A chemist in the 1950s using column chromatography. The Erlenmeyer receptacles are on the floor.
Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents (normal phase, reversed phase, or otherwise) can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.
A thin-layer chromatograph can show how a mixture of compounds will behave when purified by column chromatography. The separation is first optimised using thin-layer chromatography before performing column chromatography.
Column preparation
A column is prepared by packing a solid adsorbent into a cylindrical glass or plastic tube. The size will depend on the amount of compound being isolated. The base of the tube contains a filter, either a cotton or glass wool plug, or glass frit to hold the solid phase in place. A solvent reservoir may be attached at the top of the column.
Two methods are generally used to prepare a column: the dry method and the wet method. For the dry method, the column is first filled with dry stationary phase powder, followed by the addition of mobile phase, which is flushed through the column until it is completely wet, and from this point is never allowed to run dry.[1] For the wet method, a slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. The top of the silica should be flat, and the top of the silica can be protected by a layer of sand. Eluent is slowly passed through the column to advance the organic material.
The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. At the end of the column they elute one at a time. During the entire chromatography process the eluent is collected in a series of fractions. Fractions can be collected automatically by means of fraction collectors. The productivity of chromatography can be increased by running several columns at a time. In this case multi stream collectors are used. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption spectra, or fluorescence. Colored compounds (or fluorescent compounds with the aid of a UV lamp) can be seen through the glass wall as moving bands.
Stationary phase
Automated fraction collector and sampler for chromatography techniques
The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is silica gel, the next most common being alumina. Cellulose powder has often been used in the past. A wide range of stationary phases are available in order to perform ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in EBA a fluidized bed is used. There is an important ratio between the stationary phase weight and the dry weight of the analyte mixture that can be applied onto the column. For silica column chromatography, this ratio lies within 20:1 to 100:1, depending on how close to each other the analyte components are being eluted.[2]
Mobile phase (eluent)
Column chromatography proceeds by a series of steps.
The mobile phase or eluent is a solvent or a mixture of solvents used to move the compounds through the column. It is chosen so that the retention factor value of the compound of interest is roughly around 0.2 - 0.3 in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase, using solvents of different polarity until a suitable solvent system is found. Common mobile phase solvents, in order of increasing polarity, include hexane, dichloromethane, ethyl acetate, acetone, and methanol.[3] A common solvent system is a mixture of hexane and ethyl acetate, with proportions adjusted until the target compound has a retention factor of 0.2 - 0.3. Contrary to common misconception, methanol alone can be used as an eluent for highly polar compounds, and does not dissolve silica gel.
There is an optimum flow rate for each particular separation. A faster flow rate of the eluent minimizes the time required to run a column and thereby minimizes diffusion, resulting in a better separation. However, the maximum flow rate is limited because a finite time is required for the analyte to equilibrate between the stationary phase and mobile phase, see Van Deemter's equation. A simple laboratory column runs by gravity flow. The flow rate of such a column can be increased by extending the fresh eluent filled column above the top of the stationary phase or decreased by the tap controls. Faster flow rates can be achieved by using a pump or by using compressed gas (e.g. air, nitrogen, or argon) to push the solvent through the column (flash column chromatography).[4][5]
Photographic sequence of a column chromatography
The particle size of the stationary phase is generally finer in flash column chromatography than in gravity column chromatography. For example, one of the most widely used silica gel grades in the former technique is mesh 230 – 400 (40 – 63μm), while the latter technique typically requires mesh 70 – 230 (63 – 200μm) silica gel.[6]
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.[7]
Automated systems
An automated ion chromatography system.
Column chromatography is an extremely time-consuming stage in any lab and can quickly become the bottleneck for any process lab. Many manufacturers like Biotage, Buchi, Interchim and Teledyne Isco have developed automated flash chromatography systems (typically referred to as LPLC, low pressure liquid chromatography, around 350–525kPa or 50.8–76.1psi) that minimize human involvement in the purification process. Automated systems will include components normally found on more expensive high performance liquid chromatography (HPLC) systems such as a gradient pump, sample injection ports, a UV detector and a fraction collector to collect the eluent. Typically these automated systems can separate samples from a few milligrams up to an industrial many kilogram scale and offer a much cheaper and quicker solution to doing multiple injections on prep-HPLC systems.
The resolution (or the ability to separate a mixture) on an LPLC system will always be lower compared to HPLC, as the packing material in an HPLC column can be much smaller, typically only 5 micrometre thus increasing stationary phase surface area, increasing surface interactions and giving better separation. However, the use of this small packing media causes the high back pressure and is why it is termed high pressure liquid chromatography. The LPLC columns are typically packed with silica of around 50 micrometres, thus reducing back pressure and resolution, but it also removes the need for expensive high pressure pumps. Manufacturers are now starting to move into higher pressure flash chromatography systems and have termed these as medium pressure liquid chromatography (MPLC) systems which operate above 1MPa (150psi).
Typically, column chromatography is set up with peristaltic pumps, flowing buffers and the solution sample through the top of the column. The solutions and buffers pass through the column where a fraction collector at the end of the column setup collects the eluted samples. Prior to the fraction collection, the samples that are eluted from the column pass through a detector such as a spectrophotometer or mass spectrometer so that the concentration of the separated samples in the sample solution mixture can be determined.
For example, if you were to separate two different proteins with different binding capacities to the column from a solution sample, a good type of detector would be a spectrophotometer using a wavelength of 280nm. The higher the concentration of protein that passes through the eluted solution through the column, the higher the absorbance of that wavelength.
Because the column chromatography has a constant flow of eluted solution passing through the detector at varying concentrations, the detector must plot the concentration of the eluted sample over a course of time. This plot of sample concentration versus time is called a chromatogram.
The ultimate goal of chromatography is to separate different components from a solution mixture. The resolution expresses the extent of separation between the components from the mixture. The higher the resolution of the chromatogram, the better the extent of separation of the samples the column gives. This data is a good way of determining the column's separation properties of that particular sample. The resolution can be calculated from the chromatogram.
The separate curves in the diagram represent different sample elution concentration profiles over time based on their affinity to the column resin. To calculate resolution, the retention time and curve width are required.
Retention time is the time from the start of signal detection by the detector to the peak height of the elution concentration profile of each different sample.
Curve width is the width of the concentration profile curve of the different samples in the chromatogram in units of time.
A simplified method of calculating chromatogram resolution is to use the plate model.[8] The plate model assumes that the column can be divided into a certain number of sections, or plates and the mass balance can be calculated for each individual plate. This approach approximates a typical chromatogram curve as a Gaussian distribution curve. By doing this, the curve width is estimated as 4 times the standard deviation of the curve, 4σ. The retention time is the time from the start of signal detection to the time of the peak height of the Gaussian curve.
From the variables in the figure above, the resolution, plate number, and plate height of the column plate model can be calculated using the equations:
For an adsorption column, the column resin (the stationary phase) is composed of microbeads. Even smaller particles such as proteins, carbohydrates, metal ions, or other chemical compounds are conjugated onto the microbeads. Each binding particle that is attached to the microbead can be assumed to bind in a 1:1 ratio with the solute sample sent through the column that needs to be purified or separated.
Binding between the target molecule to be separated and the binding molecule on the column beads can be modeled using a simple equilibrium reaction Keq = [CS]/([C][S]) where Keq is the equilibrium constant, [C] and [S] are the concentrations of the target molecule and the binding molecule on the column resin, respectively. [CS] is the concentration of the complex of the target molecule bound to the column resin.[8]
Using this as a basis, three different isotherms can be used to describe the binding dynamics of a column chromatography: linear, Langmuir, and Freundlich.
The linear isotherm occurs when the solute concentration needed to be purified is very small relative to the binding molecule. Thus, the equilibrium can be defined as:
[CS] = Keq[C].
For industrial scale uses, the total binding molecules on the column resin beads must be factored in because unoccupied sites must be taken into account. The Langmuir isotherm and Freundlich isotherm are useful in describing this equilibrium. The Langmuir isotherm is given by:
[CS] = (KeqStot[C])/(1 + Keq[C]), where Stot is the total binding molecules on the beads.
The Freundlich isotherm is given by:
[CS] = Keq[C]1/n
The Freundlich isotherm is used when the column can bind to many different samples in the solution that needs to be purified. Because the many different samples have different binding constants to the beads, there are many different Keqs. Therefore, the Langmuir isotherm is not a good model for binding in this case.[8]
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
Size-exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc., which have been dissolved into liquid solutions.
Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates high molecular weight or colloidal analytes on the basis of size or diameter, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964. The proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.
Paper chromatography is an analytical method used to separate coloured chemicals or substances. It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).
Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.
Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures.
Solid-phase extraction (SPE) is a solid-liquid extractive technique, by which compounds that are dissolved or suspended in a liquid mixture are separated, isolated or purified, from other compounds in this mixture, according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue.
Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.
In manufacturing, the simulated moving bed (SMB) process is a highly engineered process for implementing chromatographic separation. It is used to separate one chemical compound or one class of chemical compounds from one or more other chemical compounds to provide significant quantities of the purified or enriched material at a lower cost than could be obtained using simple (batch) chromatography. It cannot provide any separation or purification that cannot be done by a simple column purification. The process is rather complicated. The single advantage which it brings to a chromatographic purification is that it allows the production of large quantities of highly purified material at a dramatically reduced cost. The cost reductions come about as a result of: the use of a smaller amount of chromatographic separation media stationary phase, a continuous and high rate of production, and decreased solvent and energy requirements. This improved economic performance is brought about by a valve-and-column arrangement that is used to lengthen the stationary phase indefinitely and allow very high solute loadings to the process.
Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.
Micellar liquid chromatography (MLC) is a form of reversed phase liquid chromatography that uses an aqueous micellar solutions as the mobile phase.
Aqueous normal-phase chromatography (ANP) is a chromatographic technique that involves the mobile phase compositions and polarities between reversed-phase chromatography (RP) and normal-phase chromatography (NP), while the stationary phases are polar.
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent: washing of loaded ion-exchange resins to remove captured ions, or eluting proteins or other biopolymers from a gel electrophoresis or chromatography column.
Displacement chromatography is a chromatography technique in which a sample is placed onto the head of the column and is then displaced by a solute that is more strongly sorbed than the components of the original mixture. The result is that the components are resolved into consecutive "rectangular" zones of highly concentrated pure substances rather than solvent-separated "peaks". It is primarily a preparative technique; higher product concentration, higher purity, and increased throughput may be obtained compared to other modes of chromatography.
Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents.
Radial chromatography is a form of chromatography, a preparatory technique for separating chemical mixtures. It can also be referred to as centrifugal thin-layer chromatography. It is a common technique for isolating compounds and can be compared to column chromatography as a similar process. A common device used for this technique is a Chromatotron.
Thermoresponsive polymers can be used as stationary phase in liquid chromatography. Here, the polarity of the stationary phase can be varied by temperature changes, altering the power of separation without changing the column or solvent composition. Thermally related benefits of gas chromatography can now be applied to classes of compounds that are restricted to liquid chromatography due to their thermolability. In place of solvent gradient elution, thermoresponsive polymers allow the use of temperature gradients under purely aqueous isocratic conditions. The versatility of the system is controlled not only through changing temperature, but through the addition of modifying moieties that allow for a choice of enhanced hydrophobic interaction, or by introducing the prospect of electrostatic interaction. These developments have already introduced major improvements to the fields of hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, and affinity chromatography separations as well as pseudo-solid phase extractions.
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