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

柱色谱是一种使用填充柱根据化合物与固定相（通常为微珠形式）的相互作用来分离化合物的技术。珠子之间的空隙充满了溶剂。打开色谱柱可让溶剂流过固定相。当混合物上样到填充柱顶部，然后上样到更多溶剂时，混合物会进入流动相并流过固定相。
混合物的每种组分都以不同的方式与固定相相互作用。一些组分与固定相的相互作用较弱，因此在色谱柱中移动较快，而另一些组分与固定相的相互作用较强，因此移动速度较慢。这样可以将不同的化合物分离成条带，这些条带以小馏分的形式收集。这样可以纯化每种化合物。
那么，可以使用什么样的特性来分离混合物呢？最常用的特性之一是极性。为此，硅胶（二氧化硅的一种形式）通常用作固定相。硅胶通过偶极-偶极相互作用和氢键通过其表面形成的 -OH 基团与化合物相互作用。因此，极性化合物将与固定相发生强烈相互作用，而非极性化合物将与固定相发生微弱相互作用。
其他可用于分离的特性包括尺寸、电荷和疏水性。将固定相加载到色谱柱中的一种常见方法是作为固定相和溶剂的浆液。然后，通过让更多溶剂流过色谱柱来填充固定相，以压实浆料。固定相必须均匀填充，没有气泡、空通道或干斑。这些会破坏流动并导致条带混合。
选择色谱柱时，直径取决于要分离的样品的体积。样品应以薄而均匀的层覆盖色谱柱顶部。较厚的样品层会导致更宽的条带。色谱柱长度取决于化合物在固定相上的分离程度。与固定相具有相似亲和力的化合物需要较长的色谱柱才能充分分离。但是，对固定相亲和力截然不同的化合物混合物可以在较短的色谱柱上分离。
在本实验中，您将通过首先填充和制备硅胶柱来探索色谱柱色谱。然后，您将使用色谱柱分离绿色食用染料中的有色组分。

层析柱

层析柱
在有机化学实验室中，有多种技术可用于纯化和分离化合物。柱色谱法是微量分离最可靠的分离技术之一，其中可用于分析的化合物少于 10 克。该技术根据化合物的物理性质（例如溶解度、极性、疏水性、大小和电荷）分离混合物中的化合物。
<h4>色谱基础知识</h4>
任何色谱系统的主要成分都是固定相、流动相和待分析的样品混合物。在柱色谱中，固定相通常是微量微珠，它们均匀地填充在垂直柱中。将流动相（也称为溶剂）的连续流添加到色谱柱顶部，该流动相通过重力流过固定相，或通过泵以受控流速流过固定相。
将样品溶解在少量溶剂中，然后加入到色谱柱顶部。添加更多溶剂以迫使样品流过固定相。混合物中的化合物根据其不同的性质在流动相和固定相之间分配，并形成离散的条带。
与固定相相互作用较强的化合物比与固定相相互作用较弱的化合物移动得更慢。因此，具有弱相互作用的化合物将比具有强相互作用的化合物更早离开色谱柱或洗脱。化合物与固定相相互作用的强度由延迟因子 （Rf） 定义，延迟因子是组分行进的距离与流动相行进的距离之比。通过首先进行薄层色谱来确定延迟因子。
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
高延迟因子值表示样品与固定相相互作用强烈，洗脱时间长。低延迟因子值表示样品与固定相的相互作用不是很强烈，洗脱速度更快。由于化合物的 Rf 值不同，它们会分流成单独的条带并在不同的时间从色谱柱中洗脱出来，因此可以通过从色谱柱下分馏出溶剂来单独分离它们。
<h4>色谱柱色谱注意事项</h4>
色谱柱是垂直方向的玻璃管或塑料管。色谱柱的尺寸范围从几厘米（例如巴斯德移液器）到米（例如工业色谱柱）。色谱柱的直径取决于要分离的样品量，而色谱柱的长度取决于分离的难度。最有效地划分为离散波段需要较薄的样品层;因此，确定合适的直径是一个至关重要的步骤。相对于色谱柱直径而言，如果样品体积过大，则与直径较大的色谱柱中相同体积的样品相比，会产生更宽且不太明确的谱带。
在选择柱子的长度时，必须考虑组件的 Rf 值。具有相似阻滞因子的组分可能难以分离，并且需要更长的色谱柱来分离各个谱带。具有非常不同的阻滞因子的化合物可能不需要长色谱柱，因为它们应该很容易分配。选择固定相时，重要的是要选择一种能为每种组分带来不同延迟因子的固定相。
制备色谱柱是运行色谱柱最关键的部分。色谱柱中填充的固定相质量应至少为样品质量的 20 倍。如果塔底部没有多孔盘，则应用棉花和一层薄薄的沙子填充。这可以防止固定相通过色谱柱出口损失。
填料塔有两种方法:干填料法和浆料法。在干法中，固定相以粉末形式转移到色谱柱中。然后用流动相/溶剂清洗色谱柱数次，以确保溶剂渗透到硅胶色谱柱的每个部分。如果作不当，这种方法会增加色谱柱中形成干斑、通道和气泡的可能性。这些缺陷将影响色谱柱对混合物成分进行分区的能力。
浆料法提供了更均匀的填充塔，不包含气泡、干燥区域和通道。对于这种方法，固定相与溶剂混合以形成一致的浆液。然后将浆料转移到塔上，同时敲击侧面以去除形成的任何气泡。在此步骤中，色谱柱的旋塞阀（如果有）应打开，以便溶剂排出。
<h4>硅胶色谱法</h4>
利用许多不同的特性来分离化合物的混合物，例如大小、电荷和疏水性。在有机化学中用于分离化合物的一种特性是极性。为此，硅胶和氧化铝凝胶是最常用的固定相。硅胶具有极强的极性，并与极性化合物形成强偶极-偶极相互作用。此外，由于硅胶表面存在 -OH 基团，它可以与分析物形成氢键。
混合物中极性最强的组分与硅胶紧密结合并缓慢通过色谱柱，而非极性组分在流动相中更易溶解，并快速通过色谱柱。因此，混合物的组分必须具有不同的极性。与固定相相互作用强烈的组分通过使极性流动相流经色谱柱来洗脱。
<h4>引用</h4>
<ol>
	<li style="font-size:17px">Shuler， M.L.， Kargi， K.， DeLisa， M. （2017）.生物工艺工程，基本概念。新泽西州上马鞍河:Prentice Hall。</li>
	<li style="font-size:17px">哈里斯，哥伦比亚特区（2015 年）。 定量化学分析。纽约州纽约市:W.H. Freeman and Company。</li>
</ol>

层析柱
在有机化学实验室中，有多种技术可用于纯化和分离化合物。最可靠的分离之一 ...

Education

教育

JoVE Lab Manual

Chemistry

化学

Column Chromatography

separating mixtures by polarity

Column chromatography separates compounds in an organic chemistry lab by using differences in physical properties. It is especially useful for microscale separations when less than 10 grams of material is available for analysis. The method can separate a mixture based on solubility, polarity, hydrophobicity, size, and charge.

Any chromatography system has three main parts: the stationary phase, the mobile phase, and the sample mixture. In column chromatography, the stationary phase is usually made of microscale beads packed into a vertical column. The mobile phase is a solvent that flows through the column by gravity or at a controlled rate with a pump. The sample is first dissolved in a small amount of solvent, added to the top of the column, and then pushed through with more solvent.

As the mixture moves through the column, its compounds partition between the two phases. They form separate bands because each compound interacts differently with the stationary phase. Compounds that interact strongly move more slowly. Compounds with weaker interactions elute, or leave the column, earlier. The retardation factor, or Rf, describes this interaction by comparing how far a component travels with how far the mobile phase travels. A high Rf means strong interaction and slow elution. A low Rf means weaker interaction and faster elution. The Rf is determined first with thin layer chromatography, and the separated compounds are collected in fractions as they leave the column.

The size and shape of the column affect how well the separation works. A chromatography column is a vertical glass or plastic tube that can range from a few centimeters, such as a Pasteur pipette, to meters in industrial systems. The column diameter depends on the amount of sample, and the length depends on how difficult the separation is. Thin sample layers give the best band separation, so using the right diameter is important. If too much sample is loaded for the column diameter, the bands become broader and less defined. When components have similar Rf values, a longer column may be needed to separate them. If their Rf values are very different, a shorter column may still work well. The stationary phase should also be chosen so the components have different Rf values.

Packing the column correctly is one of the most important steps. The stationary phase should weigh at least 20 times more than the sample. If there is no porous disk at the bottom, cotton and a thin layer of sand are used to stop the stationary phase from leaving the column. The column can be packed by a dry method or by a slurry method. In the dry method, the powder is added directly and then washed with solvent so the mobile phase can move through the silica gel. If this step is not done well, dry patches, channels, and air bubbles can form. These defects reduce how well the mixture separates. The slurry method mixes the stationary phase with solvent first, then transfers it into the column while tapping the sides to remove bubbles. If the column has a stopcock, it should be open so the solvent can drain.

Silica gel chromatography is a common way to separate compounds by polarity. Silica gel and alumina gel are the most used stationary phases for this purpose. Silica gel is very polar and can form dipole-dipole interactions and hydrogen bonds because of its -OH groups. Polar components bind more strongly to silica gel and move slowly through the column. Non-polar components stay more soluble in the mobile phase and move faster. Because of this, the mixture must contain components with different polarities. More polar compounds are eluted by using a polar mobile phase through the column.

Separating Mixtures by Polarity

lab techniques

Measuring with Accuracy in the Lab

Measuring with accuracy in the lab starts with careful records, the right tools, and correct reading of data. Scientists use lab notebooks to record how an experiment was done. These notes include the amounts of materials used and each step of the procedure. Without that record, an experiment cannot be repeated in the same way. Balances and glassware are used to measure solids and liquids for experiments. The precision of a measurement depends on the equipment used. Digital balances show the...

scientific measurement and lab skills

Graphs, Curve Fit, and Standard Deviation

Two-dimensional graphs help students compare an independent variable with a dependent variable in science data. In a chemistry example, temperature is the independent variable and volume is the dependent variable. After data are collected, the relationship between the two measurements must be quantified so it can be evaluated and compared with other relationships. A graph can also show when no relationship exists between the two variables. To make a two-dimensional graph, each data point needs...

stoichiometry, product yield, and limiting reactants

stoichiometry-product-yield-and-limiting-reactants

Balancing Equations and Reactant Limits

Chemical equations show how reactants change into products during a reaction. Stoichiometry is the study of the relative amounts of reactants and products. It is based on the Law of Conservation of Mass, which says matter is neither created nor destroyed. Because atoms are only rearranged, the number and identity of atoms must match on both sides of a reaction. That is why a chemical equation must be balanced. A balanced equation has the same number of atoms for each element in the reactants...

redox reactions

Oxidation States in Redox Reactions

Oxidation states help identify redox reactions. In a redox reaction, oxidation is the loss of one or more electrons, and reduction is the gain of one or more electrons. These reactions are often tracked by changes in oxidation state, also called oxidation number. Each atom in a molecule has an oxidation state. This number shows how oxidized an atom is compared with its free elemental form. It is written as the charge an atom would have if every bond were purely ionic. In that model, bonding...

ideal gas law

Gas Laws Behind the Ideal Gas Equation

The ideal gas equation brings together the main gas laws used in chemistry. Gases have molecules that are far apart, so their behavior is described with four measurable variables: pressure, volume, number of moles, and temperature. The ideal gas law links these variables with the gas constant, R, which equals 8.314 J·K−1 mol−1. Boyle’s law describes the link between pressure and volume. When temperature and the number of moles stay constant, pressure and volume change in opposite directions.

acid and base concentrations

Acids, Bases, and pH in Titration

Acids, bases, and pH explain how acidic or basic a solution is. An Arrhenius acid releases hydrogen ions when it dissolves in water, while an Arrhenius base releases hydroxide ions. These reactions also create conjugate partners: an acid forms a conjugate base after losing hydrogen, and a base forms a conjugate acid after losing hydroxyl. Every acid has a conjugate base, and every base has a conjugate acid. pH measures the amount of hydrogen ions in a solution. The pH scale runs from 0 to 14...

buffers

Buffer pH and the Common Ion Effect

Buffer solutions help keep pH steady when acids or bases are added. In water, an Arrhenius acid (HA) dissociates into hydrogen ions, H+, and a conjugate base, A-. More hydrogen ions make a solution more acidic and lower its pH. An Arrhenius base (BOH) dissociates into a conjugate acid, B+, and hydroxide ions, OH-. Strong acids and strong bases usually change pH a lot because they react completely in water and raise the amount of H+ or OH-. Buffers reduce those pH changes. A buffer usually...

enthalpy of reaction

Reaction Heat, Hess’s Law, and Calorimetry

Reaction heat is explained through enthalpy, Hess’s law, and calorimetry. These ideas show how heat moves during chemical reactions and how scientists measure it. Thermodynamics is the study of heat energy and other forms of energy, such as work. Its laws are used across science, from biology to physics. The first law says energy is conserved in an isolated system. Energy can change form, such as thermal energy becoming work, but it cannot be created or destroyed. The second law of...

solubility

Solubility in Water and Other Solvents

Solubility describes how much of a solute can dissolve in a specific solvent. It is usually reported as solute mass per solvent volume or as solute mass per solvent mass. For example, sodium chloride has a solubility of 36 g per 100 mL of water at room temperature. If solubility is given by solvent mass, that mass must be converted to volume for later calculations. Temperature changes solubility. Sodium carbonate dissolves at about 7 g per 100 mL of water at 0 °C, 22 g per 100 mL at room...

metal flame emission

Flame Test Metal Color Clues

Flame testing uses a metal sample and a flame to identify a substance by its color. Each element gives off a characteristic light emission in the flame, so the color can help distinguish one element from another. This same idea appears in fireworks, where different metal components create different colors. The color comes from energy absorbed by the metallic component. When a species absorbs energy, that energy can go into translation, vibration, rotation, or electron excitement. Electron...

balmer series

Hydrogen Emission Lines

Hydrogen emission lines show how electrons move between fixed energy levels in the Bohr model. Niels Bohr proposed this model for the hydrogen atom in 1913. In this model, an electron can only occupy certain orbits, and it cannot exist between them. These energy states are labeled with the quantum number n. The ground state is n = 1. Higher values, such as n = 2, 3, 4, and so on, are excited states. An electron changes state only by absorbing or emitting energy equal to the difference between...

beer's law

Absorbance Spectra and Beer-Lambert Analysis

Absorbance spectra and the Beer-Lambert Law show how light can be used to study molecules in solution. When light hits a substance, it may be absorbed, transmitted, or reflected. A substance often interacts with a range of wavelengths, and each wavelength can affect the molecules or atoms in a different way. When a molecule absorbs light, that energy can be used for translation, vibration, rotation, or electron excitement. Electron excitement is especially important in spectroscopy because it...

concentration dependence

Reaction Order and Rate Constants

Chemical kinetics describes the speed of a chemical reaction. The rate depends on the reaction mechanism, the complexity of the reaction, and the number of reactants involved. Reactant concentration also affects how fast a reaction happens. The rate law is the experimental relationship that connects these factors to reaction rate. In the rate law, each reactant contributes through a reaction order. The general form is r = k[A]^m[B]^n for aA + bB + ... → cC. Here, r is the reaction rate, k is...

temperature dependence

Reaction Rate, Arrhenius, and Catalysts

Chemical kinetics explains how fast a reaction happens and why that speed changes. The reaction rate is the speed of a chemical reaction. It is defined as the change in concentration of a reactant or product over time. Reaction speed depends on several factors, including the concentration of the reactants and the temperature of the reaction. Each reactant affects the rate in a specific way. That relationship is described by the rate law. The rate law is an equation that connects the...

galvanic cells

Battery Power from Redox Reactions

Electrochemistry studies how chemical change and electrical energy are linked. In this branch of chemistry, electrons move from one species to another. That movement can either produce current or be driven by an applied current. The main reaction in electrochemistry is the oxidation-reduction, or redox, reaction. A redox reaction has two half-reactions. Oxidation is the loss of electrons, and reduction is the gain of electrons. These changes also shift the oxidation state of each substance. The...

electrolytic cells

Electroplating with Electrolytic Cells

Electrochemistry links electrical energy with chemical change, and electrolytic cells show how electricity can drive a nonspontaneous reaction. In these reactions, electrons move from one species to another. The key chemistry is a redox reaction, or oxidation-reduction reaction, which happens in two half-reactions. Oxidation is the loss of electrons, and reduction is the gain of electrons. The substance that loses electrons is the reducing agent, and the substance that gains electrons is the...

proper lab notebook keeping

Recording a Reproducible Lab Experiment

Accurate lab notebook records are essential in science and organic chemistry labs. Without clear notes, descriptions, quantities, and observations, an experiment cannot be repeated or checked. Good record keeping supports reproducibility, which means other people can follow the same work and get similar results. A laboratory notebook is useful in school labs, research settings, and industry. It can also become a legal record when an idea, process, substance, or technique is patented and...

basic organic chemistry techniques

Organic Lab Glassware and Safe Handling

Organic chemistry labs use specific glassware, careful weighing steps, and controlled heating to keep reactions safe and accurate. Beakers and Erlenmeyer flasks are often used for mixing or holding solvents, but they are not meant for measuring volume unless only an approximate amount is needed. Volumetric glassware is used when a precise volume matters. This group includes graduated cylinders, volumetric pipettes, and volumetric flasks. A graduated cylinder is commonly used in the organic lab...

melting points

How Impurities Change Melting Points

Melting point helps identify an organic compound by showing the temperature where a solid turns into a liquid at 1 atmosphere of pressure. It is a physical property, like solubility, density, color, and electronegativity. Because the exact start of melting is hard to measure, the result is reported as a range. The lower end is when the first drops of liquid appear. The upper end is when the last solid has melted. Scientists compare this range with literature values to help identify a compound. <br...

boiling points

Boiling Point and Vapor Pressure

Boiling point and vapor pressure are key physical properties of a liquid. For a pure compound, the boiling point can help identify the substance. In experiments, the boiling point is usually recorded as a range because exact values are hard to measure and may differ by a few degrees from the literature value. Vapor pressure explains why a liquid boils. In a sealed container, some molecules at the liquid surface gain enough energy to escape into the gas phase. At the same time, some gas...

recrystallization

Purifying Crystals by Solubility

Purifying crystals by solubility is a key organic chemistry technique. Recrystallization uses differences in solubility to separate a desired solid product from starting materials, solvent, and impurities. It is one of the three standard purification methods, along with distillation and extraction. Solubility is the maximum amount of a substance that can dissolve in a fixed volume of solvent at a given temperature. Different solutes dissolve in different solvents, and their behavior can vary...

extraction

Separating Mixtures with Solvent Extraction

Solvent extraction is a common way to separate and isolate a target compound in organic chemistry. In this process, a solute moves from one phase to another so it can be separated from unreacted starting materials or impurities. Extraction is also useful when a product is dissolved in a reaction solvent that is hard to remove by evaporation, such as a solvent with a high boiling point. There are three main types of extraction. In solid-liquid extraction, the solute moves from a solid phase...

simple distillation

Separating Liquids by Boiling Point

Separating liquids by boiling point is the goal of distillation. A miscible mixture contains liquids that mix evenly, and it can be heated so that the more volatile liquid evaporates first. Volatility is a liquid’s tendency to vaporize, and boiling begins when bubbles form in the liquid phase. In a closed container, liquid and vapor can reach equilibrium, which means the rate of vaporization equals the rate of condensation. The pressure from the vapor above the liquid is called vapor pressure.

steam distillation

Steam Distillation and Low-Boiling Separation

Steam distillation is a separation method for temperature-sensitive organic compounds. It works best when the organic compound is immiscible in water, meaning the two liquids do not mix. The method helps remove the organic molecule from a non-volatile contaminant without using the higher temperatures needed for many other separations. In steam distillation, the immiscible mixture is heated until both water and the volatile organic compound distill together. The vapor rises to a condenser,...

thin-layer chromatography

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...

column chromatography

Column chromatography separates compounds in an organic chemistry lab by using differences in physical properties. It is especially useful for microscale separations when less than 10 grams of material is available for analysis. The method can separate a mixture based on solubility, polarity, hydrophobicity, size, and charge. Any chromatography system has three main parts: the stationary phase, the mobile phase, and the sample mixture. In column chromatography, the stationary phase is usually...

hydrolysis of an ester

Soap Formation from Triglycerides

Triglycerides are lipids made of a glycerol backbone linked to three fatty acids. They are common in vegetable oils and animal fats. Plant lipids are the basis of vegetable oils, while animal lipids are used as lard or general sources of fats. A fatty acid has a long carbon chain, usually between 12 and 20 carbons, with a weak organic acid at one end. Some fatty acids contain double bonds. These are unsaturated fats. The double bond usually gives the chain a bent, cis shape. As a result,...

synthesis of luminol

Luminol Chemiluminescence in Forensics

Luminol chemiluminescence shows how a chemical reaction can produce light and help with blood detection. Chemical reactions may be endothermic or exothermic, depending on whether they absorb or release heat as reactants become products. The enthalpy of a reaction is the energy difference between reactants and products, or ΔH. A positive ΔH means the reaction is endothermic. A negative ΔH means it is exothermic. Many reactions do not happen in a single step. They can pass through intermediates...

esterification

Ester Formation and Everyday Fragrances

Esters are organic compounds with a carbonyl group attached to an oxygen that connects to another alkyl or aryl group. Their general formula is RCOOR'. They are often made by reacting a carboxylic acid with an alcohol in an esterification reaction. In this process, the hydroxyl group of the acid is replaced by an alkyl or aryl group. Many simple esters have familiar fruit and flower smells because their structures are small and less complex. Formic acid and methanol can combine to form methyl...

identification of unknown aldehydes and ketones

identification-of-unknown-aldehydes-and-ketones

Carbonyl Tests for Aldehydes and Ketones

Aldehydes and ketones are carbonyl compounds, which means they contain a C=O functional group. A ketone has two alkyl or aryl groups attached to the carbonyl carbon. Acetone is the simplest ketone, with two methyl groups. An aldehyde is similar, but it has at least one hydrogen attached to the carbonyl carbon. Formaldehyde is the simplest aldehyde, and acetaldehyde has one hydrogen and one methyl group. The carbonyl carbon in both aldehydes and ketones is electrophilic because the oxygen atom...

uv-vis spectroscopy of dyes

How Dyes Absorb Visible Light

Dyes absorb visible light because their molecular structure lets them interact with specific wavelengths. When light hits a substance, some light is absorbed and the rest is reflected or transmitted. The color we see depends on the wavelength that is reflected. A blue substance reflects blue light, around 430 to 480 nm, and absorbs the complementary orange region, around 590 to 630 nm. Compounds that do not absorb in the visible range appear colorless to the human eye. Light also has energy and...

identifying alcohols

Alcohol Tests for Primary, Secondary, and Tertiary

Alcohol tests can help sort alcohols by structure and reactivity. Alcohols are organic molecules with a hydroxyl group attached to an alkyl or aryl group, written as ROH. If the carbon with the hydroxyl group has one R group, the alcohol is primary. If it has two R groups, it is secondary. If it has three R groups, it is tertiary. Alcohols can also be aromatic when they contain a benzene ring. Phenol is the simplest aromatic alcohol. To identify an alcohol, chemists can use its chemical...

chemistry

Comprehensive, curriculum-focused videos for both instructors and students in introductory chemistry lab courses.

https://cloudfront.jove.com/files/banners/scicat/labbio.jpg

Learn how column chromatography separates mixtures by polarity, Rf values, and band movement through a packed column using silica gel and solvents.

Separating Mixtures by Polarity | JoVE K12

通过这段 3 分钟的教师指南，准备教授 层析柱。涵盖 JoVE Lab Manual 上 Chemistry 实验的关键准备步骤。

Chemistry 中的 层析柱 教师准备 - 9.4K+ 观看次数 | JoVE Lab Manual

在这段 9 分钟的学生实验流程视频中，逐步学习 层析柱 的操作步骤。属于 JoVE Lab Manual 上的 Chemistry 系列。

Chemistry 中的 层析柱 学生实验流程 - 43K+ 观看次数 | JoVE Lab Manual

Separating Mixtures by Polarity

Description

Transcript