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

Колоночная хроматография — это метод, при котором насадочная колонка используется для разделения соединений на основе их взаимодействия со стационарной фазой, которая обычно имеет форму микроскопических шариков. Промежутки между бусинами заполняются растворителем. Открытие колонны позволяет растворителю протекать через неподвижную фазу. Когда смесь наносится на верхнюю часть насадочной колонны с последующим добавлением большего количества растворителя, смесь переходит в подвижную фазу и протекает через стационарную фазу.
Каждый компонент смеси по-разному взаимодействует со стационарной фазой. Некоторые компоненты имеют слабое взаимодействие со стационарной фазой и поэтому быстро перемещаются по колонне, в то время как другие имеют сильное взаимодействие со стационарной фазой и, таким образом, движутся медленнее. При этом различные соединения разделяются на полосы, которые собираются в мелкие фракции. Это позволяет очищать каждое соединение.
Итак, какие же свойства можно использовать для разделения смесей? Одним из наиболее часто используемых свойств является полярность. Для этого в качестве стационарной фазы часто используется силикагель, который является формой диоксида кремния. Силикагель взаимодействует с соединениями посредством диполь-дипольных взаимодействий и водородных связей через группы -ОН, которые образуются на его поверхности. Таким образом, полярные соединения будут сильно взаимодействовать со стационарной фазой, в то время как неполярные соединения будут взаимодействовать слабо.
Другие свойства, которые могут быть использованы для разделения, включают размер, заряд и гидрофобность. Распространенным способом загрузки стационарной фазы в колонну является загрузка неподвижной фазы в виде суспензии и растворителя. Затем неподвижная фаза упаковывается путем пропускания большего количества растворителя через колонну для уплотнения суспензии. Важно, чтобы неподвижная фаза была равномерно упакована без пузырьков воздуха, пустых каналов или сухих участков. Это может нарушить поток и привести к смешиванию полос.
При выборе колонки диаметр определяется исходя из объема разделяемой пробы. Проба должна покрывать верхнюю часть столбика тонким ровным слоем. Более толстый слой образца приводит к более широким полосам. Длина колонки зависит от того, насколько хорошо соединения разделяются на неподвижной фазе. Соединения с одинаковым сродством к стационарной фазе требуют длинной колонки для адекватного разделения. Однако смесь соединений с очень разным сродством к стационарной фазе может быть разделена на более короткой колонне.
В этой лаборатории вы изучите колоночную хроматографию, сначала упаковав и подготовив силикагелевую колонку. Затем вы будете использовать колонку для разделения цветных компонентов зеленым пищевым красителем.

Колоночная хроматография

Колоночная хроматография
Существует множество методов очистки и разделения соединений в лаборатории органической химии. Одним из наиболее надежных методов разделения для микромасштабного разделения, когда для анализа доступно менее 10 граммов соединения, является колоночная хроматография. Этот метод разделяет соединения в смеси на основе их физических свойств, таких как растворимость, полярность, гидрофобность, размер и заряд.
<h4>Основы хроматографии</h4>
Основными компонентами любой хроматографической системы являются стационарная фаза, подвижная фаза и анализируемая смесь образцов. В колоночной хроматографии неподвижная фаза обычно представляет собой микроразмерные шарики, которые равномерно упакованы в вертикальную колонку. Непрерывный поток подвижной фазы, также известной как растворитель, добавляется в верхнюю часть колонны, которая протекает через неподвижную фазу под действием силы тяжести или с контролируемой скоростью потока с помощью насоса.
Образец растворяют в небольшом количестве растворителя, а затем добавляют в верхнюю часть колонны. Добавляется больше растворителя, чтобы образец протекал через неподвижную фазу. Соединения в смеси разделяются между подвижной и стационарной фазами в зависимости от их различных свойств и образуют дискретные полосы.
Соединения с сильным взаимодействием со стационарной фазой будут двигаться медленнее, чем соединения со слабыми взаимодействиями со стационарной фазой. Таким образом, соединения со слабыми взаимодействиями будут выходить из колонки, или элюировать, раньше, чем соединения с сильными взаимодействиями. Интенсивность взаимодействия соединения со стационарной фазой определяется коэффициентом замедления (Rf), который представляет собой отношение расстояния, пройденного компонентом, к расстоянию, пройденному подвижной фазой. Фактор запаздывания определяется путем предварительного проведения тонкослойной хроматографии.
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
Высокое значение коэффициента замедления указывает на то, что образец сильно взаимодействует со стационарной фазой и долго элюируется. Низкое значение коэффициента запаздывания указывает на то, что образец не очень сильно взаимодействует со стационарной фазой и быстрее элюирует. Поскольку соединения разделяются на отдельные полосы и вымываются из колонки в разное время из-за их различных значенийRf f, их можно выделить по отдельности, собирая растворитель из-под колонки по фракциям.
<h4>Рекомендации по колоночной хроматографии</h4>
Хроматографическая колонка представляет собой вертикально ориентированную стеклянную или пластиковую трубку. Размер колонки может варьироваться от нескольких сантиметров (например, у пастеровской пипетки) до нескольких метров (как в случае с промышленными колоннами). Диаметр колонны зависит от количества образца, который необходимо отделить, в то время как длина колонны зависит от сложности разделения. Наиболее эффективное разделение на дискретные полосы требует тонких слоев выборки; Поэтому определение подходящего диаметра является жизненно важным этапом. Загрузка слишком большого объема образца относительно диаметра колонки приведет к созданию более широких и менее определенных полос, чем тот же объем пробы в колонке большего диаметра.
При выборе длины столбца необходимо учитывать значения Rf компонентов. Компоненты с аналогичными коэффициентами запаздывания может быть сложно разделить, и для их разделения отдельных полос требуется более длинная колонка. Соединения с очень разными коэффициентами замедления могут не нуждаться в длинной колонке, так как они должны легко разделяться. При выборе стационарной фазы важно выбрать ту, которая приводит к различным коэффициентам замедления для каждого компонента.
Подготовка колонки является наиболее важной частью работы хроматографической колонки. Масса неподвижной фазы, упакованной в колонку, должна быть не менее чем в 20 раз больше массы образца. Если колонка не имеет пористого диска внизу, ее следует набить ватой и тонким слоем песка. Это предотвращает потерю неподвижной фазы через выход из колонны.
Существует два метода уплотнения колонны: метод сухой упаковки и метод навозной жижи. При сухом способе неподвижная фаза переносится в колонну в виде порошка. Затем колонку несколько раз промывают подвижной фазой/растворителем, чтобы убедиться, что растворитель проникает во все части силикагелевой колонки. Этот метод, если он выполнен неправильно, может увеличить вероятность образования сухих участков, каналов и пузырьков воздуха в колонке. Эти дефекты повлияют на способность колонны разделять компоненты смеси.
Метод навозной жижи обеспечивает более однородную насадочную колонну, которая не содержит пузырьков воздуха, сухих участков и каналов. Для этого метода неподвижная фаза смешивается с растворителем для образования однородной суспензии. Затем суспензия переносится в колонну, постукивая по бокам, чтобы избавиться от образовавшихся пузырьков воздуха. Запорный кран колонны, если таковой имеется, должен быть открыт во время этого этапа, чтобы позволить растворителю стечь.
<h4>Силикагелевая хроматография</h4>
Для разделения смеси соединений используется множество различных свойств, таких как размер, заряд и гидрофобность. Одним из свойств, используемых для разделения соединений в органической химии, является полярность. Для этого чаще всего используются силикагель и оксид алюминия в виде стационарных фаз. Силикагель чрезвычайно полярен и образует сильные диполь-дипольные взаимодействия с полярными соединениями. Кроме того, силикагель может образовывать водородные связи с анализируемым веществом благодаря наличию на его поверхности -ОН-групп.
Компоненты смеси, которые являются наиболее полярными, прочно связываются с силикагелем и медленно перемещаются по колонне, в то время как неполярные компоненты более растворимы в подвижной фазе и быстро перемещаются по колонне. Таким образом, важно, чтобы компоненты смеси имели разную полярность. Компоненты, которые сильно взаимодействуют со стационарной фазой, элюируются путем пропускания полярной подвижной фазы через колонку.
<h4>Ссылки</h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017). Биотехнологическая инженерия, основные понятия. Аппер Сэддл Ривер, Нью-Джерси: Прентис Холл.</li>
	<li style="font-size:17px">Harris, D.C. (2015). Количественный химический анализ. Нью-Йорк, Нью-Йорк: W.H. Freeman and Company.</li>
</ol>

Колоночная хроматография
Существует множество методов очистки и разделения соединений в лаборатории органической химии. Один из самых надежных разделителей ...

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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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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.

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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.

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Separating Mixtures by Polarity

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