离子交换树脂层析分离混合氨基酸.doc

实验二 离子交换树脂层析分离混合氨基酸【实验目的】1. 了解离子交换树脂层析的工作原理及操作技术。2. 学会用离子交换树脂层析法分离混合氨基酸。【实验原理】离子交换树脂是一种合成的高聚物，不溶于水，能吸水膨胀。高聚物分子由能电离的性基团及非极性的树脂组成。极性基团上的离子能与溶液中的离子起交换作用，而非极性的树脂本身物性不变。通常离子交换树脂按所带的基团分为强酸(RS03H)、弱(COOH)、强碱(N+R：)和弱碱(NH2NHRNR2)。 离子交换树脂分离小分子物质如氨基酸、腺苷、腺苷酸等是比较理想的。但对生物大于物质如蛋白质是不适当的，因为它们不能扩散到树脂的链状结构中。故如分离生物大子、可选用以多糖聚合物如纤维素、葡聚糖为载体的离子交换剂。 本实验用磺酸阳离子交换树脂分离酸性氨基酸(天冬氨酸)、中性氨基酸(丙氨酸)碱性氨基酸(赖氨酸)的混合液。在特定的pH条件下，它们解离程度不同，通过改变脱液的pH或离子强度可分别洗脱分离。【实验材料】1实验器材层析柱（1.6X20cm）；恒流泵；梯度混合器；试管及试管架；紫外分光光度计、磺酸阳离子交换树脂(Dowex 50)2实验试剂 (1) 2molL HCl(2) 2molL NaOH(3) 0.1mOlL HCl(4) 0.1molL NaOH(5) pH4.2的柠檬酸缓冲液：0.lmolL柠檬酸54m1加0.1molL柠檬酸钠46ml(6) pH5的醋酸缓冲液：0.2molL NaAc 70ml加0.2molL HAc 30ml(7) 0.2中性茚三酮溶液：0.2g茚三酮加100ml丙酮(8) 氨基酸混合液：丙氨酸、天冬氨酸、赖氨酸各10m1加0.1molL HCl 3m【实验方法】1. 树脂的处理100ml烧杯中置约10g树脂，加25ml 12mo1L HCl搅拌2h，倾弃酸液，用蒸馏水充洗涤树脂至中性。加25ml 12molL NaOH至上述树脂中搅拌2h，倾弃碱液，用蒸馏水洗涤至中性。将树脂悬浮于50ml pH4.2柠檬酸缓冲液中备用。2. 装柱取直径0.8cm1.2cm、长度10cm12cm的层析柱，底部垫玻璃棉或海绵圆垫，自顶部注入经处理的上述树脂悬浮液，关闭层柱出口，待树脂沉降后，放出过量的溶液，在加入一些树脂，至树脂沉积至8cm10cm高度即可。于柱子顶部继续加入pH4.2柠檬酸缓冲液洗涤，使流出液pH为4.2为止，关闭柱子出口，保持液面高出树脂表面1cm左右。3. 加样、洗脱及洗脱液收集打开山口使缓冲液流出，待液面几乎平齐树脂表面时关闭出口(不可使树脂表面干燥)。用长滴管将15滴氨基酸混合液仔细直接加到树脂顶部，打开出口使其缓慢流入柱内。当液面刚平树脂表面时，加入0.1molL HCl 3ml，以10滴min12滴min的流速洗脱，收集洗脱液，每管20滴，逐管收存。当HCl液面刚平树脂表面时，用1m1 pH4.2柠檬酸缓冲液冲洗柱壁一次，接着用2ml pH4.2柠檬酸缓冲液洗脱，保持流速10滴min12滴min并注意勿使树脂表面干燥。在收集洗脱液的过程中，逐管用茚三酮检验氨基酸的洗脱情况，方法是：于各管洗脱液中加10滴pH5醋酸缓冲液和10滴中性茚三酮溶液，沸水浴中煮10min，如溶液呈紫蓝色，表示已有氨基酸洗脱下来。显色的深度可代表洗脱的氨基酸浓度，可比色测。在用pH4.2柠檬酸缓冲液把第二个氨基酸洗脱出来之后，再收集两管茚三酮反应阴性部分，关闭层析柱出口，将树脂顶部剩余的pH4.2柠檬酸缓冲液移去。于树脂顶部加入2ml 0.1mo1L NaOH，打开出口使其缓慢流入柱内，按上面I续用0.1mo1L NaOH洗脱并逐管收集(注意仍然保持流速10滴min12滴min)，每管20滴。做洗脱液中氨基酸检验，在第三个氨基酸用0.1mo1L NaOH洗脱下来以后，再继续收集两管茚三酮反应阴性部分。最后以洗脱液管号为横坐标，洗脱液各管光密度（以水作空白，在570nm波长读取吸光度）或颜色深浅（以-，+，+表示）为纵坐标作图，即可画出一条洗脱曲线。【注意事项】1. 在装柱时必须防止气泡、分层及柱子液面在树脂表面以下等现象发生。2. 一直保持流速10滴/min12滴min，并注意勿使树脂表面干燥。【思考题】1. 为什么混合氨基酸从磺酸阳离子交换树脂上逐个洗脱下来?2. 树脂如何保存?Experiment 13 Separating Mixed Amino Acids by Ion Exchange Chromatography【Purpose】1. Know the principle and operating techniques of ion - exchange - resin chromatography.2. Learn to use ion- exchange- resin chromatography to separate the mixed amino acids.【Principle】Ion - exchange resin is a kind of synthetic polymer. It cant be soluble in water, but can expand by absorbing water to dilate. Polymer molecule consists of polar groups that can ionize and nonpolar resin. The ions on the polar groups can be exchanged with the ion in the solution, but the physical property of the nonpolar resin will not change. Usually the groups that carried by ion- exchange resin can be divided into strong acid (RSO3H ), weak acid (COOH ), strong base (N+R3 ) weak base (NH2NHR=NR2 ).Its relatively ideal to use ion - exchange - resin to separate small molecules such as amino acid, adenosine, and adenylate and so on. But its not proper for macro-molecule substance like protein, because it cant diffuse into the chain structure of resin. So we can choose ion- exchange reagent that takes polysaccharide such as cellulose and dextran as the supporter to separate biological macromolecules.This experiment uses sulfonic acid cation - exchange resin to separate the mixture of acidic amino acid (aspartic acid), neutral amino acid (alanine) and basic amino acid (lysine). In the condition of certain pH, the degree of their dissociation is different, so they can be respectively eluted and separated by changing the pH and the ion strength of eluting solution.【Materials】1. Apparatus:(1) Column（16/20）(2) Pump(3) Gradient maker(4) Ultraviolet meters(5) Sulfonic acid cation- exchange- resin (Dowex 50)2Reagents:(1) Hydrogen chloride (2mol/L)(2) Sodium hydroxide (2mol/L)(3) Hydrogen chloride (0.1 mol/L)(4) Sodium hydroxide (0. 1mol/L)(5) Citric acid buffer (pH4.2): 54ml of 0.1mol/L Citric acid and 46ml of 0.1mol/L sodium citrate(6) Acetate acid buffer (pH5): 70mi of 0.2mol/L sodium acetate and 30ml 0.2mol/L acetate acid(7) Neutral triketohydrindene hydrate solution (0.2%): 0.2g of triketohydrindene hydrate and 100ml of acetone(8) Mixture of amino acids: 10ml of each of alanine, aspartic acid, lysine and 3ml of 0.lmol/L hydrogen chloride【Procedures】1. The disposal of resin: Put about 10g of resin in a 100ml beaker, add 25 of 12mol/L hydrogen chloride, and stir them for 2 hours. Eliminate the acid liquor, and wash the resin completely to neutral with distilled water. Add 25ml of 12mol/L sodium hydroxide to the above resin and stir for 2 hours. Eliminate the basic liquor, and wash it to neutral with distilled water. Suspense the resin in 50ml, pH4.2 sodium citric acid buffer, and keep it for use.2. Packing: Take a chromatography column with the diameter of 0.8 cm1.2cm and the length of 10 cm12cm, put a piece of glass cotton or sponge circular cushion at the bottom, and pour the above prepared resin from the top. Close the exit of chromatography column. When the resin sediment appears, let out the excessive solution, add more resin until the height of the resin sediment is about 8 cm10cm. Add pH4.2 citric buffer from the top to wash it until the flowing liquor reaches pH4.2. Close the exit of the column, and keep the level of the liquor surface about 1cm higher than that of the resin.3. Loading, elution and collection of eluent: Open the exit to make the buffer flow out. When the level of the liquor is at almost the same height with that of resin, close the exit (dont make the surface of resin dry). Use a long dropper to add 15 drops of mixture of amino acid to the top of resin directly and carefully, and open the exit to make it flow into the column slowly. When the level of the liquor is just at the same height as that of resin, add 3ml of 0.lmol/L hydrogen chloride, and elute it at the flowing rate of 10drops/minute12drops /minute. Collect the eluent, 20 drops per tube and collect it one by one. When the level of hydrogen chloride solution is just at the same height as that of resin, use lml of pH4.2 citric acid buffer to wash the column wall once, then elute with 2ml of pH4.2 citric acid buffer. Keep the flowing rate at 10 drops/minute12 drops/minute. Pay attention not to make the surface of resin dry.While collecting the eluent, check the amino acid in the eluent with triketohydrindene hydrate tube by tube. The process is: Add 10 drops of pH5 acetate acid buffer and 10 drops of neutral triketohydrindene hydrate solution into every tube of eluent. Heat for 10 minutes in the boiling water bath. The solution showing royal blue means some amino acid has been eluted out. The degree of the color can show the concentration of amino acid, and it can be determined by color matching.After eluting out the second amino acid with pH4.2 critic acid buffer, collect two tubes of the negative parts of the action of triketohydrindene hydrate. Close the exit of chromatography column, and transfer the pH4.2 critic acid buffer left on the top of the resin.Add 2ml of 0.1mol/L sodium hydroxide from the top of the resin, open the exit to make it flow into the column slowly. Elute with 0.lmol/L sodium hydroxide according to the above operation, and collect it tube by tube (caution to keep the flowing rate of 10 drops/minute12 drops/minute) by 20 drops per tube. Check up the amino acid in the eluent. After the third amino acid has been eluted out by