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- North Clin Istanb
- v.3(2); 2016
Separation techniques: Chromatography
Department of Biophysics, Canakkale Onsekiz Mart University, Canakkale, Turkey
Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification.
Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights [ 1 , 2 ]. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster [ 3 ].
Based on this approach three components form the basis of the chromatography technique.
- Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface a solid support”.
- Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
- Separated molecules
The type of interaction between stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on separation of molecules from each other. Chromatography methods based on partition are very effective on separation, and identification of small molecules as amino acids, carbohydrates, and fatty acids. However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins. Paper chromatography is used in the separation of proteins, and in studies related to protein synthesis; gas-liquid chromatography is utilized in the separation of alcohol, esther, lipid, and amino groups, and observation of enzymatic interactions, while molecular-sieve chromatography is employed especially for the determination of molecular weights of proteins. Agarose-gel chromatography is used for the purification of RNA, DNA particles, and viruses [ 4 ].
Stationary phase in chromatography, is a solid phase or a liquid phase coated on the surface of a solid phase. Mobile phase flowing over the stationary phase is a gaseous or liquid phase. If mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). Gas chromatography is applied for gases, and mixtures of volatile liquids, and solid material. Liquid chromatography is used especially for thermal unstable, and non-volatile samples [ 5 ].
The purpose of applying chromatography which is used as a method of quantitative analysis apart from its separation, is to achive a satisfactory separation within a suitable timeinterval. Various chromatography methods have been developed to that end. Some of them include column chromatography, thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, high-pressure liquid chromatography, and affinity chromatography [ 6 ].
- Ion-exchange chromatography
- Gel-permeation (molecular sieve) chromatography
- Affinity chromatography
- Paper chromatography
- Thin-layer chromatography
- Gas chromatography
- Dye-ligand chromatography
- Hydrophobic interaction chromatography
- Pseudoaffinity chromatography
- High-pressure liquid chromatography (HPLC)
Since proteins have difference characteristic features as size, shape, net charge, stationary phase used, and binding capacity, each one of these characteristic components can be purified using chromatographic methods. Among these methods, most frequently column chromatography is applied. This technique is used for the purification of biomolecules. On a column (stationary phase) firstly the sample to be separated, then wash buffer (mobile phase) are applied ( Figure 1 ). Their flow through inside column material placed on a fiberglass support is ensured. The samples are accumulated at the bottom of the device in a tme-, and volume-dependent manner [ 7 ].
Ion- exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved with ionic ties. Proteins are separated from the column either by changing pH, concentration of ion salts or ionic strength of the buffer solution [ 8 ]. Positively charged ion- exchange matrices are called anion-exchange matrices, and adsorb negatively charged proteins. While matrices bound with negatively charged groups are known as cation-exchange matrices, and adsorb positively charged proteins ( Figure 2 ) [ 9 ].
Ion- exchange chromatography.
The basic principle of this method is to use dextran containing materials to separate macromolecules based on their differences in molecular sizes. This procedure is basically used to determine molecular weights of proteins, and to decrease salt concentrations of protein solutions [ 10 ]. In a gel- permeation column stationary phase consists of inert molecules with small pores. The solution containing molecules of different dimensions are passed continuously with a constant flow rate through the column. Molecules larger than pores can not permeate into gel particles, and they are retained between particles within a restricted area. Larger molecules pass through spaces between porous particles, and move rapidly through inside the column. Molecules smaller than the pores are diffused into pores, and as molecules get smaller, they leave the column with proportionally longer retention times ( Figure 3 ) [ 11 ]. Sephadeks G type is the most frequently used column material. Besides, dextran, agorose, polyacrylamide are also used as column materials [ 12 ].
Gel-permeation (molecular sieve) chromatography.
This chromatography technique is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins [ 13 ]. A ligand which can make a complex with specific protein (dextran, polyacrylamide, cellulose etc) binds the filling material of the column. The specific protein which makes a complex with the ligand is attached to the solid support (matrix), and retained in the column, while free proteins leave the column. Then the bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution ( Figure 4 ) [ 14 ].
In paper chromatography support material consists of a layer of cellulose highly saturated with water. In this method a thick filter paper comprised the support, and water drops settled in its pores made up the stationary “liquid phase.” Mobile phase consists of an appropriate fluid placed in a developing tank. Paper chromatography is a “liquid-liquid” chromatography [ 15 ].
Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this method stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all solid substances used. in column chromatography (alumina, silica gel, cellulose) can be utilized. In this method, the mobile phase travels upward through the stationary phase The solvent travels up the thin plate soaked with the solvent by means of capillary action. During this procedure, it also drives the mixture priorly dropped on the lower parts of the plate with a pipette upwards with different flow rates. Thus the separation of analytes is achieved. This upward travelling rate depends on the polarity of the material, solid phase, and of the solvent [ 16 ].
In cases where molecules of the sample are colorless, florescence, radioactivity or a specific chemical substance can be used to produce a visible coloured reactive product so as to identify their positions on the chromatogram. Formation of a visible colour can be observed under room light or UV light. The position of each molecule in the mixture can be measured by calculating the ratio between the the distances travelled by the molecule and the solvent. This measurement value is called relative mobility, and expressed with a symbol R f . R f . value is used for qualitative description of the molecules [ 17 ].
In this method stationary phase is a column which is placed in the device, and contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Gas chromatography is a “gas-liquid” chromatography. Its carrier phase consists of gases as He or N 2 . Mobile phase which is an inert gas is passed through a column under high pressure. The sample to be analyzed is vaporized, and enters into a gaseous mobile phase phase. The components contained in the sample are dispersed between mobile phase, and stationary phase on the solid support. Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly applied technique for the extremely excellent separation of very minute molecules. It is used in the separation of very little amounts of analytes [ 18 ].
Development of this technique was based on the demonstration of the ability of many enzymes to bind purine nucleotides for Cibacron Blue F3GA dye [ 19 ]. The planar ring structure with negatively charged groups is analogous to the structure of NAD. This analogy has been evidenced by demonstration of the binding of Cibacron Blue F3GA dye to adenine, ribose binding sites of NAD. The dye behaves as an analogue of ADP-ribose. The binding capacity of this type adsorbents is 10–20-fold stronger rhat that of the affinity of other adsorbents. Under appropriate pH conditions, elution with high-ionic strength solutions, and using ion-exchange property of adsorbent, the adsorbed proteins are separated from the column [ 20 , 21 ].
In this method the adsorbents prepared as column material for the ligand binding in affinity chromatography are used. HIC technique is based on hydrophobic interactions between side chains bound to chromatography matrix [ 22 , 23 ].
Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands because of their affinity especially for dehydrogenases, kinases, transferases, and reductases The mostly known type of this kind of chromatography is immobilized metal affinity chromatography (IMAC) [ 24 ].
Using this chromatography technique it is possible to perform structural, and functional analysis, and purification of many molecules within a short time, This technique yields perfect results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active molecules, In HPLC, mobile phase passes throuıgh columns under 10–400 atmospheric pressure, and with a high (0.1–5 cm//sec) flow rate. In this technique, use of small particles, and application of high presure on the rate of solvent flow increases separation power, of HPLC and the analysis is completed within a short time.
Essential components of a HPLC device are solvent depot, high- pressure pump, commercially prepared column, detector, and recorder. Duration of separation is controlled with the aid of a computerized system, and material is accrued [ 25 ].
Chromatography technique is a valuable tool for biochemists, besides it can be applied easily during studies performed in clinical laboratories For instance, paper chromatography is used to determine some types of sugar, and amino acids in bodily fluids which are associated with hereditary metabolic disorders. Gas chromatography is used in laboratories to measure steroids, barbiturates, and lipids. Chromatographic technique is also used in the separation of vitamins, and proteins.
Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal pigments. With time its application area was extended considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective separation method. Column chromatography is one of the useful separation, and determination methods. Column chromatography is a protein purification method realized especially based on one of the characteristic features of proteins. Besides, these methods are used to control purity of a protein. HPLC technique which has many superior features including especially its higher sensitivity, rapid turnover rate, its use as a quantitative method, can purify amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, antibiotics, and steroids.
Conflict of Interest: None declared.
Financial Disclosure: The authors declared that this study has received no financial support.
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E. Paper Chromatography
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- Page ID 3754
- Truro School in Cornwall
Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle. They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. We'll look at the reasons for this further down the page. In paper chromatography, the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.
Producing a paper chromatogram
You probably used paper chromatography as one of the first things you ever did in chemistry to separate out mixtures of colored dyes - for example, the dyes which make up a particular ink. That's an easy example to take, so let's start from there.
Suppose you have three blue pens and you want to find out which one was used to write a message. Samples of each ink are spotted on to a pencil line drawn on a sheet of chromatography paper. Some of the ink from the message is dissolved in the minimum possible amount of a suitable solvent, and that is also spotted onto the same line. In the diagram, the pens are labeled 1, 2 and 3, and the message ink as M.
The paper is suspended in a container with a shallow layer of a suitable solvent or mixture of solvents in it. It is important that the solvent level is below the line with the spots on it. The next diagram doesn't show details of how the paper is suspended because there are too many possible ways of doing it and it clutters the diagram. Sometimes the paper is just coiled into a loose cylinder and fastened with paper clips top and bottom. The cylinder then just stands in the bottom of the container.
The reason for covering the container is to make sure that the atmosphere in the beaker is saturated with solvent vapour. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the paper.
As the solvent slowly travels up the paper, the different components of the ink mixtures travel at different rates and the mixtures are separated into different colored spots.
The diagram shows what the plate might look like after the solvent has moved almost to the top.
It is fairly easy to see from the final chromatogram that the pen that wrote the message contained the same dyes as pen 2. You can also see that pen 1 contains a mixture of two different blue dyes - one of which might be the same as the single dye in pen 3.
Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. The distance travelled relative to the solvent is a constant for a particular compound as long as you keep everything else constant - the type of paper and the exact composition of the solvent, for example.
The distance travelled relative to the solvent is called the R f value. For each compound it can be worked out using the formula:
For example, if one component of a mixture travelled 9.6 cm from the base line while the solvent had travelled 12.0 cm, then the R f value for that component is:
In the example we looked at with the various pens, it wasn't necessary to measure R f values because you are making a direct comparison just by looking at the chromatogram.
You are making the assumption that if you have two spots in the final chromatogram which are the same color and have travelled the same distance up the paper, they are most likely the same compound. It isn't necessarily true of course - you could have two similarly colored compounds with very similar R f values. We'll look at how you can get around that problem further down the page.
What if the substances you are interested in are colorless?
In some cases, it may be possible to make the spots visible by reacting them with something which produces a colored product. A good example of this is in chromatograms produced from amino acid mixtures.
Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids. A small drop of a solution of the mixture is placed on the base line of the paper, and similar small spots of the known amino acids are placed alongside it. The paper is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labeled 1 to 5.
The position of the solvent front is marked in pencil and the chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with amino acids to give colored compounds, mainly brown or purple.
The left-hand diagram shows the paper after the solvent front has almost reached the top. The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin.
There is no need to measure the R f values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colors. In this example, the mixture contains the amino acids labeled as 1, 4 and 5. And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re-run the experiment using other amino acids for comparison.
Two way paper chromatography
Two way paper chromatography gets around the problem of separating out substances which have very similar R f values. I'm going to go back to talking about colored compounds because it is much easier to see what is happening. You can perfectly well do this with colorless compounds - but you have to use quite a lot of imagination in the explanation of what is going on!
This time a chromatogram is made starting from a single spot of mixture placed towards one end of the base line. It is stood in a solvent as before and left until the solvent front gets close to the top of the paper.
In the diagram, the position of the solvent front is marked in pencil before the paper dries out. This is labeled as SF1 - the solvent front for the first solvent. We shall be using two different solvents.
If you look closely, you may be able to see that the large central spot in the chromatogram is partly blue and partly green. Two dyes in the mixture have almost the same R f values. They could equally well, of course, both have been the same color - in which case you couldn't tell whether there was one or more dye present in that spot.
What you do now is to wait for the paper to dry out completely, and then rotate it through 90°, and develop the chromatogram again in a different solvent.
It is very unlikely that the two confusing spots will have the same R f values in the second solvent as well as the first, and so the spots will move by a different amount.
The next diagram shows what might happen to the various spots on the original chromatogram. The position of the second solvent front is also marked.
You wouldn't, of course, see these spots in both their original and final positions - they have moved! The final chromatogram would look like this:
Two way chromatography has completely separated out the mixture into four distinct spots. If you want to identify the spots in the mixture, you obviously can't do it with comparison substances on the same chromatogram as we looked at earlier with the pens or amino acids examples. You would end up with a meaningless mess of spots. You can, though, work out the R f values for each of the spots in both solvents, and then compare these with values that you have measured for known compounds under exactly the same conditions.
How does paper chromatography work?
Although paper chromatography is simple to do, it is quite difficult to explain compared with thin layer chromatography. The explanation depends to some extent on what sort of solvent you are using, and many sources gloss over the problem completely. If you haven't already done so, it would be helpful if you could read the explanation for how thin layer chromatography works (link below). That will save me a lot of repetition, and I can concentrate on the problems.
The essential structure of paper
Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose.
The key point about cellulose is that the polymer chains have -OH groups sticking out all around them. To that extent, it presents the same sort of surface as silica gel or alumina in thin layer chromatography.
It would be tempting to try to explain paper chromatography in terms of the way that different compounds are adsorbed to different extents on to the paper surface. In other words, it would be nice to be able to use the same explanation for both thin layer and paper chromatography. Unfortunately, it is more complicated than that!
The complication arises because the cellulose fibres attract water vapour from the atmosphere as well as any water that was present when the paper was made. You can therefore think of paper as being cellulose fibres with a very thin layer of water molecules bound to the surface.
It is the interaction with this water which is the most important effect during paper chromatography.
Paper chromatography using a non-polar solvent
Suppose you use a non-polar solvent such as hexane to develop your chromatogram.
Non-polar molecules in the mixture that you are trying to separate will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high R f values.
On the other hand, polar molecules will have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibres much more than in the moving solvent.
Because they spend more time dissolved in the stationary phase and less time in the mobile phase, they aren't going to travel very fast up the paper.
The tendency for a compound to divide its time between two immiscible solvents (solvents such as hexane and water which won't mix) is known as partition . Paper chromatography using a non-polar solvent is therefore a type of partition chromatography.
Paper chromatography using a water and other polar solvents
A moment's thought will tell you that partition can't be the explanation if you are using water as the solvent for your mixture. If you have water as the mobile phase and the water bound on to the cellulose as the stationary phase, there can't be any meaningful difference between the amount of time a substance spends in solution in either of them. All substances should be equally soluble (or equally insoluble) in both.
And yet the first chromatograms that you made were probably of inks using water as your solvent.
If water works as the mobile phase as well being the stationary phase, there has to be some quite different mechanism at work - and that must be equally true for other polar solvents like the alcohols, for example. Partition only happens between solvents which don't mix with each other. Polar solvents like the small alcohols do mix with water.
In researching this topic, I haven't found any easy explanation for what happens in these cases. Most sources ignore the problem altogether and just quote the partition explanation without making any allowance for the type of solvent you are using. Other sources quote mechanisms which have so many strands to them that they are far too complicated for this introductory level. I'm therefore not taking this any further - you shouldn't need to worry about this at UK A level, or its various equivalents.
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