实验六：薄层层析与柱层析

实验六：薄层层析与柱层析 实验六 薄层层析与柱层析 实验目的 1. 了解偶氮苯的光学异构反应，加深对光化学反应的理解。 2. 掌握薄层层析的基本操作,薄层层析分离顺、反式偶氮苯。 3. 了解柱层析分离有机化合物的原理，初步掌握层析柱装填和洗脱的操作方法 4. 采用柱层析分离反式偶氮苯与靛红的混合物 实验原理 偶氮苯是最简单的芳香偶氮化合物，众多偶氮染料的母体结构。含有两个苯基分别与偶氮基–N=N–两端相连的结构。偶氮苯有毒，易燃。偶氮苯有顺(Z)-反(E)-异构体。反式为橙红色棱形晶体，蒸气为深红色，溶于乙醇、乙醚、醋酸和水。反式的热力学性质稳定。当溶于乙醇的偶氮苯用一定强度的紫外光照射时，顺式的比例逐渐增大，直至达到平衡，形成顺反异构体的混合物。偶氮苯的光致异构是很多偶氮类功能材料光响应的基础。顺式为橙红色片状晶体，不稳定，在加热或可见光照射下能够变成反式。 色谱方法是通过在固定相和流动相间分配的不同而将物质分离开。待分离混合物各组分在固定相上的吸附强度不同，与流动相一起移动的速度也不同，因此被分离开。 薄层色谱属于固-液吸附色谱。薄层色谱板中的固定相中的微孔结构使得溶剂在毛细管作用下能够沿着色谱板向上移动。由于混合物中各组分对吸附剂(固定相)的吸附能力不同，当展开剂(流动相)流经吸附剂时发生无数次吸附解附过程，吸附力弱的组分随流动相迅速向前移动，吸附力强的组分滞留在后，由于intensive development. (5) have different emphasis: for development, "with development, the emphasis on" way, from space, multiple groups from a single radiation-belt open development. Spatial structure of 2, the prominent cultural city protection elements on the development of the whole city and culture series supporting role, with natural landscapes and green for the background, according to the combined city, urban Group organization structure, set up a decentralized development patterns, building ... Three rivers: the Yangtze, jinsha River and minjiang rivers, cities in three rivers based on development along the Yangtze River, city on the River, River in the city. Multiple groups: the relatively compact area layout, each district consists of multiple groups, and the area has both integrated urban functions, and have their own characteristics and perimeter formation Li Zhuang, Li Zhuangdong characteristic tour area and Hong Kong in group, consists of 17 groups. Band: modern features gathering along the Yangtze River belt. Dvn group in the upper reaches of the jinsha River along the river continues to the East to South stream groups, layout along the city's main commercial, business, administrative, cultural and creative research and development in science and education function of modern service and port equipment manufacturing, liquor, and other features and modern industry. 2.5-2 spatial planning figure 2.6 in Yibin city, Central City urban development vision vision 各组分具有不同的移动速度，最终在固定相薄层析上分离。TLC除了用于分离外，还可以通过与已知结构的的化合物比对，鉴定少量有机混合物的组成，它也是柱色谱寻求最佳展开剂的手段。上图中红色化合物的R等于竖直红线的长度除以竖直蓝线的f 长度。 柱层析也属于固-液吸附色谱。在一根玻璃“分离柱”中进行。管中装上适当的粉末作为国定相。待分离或纯化物质的溶液(流动相)在重力作用下流经吸附剂时，不同物质对溶剂和吸附剂的亲和力不同，因而被吸附的程度不同，从而以不同速度流动，使化合物得以分离。靛红与偶氮苯在极性上具有较大差异，从而可以使用极性适中的展开剂，通过柱层析将二者分离。 仪器与试剂 分析天平，TLC，锥形瓶，毛细管，层析柱，棉花，量筒，硅胶，蒸发皿，展缸;EtOAc, CHCl, 石油醚，靛红;请自带尺子(用于计算R值) f22 颜老师在实验开始之前已经准备好的试剂:a. 偶氮苯的溶液(15 mg in 1mL EtOH);b.靛红的溶液(15 mg in 1mL CHCl);c. 靛红与偶氮苯的均匀混合物22 (靛红1.4 g + 偶氮苯3.5 g)。 实验 a) 光照异构化 取0.1 g反式偶氮苯溶于5 mL无水乙醇中，将此溶液放于试管中，密封，置于可见光下二星期，以便与光照前的溶液进行对比。 b) 薄层层析 取管口平整的毛细管吸取光照后的偶氮苯溶液，在离薄层板边沿约0.5 cm的起点线上点样。再用另一毛细管吸取未经光照的反式偶氮苯溶液点样。待乙醇挥发后，将点好样品的薄层板放入内衬滤纸的展缸中。展缸内已放置展开剂(乙酸乙酯/石油醚=1/40)。薄层板的点样端在下方，浸入展开剂，展开剂不要没过起点线，也不要碰到样品点。待展开剂前沿上升到离板的上端约1 cm处时，取 2 spatial planning figure 2.6 in Yibin city, Central City urban development vision vision-odern industry. 2.5and mdevelopment in science and education function of modern service and port equipment manufacturing, liquor, and other features to South stream groups, layout along the city's main commercial, business, administrative, cultural and creative research andEast gathering along the Yangtze River belt. Dvn group in the upper reaches of the jinsha River along the river continues to theation Li Zhuang, Li Zhuangdong characteristic tour area and Hong Kong in group, consists of 17 groups. Band: modern features r formonsists of multiple groups, and the area has both integrated urban functions, and have their own characteristics and perimetethe Yangtze River, city on the River, River in the city. Multiple groups: the relatively compact area layout, each district calong terns, building ... Three rivers: the Yangtze, jinsha River and minjiang rivers, cities in three rivers based on development or the background, according to the combined city, urban Group organization structure, set up a decentralized development pate city and culture series supporting role, with natural landscapes and green fbelt open development. Spatial structure of 2, the prominent cultural city protection elements on the development of the whol-from space, multiple groups from a single radiation intensive development. (5) have different emphasis: for development, "with development, the emphasis on" way,2 大学化学实验 出薄层板，立即用铅笔在展开剂上升的前沿处划一记号，置于空气中晾干。可观察到薄层板上经光照后的偶氮苯溶液点样处上端有两个黄色斑点(哪一个斑点是顺式的,哪一个斑点是反式的,)。计算异构体的值。 Rf 用上述相同的方法，采用Ethyl Acetate : Petroleum Ether = 1:1为展开剂，对靛红与偶氮苯的混合物进行TLC，为下面的柱层析选择展开剂。 c) 柱层析 1(将层析柱固定在铁架台上，一定要保持柱子竖直。 2(将层析柱洗净后在柱底部放入一小团脱脂棉花。 3(在锥形瓶中称重7g 硅胶(SiO)，并用Ethyl Acetate : Petroleum Ether 2 (1:40)调匀。在层析柱的下方放置一个锥形瓶，以收集流下的液体。加入 少量洗脱剂于层析柱中，以润湿棉花，使其贴在柱子下端。 4(打开层析柱活塞，控制溶剂下流，将硅胶悬浊液沿柱子侧壁加入，并用 少量的溶剂洗去残余的硅胶，并加入柱中。用装有橡皮管或塞子由下向 上轻轻敲击柱子外壁，将气泡赶出，使硅胶填装均匀。并加压使硅胶紧 密，以获得较好的分离效果。轻轻敲击，使硅胶最上层成均匀平面，然 后用药匙沿柱子侧壁，轻轻地、慢慢地在硅胶表面覆盖一层海砂(注意: 不要破坏硅胶的上平面)。关闭活塞，备用。 5(打开活塞，当溶剂液面降至海砂表面时，关闭活塞。用滴管沿柱子侧壁 加入靛红与偶氮苯混合物的溶液(称取40 mg，置于1.5mL的塑料样品 管，加入1 mL二氯甲烷(CHCl)，超声振荡使其溶解)。打开活塞，22 使溶剂液面降至海砂表面，关闭活塞。再用少量的展开剂洗塑料样品管， 加入层析谱中，并注意洗去粘附在柱壁上的液滴，打开活塞，流到液面， 关闭活塞。重复上述操作，直到将所有化合物冲到硅胶里面。 6(沿侧壁加入大量的洗脱剂Ethyl Acetate : Petroleum Ether (1:40)，打开活 塞，加压保持一定的流速，观察色带的形成和分离。 7(当第一个有色带到达柱底时，并且没有流出之前，关闭活塞，倒空锥形 瓶，打开活塞，收集全部色带。然后，改用Ethyl Acetate : Petroleum Ether (1:1)作为洗脱剂。关闭活塞，换一个空的且干净的锥形瓶，打开活塞。 当第二个有色带到达柱底时，并且没有流出之前，关闭活塞，倒空锥形 ial planning figure 2.6 in Yibin city, Central City urban development vision vision2 spat-d education function of modern service and port equipment manufacturing, liquor, and other features and modern industry. 2.5yout along the city's main commercial, business, administrative, cultural and creative research and development in science anps, lae River belt. Dvn group in the upper reaches of the jinsha River along the river continues to the East to South stream groudong characteristic tour area and Hong Kong in group, consists of 17 groups. Band: modern features gathering along the Yangtzuangand the area has both integrated urban functions, and have their own characteristics and perimeter formation Li Zhuang, Li Zh the River, River in the city. Multiple groups: the relatively compact area layout, each district consists of multiple groups, ity onrivers: the Yangtze, jinsha River and minjiang rivers, cities in three rivers based on development along the Yangtze River, cng to the combined city, urban Group organization structure, set up a decentralized development patterns, building ... Three e city and culture series supporting role, with natural landscapes and green for the background, accordibelt open development. Spatial structure of 2, the prominent cultural city protection elements on the development of the whol-ps from a single radiationtiple grouintensive development. (5) have different emphasis: for development, "with development, the emphasis on" way, from space, mul3 瓶，打开活塞，收集全部色带。柱层析分离结束。 8(柱层析分离结束后，采用TLC对上述分离的两个溶液进行分析，总结分 离效果。同时，打开层析柱活塞，在加压下让展开剂流干，然后将硅胶 倒入指定的固体废物桶中。切勿倒入水槽或普通垃圾桶。 预习时的问题 1.在薄层层析实验中，为什么点样的样品斑点不可浸入展开剂的溶液中,为什么进行薄层层析时展缸要盖上盖, 2.当用混合物进行薄层层析时，如何判断各组分的在薄层板上的位置,靛红与偶氮苯，哪一个的极性较强,为什么, 3.柱层析时柱中若留有空气或者是填装不均匀，对分离效果有何影响,如何避免, 4.偶氮苯和靛红物理化学性质,毒性,有什么应用,偶氮苯光照异构化的机理,why is trans-azobenzene more stable than cis-azobenzene? 其它阅读材料 1.光化学 由光的作用引起的化学反应近年来日益受到人们的重视，光合作用就是最重要的光化学反应。研究激发态分子化学行为的光化学反应已经成为有机化学的一个重要分支。光不仅可以引起多种多样的化学反应，合成各种前所未有的奇妙反应分子，而且与我们的日常生活及生命现象有着密切的联系。 2. 薄层色谱 1. 固定相的选择 氧化铝和硅胶是薄层层析色谱常用的固定相。氧化铝多用于分离碱性或中性有机物;而硅胶的吸附性源于表面的Si-OH基，主要用于分离酸性、中性有机物质。 薄层色谱用的硅胶有薄层色谱用的硅胶有60G、6OGF254、60H、60HF254和60HF254+366等类型，其中G表示含有13,硫酸钙(作为黏合剂);H表示不含硫酸钙;F254表示含有2,无机荧光物质，在254 nm的紫外光照射下发出绿色荧光。与硅胶相似，氧化铝也因含有黏合剂或荧光剂而分为氧化铝H、氧化铝G、 2 spatial planning figure 2.6 in Yibin city, Central City urban development vision vision-odern industry. 2.5and mdevelopment in science and education function of modern service and port equipment manufacturing, liquor, and other features to South stream groups, layout along the city's main commercial, business, administrative, cultural and creative research andEast gathering along the Yangtze River belt. Dvn group in the upper reaches of the jinsha River along the river continues to theation Li Zhuang, Li Zhuangdong characteristic tour area and Hong Kong in group, consists of 17 groups. Band: modern features r formonsists of multiple groups, and the area has both integrated urban functions, and have their own characteristics and perimetethe Yangtze River, city on the River, River in the city. Multiple groups: the relatively compact area layout, each district calong terns, building ... Three rivers: the Yangtze, jinsha River and minjiang rivers, cities in three rivers based on development or the background, according to the combined city, urban Group organization structure, set up a decentralized development pate city and culture series supporting role, with natural landscapes and green fbelt open development. Spatial structure of 2, the prominent cultural city protection elements on the development of the whol-from space, multiple groups from a single radiation intensive development. (5) have different emphasis: for development, "with development, the emphasis on" way,4 大学化学实验 氧化铝HF254和氧化铝GF254等类型。黏合剂除硫酸钙外，还可用淀粉、羧甲基纤维素(CMC)。 2. 操作方法 ?点样 在薄层板一端约1cm处，用铅笔轻轻划一条作为起点线。样品用易挥发性溶剂溶解后，用毛细管吸取样品溶液，轻轻接触到起点线的某一位置上。如果溶液太稀，可多点几次，但要等第一次样品溶剂挥发后，再点第二次。 点好样品后，待溶剂挥发干净，才可以进行下面的展开过程。 ?展开剂的选择 选择展开剂，首先应考虑对被分离物有一定的溶解度和解吸能力。由于硅胶和氧化铝都是极性吸附剂，所以展开剂的极性越大，试样在薄板上移动的距离越远，值(值是一个化合物在薄层板上上升的高度与展开剂上升的高度的比RRff 值)越大。例如，在分离过程中常发现太小，说明展开剂极性不够，需要考Rf 虑加入一种极性强的展开剂进行调控。发现值太小，说明展开剂极性不够，Rf 需要考虑加入一种极性强的展开剂进行调控。 常用展开剂的洗脱力由小到大的顺序为:石油醚、环己烷、四氯化碳、二氯甲烷、氯仿、乙醚、四氢呋喃、乙酸乙醋、丙酮、正丁醇、乙醇、甲醇、水、冰乙酸、吡啶、有机酸等。 此外，在展开过程中，展开缸内展开剂的蒸气须始终处于饱和状态。一般可用一块方型滤纸贴于缸壁上(下端浸于展开剂中)，盖好密封一段时间。取放薄层板应迅速。 ?显色 yout along the city's main commercial, business, administrative, cultural and creative research and development in science anps, lae River belt. Dvn group in the upper reaches of the jinsha River along the river continues to the East to South stream groudong characteristic tour area and Hong Kong in group, consists of 17 groups. Band: modern features gathering along the Yangtzuangand the area has both integrated urban functions, and have their own characteristics and perimeter formation Li Zhuang, Li Zh the River, River in the city. Multiple groups: the relatively compact area layout, each district consists of multiple groups, ity onrivers: the Yangtze, jinsha River and minjiang rivers, cities in three rivers based on development along the Yangtze River, cng to the combined city, urban Group organization structure, set up a decentralized development patterns, building ... Three e city and culture series supporting role, with natural landscapes and green for the background, accordibelt open development. Spatial structure of 2, the prominent cultural city protection elements on the development of the whol-ps from a single radiationtiple grouintensive development. (5) have different emphasis: for development, "with development, the emphasis on" way, from space, mulial planning figure 2.6 in Yibin city, Central City urban development vision vision2 spat-d education function of modern service and port equipment manufacturing, liquor, and other features and modern industry. 2.55 展开后，要等溶剂挥发完才能显色。若被分离的是有色组分，展开板上即呈现出有色斑点。若化合物本身无色，则可以在紫外灯观察有无荧光斑点;或用碘蒸气熏的方法显色。显色后，用铅笔轻轻画出斑点位置，计算值。 Rf 3(柱色谱 吸附层析柱的分离效果不仅依赖于吸附剂和洗脱溶剂的选择，而且与制成的层析柱有关;要求柱中的吸附剂用量为被分离样品量的30-40倍，若需要可增至100倍。 柱高与直径之比为4:1至10:1，一般为7.5:1。实验室常用的层析柱,其直径在0.5-10 cm之间。当吸附物的色带占吸附剂高度的1/10—1/4时，此层析柱已经可作层析分离了。 柱层析常用吸附剂有氧化铝、硅胶、氧化镁、碳酸钙及活性炭等，吸附剂要求颗粒大小均匀，越细分离效果越佳，但此时洗脱阻力会增大，所以要选择合适大小的吸附剂。氧化铝分酸性、中性和碱性三种，本实验应用中性氧化铝。 化合物的吸附能力与它的极性成正比，具有较大极性基团的化合物，其在吸附剂上吸附能力较强。氧化铝对各化合物的吸附性按以下次序递减:酸>醇，胺，硫醇>酯，醛，酮>烃>卤化物，醚>烯>饱和烃。强吸附性的化合物要用强极性溶剂来洗脱。 作为洗脱剂的溶剂要求其具有一定极性，适合于被分离物的分离，体积尽量要小，此外要求易挥发，易除去(乙醚等挥发性太大的溶剂不合适)。洗脱剂的极性按下列次序递增:己烷或石油醚<环己烷<四氯化碳<三氯乙烯<二硫化碳<甲苯<苯<二氯甲烷<氯仿<乙醚<乙酸乙酯<丙酮<丙醇<乙醇<甲醇<水<吡啶<乙酸。 装柱要求吸附剂必须均匀填充于柱内，不能有气泡和裂缝，否则将影响洗脱和分离。在装柱时，通常把柱子竖直固定好，关闭下端活塞，底部用少量脱脂棉轻轻塞紧，加入0.5 cm厚的海砂，然后加入溶剂到柱子体积的1/4，用一定量的溶剂与吸附剂在烧杯中调成糊状，打开柱下端的活塞，让溶剂一滴一滴地滴入锥形瓶中，同时把糊状物快速倒入柱中，吸附剂通过溶剂慢慢下沉，进行均匀填充。柱顶部1/4处一般不填充吸附剂:以便使吸附剂上面始终保持一液层。柱填充好后，上面再覆盖0.5 cm厚的海砂。 过柱时，首先把试样溶解在最小体积的溶剂中，用滴管将试液加到吸附剂的 to South stream groups, layout along the city's main commercial, business, administrative, cultural and creative research andEast gathering along the Yangtze River belt. Dvn group in the upper reaches of the jinsha River along the river continues to theation Li Zhuang, Li Zhuangdong characteristic tour area and Hong Kong in group, consists of 17 groups. Band: modern features r formonsists of multiple groups, and the area has both integrated urban functions, and have their own characteristics and perimetethe Yangtze River, city on the River, River in the city. Multiple groups: the relatively compact area layout, each district calong terns, building ... Three rivers: the Yangtze, jinsha River and minjiang rivers, cities in three rivers based on development or the background, according to the combined city, urban Group organization structure, set up a decentralized development pate city and culture series supporting role, with natural landscapes and green fbelt open development. Spatial structure of 2, the prominent cultural city protection elements on the development of the whol-from space, multiple groups from a single radiation intensive development. (5) have different emphasis: for development, "with development, the emphasis on" way,2 spatial planning figure 2.6 in Yibin city, Central City urban development vision vision-odern industry. 2.5and mdevelopment in science and education function of modern service and port equipment manufacturing, liquor, and other features 6 大学化学实验 上面;试液加完并流到吸附剂上端时，立即加入展开剂进行展开，自始至终保持柱内液面高于吸附剂的顶端，否则柱内将出现气泡或裂缝。 试样中极性小的组分首先被洗脱下来，极性大的组分吸附较强，后被洗脱下来。 and the area has both integrated urban functions, and have their own characteristics and perimeter formation Li Zhuang, Li Zh the River, River in the city. Multiple groups: the relatively compact area layout, each district consists of multiple groups, ity onrivers: the Yangtze, jinsha River and minjiang rivers, cities in three rivers based on development along the Yangtze River, cng to the combined city, urban Group organization structure, set up a decentralized development patterns, building ... 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