Chromatography-2

CHROMATOGRAPHY

ABSTRACT

Few methods of chemical analysis are truly specific to a particular analyte. It is often found that the analyte of interest must be separated from the myriad of individual compounds that may be present in a sample. As well as providing the analytical scientist with methods of separation, chromatographic techniques can also provide methods of analysis.

Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.

Techniques such as H.P.L.C. (High Performance Liquid Chromatography) and G.C. (Gas Chromatography) use columns - narrow tubes packed with stationary phase, through which the mobile phase is forced. The sample is transported through the column by continuous addition of mobile phase. This process is called elution. The average rate at which an analyte moves through the column is determined by the time it spends in the mobile phase.

INTRODUCTION

Pictured is a sophisticated gas chromatography system. This instrument records concentrations of acrylonitrile in the air at various points throughout the chemical laboratory.

Chromatography is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated.

Chromatography may be preparative or analytical. Preparative chromatography seeks to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture.

History

The history of chromatography spans from the mid-19th century to the 21st. Chromatography, literally "color writing", was used—and named— in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll. New forms of chromatography developed in the 1930s and 1940s made the technique useful for a wide range of separation processes and chemical analysis tasks, especially in biochemistry.

Some related techniques were developed in the 19th century (and even before), but the first true chromatography is usually attributed to Russian botanist Mikhail Semyonovich Tsvet, who used columns of calcium carbonate for separating plant pigments in the first decade of the 20th century during his research on chlorophyll.

Chromatography began to take its modern form following the work of Archer John Porter Martin and Richard Laurence Millington Synge in the 1940s and 1950s. They laid out the principles and basic techniques of partition chromatography, and their work spurred the rapid development of several lines of chromatography methods: paper chromatography, gas chromatography, and what would become known as high performance liquid chromatography. Since then, the technology has advanced rapidly. Researchers found that the principles underlying Tsvet's chromatography could be applied in many different ways, giving rise to the different varieties of chromatography described below. Simultaneously, advances continually improved the technical performance of chromatography, allowing the separation of increasingly similar molecules

Chromatography terms

• The analyte is the substance that is to be separated during chromatography.
• Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.
• A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
• A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

On the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated

• A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
• Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
• The effluent is the mobile phase leaving the column.
• An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
• The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A better definition: The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In one case of HPLC the solvent consists of a carbonate/bicarbonate solution and the sample is the anions being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.
• Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis.
• The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
• The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.

The solute refers to the sample components in partition.

Techniques by chromatographic bed shape

Column Chromatography

• The solvent refers to any substance capable of solubilizing other substance, and especially the liquid mobile phase in LC.
• The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in Chromatography & Thin layer chromatography

Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.

In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash). The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.

A spreadsheet that assists in the successful development of flash columns has been developed. The spreadsheet estimates the retention volume and band volume of analytes, the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted.

In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.

Planar Chromatography

Thin layer chromatography is used to separate components of chlorophyll

Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase . The specific Retardation factor (R_f) of each chemical can be used to aid in the identification of an unknown substance.

Paper Chromatography.

Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.

Thin layer chromatography

Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantitation, high-performance TLC can be used.

Techniques by physical state of mobile phase

Gas chromatography

Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see below) .

Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.

Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC).

In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.

Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography.

Affinity chromatography

Affinity chromatography^[4] is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labelled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.

Supercritical fluid chromatography

is commonly used to purify proteins using FPLC. Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. It is usually performed in columns but the mechanism can be benefited also in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography

TECHNIQUES BY SEPARATION MECHANISM

Ion exchange chromatography

Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.

PRINCIPLE

Stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase.

Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group:

Anion exchange chromatography retains anions using positively charged functional group:Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position and thus retention time.The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.

Size exclusion chromatography

Size exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to enter the pores of the media and, therefore, take longer to elute, whereas larger molecules are excluded from the pores and elute faster. It is generally a low resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.

SPECIAL TECHNIQUES

Reversed-phase chromatography

Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase. Reversed-phase chromatography (RPC) includes any chromatographic method that uses a non-polar stationary phase. The name "reversed phase" has a historical background. In the 1970s most liquid chromatography was done on non-modified silica or alumina with a hydrophilic surface chemistry and a stronger affinity for polar compounds - hence it was considered "normal". The introduction of alkyl chains bonded covalently to the support surface reversed the elution order. Now polar compounds are eluted first while non-polar compounds are retained - hence "reversed phase". All of the mathematical and experimental considerations used in other chromatographic methods apply (ie separation resolution proportional to the column length). Today, reversed-phase column chromatography accounts for the vast majority of analysis performed in liquid chromatography.

Stationary Phases

* Silica Based Stationary Phases

Any inert non-polar substance that achieves sufficient packing can be used for reversed-phase chromatography. The most popular column is a C18 bonded silica (USP classification L1) with 297 columns commercially available ^[3] This is followed by C8 bonded silica (L7 - 166 columns), pure silica (L3 - 88 columns), cyano bonded silica (L10 - 73 columns) and phenyl bonded silica (L11 - 72 columns). Note that C18, C8 and phenyl are dedicated reversed phase packings while cyano columns can be used in a reversed phase mode depending on analyte and mobile phase conditions. It should be noted at this point that not all C18 columns have identical retention properties. Surface functionalization of silica can be performed in a monomeric or a polymeric reaction with different short-chain organosilanes used in a second step to cover remaining silanol groups (end-capping). While the overall retention mechanism remains the same subtle differences in the surface chemistries of different stationary phases will lead to changes in selectivity.

* Mobile Phase Considerations

Mixtures of water or aqueous buffers and organic solvents are used to elute analytes from a reversed phase column. The solvents have to be miscible with water and the most common organic solvents used are acetonitrile, methanol or tetrahydrofuran (THF). Other solvents can be used such as ethanol, 2-propanol (iso-propyl alcohol). Elution can be performed isocratic (the water-solvent composition does not change during the separation process) or by using a gradient (the water-solvent composition does change during the separation process). The pH of the mobile phase can have an important role on the retention of an analyte and can change the selectivity of certain analytes. Charged analytes can be separated on a reversed phase column by the use of ion-pairing (also called ion-interaction). This technique is known as reversed phase ion-pairing chromatography.

Two-dimensional chromatography

In some cases, the chemistry within a given column can be insufficient to separate some analytes. It is possible to direct a series of unresolved peaks onto a second column with different physico-chemical (Chemical classification) properties. Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds that are indistinguishable by one-dimensional chromatography.

Simulated Moving-Bed Chromatography

In chromatography, the simulated moving bed (SMB) technique is a a variant of high performance liquid chromatography; it is used to separate particles and/or chemical compounds that would be difficult or impossible to resolve otherwise. This increased separation is brought about by a valve-and-column arrangement that is used to lengthen the stationary phase indefinitely.

In the moving bed technique of preparative chromatography the feed entry and the analyte recovery are simultaneous and continuous, but because of practical difficulties with a continuously moving bed in the simulated moving bed technique instead of moving the bed the sample inlet and the analyte exit positions are moved continuously, giving the impression of a moving bed.

True moving bed chromatography (MBC) is only a theoretical concept. Its simulation, SMBC is achieved by the use of a multiplicity of columns in series and a complex valve arrangement, which provides for sample and solvent feed, and also analyte and waste takeoff at appropriate locations of any column, whereby it allows switching at regular intervals the sample entry in one direction, the solvent entry in the opposite direction, whilst changing the analyte and waste takeoff positions appropriately as well.

Ref 3 explains that the advantage of the SMBC is high speed, because a system could be near continuous, whilst its disadvantage is that it only separates binary mixtures. It does not say, but perhaps it can be assumed that this is equivalent with the separation of a single component from a group of compounds. With regard to efficiency it compares with simple chromatography technique like continuous distillation does with batch distillation.

Advantages

When affinity differences between molecules are very small, it is sometimes not possible to improve resolution via mobile- or stationary-phase changes. In these cases, the multi-pass approach of SMB can separate mixtures of those compounds by allowing their small retention time differences to accumulate.

At industrial scale an SMB chromatographic separator is operated continuously, requiring less resin and less solvent than batch chromatography. The continuous operation facilitates operation control and integration into production plants.

Drawbacks

The drawbacks of the SMB are higher investment cost compared to single column operations, a higher complexity, as well as higher maintenance costs. But these drawbacks are effectively compensated by the better yield and a much lower solvent consumption as well as a much higher productivity compared to simple batch separations.

For purifications, in particular the isolation of an intermediate single component or a fraction out of a multicomponent mixture, the SMB is not suited in general. It can only separate two fractions from each other and it does not implement linear solvent gradients as required for the purification of biomolecules.

Applications

In size exclusion chromatography, where the separation process is driven by entropy, it is not possible to increase the resolution attained by a column via temperature or solvent gradients. Consequently, these separations often require SMB, to create usable retention time differences between the molecules or particles being resolved. SMB is also very useful in the pharmaceutical industry, where resolution of molecules having different chirality must be done on a very large scale.

For the production of Fructose e.g. in High fructose corn syrup or amino-acids, biological-acids, etc. industrial scale chromatography is used.

Fast protein liquid chromatography

Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques which are used to purify proteins. Many of these techniques are identical to those carried out under high performance liquid chromatography, however use of FPLC techniques are typically for preparing large scale batches of a purified product.Basically Fast Protein Liquid Chromatography, usually referred to as FPLC, is a form of column chromatography used to separate or purify proteins from complex mixtures. It is very commonly used in biochemistry and enzymology. Columns used with an FPLC can separate macromolecules based on size, charge distribution (ion exchange), hydrophobicity, or biorecognition (as with affinity chromatography).

The system setup is very similar to that of an HPLC, although the materials, buffers, and pressures used are usually different.

Technically, FPLC is the trade name for the protein chromatography system developed by Pharmacia (now GE Healthcare), and is now sold under the ÄKTA brand. However, it is often used as a genericized trademark to describe high-pressure chromatography purification of proteins.

Countercurrent chromatography

Countercurrent chromatography (CCC) or partition chromatography is a category of liquid-liquid chromatography techniques.^[1] Chromatography in general is used to separate components of a mixture based on their differing affinities for mobile and stationary phases of a column. The components can then be analyzed separately by various sorts of detectors which may or may not be integrated into an apparatus. Partition chromatography is based on differences in capacity factor,k, and distribution coefficient,Kd.of the analytes using liquid stationary and mobile liquid phase.In liquid-liquid chromatography, both the mobile and stationary phases are liquid. In contrast, standard column chromatography uses a solid stationary phase and a liquid mobile phase, while gas chromatography uses a liquid stationary phase on a solid support and a gaseous mobile phase. By eliminating solid supports, permanent adsorption of the analyte onto the column is avoided, and a near 100% recovery of the analyte can be achieved. The instrument is also easily switched between various modes of operation simply by changing solvents. With liquid-liquid chromatography, researchers are not limited by the composition of the columns commercially available for their instrument. Nearly any pair of immiscible solutions can be used in liquid-liquid chromatography, and most instruments can be operated in standard or reverse-phase modes. Solvent costs are also generally cheaper than for HPLC, and the cost of purchasing and disposing of solid adsorbents is completely eliminated. Another advantage is that experiments conducted in the lab can easily be scaled to industrial volumes interface between them has a large area, and the analyte can move between the phases according to its partition coefficient. A partition coefficient is a ratio of the amount of analyte found in each of the solvents at equilibrium and is related to the analyte's affinity for one over the other. The mobile phase is mixing with then settling from the stationary phase throughout the column. The degree of stationary phase retention (inversely proportional to the amount of stationary phase loss or "bleed" in the course of a separation) is a crucial parameter. Higher quality instruments have greater stationary phase retention. The settling time is a property of the solvent system and the sample matrix, both of which. When GC or HPLC is done with large volumes, resolution is lost due to issues with surface-to-volume ratios and flow dynamics; this is avoided when both phases are liquid.

CCC can be thought of as occurring in three stages: mixing, settling, and separation (although they often occur continuously). Mixing of the phases is necessary so that the greatly influence stationary phase retention

Chiral chromatography

Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being three dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes. Chiral chromatography HPLC columns (with a chiral stationary phase) in both normal and reversed phase are commercially available.

CONCLUSIONS

In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Today, there exists a wide market of methods in which industries can accomplish these goals. Chromatography is a very special separation process for a multitude of reasons! First of all, it can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography. In fact, chromatography can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed. Second, chromatography can be used to separate delicate products since the conditions under which it is performed are not typically severe. For these reasons, chromatography is quite well suited to a variety of uses in the field of biotechnology, such as separating mixtures of proteins

REFERENCES

1. http://en.wikipedia.org/wiki/Chromatography

2. http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chromintro.html

3.http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm