Column chromatography
Overview
 
Column chromatography in chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....

 is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications 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.

The classical preparative chromatography column, is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug – to prevent the loss of the stationary phase) at the bottom.
Encyclopedia
Column chromatography in chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....

 is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications 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.

The classical preparative chromatography column, is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug – to prevent the loss of the stationary phase) at the bottom. 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.
  • For the wet method, a slurry
    Slurry
    A slurry is, in general, a thick suspension of solids in a liquid.-Examples of slurries:Examples of slurries include:* Lahars* A mixture of water and cement to form concrete* A mixture of water, gelling agent, and oxidizers used as an explosive...

     is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the organic material is pipetted on top of the stationary phase. This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column.


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 fraction
Fractionation
See also: Fractionated spacecraftFractionation is a separation process in which a certain quantity of a mixture is divided up in a number of smaller quantities in which the composition changes according to a gradient. Fractions are collected based on differences in a specific property of the...

s. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

 absorption, or fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

. Colored compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the glass wall as moving bands.



Stationary phase

The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is silica gel
Silica gel
Silica gel is a granular, vitreous, porous form of silica made synthetically from sodium silicate. Despite its name, silica gel is a solid. It is a naturally occurring mineral that is purified and processed into either granular or beaded form...

, followed by alumina
Aluminium oxide
Aluminium oxide is an amphoteric oxide with the chemical formula 23. It is commonly referred to as alumina, or corundum in its crystalline form, as well as many other names, reflecting its widespread occurrence in nature and industry...

. Cellulose
Cellulose
Cellulose is an organic compound with the formula , a polysaccharide consisting of a linear chain of several hundred to over ten thousand β linked D-glucose units....

 powder has often been used in the past. Also possible are ion exchange chromatography
Ion exchange chromatography
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. 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...

, reversed-phase chromatography
Reversed-phase chromatography
Reversed-phase chromatography 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...

 (RP), affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...

 or expanded bed adsorption
Expanded bed adsorption
Expanded bed adsorption is a preparative chromatographic technique which makes processing of viscous and particulate liquids possible.-Principle:...

 (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.

Mobile phase (eluent)

The mobile phase or eluent is either a pure solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...

 or a mixture of different solvents. 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
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....

 (TLC) with the same stationary phase.

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 analyte to equilibrate between stationary phase and mobile phase, see Van Deemter's equation
Van Deemter's equation
The Van Deemter equation in chromatography relates the variance per unit length of a separation column to the linear mobile phase velocity by considering physical, kinetic, and thermodynamic properties of a separation. These properties include pathways within the column, diffusion , and mass...

. 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
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...

, or argon
Argon
Argon is a chemical element represented by the symbol Ar. Argon has atomic number 18 and is the third element in group 18 of the periodic table . Argon is the third most common gas in the Earth's atmosphere, at 0.93%, making it more common than carbon dioxide...

) to push the solvent through the column (flash 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 – 63 µm), while the latter technique typically requires mesh 70 – 230 (63 – 200 µm) silica gel.

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.

Automated Systems

Column chromatography is an extremely time consuming stage in any lab and can quickly become the bottleneck for any process lab. Therefore, several manufacturers have developed automated flash chromatography systems (typically referred to as LPLC, low pressure liquid chromatography, around 50-75 psi) that minimize human involvement in the purification process. Automated systems will include components normally found on more expensive 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 kg 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 150 psi.

The software controlling an automated system will coordinate the components, allow a user to only collect the fractions that contain their target compound (assuming they are detectable on the system's detector) and help the user to find the resulting purified material within the fraction collector. The software will also save the resulting chromatograph from the process for archival and/or later recall purposes.

A representative example of column chromatography as part of an undergraduate laboratory exercise is the separation of three components (out of 28) in the oil of spearmint
Spearmint
Mentha spicata syn. M. cordifolia is a species of mint native to much of Europe and southwest Asia, though its exact natural range is uncertain due to extensive early cultivation. It grows in wet soils...

: carvone
Carvone
Carvone is a member of a family of chemicals called terpenoids. Carvone is found naturally in many essential oils, but is most abundant in the oils from seeds of caraway and dill.-Stereoisomerism and odor:...

, limonene
Limonene
Limonene is a colourless liquid hydrocarbon classified as a cyclic terpene. The more common D isomer possesses a strong smell of oranges. It is used in chemical synthesis as a precursor to carvone and as a renewably-based solvent in cleaning products....

 and dehydrocarveol. A microscale
Microscale chemistry
Microscale Chemistry is a teaching method widely used at school and at university levels, working with small quantities of chemical substances...

 setup consisting of a Pasteur pipette
Pasteur pipette
Pasteur pipettes, also known as droppers or eye droppers, are used to transfer small quantities of liquids. They are usually glass tubes tapered to a narrow point, and fitted with a rubber bulb at the top. The combination of the Pasteur pipette and rubber bulb has also been referred to as a teat...

 as column with silica gel stationary phase can suffice. The starting eluent is hexane
Hexane
Hexane is a hydrocarbon with the chemical formula C6H14; that is, an alkane with six carbon atoms.The term may refer to any of four other structural isomers with that formula, or to a mixture of them. In the IUPAC nomenclature, however, hexane is the unbranched isomer ; the other four structures...

 and solvent polarity
Chemical polarity
In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in...

 is increased during the process by adding ethyl acetate
Ethyl acetate
Ethyl acetate is the organic compound with the formula CH3COOCH2CH3. This colorless liquid has a characteristic sweet smell and is used in glues, nail polish removers, and cigarettes...

.

Column Chromatogram Resolution Calculation

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 280 nm. 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: 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: 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. 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:

Resolution (Rs)

Rs = 2(tRB – tRA)/(wB + wA)

Where:

tRB = retention time of solute B

tRA = retention time of solute A

wB = Gaussian curve width of solute B

wA = Gaussian curve width of solute A

Plate Number (N):

N = (tR)2/(w/4)2

Plate Height (H):

H = L/N

Where L is the length of the column.

Column Adsorption Equilibrium

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.

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.
Langmuir Isotherm:

[CS] = (KeqStot[C])/(1 + Keq[C]), where Stot is the total binding molecules on the beads.

Freundlich Isotherm:

[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 Keq’s. Therefore, the Langmuir isotherm is not a good model for binding in this case.

See also

  • Chromatography
    Chromatography
    Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....

    , an overview article covering all chromatographic techniques.
  • High performance liquid chromatography
    High performance liquid chromatography
    High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...

     (HPLC) for column chromatography using high pressure.
  • Fast protein liquid chromatography
    Fast protein liquid chromatography
    Fast protein liquid chromatography , is a form of liquid chromatography similar to high-performance liquid chromatography that is used to separate or purify proteins and other polymers from complex mixtures. FPLC system is a complete system for laboratory scale chromatographic separations of...

    (FPLC) for separation of proteins using column chromatography.

External links

The source of this article is wikipedia, the free encyclopedia.  The text of this article is licensed under the GFDL.
 
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