The Three Techniques of Chromatography - Research Paper Example

Unit: Introduction In chemistry, scientists may need to separate mixtures of multiple compounds. Chromatography provides a technique of separating and analyzing the components of various mixtures. It has been widely applied in the industry and for study purposes. This research paper examines the three techniques of chromatography, namely; gas chromatography, High performance liquid chromatography and Fourier infrared spectroscopy. Gas Chromatography GC This is a physical separation technique for separating volatile mixtures. It is applied in fields such as pharmaceuticals, environmental conservation and cosmetics. Due to their volatility, human breath, secretions, and other body fluids can be analyzed using this technique. It can also analyze air samples for various compounds. This technique came up in early 60s. Among the various forms of GC, gas-liquid chromatography is the most popular method. Combined with techniques such as mass spectrometry, it becomes invaluable to separation and identification of molecules. Various kinds of detectors can be used for the separation process. They include flame ionization detector, thermal conductivity detector and electron capture detector. Factors influencing the separation process include the stationary phase’s polarity. The polar compounds have strong interactions during this phase. This causes polar compounds to have a longer retention times than their non-polar counterparts. The temperature also affects the process by reducing the retention time. Physical components Injection port It has a rubber cork through which the sample is injected. This point has to be maintained at a higher temperature than the boiling point of the most volatile component of the mixture (Harris 231). The injector could be either a normal packed column injector or a split column injector. For a normal packed injector, the process starts with the injection of a small amount of the liquid using a syringe. It has to be kept hot so that it vaporizes the liquid sample. The vapor is pushed into the column by a high pressure inert gas. To keep the injector hot, a thermostatically controlled large metal block is used. Below is a figure of the packed column injector. The split/splitless injector has similar requirements as the one described above. These requirements are a carrier gas inlet, injector insert and a rubber septum. The main difference with the one above is that the flow of the carrier gas is constant and able to maintain the requirements of the chromatographic process. Carrier gas This plays an important part and has to possess several characteristics. It should be free of oxygen, inert and dry. Hydrogen and helium are the best choices since they possess these characteristics. Helium, being safer than hydrogen is the most commonly used gas. However, depending on the detector in use, nitrogen and argon may also be used. Detectors such as the flame ionization detector, thermal conductivity and electron capture detectors are best suited for use with argon (Higson 87). Column oven The oven contains a thermostat to control its temperature. It operates in two modes; isothermal or temperature programming. Isothermal programing holds the temperatures constant for the whole process. The optimum temperature for the column is ideally the midpoint boiling range of the sample. Detection systems The detector is located at the end of the column to provide a measurement of the components of the mixture. The detection method used is usually any property of the carrier gas which is different from the gas. The detection properties can be classified into two major groups; bulk properties and specific properties. The bulk properties are general and possessed by the carrier gas and the one under study. Specific properties have less functionality. A detector has two main parts used to transform detected property changes into electrical signals, registered on the chromatogram (Pavia 789). The first of these parts is the sensor. It is placed near the exit for maximum detection. The second is an electronic device used to transform the analog changes in magnitude into electrical signals. Each of the detectors has its strengths and weaknesses. Flame ionization detectors They are the most generally applicable and used detectors. In these detectors, the sample exits the column into an air-hydrogen flame. The intense heat causes the sample to chemically decompose. The hydrocarbons release ions that carry high current. This current is then measured using a high impedance picoameter. Thermal conductivity detectors These were the earliest developments in gas chromatography. They measure the changes in carrier gas thermal conductivity. This is caused by the sample having a different thermal conductivity than that of the gas. The thermal conductivity of the surrounding gases determines the temperature of the source. The main advantages offered by the TCD include ease of use, being applicable to both organic and inorganic compounds and the opportunity to collect the sample after analysis. Electron capture detectors These detectors are highly selective and used for detecting environmental changes. They can detect organic compounds with moieties such as peroxides, quinones and others. When dealing with trace elements of such compounds, these are the best option to use. They can also be used in sampling radioactive fields and separation of various radioactive materials. The main advantage of these detectors is their high sensitivity for certain organic species. High performance liquid chromatography (HPLC) HPLC works in a similar way to techniques such as column and layer chromatography. The difference is that rather than the solvent being passed through a column under the effects of gravitational pull, a force of up to 400 atmospheres is applied to it to make the process faster (L. R. Snyder 567). The technique also allows the use of small particles in packing material for the column. This effectively increases the surface area for separation of components. The detection methods used are also more sensitive. Specifications Column dimensions are typically an internal diameter of 4.66mm and a length of 250mm. The columns are shorter because they generate lower back pressure, allowing the user the flexibility of adjusting the flow rate. Particle size is the next specification. Columns are stacked with silica particles, with the size ranging between 3 and 5 microns. Larger particles generate less pressure within the system. The smaller ones generate more pressure resulting in more efficiency in separation (J.J. Kirkland 571). The stationary phase is the next component of the system. It is made of alkyl chains. C4, C8 and C18 are the common chain lengths for this component. C4 is used for large protein molecules while C18 is for small peptide molecules. Depending on the relative polarity of the solvent and the stationary phase, there are two variations of the technique. These are the normal and reverse phase HPLC. Normal phase HPLC This is the less popular of the two variations. Here, the columns have to be filled with tiny particles of silica, with a non-polar solvent. It relies on the properties of the polar compounds of the mixture sticking to the silica for longer than the non-polar compounds. Separation is achieved by the non-polar ones passing faster than the polar ones. Reverse phase HPLC This uses the same column size as the normal phase HPLC. However, the silica particles are made non-polar by the attachment of hydrocarbon chains. The solvent used for this technique is polar. A strong attraction exists between the solvent and polarized molecules of the mixture passing through. The hydrocarbons attached to the silica will not have as much attraction to the polar molecules of the mixture and therefore they spend most of their time moving around in the solvenstrt. Because of the Verderwall’s forces of dispersion, the non-polarized compounds in the mixture be attracted to the hydrocarbons (Neue 578). Due to the need to squeeze in between the solvent molecules, the compounds will be less soluble. They therefore spend more time in the solution slowing their movement to the column. HPLC flow diagram The sample is injected automatically. The pressure involved is the main factor differentiating it from gas chromatography Retention time This is the time it takes the compound to travel from the injection point to the detector. It is taken when the display shows the maximum peak for a sample. Various factors determine the retention time for different compounds. They include; Amount of pressure used. As the pressure increases, the flow rate of the compound increases and therefore the retention time reduces. The nature of the stationary phase also determines the retention time. The particle size is the most important factor of this nature. The components of the solvent The column’s temperature If retention time is going to be used as the parameter for identification of different components, then these factors have to be taken into account. Detector Various techniques can be used to detect presence of a substance passing through the column. However, ultra-violet absorption is a commonly used method. Organic compounds absorb UV light of different wavelengths. If a beam of this kind of light is shone through a liquid flowing out of the column, a UV detector on the opposite side can be used to measure the amount of light absorbed. The volume of the compound passing through at that time also determines the amount of UV light absorbed. The output of the detector is a series of peaks. Each peak represents a compound in the mixture that passes through the detector and absorbs UV light. The retention time of different compounds can be used to make conclusions of the different compounds present in the mixture. The peaks can be used as indicators of the quantities of different compounds present. The area under a peak is directly proportional to the amount of a particular compound. Fourier transformation infrared spectroscopy (FTIR) This technique involves passing radiation through a sample which absorbs some of it while the rest is transmitted through it. The result is a spectrum that represents the molecular absorption and transmission and a unique pattern for the sample. All molecular structures have a unique spectrum that identifies them (Jef Poortmans 789). The spectrometers collect all wavelengths simultaneously. Below is a diagram of the basic structure of the spectrometer FTIR can be useful for various kinds of analysis. It identifies unknown materials, determines quality and consistency of a sample, and the magnitude of the composition of a mixture. It has several advantages including; It does not destroy the sample It does not require external calibration since it is precise It is a fast method of collecting scans It is highly sensitive Its optical throughput is higher It has only one moving part eliminating mechanical complexity The main reason behind the development of this technique was to enable the measurement of infrared frequencies simultaneously. An optical device called an interferometer was invented for this purpose. It encodes all the infrared frequencies unto a single beam. The sample beam can be taken in a matter of seconds. Most of the interferometers in use are equipped with a device called a beamspliter. It divides the infrared signal into two beams of light. Of the beams, one is bounced off a flat mirror at a fixed point. The other beam is bounced off a mirror that moves over short distances on a mechanism built for that. The two beams come back to the splitter where they recombine. The new signal is called an interferogram. As the optical path difference between the two beams increases, the readings of different wavelengths peak (Michael Gaft 871). This gives the interferogram an oscillatory appearance. As the distance between the mirror and the splitter changes, various radiations will be in and out of phase. The frequencies of these radiations depend on frequency of movement of the mirror and that of the source (Michael Gaft 871). The interferogram contains information about every signal coming from the source. Therefore, when taking measurements of the interferogram, one takes measurement of all the infrared signals combined. A frequency spectrum is needed for success of this task. This is the plot of individual intensity at each of the points. The measured interferogram has to be decoded to take measurements of all individual frequencies. A mathematical method by the name Fourier transformation is used for this purpose. It is usually a computerized process. Process of sample analysis Source An emitter is used to emit the infrared energy. An aperture is used to control the amount of energy that hits the sample. Interferometer The beam is reflected off the two mirrors where the infrared frequencies are combined into an interferogram. Sample In the sample compartment, the beam is transmitted through or is reflected off the surface. The action taken depends on the analysis being carried out. The specific frequencies unique to the sample are absorbed. Detector The devices are built specifically for one kind of spectrogram. The computer The measurements are digitized and a computer performs Fourier transformation on them. The final output is presented to the user for his/her interpretation. For purposes of control, a background spectrum should be taken without any sample. It can then be used for comparison with the measurement of the spectrum with a sample. This comparison allows computation of the percent transmittance. This way, instrumental characteristics can be removed ensuring that all the features present are due to the sample. The instruments used for FTIR do not require to have slits for resolution (White 761). This gives them a higher resolution that would be achieved using other techniques such as a dispersive instrument. A spectrometer possesses a characteristic called spectral resolution. This is its ability to distinguish between closely related features in a spectrum. The optical path difference in FTIR is the main determinant of spectral resolution. The instruments also have a short wavelength limit. The advantages outlined are responsible for giving the measurements of FTIR spectrometers highly accurate and reproducible. The technique can be relied upon for positive identification of any sample compounds (Griffiths and de Hasseth 995). With the high sensitivity, any contaminants in a sample can be easily identified. These characteristics make the techniques ideal for quality control functions in a production system or a plant. It has also been widely used in quantitative analysis tasks in many situations. This technique has gained a lot of popularity and a lot of studies are being done on its improvement. In experimental laboratories, scientists are coming up with new ways of coming up with more precise analysis of components. In the field, it is being used for qualitative and quantitative analysis for various industries. The quality control department of any plant plays an important role in its development and performance. Work cited Griffiths, P. and J.A. de Hasseth. Fourier Transform Infrared Spectrometry. Wiley-Blackwell, 2007. Harris, Daniel C. Quantitative chemical analysis. W. H. Freeman and Company, 1999. Higson, S. Analytical Chemistry. OXFORD University Press, 2004. J.J. Kirkland, L. R. Snyder, and J. L. Glajch. Practical HPLC Method Development. New York: John Wiley & Sons, 1997. Jef Poortmans, Vladimir Arkhipov. Thin film solar cells: fabrication, characterization and applications. John Wiley and Sons, 2006. L. R. Snyder, J.J. Kirkland, and J. W. Dolan. Introduction to Modern Liquid Chromatography. New York: John Wiley & Sons, 2009. Michael Gaft, Renata Reisfeld, Gérard Panczer. Luminescence spectroscopy of vminerals and materials. Springer, 2005. Neue, U. D. Theory, Technology, and Practice. New York: Wiley-VCH, 1997. Pavia, Donald L., Gary M. Lampman, George S. Kritz, Randall G. Engel. Introduction to Organic Laboratory Techniques. Thomson Brooks/Cole, 2006. White, Robert. Chromatography/Fourier transform infrared spectroscopy and its applications. Marcel Dekker., 1990. Read More