Chromatography
In
chemistry,
chromatography is a process for the separation of mixtures. This is achieved by passing a sample mixture (the "analyte") in a stream of
solvent (the "mobile phase") through some form of material (the "stationary phase") that will provide resistance by virtue of chemical interactions (
not reactions) between the components of the sample and the material. Usually, each component has a characteristic separation rate that can be used to identify it and thus the composition of the original mixture.
IUPAC definition of Chromatography: it is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction .
It was the Russian botanist Mikhail Tswett (Mikhail Semenovich Tsvett, 1872-1919) who in 1906 first used the term chromatography.
He used the technique to separate differntly colored plant pigments (which explains why 'chroma' is part of the name). The modern concept of chromatography has nothing to do with color.
Analytical chromatography is used to determine to which chemicals are in a mixture and their concentrations. Preparative chromatography is used to purify larger quantities of a chemical. Most of the following refers to analytical chromatography.
Paper chromatography
A small spot of the solution containing the sample is applied to a strip of
chromatography paper about one centimetre from the base. This sample is
adsorbed onto the paper. This means that the sample will contact the paper and may form interactions with it. Any substance that will react with (and thus bond to) the paper cannot be measured using this technique. The paper is then dipped in to a suitable
solvent (such as
ethanol or
water) and placed in a sealed container. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different
compoundss in the sample mixture travel different distances according to how strongly they interact with the paper. Paper chromatography takes some time and the experiment is usually left to complete for some hours.
The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes. Whereas compounds can be identified by calculating Rf values which can be compared with values in a data book. These can be calculated by:
Rf = (distance moved by spot) / (distance moved by solvent)
Two-way paper chromatography involves using two solvents and rotating the paper 90
o inbetween. This is useful for separating complex mixtures of similar compounds.
Thin layer chromatography
This involves the adsorbent (the solid which the sample is adsorbed to) being in a thin layer on the surface of glass plate. The adsorbent (eg silica gel or calcium sulphate) is pasted on to the glass and baked. The process if the same for paper chromatography. The advantage is that wider seperations can be achieved in less distance, and different adsorbents can be used.
Gas-liquid chromatography
In gas-liquid chromatography (GC or GLC) the mobile phase is a carrier gas, usually an inert gas such as helium or nitrogen. The stationary phase microscopic layer of liquid on an inert solid support. The stationary phase is in a lond thin tube known as a column. A known volume of gaseous or liquid analyate is injected into the entrance of the column. The carrier gas sweeps the analyate molecules through the column, but this motion is inhibited by adsorption of the analyate molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the column materials. Since each type of molecule has a different rate of progression, the various components of the analyate mixture are separated as they progress along the column and reach the end of the column at different times. A detector is used to monitor the outlet stream from the column, and thus the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified by the order in
which they emerge from the column.
Two types of columns are used in GC. Packed columns contain a finely divided, inert, solid support material (eg. diatomaceous earth) coated with a liquid or solid stationary phase. The nature of the coating material determines what type of materials will be most strongly adsorbed. Thus numerous columns are available that are designed to separate specific types of compounds. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. The outer tubing is usually made of stainless steel or glass. Capillary columns have a very small internal diameter, on the order of a few tenths of millimeters. The column walls are coated with the active materials. Most capillary columns are made of fused-silica with a polyimide outer coating. These columns are flexible, so a very long column can be wound into a small coil.
Molecular adsorption and thus the rate of progression along the column depends on the temperature, thus for precise work the column temeprature is carefully controlled to within a few tenths of a degree. Reducing the temperature produces the greatest level of separation, but can result in very long elution times. For some cases temperature is ramped either continuously or in steps to provide the desired separation.
A number of detetectors are used in gas chromatography. The most common is the thermal conductivity detector (TCD), which monitors changes in the thermal conductivity of the effluent. The main advantage of the TCD is that it can detect any substance (except the carrier gas). Some of the other detectors are only sensitive to specific types of substances. Other detectors include the Flame ionization detector (FID), Electron capture detector (ECD), Flame photometric detector (FPD), Photo-ionization detector (PID) and Hall electrolytic conductivity detector.
An example of the use of gas chromatography is in the study of the selectivity of Fischer-Tropsch synthesis catalysts. The outlet from this process contains a number of light gases including N2, H2, CO, CO2, H2, CH4, and Ar, as well as heavier parafinic and olefinic hydrocarbons (C2-C40). In a typical experiment, a packed column is used to separate the light gases, which are then detected with a TCD. The hydrocarbons are separated using a capillary column and detected with an FID.
Column chromatography
Immobilized Metal Ion Chromatography
IMAC is a popular and powerful way to purify proteins. It is based on the specific coordinate covalent binding (see coordinate covalent bond) between histidine or other unique amino acids (either naturally present on the surface of the protein or grafted with recombinant DNA techniques) and various immobilized metal ions, such as copper, nickel, zinc, or iron.
High performance or high pressure liquid chromatography
Frequently referred to simply as HPLC, this form of column chromatography is used frequently in biochemistry. The analyte is forced through a column by liquid at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to spread out within the column, leading to broader peaks. Less time on the column then translates to narrower peaks in the resulting chromatogram and thence to better selectivity (it's easier to differentiate one peak from another) and sensitivity (tall, narrow peaks can be easier to discriminate from noise than shorter, broader peaks). Solvents used include any miscible combination of water or various organic liquids (alcohols, acetonitrile, dichloromethane). Often, a gradient over time in the solvent composition passing through the column is used to separate analyte mixtures, as a function of how well the changing solvent composition differentially mobilizes the analyte. For instance, using a
water/methanol gradient, the more hydrophobic components will elute under conditions of relatively high methanol, whereas the more hydrophilic will elute under conditions of relatively low methanol. Whether one starts with high methanol or low methanol depends on the nature of the stationary phase. Traditionally HPLC stationary phases are polar, whereas so-called "reverse" phase (RP-HPLC) stationary phases are hydrophobic. On an RP-HPLC column, then, hydrophobic analytes would tend to be retained on the column, eluting more readily as the proportion of the hydrophobic component of the stationary phase is increased.
