one,    principle: Some high molecular substances contain some genes that can be separated, such as -SO 3 H , -COOH, etc., so they can exchange reactions with ions in solution. Such as: R- SO 3 H + M + ———— R- S 3 M + H + Or R -NH 3 OH + CL -  —————  R- NH 3 CL + OH - Such high molecular substances are generally referred to as ion exchangers, and the most common ones are ion exchange resins. Since certain ion exchangers have different affinities for different ions, the migration speed of different ions on the ion exchange column is different during the elution process, and finally separation is obtained. two,    Purpose and requirements: In this experiment , a column packed with a Zerolit 225 cation exchange resin was selected, and a specific pH buffer eluent was selected to separate two amino acid solutions having different properties. Through experiments, it is required to grasp the main points of ion exchange column chromatography technology such as packing, loading, elution and collection. Third, instruments and devices: Glass chromatography column: 19 cm long , 1.2 cm inner diameter , 3# sand core. HL-2B constant current pump. HD-4 computer nucleic acid protein detector. BS-100A automatic partial collector. 250ml beaker. 1ml straw. Water bath. Type 72 (or 721 type) spectrophotometer. four,    Reagents and drugs: Resin: Zerolit 225 type cation exchange resin. Eluent: 0.45N, PH5.3 citrate buffer, citrate take 285g (C 6 O 7 H 8 · H 2 O); 186g 97 ℅ NaOH; 105ml concentrated sulfuric acid dissolved in water and diluted to 10 liters. Sample solution: 0.005 M ASP and LYs in 0.02 N HCL mixed solution. Developer: Developers List two of the optional ones. Developer ( I) ninhydrin- TiCL 3 solution. 10 g ninhydrin is dissolved in 500 ml of ethylene glycol methyl ether, and then 0.85 ml of TiCL 3 ( 15% ) Developer (II): ninhydrin- KCN solution. 0.1M KCN: 0.1628g KCN is diluted in water to 250ml A, and 1.25g ninhydrin dissolved in 25ml of ethylene glycol monomethyl ether, dubbed the concentration of 5% (W / V) solution. B, and 2.5ml 0.01M KCN solution was mixed with 125ml of ethanol ether. The combined set A and B can be used brown bottle overnight. When this solvent is used, the two solutions A and B are combined on the previous day, and the prepared solution can only be used within 1-2 days, and the overdose failure must be re-matched. V. Methods and steps:    1 , resin treatment: For the treatment of commercially available new resins, see 7 , and the treated resin is used in this experiment. 2. Packing column: Install the column vertically, close the bottom outlet of the column, and inject a citrate buffer of about 1 cm in the column . The treated become the sodium form resin placed in a beaker, add 1 volume citrate buffer, a suspension churned column along the inner wall of the column filled carefully. Don't be too fast to avoid foaming. When the resin is gradually deposited to a height of about 1 cm at the bottom of the column, the supernatant present in the upper layer of the column is sucked up with a pipette, and the outlet of the column bottom is slowly opened, and the resin suspension is continuously filled until the column is attached to a height of 8 cm . When packing the column, avoid circulating the liquid in the column to cause the column to fail, and the temperature of the resin suspension should be relatively constant (especially for the high temperature method). The installed cylinder should have no grain, no streaks, no air bubbles, and the top surface of the column is flat and uniform. In this way, it can be put into use, otherwise it will need to be reinstalled. 3 , balance: Good pick up of the current pump flow rate or constant pressure column after elution vial installed, using a citrate buffer eluent 24ml / hr and a flow rate balance, until the PH PH same eluent until the effluent, which is approximately It takes 2-3 times the bed volume. 4 , loading and elution: Remove the pipette plug from the column, open the bottom outlet of the column, and carefully close the liquid in the column as it flows to the surface of the body. Aspirate 0.5 ml of the amino acid acid mixed sample solution with a pipette . Carefully add into the column along the column wall, do not add too fast, so as not to damage the resin surface, slowly open the bottom valve after loading, close the liquid surface when it is aligned with the resin surface, and then use a pipette to absorb the appropriate amount of elution. Liquid, so clean the inner wall of the column 2-3 times. After washing, the solution was added to the 2-3 cm high liquid layer in the column with a buffer , and then the pipette was attached, and the flow rate ( 24 ml/hr ) was adjusted to start elution. Note that all air bubbles in the connecting tube between the constant current pump or the constant pressure bottle and the column should be excluded when adjusting the flow rate. And do not be too aggressive when adjusting, so as not to affect the chromatographic behavior. 5. Collection: The column fluid can be used to collect 20 tubes from a part of the collector . 6 , measurement: After collecting the collected tubes, take 0.5 ml of the collected solution in a clean dry test tube, add 1 ml of elution buffer; 0.5 ml of KCN ninhydrin reagent, mix and heat in a water bath at 100 ° C for 25 minutes, then water to cool 5 -10 min, add 3 ml of 60% ethanol to dilute, shake well and colorimetrically at 570 nm with a 72 or 721 spectrophotometer . After the measurement, the light absorption is plotted on the ordinate, and the number of tubes or ml collected is plotted on the abscissa. 7 , regeneration: After using the packed column several times, it needs 0.2N NaOH solution to be eluted, and then washed with distilled water until it is neutral and then reused. For the commercially available dry resin, the treatment method is as follows: after the water is sufficiently dissolved, the fine particles are removed and the water is clarified. Then, by flotation, a resin with a suitable particle size is obtained, and the suspension is sequentially washed with 4 times of 2N HCL and 2N NaOH for half an hour. When changing the acid or alkali, the resin is washed to neutral with water, and finally the resin should be treated. No yellow to the solution. At this time, the resin was converted to a sodium form by using 1 N NaOH (depending on the experiment and the resin for the transformation), and washed with distilled water until neutral. For sample chromatography in which chromatographic behavior is unknown or difficult to estimate, a tube-by-tube collection assay is used to capture the results of the chromatogram. The amino acid measurement is generally around 1PH . In this experiment, the pH of the buffer is 5.3 , so it can be directly measured. However, if the pH deviation is too large, you can add 1ml , 2N , PH5.5 acetate buffer to ensure the measurement conditions. Ultrasound System,Ultrasound General Images,Ultrasound Imaging Device,Ic Enter Integrated Ultrasound MinFound Medical Systems Co., Ltd , https://www.minfoundmed.com
Ion exchange column chromatography (introduction to chromatographic experiments)
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