled to room temperature, and then added 1.5mL of 50% ethanol solution. Leave for 10 minutes. The light absorption value of A570 wavelength is measured with the blank of the 2nd tube of the collecting liquid, the light absorption value is the ordinate, and the escape curve is drawn with the liquid product as the horizontal coordinate. With a pure solution sample of 3 known amino acids, operated separately according to the above methods and conditions, the resulting desulfurized curve is compared with the purification curve of the mixed amino acids, and the approximate position of the 3 peaks and what amino acids are the peaks can be determined.