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

Kolon kromatografisi, bileşikleri, genellikle mikroskobik boncuklar şeklinde olan sabit bir faz ile etkileşimlerine dayalı olarak ayırmak için paketlenmiş bir kolon kullanan bir tekniktir. Boncuklar arasındaki boşluklar çözücü ile doldurulur. Kolonun açılması, çözücünün sabit fazdan akmasına izin verir. Paketlenmiş kolonun tepesine bir karışım uygulandığında ve ardından daha fazla çözücü uygulandığında, karışım mobil faza geçer ve sabit fazdan akar.
Karışımın her bir bileşeni, sabit faz ile farklı bir şekilde etkileşime girer. Bazı bileşenler durağan faz ile zayıf etkileşimlere sahiptir ve bu nedenle kolon boyunca hızlı hareket ederken, diğerleri durağan faz ile güçlü etkileşimlere sahiptir ve bu nedenle daha yavaş hareket eder. Bu, farklı bileşikleri küçük fraksiyonlar halinde toplanan bantlara ayırır. Bu, her bir bileşiğin saflaştırılmasını sağlar.
Peki, karışımları ayırmak için ne tür özellikler kullanılabilir? En sık kullanılan özelliklerden biri polaritedir. Bunun için bir silikon dioksit formu olan silika jel genellikle sabit faz olarak kullanılır. Silika jel, yüzeyinde oluşan -OH grupları aracılığıyla dipol-dipol etkileşimleri ve hidrojen bağı ile bileşiklerle etkileşime girer. Bu nedenle, polar bileşikler durağan faz ile güçlü bir şekilde etkileşime girerken, polar olmayan bileşikler zayıf bir şekilde etkileşime girecektir.
Ayırma için kullanılabilecek diğer özellikler arasında boyut, yük ve hidrofobiklik bulunur. Sabit fazı kolona yüklemenin yaygın bir yolu, sabit faz ve çözücünün bir bulamacıdır. Daha sonra, sabit faz, bulamacı sıkıştırmak için kolondan daha fazla çözücü akıtılarak paketlenir. Sabit fazın hava kabarcıkları, boş kanallar veya kuru lekeler olmadan düzgün bir şekilde paketlenmesi önemlidir. Bunlar akışı bozabilir ve bantların karışmasına neden olabilir.
Bir kolon seçerken, çap ayrılacak numunenin hacmine bağlıdır. Numune, kolonun üst kısmını ince, eşit bir tabaka halinde kaplamalıdır. Daha kalın bir numune tabakası daha geniş bantlara yol açar. Kolon uzunluğu, bileşiklerin sabit fazda ne kadar iyi ayrıldığına bağlıdır. Sabit faz için benzer afiniteye sahip bileşikler, yeterli ayırma için uzun bir kolon gerektirir. Bununla birlikte, durağan faz için çok farklı afinitelere sahip bileşiklerin bir karışımı, daha kısa bir kolon üzerinde ayrılabilir.
Bu laboratuvarda, önce bir silika jel sütunu paketleyip hazırlayarak sütun kromatografisini keşfedeceksiniz. Ardından, yeşil gıda boyasındaki renkli bileşenleri ayırmak için sütununuzu kullanacaksınız.

kolon kromatografisi

Kolon Kromatografisi
Bir organik kimya laboratuvarında bileşikleri saflaştırmak ve ayırmak için birden fazla teknik mevcuttur. Analiz için 10 gramdan daha az bileşiğin mevcut olduğu mikro ölçekli ayırmalar için en güvenilir ayırma tekniklerinden biri kolon kromatografisidir. Bu teknik, bir karışımdaki bileşikleri çözünürlükleri, polariteleri, hidrofobiklikleri, boyutları ve yükleri gibi fiziksel özelliklerine göre ayırır.
<h4>Kromatografi Temelleri</h4>
Herhangi bir kromatografi sisteminin ana bileşenleri, sabit faz, mobil faz ve analiz edilecek numune karışımıdır. Kolon kromatografisinde, sabit faz tipik olarak dikey bir kolonda düzgün bir şekilde paketlenen mikro ölçekli boncuklardır. Çözücü olarak da bilinen mobil fazın sürekli bir akışı, sabit faz boyunca yerçekimi yoluyla veya bir pompa tarafından kontrollü bir akış hızında akan kolonun tepesine eklenir.
Numune, az miktarda çözücü içinde çözülür ve daha sonra kolonun tepesine eklenir. Numuneyi sabit fazdan akmaya zorlamak için daha fazla çözücü eklenir. Karışımdaki bileşikler, farklı özelliklerine bağlı olarak mobil faz ile sabit faz arasında bölünür ve ayrı bantlar oluşturur.
Durağan faz ile güçlü etkileşimleri olan bileşikler, durağan faz ile zayıf etkileşimleri olan bileşiklerden daha yavaş hareket edecektir. Bu nedenle, zayıf etkileşimlere sahip bileşikler, güçlü etkileşimlere sahip olanlardan daha erken kolondan çıkacak veya elute edecektir. Bir bileşiğin durağan faz ile etkileşiminin gücü, bir bileşen tarafından kat edilen mesafenin mobil faz tarafından kat edilen mesafeye oranı olan geciktirme faktörü (Rf) ile tanımlanır. Geciktirme faktörü, önce ince tabaka kromatografisi yapılarak belirlenir.
<img src="/CDNSource/lm/labs/63/63_Concepts_1.jpg">
Yüksek bir geciktirme faktörü değeri, numunenin durağan faz ile güçlü bir şekilde etkileşime girdiğini ve yükselmesinin uzun sürdüğünü gösterir. Düşük bir geciktirme faktörü değeri, numunenin durağan faz ile çok güçlü bir şekilde etkileşime girmediğini ve daha hızlı elüte ettiğini gösterir. Bileşikler, farklı Rf değerleri nedeniyle ayrı bantlara ayrıldığından ve farklı zamanlarda kolondan elüte olduklarından, çözücüyü kolonun altından fraksiyonlar halinde toplayarak ayrı ayrı izole edilebilirler.
<h4>Kolon Kromatografisi ile İlgili Dikkat Edilmesi Gerekenler</h4>
Bir kromatografi kolonu, dikey olarak yönlendirilmiş bir cam veya plastik tüptür. Kolonun boyutu birkaç santimetreden (Pasteur pipeti gibi) metrelere (endüstriyel kolonlarda olduğu gibi) kadar değişebilir. Kolonun çapı ayrılacak numune miktarına bağlıdır, kolonun uzunluğu ise ayırma zorluğuna bağlıdır. Ayrık bantlara en verimli bölme, ince numune katmanları gerektirir; Bu nedenle, uygun çapın belirlenmesi hayati bir adımdır. Kolonun çapına göre çok fazla numune hacmi yüklemek, daha büyük çaplı bir kolondaki aynı numune hacminden daha geniş ve daha az tanımlanmış bantlar oluşturacaktır.
Kolonun uzunluğunu seçerken, bileşenlerin Rf değerleri dikkate alınmalıdır. Benzer geciktirme faktörlerine sahip bileşenlerin ayrılması zor olabilir ve tek tek bantları çözmek için daha uzun bir sütun gerektirebilir. Çok farklı geciktirme faktörlerine sahip bileşikler, kolayca bölünmeleri gerektiği için uzun bir sütuna ihtiyaç duymayabilir. Sabit fazı seçerken, her bileşen için farklı geciktirme faktörlerine neden olan bir faz seçmek önemlidir.
Kolonu hazırlamak, bir kromatografi kolonunu çalıştırmanın en kritik kısmıdır. Kolonda paketlenen sabit fazın kütlesi, numunenin kütlesinin en az 20 katı olmalıdır. Kolonun altında gözenekli bir disk yoksa, pamuk ve ince bir kum tabakası ile doldurulmalıdır. Bu, kolonun çıkışı yoluyla sabit fazın kaybını önler.
Sütunu paketlemek için iki yöntem vardır: kuru paketleme yöntemi ve bulamaç yöntemi. Kuru yöntemde, sabit faz toz halinde kolona aktarılır. Sütun daha sonra çözücünün silika jel kolonunun her parçasına nüfuz etmesini sağlamak için mobil faz/çözücü ile birkaç kez yıkanır. Bu yöntem, doğru şekilde yapılmazsa, kolonda kuru lekeler, kanallar ve hava kabarcıkları oluşma olasılığını artırabilir. Bu kusurlar, kolonun bir karışımın bileşenlerini bölme yeteneğini etkileyecektir.
Bulamaç yöntemi, hava kabarcıkları, kuru alanlar ve kanallar içermeyen daha düzgün bir paketlenmiş kolon sağlar. Bu yöntem için, sabit faz, tutarlı bir bulamaç oluşturmak için bir çözücü ile karıştırılır. Bulamaç daha sonra, oluşan hava kabarcıklarından kurtulmak için yanlara vurulurken kolona aktarılır. Çözücünün boşalmasına izin vermek için bu adım sırasında kolonun vanası, varsa, açık olmalıdır.
<h4>Silika Jel Kromatografisi</h4>
Boyut, yük ve hidrofobiklik gibi bileşiklerin bir karışımını ayırmak için birçok farklı özellikten yararlanılır. Organik kimyada bileşikleri ayırmak için kullanılan özelliklerden biri polaritedir. Bunun için silika jel ve alümina jel en yaygın kullanılan sabit fazlardır. Silika jel son derece polardır ve polar bileşiklerle güçlü dipol-dipol etkileşimleri oluşturur. Ek olarak, silika jel, yüzeyinde -OH gruplarının varlığından dolayı analit ile hidrojen bağları oluşturabilir.
Karışımın en polar olan bileşenleri silika jele güçlü bir şekilde bağlanır ve kolon boyunca yavaşça hareket ederken, polar olmayan bileşenler mobil fazda daha fazla çözünür ve kolon boyunca hızlı bir şekilde hareket eder. Bu nedenle, karışımın bileşenlerinin farklı polaritelere sahip olması esastır. Sabit faz ile güçlü bir şekilde etkileşime giren bileşenler, kolon boyunca polar bir mobil faz akıtılarak ayrıştırılır.
<h4>Başvuru</h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017). Biyoproses Mühendisliği, Temel Kavramlar. Yukarı Saddle Nehri, NJ: Prentice Salonu.</li>
	<li style="font-size:17px">Harris, D.C. (2015). Kantitatif Kimyasal Analiz. New York, NY: W.H. Freeman ve Şirketi.</li>
</ol>

Kolon Kromatografisi

Kolon Kromatografisi
Bir organik kimya laboratuvarında bileşikleri saflaştırmak ve ayırmak için birden fazla teknik mevcuttur. En güvenilir separasyonlardan biri ...

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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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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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Separating Mixtures by Polarity

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