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

컬럼 크로마토그래피는 패킹된 컬럼을 사용하여 일반적으로 미세한 비드 형태의 고정상과의 상호 작용을 기반으로 화합물을 분리하는 기술입니다. 구슬 사이의 공간은 용제로 채워져 있습니다. 컬럼을 열면 용매가 고정상을 통해 흐를 수 있습니다. 충진된 컬럼의 상단에 혼합물을 적용한 다음 더 많은 용매를 주입하면 혼합물이 이동상으로 이동하여 고정상을 통해 흐릅니다.
혼합물의 각 성분은 다른 방식으로 고정상과 상호 작용합니다. 일부 구성 요소는 고정상과의 상호 작용이 약하여 컬럼을 통해 빠르게 이동하는 반면, 다른 구성 요소는 고정상과 강한 상호 작용을 가지므로 더 느리게 이동합니다. 이것은 서로 다른 화합물을 띠로 분리하여 작은 조각으로 수집합니다. 이를 통해 각 화합물을 정제할 수 있습니다.
그렇다면 혼합물을 분리하는 데 어떤 종류의 속성을 사용할 수 있습니까? 가장 일반적으로 사용되는 속성 중 하나는 극성입니다. 이를 위해 이산화규소의 한 형태인 실리카겔이 고정상으로 자주 사용됩니다. 실리카겔은 쌍극자-쌍극자 상호 작용 및 표면에 형성되는 -OH 그룹을 통한 수소 결합에 의해 화합물과 상호 작용합니다. 따라서 극성 화합물은 고정상과 강하게 상호 작용하는 반면 비극성 화합물은 약하게 상호 작용합니다.
분리를 위해 악용될 수 있는 다른 특성으로는 크기, 전하 및 소수성이 있습니다. 고정상을 컬럼에 로드하는 일반적인 방법은 고정상과 용매의 슬러리로 사용하는 것입니다. 그런 다음, 고정상은 슬러리를 압축하기 위해 컬럼을 통해 더 많은 용매를 유동시켜 패킹됩니다. 고정상은 기포, 빈 채널 또는 건조한 패치 없이 균일하게 충전되는 것이 중요합니다. 이는 흐름을 방해하고 밴드의 혼합을 유발할 수 있습니다.
컬럼을 선택할 때 직경은 분리할 샘플의 부피를 기준으로 합니다. 샘플은 컬럼 상단을 얇고 균일한 층으로 덮어야 합니다. 샘플 레이어가 두꺼울수록 밴드가 넓어집니다. 컬럼 길이는 화합물이 고정상에서 얼마나 잘 분리되는지에 따라 달라집니다. 고정상에 대해 유사한 친화도를 가진 화합물은 적절한 분리를 위해 긴 컬럼이 필요합니다. 그러나 고정상에 대한 친화도가 매우 다른 화합물의 혼합물은 더 짧은 컬럼에서 분리될 수 있습니다.
이 실험실에서는 먼저 실리카겔 컬럼을 패킹하고 준비하여 컬럼 크로마토그래피를 살펴봅니다. 그런 다음 컬럼을 사용하여 녹색 식품 염료의 착색 성분을 분리합니다.

컬럼 크로마토그래피

컬럼 크로마토그래피
유기 화학 실험실에서 화합물을 정제하고 분리하기 위한 여러 기술이 존재합니다. 10g 미만의 화합물을 분석에 사용할 수 있는 마이크로 스케일 분리를 위한 가장 신뢰할 수 있는 분리 기술 중 하나는 컬럼 크로마토그래피입니다. 이 기술은 용해도, 극성, 소수성, 크기 및 전하와 같은 물리적 특성을 기반으로 혼합물의 화합물을 분리합니다.
<h4>크로마토그래피의 기초</h4>
모든 크로마토그래피 시스템의 주요 구성 요소는 고정상, 이동상 및 분석할 시료 혼합물입니다. 컬럼 크로마토그래피에서 고정상은 일반적으로 마이크로 스케일 비드이며, 수직 컬럼에 균일하게 충전되어 있습니다. 용매라고도 하는 이동상의 연속적인 흐름이 컬럼 상단에 추가되며, 이는 중력을 통해 또는 펌프에 의해 제어된 유속으로 고정상을 통해 흐릅니다.
샘플은 소량의 용매에 용해된 다음 컬럼 상단에 추가됩니다. 샘플이 고정상을 통해 흐르도록 하기 위해 더 많은 용매가 추가됩니다. 혼합물의 화합물은 서로 다른 특성에 따라 이동상과 고정상을 분할하고 개별 띠를 형성합니다.
고정상과 강한 상호 작용을 하는 화합물은 고정상과의 약한 상호 작용을 가진 화합물보다 느리게 움직입니다. 따라서 약한 상호 작용이 있는 화합물은 강한 상호 작용이 있는 화합물보다 더 일찍 컬럼을 빠져나가거나 용리됩니다. 화합물과 고정상의 상호 작용 강도는 지연 계수(R, f)에 의해 정의되며, 이는 이동상이 이동한 거리에 대한 구성 요소가 이동한 거리의 비율입니다. 지연 계수는 먼저 박막 크로마토그래피를 수행하여 결정됩니다.
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
지연 계수 값이 높으면 샘플이 고정상과 강하게 상호 작용하고 용리하는 데 시간이 오래 걸린다는 것을 나타냅니다. 지연 계수 값이 낮으면 샘플이 고정상과 매우 강하게 상호 작용하지 않고 더 빨리 용리된다는 것을 나타냅니다. 화합물은 개별 띠로 분할하고R-f 값이 다르기 때문에 서로 다른 시간에 컬럼에서 용리되기 때문에 컬럼 아래에서 분획 단위로 용매를 수집하여 개별적으로 분리할 수 있습니다.
<h4>컬럼 크로마토그래피 고려 사항</h4>
크로마토그래피 컬럼은 수직 방향의 유리 또는 플라스틱 튜브입니다. 컬럼의 크기는 몇 센티미터(예: 파스퇴르 피펫)에서 미터(예: 산업용 칼럼)까지 다양합니다. 컬럼의 직경은 분리할 샘플의 양에 따라 달라지는 반면, 컬럼의 길이는 분리 난이도에 따라 달라집니다. 개별 밴드로 가장 효율적으로 분할하려면 얇은 샘플 레이어가 필요합니다. 따라서 적절한 직경을 결정하는 것은 중요한 단계입니다. 컬럼의 직경에 비해 너무 많은 시료를 주입하면 직경이 더 큰 컬럼에서 동일한 부피의 시료보다 더 넓고 덜 정의된 밴드가 생성됩니다.
열의 길이를 선택할 때 구성 요소의 Rf 값을 고려해야 합니다. 유사한 지연 계수를 가진 구성 요소는 분리하기 어려울 수 있으며 개별 밴드를 해결하기 위해 더 긴 컬럼이 필요할 수 있습니다. 지연 계수가 매우 다른 화합물은 쉽게 분할되어야 하므로 긴 컬럼이 필요하지 않을 수 있습니다. 고정상을 선택할 때는 각 구성요소에 대해 서로 다른 지연 계수를 생성하는 위상을 선택하는 것이 중요합니다.
컬럼 준비는 크로마토그래피 컬럼 실행에서 가장 중요한 부분입니다. 컬럼에 충전된 고정상의 질량은 샘플의 질량의 20배 이상이어야 합니다. 기둥 바닥에 다공성 디스크가 없으면 면과 얇은 모래 층으로 포장해야 합니다. 이렇게 하면 컬럼의 출구를 통해 고정상이 손실되는 것을 방지할 수 있습니다.
컬럼을 패킹하는 방법에는 건식 패킹 방법과 슬러리 방법의 두 가지가 있습니다. 건식 방법에서는 고정상이 분말 형태로 컬럼으로 전달됩니다. 그런 다음 용매가 실리카겔 컬럼의 모든 부분에 침투하도록 이동상/용매로 컬럼을 여러 번 세척합니다. 이 방법을 올바르게 수행하지 않으면 컬럼에 건조한 패치, 채널 및 기포가 형성될 확률이 높아질 수 있습니다. 이러한 결함은 혼합물의 성분을 분할하는 컬럼의 기능에 영향을 미칩니다.
슬러리 방법은 기포, 건조 영역 및 채널을 포함하지 않는 보다 균일한 충전 컬럼을 제공합니다. 이 방법의 경우, 고정상을 용매와 혼합하여 일관된 슬러리를 형성합니다. 그런 다음 슬러리는 측면을 두드리면서 형성된 기포를 제거하면서 컬럼으로 옮겨집니다. 컬럼의 마개가 있는 경우 이 단계에서 용매가 배출될 수 있도록 열어야 합니다.
<h4>실리카겔 크로마토그래</h4>피
크기, 전하 및 소수성과 같은 화합물의 혼합물을 분리하기 위해 다양한 특성이 활용됩니다. 유기 화학에서 화합물을 분리하는 데 사용되는 한 가지 특성은 극성입니다. 이를 위해 실리카겔과 알루미나 겔이 가장 일반적으로 사용되는 고정상입니다. 실리카겔은 극성이 매우 높으며 극성 화합물과 강력한 쌍극자-쌍극자 상호 작용을 형성합니다. 또한 실리카겔은 표면에 -OH 기가 존재하기 때문에 분석물과 수소 결합을 형성할 수 있습니다.
가장 극성이 높은 혼합물의 성분은 실리카겔에 강하게 결합하여 컬럼을 통해 천천히 이동하는 반면, 비극성 성분은 이동상에서 더 잘 용해되고 컬럼을 통해 빠르게 이동합니다. 따라서 혼합물의 성분이 서로 다른 극성을 갖는 것이 중요합니다. 고정상과 강하게 상호 작용하는 성분은 컬럼을 통해 극성 이동상을 유동시켜 용리됩니다.
<h4>참조</h4>
<ol>
	<li style="font-size:17px">Shuler, ML, Kargi, K., DeLisa, M. (2017). 바이오 프로세스 공학, 기본 개념. 어퍼 새들 리버, 뉴저지 : 프렌티스 홀.</li>
	<li style="font-size:17px">Harris, D.C. (2015). 정량적 화학 분석. 뉴욕, 뉴욕 : WH 프리먼 앤 컴퍼니.</li>
</ol>

컬럼 크로마토그래피
유기 화학 실험실에서 화합물을 정제하고 분리하기 위한 여러 기술이 존재합니다. 가장 신뢰할 수 있는 분리 중 하나 ...

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JoVE Lab Manual

Chemistry

화학

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

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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.

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Acids, Bases, and pH in Titration

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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

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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

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Separating Mixtures by Polarity

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