DNS- 氨基酸的双向聚酰胺薄膜层析

1. DNS-氨基酸的双向聚酰胺薄膜层析 检验系生化教研室 李莉

2. 一、吸附层析的概念及原理 • 吸附：一种物质被聚集在另一种物质表面，这种现象是吸附。 • 吸附剂：凡是能够将其他物质聚集到本身分子表面的物质称为吸附剂，如氧化铝、硅胶等。 • 被吸附物：聚集在吸附剂表面的分子就称为被吸附物。

3. 吸附层析（Absorption Chromatography): 指混合物组分随流动相经过由吸附剂组成的固定相时，由于吸附剂对不同物质的吸附能力的不同以及各组分在流动相中的溶解度不同而加以分离的方法。

4. 二、薄层层析（Thin-layer chromatography,TLC) • 薄层层析法是在平滑的玻璃板上或在聚脂薄膜上，将吸附剂铺在上面成薄层作为固定相，以溶剂作为流动相，把样品中各组分分离。 分离原理：吸附层析、分配层析和离子交换层析。

5. （一）吸附剂 吸附剂主要是利用溶质在吸附剂和溶剂中的可逆平衡以及吸附剂对不同物质吸附力的不同而达到分离的目的。因此在薄层层析中吸附剂是关键的材料。

6. 1、对吸附剂的要求 （1）应不溶于所使用的溶剂以及与所使用的溶剂和样品中各组分不起化学反应； （2）应具有较大的吸附表面（吸附容量）和一定的吸附力，对被分离物质应有足够的分辨力； （3）吸附剂颗粒大小要均匀，保持良好的重复性。

7. 吸附剂的吸附能力常称为活度，用罗马字母Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ表示。吸附剂的吸附能力常称为活度，用罗马字母Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ表示。 活度 减少 含水量 增大

8. 2、常用吸附剂 极性：氧化铝 硅胶 非极性：纤维素 聚酰胺：a.合成：己二酸、己二胺 b.特点：氢键吸附剂

9. 聚酰胺是—类化学纤维原料，即锦纶 (又称尼龙)。由己二酸与己二胺聚合 而成的称锦纶66 。 因为在这类物质分子中都含有大量酰胺 基团，故统称聚酰胺。

10. 聚酰胺对很多极性物质有吸附作用，这是由于聚酰胺的一C＝O及>NH基能与被分离物质之间形成氢键。如酚类(包括黄酮类、鞣质等)和酸类<如核苷酸、氨基酸等)是以其羟基与酰胺键的羰基形成氢键；硝基化合物和醌类等物质与酰胺键的氨基形成氢键。被分离物质形成氢键能力的强弱，确定吸附能力的差异。在层析过程中，层层溶剂与被分离物质在聚酰胺表面竞相形成氢键。因此选择适当的展层溶剂，使被分离物质在溶剂与聚酰胺表面之间的分配系数能有较大差异，经过吸附与解吸的展层过程，可以一一分离.

11. （二）溶剂 不同溶剂具有不同的结构性质，依极性大小各种溶剂的洗脱能力各不相同。 一般所选溶剂要求： a.纯度合格 b.黏度要小，易与样品中各组分相分离 c.与所欲分离样品和吸附剂不起化学反应

12. 溶剂选择考虑因素： 1、溶剂与吸附剂之间的相互作用力 2、溶剂与样品之间的作用因素 溶剂的选择可通过实验来确定。

13. （三）薄层层析的操作 1、点样 样品最好溶解在挥发性的溶剂中，如氯仿、丙酮等，避免用水每次点少量样品，可在溶剂挥发后反复进行至点完为止。 样品原点直径＜3mm

14. 2.平衡 3.展开 方法：上行层析法 下行层析法 双向展开法

15. 4、显色 1）有色物质（如黄体酮）展开后即可显出斑点 2）紫外灯下显出荧光斑点，如核苷酸和某些生物碱 3）显色剂显色

16. 荧光试剂：DNS-Cl二甲氨基萘磺酰氯 蛋白质、多肽、氨基酸可与其游离氨基结合，DNS-aa发出黄绿色荧光

17. DNS-C1在pH过高时，水解产生 副产物DNS-OH，即：

18. 在DNS-C1过量时，会产生DNS—NH2，即：

19. DNS-氨基酸在紫外光照射下呈现黄色荧光，而DNS-OH和DNS-NH2产生蓝色荧光，可彼此区分开。

20. 三、薄层层析的应用 （一）定性分析：将已知化合物作为标准品与样品一起进行层析后对照，可初步确定未知化合物的组成。 （二）定量分析：可直接在薄板上测定，无须破坏薄层； 用工具将斑点从薄层上取下，用溶剂洗脱，再用其他方法测定。

21. （三）临床生化上的应用 1.氨基酸的分离 2.核苷、核苷酸和核酸的分析

22. DNS-氨基酸的双向聚酰胺薄膜层析 二甲氨基萘磺酰氯 （1-Dimethylaminonaphtalene-5-sulfonyl chloride）简称DNS-Cl，可与氨基酸的游离氨基结合成DNS-氨基酸，形成的DNS-氨基酸在紫外线（260nm或365nm）照射下发出强烈的黄色荧光，因此可用荧光检测DNS-氨基酸的存在。反应过程如下：

24. Procedure for TLC 1. Prepare the developing container. The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top:

25. Pour solvent into the beaker to a depth of just less than 0.5 cm.

26. To aid in the saturation of the TLC chamber with solvent vapors, line part of the inside of the beaker with filter paper

27. Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate.

28. 2. Prepare the TLC plate. TLC plates used in the organic chem teaching labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be.

29. Shown in the photo to the left is a box of TLC plates, a large un-cut TLC sheet, and a small TLC plate which has been cut to a convenient size. Plates will usually be cut and ready for you when you come to lab. Handle the plates carefully so that you do not disturb the coating of adsorbent or get them dirty.

30. Measure 0.5 cm from the bottom of the plate. Take care not to press so hard with the pencil that you disturb the adsorbent.

31. Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin: the line on which you will "spot" the plate.

32. It's kind of hard to see the pencil line in the above photos, so here is a close-up of how the plate looks after the line has been drawn.

33. Under the line, mark lightly the name of the samples you will spot on the plate, or mark numbers for time points. Leave enough space between the samples so that they do not run together, about 4 samples on a 5 cm wide plate is advised. Use a pencil and do not press down so hard that you disturb the surface of the plate. A close-up of a plate labeled "1 2 3" is shown to the right.

34. 3. Spot the TLC plate

35. dip the microcap into solution - the arrow points to the microcap, it is tiny and hard to see make sure it is filled - hold it up to the light if necessary

36. touch the filled microcap to TLC plate to spot it - make sure you watch to see that all the liquid has drained from the microcap

37. rinse the microcap with clean solvent by first filling it . . . . . . and then draining it by touching it to a paper towel

38. here's the TLC plate, spotted and ready to be developed

39. 4. Develop the plate. place the TLC plate in the developing container - make sure the solvent is not too deep

40. The solvent will rise up the TLC plate by capillary action. In this photo, it is not quite halfway up the plate. In this photo, it is about 3/4 of the way up the plate.

41. Remove the plate from the beaker.

42. Allow the solvent to evaporate completely from the plate. If the spots are colored, simply mark them with a pencil. quickly mark a line across the plate at the solvent front with a pencil

43. 5. Visualize the spots Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over the plate and mark any spots which you see lightly with a pencil. this is a UV lamp

44. here are two proper sized spots, viewed under a UV lamp (you would circle these while viewing them)

45. The plate shows three compounds run at three different concentrations. The middle and right plate show reasonable spots; the left plate is run too concentrated and the spots are running together, making it difficult to get a good and accurate Rf reading.

46. Here's what overloaded plates look like compared to well-spotted plates. The plate on the left has a large yellow smear; this smear contains the same two compounds which are nicely resolved on the plate next to it. The plate to the far right is a UV visualization of the same overloaded plate.

47. The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.