About this sample
About this sample
4 pages /
4 pages /
Gas Chromatography (GC or GLC) is a commonly used analytic technique in many research and industrial laboratories for quality control as well as identification and quantitation of compounds in a mixture. GC is also a frequently used technique in many environmental and forensic laboratories because it allows for the detection of very small quantities. A broad variety of samples can be analyzed as long as the compounds are sufficiently thermally stable and reasonably volatile.
Gas-liquid chromatography requires a sample to vaporise and then injected through a layer into a chromatographic column. The vapourised sample is moved up the column by a mobile phase. The mobile phase used in gas-liquid chromatography is an inert gas. Whereas the column contains a liquid stationary phase which has been adsorbed onto an inert solid surface. The mobile phase is required to be chemically inert so that it doesn’t react with the vapourised sample or other chemicals present. In capillary gas chromatography the capillary columns have a very small internal diameter. This is mainly a few tenths of a millimeter. Two types of capillary columns can be used. A wall coated open tubular capillary column or a support coated open tubular capillary column. The wall coated columns are coated with the liquid stationary phase whereas in the support coated column the inner wall is lined with a thin layer of material which absorbs the liquid stationary phase. Both columns are more efficient than packed columns however support coated columns are less efficient than wall coated columns.
The stages of gas chromatography would be the injection of the sample through a rubber septum and into a vapourisation chamber where a carrier gas inlet will flood the vapourisation chamber with the carrier gas. The chamber is surrounded by a heated metal block with two glass liners that direct the gas mixture to the opening of the capillary column. The inert gas carries the vapourised sample through capillary column that has its inner wall coated with the liquid stationary phase. In the column the temperature is managed to within tenths of a degree. The optimum temperature for the column should be the midway between its upper bound and lower bound values for the sample’s boiling point. If the sample’s boiling point varies too much for the column temperature to be managed manually a computer programme can be used to automatically make the temperature changes needed.
At this point the sample will have reached a detector. Initially the retention time can be calculated which is the time taken for a sample to reach the detector from injection point to arrival point. Furthermore more detectors can be attached to the retention time detector however there are many types of detectors that have been specialised for different purposes. There are two categorisations for all detectors in gas chromatography. These are: mass flow dependant detector which will destroy the sample however it determines the rate at which solute molecules enter the detector. The other would be concentration dependant detectors. The concentration dependent detectors don’t destroy the solute. HPLCHigh performance liquid chromatography is an improved version of column chromatography that revolves around forcing the solvent via very high pressures up to 400 atmospheres through the column which makes it much faster. Not only that but it allows the column have smaller particle size packing material to provide greater surface area for more interactions between the stationary phase and the molecules moving past it.
There are different types of high performance liquid chromatography that can be used which depends on the polarity of the solvent and the stationary phase. These are normal phase high performance liquid chromatography and reversed phase high performance liquid chromatography.
Normal phase HPLC is the standard textbook HPLC that is used which is just a modified variant of column chromatography. Whereas reversed phase HPLC has the silica coating modified to that it is non-polar. This is done by attaching long chains of hydrocarbons to the surface. A polar solvent is then used. This means that less molecules will be attracted to the coating resulting in more molecules progressing faster through the column. On the other hand due to Van Der Waal’s dispersion forces nonpolar compounds in the mixture will form attractions to the hydrocarbon coating on the silica layer. Due to this attraction the non-polar compounds spend more time being attracted to the hydrocarbons and less time progressing through the column.
Due to the 400 atmospheres of pressure the sample will be injected automatically from a solvent reservoir. There are several factors that affect the retention time of a compound. These are the pressures used which dictates the flow rate of the solvent, the material and particle size of the stationary phase coating within the column and the temperature of the column.
The detectors usable remain the same as in gas chromatography. However a combination can be used for example a UV detector to a mass spectrometer. Once the UV detector has recognised the missing wavelengths of UV radiation that has been absorbed by the solvent passing through the column a peak is generated on the graph which is dependant on the wavelength absorbed. Once a peak has been registered some of the sample is automatically siphoned and channeled to a mass spectrometer where it will produce a fragmentation pattern and destroy the small sample that was siphoned.
A qualitative analysis involves the identification of a component by means of peak data on the chromatogram. The retention of a component is the result of a specific interaction of that component with the stationary phase and the mobile phase. Since the retention time is a specific property of a component, it may be used as a means to identify the component. The retention time of the unknown component is compared to the retention time of a so-called standard. This is a compound of which the identity is known and which is likely to possess the same identity as the unknown component. When the retention times of both compounds are similar, the unknown is considered identified. If the analyte itself is not available as a pure substance, identification based on chromatographic results only is not possible.
The analyst should always realize that the retention time is not only dependent on the component but also on the system (column, stationary phase, conditions and the instrumental settings and performance). It means that a correct comparison is only possible when two chromatographic runs are performed under identical conditions on the same GC system. Only under these circumstances the differences in retention times due to the stationary phase, the mobile phase, the flow, the column length, the column temperature etc. can be ignored. Validation of the system can prove the performance and stability of the instrument, using statistics to show its robustness and reproducibility.
Gas Chromatogram (mass spectrometry)HPLC ChromatogramThe concentration from a mass spectrogram can be calculated by comparing the peak area from the sample with peak area from a standard of known concentration. The areas under the peaks are directly proportional to the amount of the compound present in the mixture. These figures can be calculated automatically by a computer. To identify the compounds present a mass spectrometer can generate a fragmentation pattern for each peak which is comparable to a computer database for identification. If individual peaks are needed to be know the mass: charge ratio can be used to identify the respective peak.The peaks can be used to measure relative quantities of the compounds present in the mixture however, it is known to be more accurate if there are similar compounds in the mixture.Beginning with principles of factors affecting retention times of compounds on a mixture, a method can be made. The factors affecting retention time are the polarity of the sample/stationary phase, the temperature in the column, the pressure used to control the flow rate and the particle size of the coating.
The base method of HPLC that will be used will be reverse high performance liquid chromatography. The specific sugar compound that is being sought for can have special properties unique to that sugar compound. For example it has a long chain or unusual bonding. The compound could be slightly polar or remains nonpolar whilst the other sugar compounds are different. These could all act as a filter criteria which will separate the mixture so that the specific compound could be gained. Another theory would be to use the boiling point for the specific sugar compound. Using the optimum boiling point for that specific sugar compound will result in that compound progressing faster through the column towards the detector.
Regarding the amount of the sugar compound present isn’t too tough. This is because the detector sequence would be a UV tester then a mass spectrometry. The UV test will check the wavelengths of ultraviolet radiation absorbed which is unique to each compound. Once the desired wavelength has been found a portion of that compound is sent to through mass spectrometry where the sample will be destroyed but a fragmentation pattern will be produced along with peaks. The area underneath the peaks represents the amount present in the sample. Comparing this with a mass spectrogram of the pure sugar compound sought for increases accuracy and confidence.
There are other tested and more commonly used methods of separating sugar compounds in HPLC. These are ligand conversion, size exclusion, borate complex anion exchange, anion exchange and partition. Size exclusion revolves around separating the sugar compounds by their molecular weight. This is based on the molecular size of the compound so that any other compounds that have the same weight but a different molecular size can’t be separated.
The packing material used would be a hydrophilic polymer whilst water would act as the mobile phase. Ligand conversion revolves around the sugar compounds forming complexes with a metal counterion. For this circumstance sulfonated polystyrene gel with a metal counterion can be used as packing material. This can be specific types of sodium, calcium or lead. This is method is a good process for separating sugars up to disaccharides but not beyond. The retention of the complexes formed is entirely dependant the molecular size of the compound. Water is also used as a mobile phase in this method. Partition can only be done for normal phase HPLC and not reverse phase but it is still a method that can be used. This method can be used to separate all the sugar compounds and view the differences in between oligosaccharides. This is done by using aminopropyl bonded to a silica polymer support. The mobile phase consists of acetonitrile and water however the aldehyde radicals in sugars can react with the amino radicals in the stationary phase which causes significant tailing on peaks for pentasaccharides. This can be prevented by adding some salt to the mobile phase. Borate complex anion exchange is based on sugars compounds reacting with borate/borate salts to form negatively charged complexes. Resulting in a mixture that can be separated by anion exchange if a borate buffer solution is used as a substitute for the mobile phase. By varying the concentration and pH of the borate buffer solution the sugar complexes can be separated more efficiently.
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