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

カラムクロマトグラフィーは、パックドカラムを使用して、通常は顕微鏡ビーズの形をした固定相との相互作用に基づいて化合物を分離する手法です。ビーズ間のスペースは溶媒で満たされています。カラムを開くと、溶媒が固定相を流れるようになります。混合物を充塡カラムの上部に塗布し、続いてさらに多くの溶媒を塗布すると、混合物は移動相に移動し、固定相を流れます。
混合物の各成分は、異なる方法で固定相と相互作用します。一部の成分は固定相との相互作用が弱いためカラム内を速く移動しますが、他の成分は固定相との相互作用が強いため、移動が遅くなります。これにより、さまざまな化合物がバンドに分離され、バンドが小さなフラクションに収集されます。これにより、各化合物の精製が可能になります。
では、どのような特性で混合物を分離できるのでしょうか。最も一般的に使用されるプロパティの1つは極性です。このために、二酸化ケイ素の一種であるシリカゲルが固定相としてよく使用されます。シリカゲルは、双極子-双極子相互作用と、その表面に形成される-OH基を介した水素結合により、化合物と相互作用します。したがって、極性化合物は固定相と強く相互作用し、非極性化合物は弱く相互作用します。
分離に利用できるその他の特性には、サイズ、電荷、疎水性などがあります。固定相をカラムにロードする一般的な方法は、固定相と溶媒のスラリーとしてロードすることです。次に、カラムにより多くの溶媒を流すことにより固定相を充填し、スラリーを圧縮します。固定相は、気泡、空のチャネル、または乾燥パッチなしで均一に充填されていることが不可欠です。これらは流れを乱し、バンドの混合を引き起こす可能性があります。
カラムを選択する場合、直径は分離するサンプルの体積に基づいています。サンプルは、カラムの上部を薄く均一な層で覆う必要があります。サンプル層が厚いほど、バンドが広くなります。カラムの長さは、化合物が固定相でどれだけうまく分離するかによって異なります。固定相に対して同様の親和性を持つ化合物は、適切な分離のために長いカラムが必要です。ただし、固定相に対して非常に異なる親和性を持つ化合物の混合物は、より短いカラムで分離できます。
このラボでは、まずシリカゲルカラムを充塡して調製することにより、カラムクロマトグラフィーについて検討します。次に、カラムを使用して、緑色の食品染料で着色された成分を分離します。

カラムクロマトグラフィー

カラムクロマトグラフィー
有機化学実験室で化合物を精製および分離するための複数の技術が存在します。分析に利用できる化合物が10g未満であるミクロスケールの分離において、最も信頼性の高い分離手法の1つがカラムクロマトグラフィーです。この手法では、溶解度、極性、疎水性、サイズ、電荷などの物理的特性に基づいて、混合物中の化合物を分離します。
<h4>クロマトグラフィーの基礎</h4>
クロマトグラフィーシステムの主な構成要素は、固定相、移動相、および分析するサンプル混合物です。カラムクロマトグラフィーでは、固定相は通常、マイクロスケールのビーズであり、垂直カラムに均一に充填されています。移動相(溶媒とも呼ばれる)の連続的な流れがカラムの上部に追加され、重力を介して、またはポンプによって制御された流量で固定相を流れます。
サンプルを少量の溶媒に溶解し、カラムの上部に添加します。サンプルが固定相を流れるように強制するために、さらに溶媒が追加されます。混合物中の化合物は、移動相と固定相の異なる特性に基づいて分配され、離散的なバンドを形成します。
固定相との相互作用が強い化合物は、固定相との相互作用が弱い化合物よりもゆっくりと移動します。したがって、相互作用が弱い化合物は、相互作用が強い化合物よりも早くカラムから出る、または溶出します。化合物と固定相との相互作用の強度は、リタデーション係数(Rf)によって定義され、これは移動相が移動した距離に対する成分の移動距離の比です。遅延係数は、最初に薄層クロマトグラフィーを実行することによって決定されます。
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
遅延係数の値が高い場合は、サンプルが固定相と強く相互作用し、溶出に時間がかかることを示します。遅延係数の値が低い場合は、サンプルが固定相とあまり強く相互作用せず、より迅速に溶出することを示します。化合物は個々のバンドに分裂し、Rf値が異なるため、異なるタイミングでカラムから溶出するため、カラムの下から溶媒をフラクションで収集することで個別に単離できます。
<h4>カラムクロマトグラフィーに関する考慮事項</h4>
クロマトグラフィーカラムは、垂直方向のガラス管またはプラスチック管です。カラムのサイズは、数センチメートル(パスツールピペットなど)からメートル(工業用カラムなど)までさまざまです。カラムの直径は分離するサンプルの量に依存しますが、カラムの長さは分離の難しさによって異なります。離散バンドへの最も効率的な分割には、薄いサンプル層が必要です。したがって、適切な直径を決定することは重要なステップです。カラムの直径に対してサンプル量が多すぎると、直径が大きいカラム内の同じ量のサンプルよりも幅が広く、定義が不十分なバンドが作成されます。
列の長さを選択するときは、コンポーネントのR f 値を考慮する必要があります。リタデーション係数が類似している成分は、分離が難しく、個々のバンドを分離するために長いカラムが必要になる場合があります。リターデーション係数が大きく異なる化合物は、簡単に分割できるため、長いカラムは必要ないかもしれません。固定相を選択する際には、各成分で遅延係数が異なるものを選択することが重要です。
カラムの調製は、クロマトグラフィーカラムを運転する上で最も重要な部分です。カラムに充塡された固定相の質量は、サンプルの質量の20倍以上である必要があります。カラムの底に多孔質のディスクがない場合は、綿と薄い砂の層を詰める必要があります。これにより、カラムの出口を通る固定相の損失が防止されます。
カラムを充填する方法には、ドライパッキング法とスラリー法の2つの方法があります。乾式法では、固定相は粉末の形でカラムに移されます。次に、カラムを移動相/溶媒で数回洗浄し、溶媒がシリカゲルカラムの隅々まで浸透するようにします。この方法を正しく行わないと、カラム内にドライパッチ、チャネル、気泡が形成される可能性が高くなる可能性があります。これらの欠陥は、カラムが混合物の成分を分配する能力に影響を与えます。
スラリー法は、気泡、乾燥領域、チャネルを含まない、より均一な充填カラムを提供します。この方法では、固定相を溶媒と混合して一貫したスラリーを形成します。次に、スラリーは側面を軽くたたいてカラムに移され、形成された気泡を取り除きます。カラムのストップコックがある場合は、このステップ中に開いて、溶媒を排出できるようにする必要があります。
<h4>シリカゲルクロマトグラフィー</h4>
化合物の混合物を分離するために、サイズ、電荷、疎水性など、さまざまな特性が利用されます。有機化学で化合物を分離するために使用される特性の1つは極性です。このために、シリカゲルとアルミナゲルが最も一般的に使用される固定相です。シリカゲルは非常に極性が高く、極性化合物と強い双極子-双極子相互作用を形成します。さらに、シリカゲルは、その表面に-OH基が存在するため、分析対象物と水素結合を形成することができます。
混合物の成分のうち、最も極性が高い成分はシリカゲルに強く結合してカラム内をゆっくりと移動しますが、非極性成分は移動相に溶解しやすく、カラム内を素早く移動します。したがって、混合物の成分が異なる極性を持つことが不可欠です。固定相と強く相互作用する成分は、極性移動相をカラムに流すことによって溶出されます。
<h4>参照</h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017).バイオプロセス工学、基本概念。ニュージャージー州アッパーサドルリバー:プレンティスホール。</li>
	<li style="font-size:17px">ハリス、ワシントンD.C.(2015年)。 定量的化学分析。ニューヨーク州ニューヨーク:WHフリーマンアンドカンパニー。</li>
</ol>

カラムクロマトグラフィー
有機化学実験室で化合物を精製および分離するための複数の技術が存在します。最も信頼性の高い分離の1つ...

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Chemistry

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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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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...

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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

カラムクロマトグラフィー を教える準備を、この 3 分間の教員向けガイドで行いましょう。JoVE Lab Manual の Chemistry ラボの主要な準備手順を解説します。

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Chemistry の カラムクロマトグラフィー 学生プロトコル - 43K+ 回の視聴 | JoVE Lab Manual

Separating Mixtures by Polarity

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