Sameer - Thesis

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1 History 2 Chromatography terms 3 Techniques by chromatographic bed shape o 3.1 Column chromatography o 3.2 Planar chromatography  3.2.1 Paper chromatography  3.2.2 Thin layer chromatography 4 Displacement Chromatography 5 Techniques by physical state of mobile phase o 5.1 Gas chromatography o 5.2 Liquid chromatography 6 Affinity chromatography o 6.1 Supercritical fluid chromatography 7 Techniques by separation mechanism o 7.1 Ion exchange chromatography o 7.2 Size exclusion chromatography 8 Special techniques o 8.1 Reversed-phase chromatography o 8.2 Two-dimensional chromatography o 8.3 Simulated moving-bed chromatography o 8.4 Pyrolysis gas chromatography o 8.5 Fast protein liquid chromatography o 8.6 Countercurrent chromatography o 8.7 Chiral chromatography

Chromatography (from Greek χρώμα:chroma, color and γραφειν:graphein to write) is the collective term for a set 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 based on differential partitioning between the mobile and stationary phases. Subtle differences in compounds partition coefficient results in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.

History The history of chromatography begins during the mid-19th century. 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 types of chromatography developed during the 1930s and 1940s made the technique useful for many types of separation process. Some related techniques were developed during 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 during the first decade of the 20th century during his research of chlorophyll. Chromatography became developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several types of chromatography method: 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 main principles of Tsvet's chromatography could be applied in many different ways, resulting in 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

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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.

Plotted 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.

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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 the case of HPLC the mobile phase consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in reverse phase chromotagraphy and the sample 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. See The sample is the matter analyzed 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 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 Submitted by James Nguyen

Techniques by chromatographic bed shape Column 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. 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 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 (Rf) 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 or line 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 quantification, high-performance TLC can be used.

Displacement Chromatography The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities. There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.

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 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 labeled 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 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.

Techniques by separation mechanism Ion exchange chromatography Ion exchange chromatography uses ion exchange mechanism to separate analytes. It is usually performed in columns but can also be useful 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 is commonly used to purify proteins using FPLC.

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.

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 Pyrolysis gas chromatography 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.

Countercurrent chromatography Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing them to settle into layers and then separating the layers.

Chiral chromatography Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being threedimensional 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.

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1 Technique of Paper Chromatography o 1.1 Ascending chromatography o 1.2 Descending chromatography 2 Chromatography as art 3 Rƒ value Applications

Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be coloured, especially pigments. This can also be used in secondary or primary colours in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Twoway paper chromatography, also called two-dimensional chromatography , involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.

Technique of Paper Chromatography A small concentrated spot of solution that contains the sample of the solute is applied to a strip of chromatography paper about two centimetres away from the base of the plate, usually using a capillary tube for maximum precision. This sample is absorbed onto the paper and may form interactions with it. Any substance that reacts or bonds with the paper cannot be measured using this technique. The paper is then dipped into a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper; this can also be explained as differential adsorption of the solute components into the solvent. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibres in the paper. The more soluble the component the further it goes. Paper chromatography takes anywhere from several minutes to several hours.In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent. In these cases, twoway chromatography is used to separate the multiple-pigment spots.

Ascending chromatography In this method, the solvent is in pool at the bottom of the vessel in which the paper is supported.

Descending chromatography In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the gravitational force, thus this method is also known as the gravitational method.In this case, the flow is more rapid as compared to the ascending method, and the chromatography is completed more quickly. The apparatus needed for this case is more

sophisticated. The developing solvent is placed in a trough at the top which is usually made up of an inert material. The paper is then suspended in the solvent. Substances that cannot be separated by ascending method can sometimes be separated by the descending method.

Chromatography as art Chromatography can be used as an art alternative because of certain colours that it creates, but paper chromatography rarely produces decipherable colours if executed incorrectly. Sometimes, chromatographic results may start with undesirable "leafy" colours, but if done correctly and completely, magnificent hues can be produced.

Paper chromatography

Chromatography jar

Rƒ value Rƒ value may be defined as the ratio of the distance travelled by the substance to the distance travelled by the solvent. Rƒ values are usually expressed as a fraction of two decimal places but it was suggested by Smith that a percentage figure should be used instead. If Rƒ value of a solution is zero, the solute remains in the stationary phase and

thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front

Applications:

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1 Plate preparation 2 Technique 3 Preparative TLC 4 Analysis 5 Applications

Thin layer chromatography

Separation of black ink on a TLC plate

Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. Thin layer chromatography finds many applications, including: • • • • • •

assaying the radiochemical purity of radiopharmaceuticals determination of the pigments a plant contains detection of pesticides or insecticides in food analyzing the dye composition of fibers in forensics, or identifying compounds present in a given substance monitoring organic reactions.

A number of enhancements can be made to the original method to automate some steps, to increase the resolution achieved with TLC and to allow more accurate quantitation. This method is referred to as HPTLC, or "high performance TLC".

Plate preparation TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC.[3]

Technique Development of a TLC plate, a purple spot separates into a red and blue spot.

Chromatogram of 10 essential oils coloured with vanillin reagent. The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.A small spot of solution containing the sample is applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a sealed container. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be

adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column.[4] Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the most polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. Practically this means that if you use a mixture of ethyl acetate and heptane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will not result in reversed order of running of the compounds on the TLC plate. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18-functionalized silica.

Preparative TLC TLC can also be used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. The mixture is not “spotted” on the TLC plate as dots, but rather is applied to the plate as a thin even layer horizontally to and just above the solvent level. When developed with solvent the compounds separate in horizontal bands rather than horizontally separated spots. Each band (or a desired band) is scraped off the backing material. The backing material is then extracted with a suitable solvent (e.g. DCM) and filtered to give the isolated material upon removal of the solvent. For small-scale reactions with easily separated products, preparative TLC can be a far more efficient in terms of time and cost than doing column chromatography. Obviously, the whole plate can not be chemically developed or the product will be chemically destroyed. Thus this technique is best used with compounds that are coloured, or visible under UV light. Alternatively, a small section of the plate can be chemically developed e.g. cutting a section out and chemically developing it, or masking most of the plate and exposing a small section to a chemical developer like iodine.

Analysis As the chemicals being separated may be colorless, several methods exist to visualize the spots: •

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Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence. Iodine vapors are a general unspecific color reagent Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate[5] In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for example mass spectrometry, a technique known as Far-Eastern blotting.

Once visible, the Rf value , or Retention factor, of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants. Eluent on the thin layer is put on top of the plate

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Applications In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a "co-spot" with both. A small (3 by 7 cm) TLC plate takes a couple of minutes to run. The analysis is qualitative, and it will show if the starting

material has disappeared, i.e. the reaction is complete, if any product has appeared, and how many products are generated (although this might be under-estimated due to coelution). Unfortunately, TLCs from low-temperature reactions may give misleading results, because the sample is warmed to room temperature in the capillary, which can alter the reaction—the warmed sample analyzed by TLC is not the same as what is in the low-temperature flask. One such reaction is the DIBALH reduction of ester to aldehyde. As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow. In one study TLC has been applied in the screening of organic reactions[6] for example in the fine-tuning of BINAP synthesis from 2-naphthol. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are place separately on the base line, then reacted and then instantly analyzed.

History of HPLC Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to

decrease flowthrough time, thus reducing purification times of compounds being isolated by column chromatogaphy. However, flow rates were inconsistant, and the question of whether it was better to have constant flow rate or constant pressure was debated. (Analytical Chem. vol 62, no. 19, oct 1 1990). High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds. By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. Improvements in type of columns and thus reproducibility were made as such terms as micro-column, affinity columns, and Fast HPLC began to immerge. The past decade has seen a vast undertaking in the development of the micro-columns, and other specialized columns. The dimensions of the typical HPLC column are: XXX mm in length with an internal diameter between 3-5 mm. The usual diameter of microcolumns, or capillary columns, ranges from 3 µm to 200 µm. Fast HPLC utilizes a column that is shorter than the typical column, with a length of about 3 mm long, and they are packed with smaller particles. Currently, one has the option of considering over x# types of columns for the separation of compounds, as well as a variety of detectors to interface with the HPLC in order to get optimal analysis of the compound. We hope this review will provide a reference which all levels of HPLC users will be able to find quick answers to their HPLC problems. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry, these fields corrently comprise only about 50% of HPLC users.(Analytical Chem. vol 62, no. 19, oct 1 1990). Currently HPLC is used by a variety of fields including cosmetics, energy, food, and environmental industries.Return to table of contents

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1 Operation 2 Types of HPLC o 2.1 Partition chromatography o 2.2 Normal phase chromatography o 2.3 Displacement Chromatography o 2.4 Reverse Phase Chromatography (RPC) o 2.5 Size exclusion chromatography o 2.6 Ion exchange chromatography o 2.7 Bioaffinity chromatography o 2.8 Aqueous Normal Phase Chromatography 3 Isocratic flow and gradient elution 4 Parameters o 4.1 Internal diameter o 4.2 Particle size o 4.3 Pore size o 4.4 Pump pressure 5 Applications:

High performance liquid chromatography High-performance liquid chromatography (or high pressure liquid chromatography, HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used.

In this method, high pressure (rather than gravity forces) can propel mobile phase through the column packed with a stationary phase of much smaller particle sizes. This allows for a quicker and more precise analysis on columns of shorter length, when compared to plain column chromatography.The sample to be analyzed is introduced in small volume to the stream of mobile phase. The analyte's motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent. A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as gradient elution. A normal gradient for reversed phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes; the gradient chosen depends on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not stepwise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) when the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase). The more hydrophilic compounds will elute under conditions of relatively low methanol/high water. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of trial runs may be processed in order to find the HPLC method which gives the best separation of peaks.

Operations:

New HPLC Accessories

Piston Seals Preventive Maint. Kit for Agilent 1050 Autosampler,

Needle Seat Assembly

Outlet Ball Valve,

Outlet Cap & Gold Seal,.

Connecting Tube

HPLC Chromatogram of an Ethanol Extract of Kava Hawaii Kavalactone Distribution (%) Methysticin

17.14

Dihydromethysticin

13.39

Kavain

22.15

Dihydrokavain

29.48

Yangonin

10.29

Dehydrokavain

7.54

Types of HPLC Partition chromatography

A modern self contained HPLC. In this picture; an Agilent 1200 connected to a PC Partition chromatography was the first kind of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid-liquid applications. The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids. Partition chromatography uses a retained solvent, on the surface or within the grains or fibres of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some additional coulombic and/or hydrogen donor interaction with the solid support. Molecules equilibrate (partition) between a liquid stationary phase and the eluent. Known as Hydrophilic Interaction Chromatography (HILIC) in HPLC, this method separates analytes based on polar differences. HILIC most often uses a bonded polar stationary phase and a non-polar, water miscible, mobile phase. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences, however, HILIC has the advantage of separating acidic, basic and neutral solutes in a single chromatogram. The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. Retention strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength depends on the functional groups in the analyte molecule which promote partitioning but can also include coulombic (electrostatic) interaction and hydrogen donor capability. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times.

Partition and NP-HPLC had fallen out of favor in the 1970s with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HILIC bonded phases which improve reproducibility.

Normal phase chromatography Also known as Normal phase HPLC (NP-HPLC), or adsorption chromatography, this method separates analytes based on adsorption to a stationary surface chemistry and by polarity. It was one of the first kinds of HPLC that chemists developed. NP-HPLC uses a polar stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength depends not only on the functional groups in the analyte molecule, but also on steric factors. The effect of sterics on interaction strength allows this method to resolve (separate) structural isomers. The use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times. Very polar solvents in a mixture tend to deactivate the stationary phase by creating a stationary bound water layer on the stationary phase surface. This behavior is somewhat peculiar to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather than a soft layer on a surface). NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HILIC bonded phases which improve reproducibility.

Displacement Chromatography The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.[1] There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with

gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.

Reverse Phase Chromatography (RPC)

A chromatogram of complex mixture (perfume water) obtained by reversed phase HPLC Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With these stationary phases, retention time is longer for molecules which are more non-polar, while polar molecules elute more readily. An investigator can increase retention time by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RPC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RPC to qualify drugs before their release. RPC operates on the principle of hydrophobic forces, which originate from the high symmetry in the dipolar water structure and play the most important role in all processes in life science. RPC is allowing the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand in the aqueous eluent. This solvophobic effect is dominated by the force of water for "cavityreduction" around the analyte and the C18-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 7.3 × 10-6 J/cm², methanol: 2.2 × 10-6 J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent

(methanol, acetonitrile) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis. Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) results in a longer retention time because it increases the molecule's non-polar surface area, which is noninteracting with the water structure. On the other hand, polar groups, such as -OH, -NH2, COO- or -NH3+ reduce retention as they are well integrated into water. Very large molecules, however, can result in an incomplete interaction between the large analyte surface and the ligand's alkyl chains and can have problems entering the pores of the stationary phase. Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond. Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5 × 10-7 J/cm² per Mol for NaCl, 2.5 × 10-7 J/cm² per Mol for (NH4)2SO4), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC). Another important component is the influence of the pH since this can change the hydrophobicity of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of ammonium adducts. A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is one of the strongest organic acids. The effects of acids and buffers vary by application but generally improve the chromatography. Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the

underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine. Because the 2,2'-bipy can chelate the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) when metal ions are present on the surface of the silica..

Size exclusion chromatography Size exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of size. It is generally a low resolution chromatography 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. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time. This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight heparins.

Ion exchange chromatography In ion-exchange chromatography, retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded. Types of ion exchangers include: •

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Polystyrene resins – These allow cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity. Cellulose and dextran ion exchangers (gels) – These possess larger pore sizes and low charge densities making them suitable for protein separation. Controlled-pore glass or porous silica

In general, ion exchangers favor the binding of ions of higher charge and smaller radius. An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. An increase in pH reduces the retention time in cation exchange while a decrease in pH reduces the retention time in anion exchange. This form of chromatography is widely used in the following applications: water purification, preconcentration of trace components, ligand-exchange chromatography, ion-exchange chromatography of proteins, high-pH anion-exchange chromatography of carbohydrates and oligosaccharides, and others.

Bioaffinity chromatography This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of these complexes involves the participation of common molecular forces such as the Van der Waals interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.

Aqueous Normal Phase Chromatography Aqueous normal phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reverse-phase solvents

Isocratic flow and gradient elution A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (meaning constant composition). The word was coined by Csaba Horvath from Yale University[citation needed], who was one of the pioneers of HPLC. The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution.[2] One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column. Solvent A is often water, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol. In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that lateeluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks. Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times - all according to the desire for optimum separation in minimum time. In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change - that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.[citation needed] The driving force is originated in reversed phase chromatography in the high order of the water structure. The role of the organic mobile phase is to reduce this high order by reducing the retarding strength of the aqueous component.

Parameters Internal diameter The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity. • •

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Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity. Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector. Narrow-bore columns (1-2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, fluorescence detection or with other detection methods like liquid chromatography-mass spectrometry Capillary columns (under 0.3 mm) are used almost exclusively with alternative detection means such as mass spectrometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ.

Particle size Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 μm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared. This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (column diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase extraction.

Pore size Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.

Pump pressure Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure may reach as high as 40 MPa (6000 lbf/in2), or about 400 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography" systems or RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in²), or about 1000 atmospheres. The term "UPLC", though sometimes used is a trademark of Waters Corporation and not the name for the technique in general.

Applications:

References 1. ^ IUPAC Nomenclature for Chromatography IUPAC Recommendations 1993, Pure & 2. 3. 4. 5.

Appl. Chem., Vol. 65, No. 4, pp.819-872, 1993. ^ Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43(14), 2923-2925. doi:10.1021/jo00408a041 ^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles and Practice (Illustrated edition ed.). pp. 180-185. ^ Displacement Chromatography 101. Sachem, Inc. Austin, TX 78737 ^ Pascal Bailon, George K. Ehrlich, Wen-Jian Fung and Wolfgang Berthold, An Overview of Affinity Chromatography, Humana Press, 2000. ISBN 978-0-89603-694-9, ISBN 978-1-60327-261-2

6. Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles and Practice (Illustrated edition ed.). pp. 159-173. 7. Vogel's Textbook of Practical Organic Chemistry (5th Edition) (Hardcover) by A.I. Vogel (Author), A.R. Tatchell (Author), B.S. Furnis (Author), A.J. Hannaford (Author), P.W.G. Smith ISBN 0582462363 8 Tables showing the thickness value of commercial regular and preparative Thin Layer Chromatography plates 9. Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54. (doi:10.1016/j.chroma.2008.09.085) 10.Stains for Developing TLC Plates http://www.ux1.eiu.edu/~cfthb/research/handbook/TLCstains.htm 11. Displacement Chromatography 101. [1] Sachem, Inc. Austin, TX 78737 12. A recent book provides a comprehensive treatment of the theory of high-performance gradient chromatography: Lloyd R. Snyder and John W. Dolan (2006). High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model. Wiley Interscience. ISBN 0471706469.. 13. Fast and Ultrafast HPLC on sub-2 μm Porous Particles — Where Do We Go From Here? - LC-GC Europe 14. Xiang, Y.; Liu Y. and Lee M.L. (2006). "Ultrahigh pressure liquid chromatography using elevated temperature". Journal of Chromatography A 1104 (1-2): 198–202. doi:10.1016/j.chroma.2005.11.118. 15. Horváth, Cs.; Preiss B.A. and Lipsky S.R. (1967). "Fast liquid chromatography. Investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers". Analytical Chemistry 39: 1422–1428. doi:10.1021/ac60256a003.