Chromatography. Gas Chromatography
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- Prosper Powell
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1 Chromatography Chromatography is essentially the separation of a mixture into its component parts for qualitative and quantitative analysis. The basis of separation is the partitioning of the analyte mixture between two immiscible phases viz. a miscible phase and an immiscible phase. The sample is dissolved in one phase, called the mobile phase. This phase can be a gas (gas chromatography) or liquid (liquid chromatography). The mobile phase is then passed through the other phase, called the stationary phase. This phase can be a gas or liquid but is typically a solid and is packed into a column. The stationary phase is made up of materials for which the mobile phase has differing affinities. As the mixture dissolved in the mobile phase interacts with the stationary phase, the components of the mixture separate and are eluted from the column. Each component can then be collected for analysis and quantification. The figure alongside shows a simple chromatographic separation process of a component ( mixture. The time taken for a compound to elute and be detected by the detector is called the retention time (R t ). Generally a longer column will allow for better separation as the compounds interact more with the stationary phase. There are two main types of chromatography that we will be looking at viz. gas chromatography and liquid chromatography which are used for volatile and non-volatile compounds respectively. Gas Chromatography In gas chromatography, the molecules partition between a gaseous mobile phase (carrier gas) and a stationary phase. The sample is injected into an injector port where it is volatilised and transported through the column by the carrier gas. Separation occurs through a process of adsorption onto the solid stationary phase or dissolving in the liquid stationary phase. The samples must be volatile and thermally stable over the period of the analysis. 1
2 ( The diagram shows a simple schematic of a typical gas chromatograph. The carrier gas is an inert gas e.g. nitrogen, argon, helium. The carrier gas should not interact with the sample in any way other than to transport the sample through the column. The carrier gas must be maintained at the appropriate flow rate to ensure that there is sufficient time for the sample to interact with the stationary phase (if the flow rate is too high, the sample will not separate effectively). The carrier gas flows through an injector port through which the sample is introduced into the instrument. A small volume (1 10 µl) of sample which has been dissolved in a volatile solvent is injected via an injector port onto the column. There are two modes in which an injector can operate; split and split-less mode (see more detail after columns). The injector port is at a higher temperature (~ 50 C higher) than the boiling point of the least volatile component in the sample. This ensures that the sample enters the column (i.e. stationary phase) as a vapour. The volume injected onto the column depends on the type of column being used. The column is housed in an oven to ensure that the temperature is controlled and to ensure that the sample remains in the gas phase. The column is at a lower temperature than the injector port, this may cause some components of your sample to condense. There are two different types of column; a packed column and a capillary column. A packed column has small support particles (finely ground diatomaceous earth) which may be coated with a non-volatile liquid. The columns can range in length up to 10 m and have an internal diameter that is between 1-5 mm. Packed Column Capillary columns are more commonly used in modern chromatography. The columns can range between m and have a significantly smaller diameter ( mm). The longer column length generally means that the capillary column show better separation than the packed column. A capillary column is a fused silica column that has been coated on the outside with a polymer which
3 improves its strength and flexibility. In a capillary column; the stationary phase is coated directly onto the walls of a column and not packed to fill up the column. There are 3 different types of capillary columns. The wall coated open tubular column (WCOT) is the most commonly used and has a liquid stationary phase film coated onto the column walls. Support coated open tubular (SCOT) columns have a thin layer of solid particles which are then coated with a liquid. This results in more separation efficiency than WCOT columns because of the higher surface area of the column. The inner surface of porous layer open tubular (PLOT) columns have an embedded layer of porous materials (alumina, molecular sieves), with the film thicker than WCOT and SCOT columns which more effectively separates gases and highly volatile compounds. Because of the differences in the structure of the two different types of columns, generally packed columns can tolerate a larger analyte volume than the capillary columns. This means that when a sample is injected onto the column via the injector port, the same volumes cannot be used for packed and capillary columns. A too high analyte concentration would result in the active sites of the column being blocked with analyte and some analyte then being un-retained by the column. To prevent continually diluting samples, an injector port can be used in two modes; split and split-less mode. Split mode is typically used when a capillary column is used and a fraction of the amount injected is automatically diverted to waste with a small amount entering the column. In split-less mode, the entire injected sample enters the column. So, when the sample is in split mode, it is injected into the injector port which is heated and it volatilises, it is carried through the injector port by the carrier gas and can either enter the column or be carried through the split valve (see diagram). When a mixture enters the column as a gas some of the analyte components may condense onto the stationary phase, some components may dissolve in the liquid film coating the surface of the stationary phase, or it may remain in the gas phase and be transported through the column without interacting with the stationary phase (i.e. be unretained this compound(s) will elute first). 3
4 The compounds that have a boiling point that is higher than the oven temperature will condense at the beginning of the column. However, what follows is a cycle of evaporation and condensation of those compounds along the length of the column, thus causing separation from other molecules with different boiling points. Molecules with a high boiling point will therefore spend a longer time on the column that those with lower boiling points. Other molecules will interact with the stationary phase depending on their solubility. The more soluble compounds will absorb onto the column more than the less soluble compounds and thus be retained for longer. The temperature of the column also plays a role in the elution of molecules. Low column temperatures will mean that the analytes spend a longer time on the column and while this leads to good separation, you will have long analysis time and peaks may not be sharp. High column temperatures will result in shorter analysis time, but if the temperature is too high then molecules may not interact sufficiently with the stationary phase. The elution of compounds is therefore dependent on a number of different factors which affect the interaction of molecules with the stationary phase; gas flow, rates of diffusion, boiling point and solubility with the stationary phase. Temperature Programming (to add) As the compounds elute from the column, a detector converts the analyte concentration into an electrical signal which can be used for quantification. There are a number of different detectors used in GC; the flame ionisation detector is commonly used for the analysis of most organic molecules. Essentially, the carrier gas and hydrogen are mixed and burned. As an organic compound elutes from the column and enter the detector, the compound will burn and produce electrons and ions. These collect at electrodes and a flow of current occurs which is detected as the signal. The output from the detector is recorded as a series of peaks, each one representing a different analyte. Gas chromatography is used for both qualitative and quantitative analysis. Compounds can be identified by matching their retention times to those of known standards. The higher the concentration of your analyte, the more ions it will generate creating a bigger signal; thus allowing for quantification. Quantification is carried out using the area under the analyte peak since this area is proportional to the concentration. 4
5 Other detectors that are widely used are the electron capture detector which is selective towards Halides, nitrates, nitriles, peroxides, anhydrides, organometallics). The thermal conductivity detector is used for most compounds since it measured thermal conductivity and it is known as a universal detector. One of the biggest sources of error in GC analysis is the injection of the sample. Because the sample injection volume is so small, any error made has a large impact. Using an internal standard allows for correction of injection errors. A known concentration of a compound is added to the samples and standards to be analysed. In this way any errors in the injection will be reflected in both the analyte and the internal standard. Because the initial concentration of the internal standard is known, correction to the data can be made using the ratio of the peak areas of the standard and sample. Gas chromatography is suited to organic compounds that are volatile. However, non-volatile compounds may be analysed if they are derivatised to produce volatile products. An example of this is the analysis of fatty acids. Fatty acids can be derivatised to form fatty acid methyl esters by the process of esterification. The methyl esters are volatile and can then be analysed by GC. Liquid Chromatography Liquid chromatography is a separation technique where the components of the sample are separated based on their affinity for a liquid mobile phase. The most widely used type of liquid chromatography is called high performance liquid chromatography (HPLC). There are a number of different types of HPLC and they are based on how separation occurs; adsorption chromatography, partition chromatography, ion-exchange chromatography and size exclusion chromatography. In adsorption chromatography, analytes are separated based on the degree of adsorption to the stationary phase which is made up of fine particles of silica or alumina. Generally non-polar compounds can be eluted and identified by adjusting the strength of the mobile phase. Partition chromatography is one of the most commonly used modes of separation and can be used for non-ionic compounds and low molecular weight compounds. A solid (e.g. silica) is coated with a thin film of stationary phase. The analytes interact with the mobile phase and equilibrate between the two. Partition chromatography can occur either in normal phase or reverse phase mode. In normal phase, the silica packing material is coated with polar/hydrophilic functional groups. Here, polar compounds will be retained on the column for 5
6 longer and are eluted using non-polar mobile phases e.g. hexane, methylene chloride. The less polar compounds elute faster (i.e. have shorter retention times). Thus the separation is based on polarity and compounds elute in order of increasing polarity. Reverse phase chromatography is the most popular modern mode of separation. Here the stationary phase is non-polar, with hydrophobic, long chain alkanes such as n-octyldecyl (C 18 ), n-decyl (C 8 ) chains and phenyl groups being the most common bonding phase onto silica particles. The mobile phase is a polar solvent such as water, methanol or acetonitrile. Reverse phase chromatography is the opposite of normal phase. The example below shows a three component mixture separated by normal and reverse phase chromatography. Note how the order of elution changes. A summary of the different response to a mixture using the different phases is presented in the table. Ion exchange (to be added) Size Exclusion Chromatography (to be added) High Performance Liquid Chromatography (HPLC) Particle size of the column packing will increase the separation efficiency. The smaller the particles, the larger the surface area for the components of the mixture to interact. This results in faster equilibrium between the mobile and stationary phases. A high pressure is used to force the mobile phase through the column to prevent issues arising from capillary action. Hence the instrumentation used for liquid chromatography is called HPLC. The brief schematic shows the process of HPLC. The mobile phase is typically a high grade, solvent with polarity that is dependent on the 6
7 molecules you are trying to separate. The solvent should be de-gassed and free of bubbles which would interfere with separation in the column. The pump forces the mobile phase and sample through the column at high pressure. As with GC, the flow rate of the mobile phase has an impact on separation and elution of the analytes. There are two different ways to enhance separation using the polarity of the mobile phase: isocratic and gradient elution. In isocratic elution, a solvent of mixture of solvents making up the mobile phase is pumped at a constant rate through the column. In gradient elution, the composition of the mobile phase is changed during the analysis. This change in polarity gradually will allow compounds to separate efficiently. The sample is introduced into the mobile phase using an injection valve. This valve is very different from the GC injection port; the sample is loaded into an injection loop. When the loop is set to load, sample can be injected into the sample loop. When the loop is switched to inject, an appropriate volume of the sample is transferred directly onto the column. This means that there are no injection errors that affect your analysis (like with GC). When the sample enters the column separation of the compounds occurs depending on the type of column present. As each compound elutes the signal is detected and plotted as a series of peaks. The retention times for each compound may be matched to standards thus allowing for qualitative analysis. The signal intensity is proportional to the concentration of the analyte and can therefore be quantified. The detector on the HPLC varies according to the type of compound being detected. In this module you will use one detector, a UV detector which you have already come across in Spectroscopy. 7
8 Some Key Equations and Definitions for Chromatography Peak Parameters t R total retention time - time between injection of sample and emergence of the peak maximum t M mobile phase hold-up time - time taken for a non-retained substance to emerge from the column t' R adjusted retention time - average time spent in the stationary phase = t R - t M k capacity factor (retention factor) = t' t R M average timespent instationaryphase averagetimespent inmobilephase no.molesinsp no.molesinmp V K V S M K K distribution constant = concentration in stationary phase concentration in mobile phase phase ratio (only applicable to liquid stationary phase) = V V M S volume of mobile phasein column volume of stationary phasein column w ½ peak width at half the peak height (the half-width w ½ or w h ) w b peak width at the base (note: w b = 1.70 x w ½ ) 8
9 Column Efficiency N plate number (number of theoretical plates) = t 5.55 w R 1 t 16 w R b H plate height (height equivalent to a theoretical plate - HETP) = N L (where L = length of the column) Separation selectivity coefficient (separation factor) = t' t' R R1 k k 1 K K 1 R S peak resolution found experimentally by: R S t w R b1 t w R1 b related to other parameters by: R N k S 4 1 k 9
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