Name: tlc separation by polarity
Uploaded: 2020-03-26T07:22:28.000Z
Duration: 3 min 7 s
Channel: JoVE

薄层色谱法（TLC）是一种根据有机化合物极性差异进行分离的技术。TLC 系统包含三个组成部分：固定相、溶质和展开溶剂。
固定相是用于分离化合物的表面，通常由极性极强的硅胶组成。待分析的化合物或混合物即为溶质。使用细毛细管将溶质的稀溶液点样于薄层板的一端边缘。
然后将板的底部浸入展开溶剂中，该溶剂通常是一种比固定相极性更小的有机溶剂。溶剂通过毛细作用沿板向上移动。当溶剂流经样品点时，会携带部分每种溶质一同移动。这种溶质与溶剂的混合物即为流动相。
流动相的顶端称为溶剂前沿。当流动相沿板上升时，溶质和溶剂分子会根据其极性暂时附着在固定相上。
极性较强的溶质与固定相之间的吸引力更强，因此它们更频繁地吸附且吸附时间较长。极性较弱的溶质吸附频率较低，且不易长时间停留。这意味着极性较弱的溶质在流动相中停留的时间比极性较强的溶质更长。换句话说，极性较弱的溶质比极性较强的溶质移动得更快。
为了实现化合物之间的良好分离，溶剂的极性应恰好足以带动极性最强的溶质。以下是一些常见溶剂，按极性由低到高排列。有时会将两种溶剂混合，以达到所需的极性。
由于大多数有机化合物为白色或无色，薄层色谱板通常含有荧光化合物。在紫外光下，有机化合物会在发光的板上呈现为暗斑。
当化合物显现后，可以测量它们移动的距离，并计算每种化合物的比移值（Rf）。该值等于溶质移动的距离除以流动相移动的总距离。
如果溶剂选择得当，每种溶质将具有明显不同的比移值（Rf）。这使得我们可以通过比对已知化合物的 Rf 值来鉴定混合物中的组分化合物。
在本实验中，您将使用薄层色谱法测定几种已知化合物的 Rf 值，然后利用这些 Rf 值来确定未知化合物的种类。

薄层色谱法

色谱法
色谱法是一种在有机化学中用于根据混合物中各化合物在两相之间溶解度差异而将其分离的技术。该原理与液-液萃取类似，但在色谱法中，这两相分别为固定相和流动相。固定相是一种固体——通常为微米级的水凝胶小球——而流动相则是携带样品的溶剂。
在传统的色谱法中，固定相被填充到一根垂直的柱子中，待分离的溶液混合物从柱子顶部加入。当混合物流经固定相时，各组分根据其结构和极性在固定相与流动相之间进行分配，从而形成不同的区带。与固定相相互作用较弱的溶质在柱中移动较快，首先流出（即洗脱）；而与固定相相互作用较强的溶质移动较慢，随后洗脱。这些区带可依次分别收集，从而实现混合物中各组分的分离与纯化。
色谱法有多种类型，每种都利用不同的化学性质来实现分离。例如，在离子交换色谱中，固定相的微珠可带正电或负电，仅吸引带有相反电荷的分子。在尺寸排阻色谱中，多孔的固定相由琼脂糖或葡聚糖聚合物等微珠组成。较小的分子更容易进入孔隙，而较大的分子则流经孔隙并更快地被洗脱出来。
<h4>薄层色谱法</h4>
薄层色谱法（TLC）是一种根据化合物极性进行分离的色谱技术。与传统色谱法类似，TLC 系统包含三个组成部分：固定相、流动相和溶质。然而，与传统色谱法不同的是，固定相以薄层形式涂布在平板上，而非填充在柱中。TLC 最常使用极性的硅胶作为固定相，硅胶是二氧化硅的一种形式。由于其表面存在羟基（OH 基团），固定相能够形成氢键。
首先，用铅笔在薄层色谱板的底部画一条起始线。使用细的毛细管将待分析的化合物或混合物点样在起始线上。然后，将色谱板的底部浸入流动相中，流动相通常为极性低于固定相的有机溶剂。溶剂通过毛细作用沿板向上移动，经过样品点，带动各组分以不同程度向上迁移。
随着溶剂沿板上升，各组分在流动相与固定相之间进行分配。如果组分具有极性，则会更多地与极性固定相发生相互作用，移动缓慢，在薄层色谱板上迁移的距离较短。如果样品中的组分极性较弱——且在流动相中的溶解度高于在固定相中的溶解度——则会更多地与流动相相互作用，在薄层色谱板上迁移得更远。组分及流动相的极性程度对于理解并预测分离效果至关重要。
薄层色谱板通常含有对紫外光有反应的荧光染料，在254纳米波长的紫外光源下会发出荧光。因此，可通过在紫外光下观察薄层色谱板来进行分析。板中的化合物，例如目标溶质，会以暗斑的形式出现在绿色背景上。使用石墨铅笔圈出这些斑点后，即可测量化合物相对于溶剂前沿的迁移距离。若有机化合物本身不具荧光性，则会遮蔽板上的荧光，从而呈现为暗斑。某些有机化合物具有紫外活性，在紫外光照射下会发光。这类化合物通常是共轭化合物，即含有交替单双键的分子，可通过其发射的光波长进行鉴定。
<h4>比移值</h4>
通过分析特定溶剂中组分的比移值（Rf），可利用薄层色谱法（TLC）确定未知溶质。比移值是指组分迁移距离与流动相迁移距离的比值。
<img src="/CDNSource/lm/labs/62/62_Concepts_1.jpg" alt="色谱法中Rf值计算公式示意图。" data-altupdatedat="1761830439000" />
溶质迁移的距离是从起始线量至斑点的中心位置，而流动相迁移的距离是从同一起始线量至溶剂前沿。化合物的比移值（Rf值）取决于所使用的流动相。当使用非极性流动相时，非极性较强的化合物具有较大的比移值；而极性组分在此非极性流动相中则表现出较低的比移值。
当流动相极性非常低时，某些极性组分可能完全不移动，导致滞留因子极低，分离效果不充分。而当流动相极性非常高时，化合物会随溶剂前沿移动，产生极高的滞留因子，结果使各组分之间的分离程度非常有限。
为了使分离有效，各组分的比移值应相差约 0.3 - 0.7。为确定高效的流动相，通常采用试错法。通常，两种溶剂的混合物效果最佳。
<h4>参考文献</h4>
<ol>
	<li style="font-size:17px">Harris, D.C. (2015). 定量化学分析。纽约，纽约州：W.H. Freeman and Company。</li>
</ol>

色谱法  
色谱法是一种在有机化学中用于分离混合物中化合物的技术，其原理基于各化合物在固定相与流动相之间的溶解度差异……

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JoVE Lab Manual

Chemistry

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Thin-Layer Chromatography

薄层色谱

tlc separation by polarity

Thin-layer chromatography (TLC) separates compounds in a mixture by polarity. It uses the same basic parts as other chromatography methods: a stationary phase, a mobile phase, and the solute. In TLC, the stationary phase is a thin coating on a plate instead of material packed into a column.

The stationary phase in most TLC systems is polar silica gel, a form of silicon dioxide. Its surface has OH groups that can form hydrogen bonds. The sample is first spotted on a pencil start line with a thin capillary. Then the bottom of the plate is placed in the mobile phase, which is usually an organic solvent that is less polar than the stationary phase.

As the solvent moves up the plate by capillary action, it carries the sample components with it. Each compound then partitions between the mobile phase and the stationary phase. A polar component interacts more with the polar stationary phase, so it moves more slowly and stays near the start. A less polar component is more soluble in the mobile phase, so it travels farther up the plate.

TLC plates often include a UV-reactive fluorescent dye that glows under 254 nm UV light. The compounds appear as dark spots against the green background, and the spots can be circled with a graphite pencil so their movement can be measured. Some organic compounds are UV-active and emit light when exposed to UV light. These are often conjugated compounds, which have alternating double and single bonds, and they can be identified by the wavelength they emit.

The retardation factor, or Rf, helps identify an unknown solute from TLC results. It is the ratio of the distance traveled by the compound to the distance traveled by the solvent front. The compound distance is measured from the start line to the center of the spot, and the solvent distance is measured from the same start line to the solvent front. Rf depends on the mobile phase used.

Rf values are high for highly non-polar compounds with a non-polar mobile phase, and low for polar compounds with a non-polar mobile phase. If the mobile phase is too non-polar, some polar compounds may not move at all. If it is too polar, the compound may move with the solvent and the separation may be poor. For an effective separation, the Rf values of the components should be about 0.3 to 0.7 apart. Choosing the best mobile phase usually takes trial and error, and a mixture of two solvents often works well.

TLC Separation by Polarity

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TLC Separation by Polarity

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