High Performance Liquid Chromatography - Assignment Example

Chromatography Chromatography is defined as a method of separating and identifying the components of a mixture. The chemical mixture is first dissolved in a mobile phase before passing through a stationary phase. The stationary phase is responsible for separating and isolating the analyte from the other components of the chemical mixture (Allman 2010). The mobile phase can be a gas, liquid or even supercritical fluid while the stationary is immobile and immiscible (Sheffield Hallam University n.d). Chromatography techniques are important in the treatment of patients who have overdosed on illicit drugs. The illicit drugs sold on the streets are usually diluted with any material by the drug dealers who want to increase the quantity and street value of their drug products. The task of separating and identifying the components of the illicit drugs and is an arduous one and chromatography is applied in such cases (Allman 2010). In both high-performance liquid chromatography and gas chromatography utilize columns, which are narrow tubes filled with the stationary phase, in which a mobile phase is passed. The chemical mixture is passed through the column by the continuous introduction of the mobile phase. This process is known as elution. The average rate that an analyte of interest is transported through a column depends on the time period it spends in the mobile phase (Sheffield Hallam University n.d). The theory of chromatography is based on the observation that when dissolved in a solution or adsorbed on a solid surface, chemical substances partially escape to the surrounding environment. Hence the solubility of a gas in a liquid determines the distribution of the gas between a liquid phase and a solid phase. When the solubility of the gas is higher, the tendency of the gas to remain in the liquid phase is greater (Allman 2010). A component that is more soluble in the stationary phase will take longer the component that is less soluble in the stationary phase and more soluble in the mobile phase to travel through it (Sheffield Hallam University n.d). Distribution of analytes of interest between the mobile phase and the stationary phase can be described as follows where each analyte is in equilibrium between the two phases; A mobile A stationary The partition coefficient is the equilibrium constant K and is defined as the analyte molar concentration in the stationary phase divided by the analyte molar concentration in the mobile phase. The retention time (tR) on the other hand is the time from when the chemical sample is introduced into the column and when the analyte peak reaches the detector at the column end. tM is the time taken by the mobile phase to move through the column. Figure 1: Retention time tR and tM on a chromatogram (Sheffield Hallam University n.d) The retention factor (k’) or capacity factor describes the rate of migration of an analyte of interest in the column. Retention factor of an analyte B can be defined as; k'B = tR - tM / tM tR and tM are obtained from the chromatogram. It is preferable for the retention factor to between 1 and 5 because a retention factor of an analyte is low ( 20) means that the elution will be very slow thereby taking a long time. The selectivity factor (α) describes the separation of two analyte species on the column; α = k 'D / k 'C For the purpose of calculating the selectivity factor species C elutes faster than species D and the selectivity factor is always greater than one (Sheffield Hallam University n.d). Resolution The measurement of the resolution describes how well the analyte species are separated. The resolution, R, of two analyte species C and D can be defined as; The baseline resolution is attained when R = 1.5 Of importance is the relation of resolution to the selectivity factor, number of plates in the column and the retention factors of the two solutes; Hence, to achieve a higher resolution, the selectivity factor, the number of plates and the retention factors must be maximized. Increasing the N or the number of plates can be achieved by lengthening the column which in turn increases the retention time and band broadening, which may be undesirable. As such, it is desirable to increase the number of plates by in reducing the height equivalent to a theoretical plate by decreasing the size of the particles of the stationary phase. Many laboratory experiments have shown that controlling the capacity factor (k’) leads to improved separations. In Gas Chromatography, this is achieved by altering the temperature while in high – performance liquid chromatography, separation is improved by changing the composition of the mobile phase (Sheffield Hallam University n.d). Increasing the temperature of the column increases the kinetic energy of the molecules making them in to move faster down the column hence reducing the retention time of the analyte of interest. Other factors that affect retention time include the type of compound, dimension of the column, the flow rate of the mobile phase, the carrier gas, the dead volumes and the active points between the injector and detector. The selectivity factor is also manipulated to improve separation. When the selectivity factor is close to one, increasing the number of plates and optimizing the capacitor factor does not result in improved separation in a reasonable duration of time. In such cases, the capacity factor is optimized first and then the selectivity factor increased in one of the following ways: Altering the composition of the mobile phase Altering the temperature of the column Altering the composition of the stationary phase Employing unique chemical effects like using a species that forms a complex with one of the solutes in the stationary phase (Sheffield Hallam University n.d). Gas Chromatography Gas chromatography (sometimes known as gas-liquid chromatography) separates chemical mixtures based on the distribution between a mobile gas phase and a stationary liquid phase. Helium or any other inert gas flows through a glass or stainless steel column. The inert carrier gas is responsible for carrying along the components of the mixture injected into the glass or stainless steel column. The separation of the chemical components will depend on their solubilities in the mobile phase (Allman 2010).Each component emerging from the glass column enters a detector. A polar mobile phase will detect that the shorter n- alkanes (with more than 8 carbon atoms) will separate faster on the chromatogram compared to the long n- alkanes (with less than 8 carbon atoms). Figure 2: Flow scheme for gas chromatography (Clark 2007) Before the sample is introduced to the gas chromatograph, 3 things must be considered; all components introduced must be volatile, the analytes present must be at a suitable concentration, the injection process should not interfere with the separation process. The chemical sample is introduced through the injection port using a syringe in a small quantity. The injector oven is heated to a temperature of at least 50 degrees Celsius above the sample component with the highest boiling point to enable instantaneous vaporization of the whole sample introduced. As the solvent flows through the column, it condenses to form a barrier trapping the solutes. Time is then allowed for solute concentration as the temperature of the column is increased and separation commences. To attain a good separation, the column is placed in an oven to control the temperature of the column. The final section of a gas chromatograph is the detector. An ideal detector should have low detection limits, the ability to respond to all solutes, unique selectivity for each class of solutes, the ability to respond to linearly to a wide range of solute concentrations for quantitative analysis. Several detectors are used including the thermal conductivity detector, Flame ionization detector and electron capture detector. The thermal conductivity however, has poor detection limits while the electron capture has excellent detection limits that extend to about two orders of magnitude. One application of gas chromatography is in the environmental analysis of many organic pollutants in the air and water. Drinking water analysis for volatile organics is achieved by purge and trap. In consumer goods, foods and beverages are analyzed using Gas chromatography following an appropriate extraction. Gas chromatography is also useful in the analysis of petroleum products such as gasoline, oil and diesel fuel (Harvey 2000, p. 571). High- Performance Liquid Phase Chromatography (HPLC) For high-performance liquid chromatography, thinly ground particulate solid is used as the stationary phase while a liquid mobile phase is pumped through the column filled with chemically treated solid particles. High-performance liquid chromatography is advantageous since it is carried out at room temperature. HPLC is preferred in materials that are highly sensitive such as the organic explosives and psychoactive drugs (Allman 2010). In high performance liquid chromatography, instead of a solvent passing down the column by the force of gravity, the solvent is pumped at high pressures reaching 400 atmospheres rendering the separation process to be much faster. HPLC also allows the scientist to use smaller particles to be packed in the column material thereby providing a much higher surface area to volume ratio for the interactions between the moving molecules and the stationary phase giving a better separation of the components of the chemical sample. The detection methods for HPLC are improved compared to other chromatographic techniques as they are automated and highly sensitive (Clark 2007). Depending on the relative polarity of the stationary phase and the solvent, there two methods of HPLC; Normal phase HPLC In normal phase HPLC, the column is packed with small sized silica particles and the solvent should be non – polar (like hexane). The column used is of length 150-250 millimeter and an internal diameter of 4.6 millimeter (or less). Polar components of the mixture flowing through the column will stick longer to the polar silica than the non – polar components. Hence the non – polar components will move faster through the column. The long n-alkanes will separate faster in the chromatogram compared to short n- alkanes. Reversed phase HPLC In reversed phase HPLC, the size of the column is similar to the normal phase HPLC except that the silica is attached to long hydrocarbon chains (8 or 18 carbons) to the surface to make it non- polar. A polar solvent such as a mixture of alcohol and water is used. Consequently, there will be a strong attraction between the polar molecules in the sample flowing through the column and the polar solvent. The attraction will be absent between the polar molecule in solvent and the silica attached with hydrocarbon chains. Therefore, polar molecules in the sample will spend a longer time duration moving with the mobile phase (solvent). Non- polar molecules in the chemical mixture will form attractions with the hydrocarbon groups due to the van der Waals dispersion forces and they will be less soluble in the solvent due to the necessity to break the hydrogen bond as the force their way between the solvent molecules (for example methanol). They will therefore spend minimal time in the solvent slowing down their rate through the column. In this case, polar molecules will travel more quickly down the column (Clark 2007). Hence the short n-alkanes will separate faster in the chromatogram compared to long n- alkanes. Figure 3: Flow scheme for High performance liquid chromatography (Clark 2007) As the operating conditions of the HPLC require high pressures, a loop injector is used to introduce the sample. The point where the sample is injected is different from the solvent and a syringe with a capacity or loop injector is used to introduce the sample. The extra sample than needed exits via the waste line indicated in the figure above. As the sample is pumped through the column, separation takes place. Several detectors are used to monitor HPLC separations including UV detectors and electrochemical detectors which have excellent detection limits. HPLC is used in the analysis of environmental, industrial, pharmaceutical, clinical and consumer products (Harvey 2000, p. 584). Compared to gas chromatography, HPLC has only slightly different in accuracy mode of operation, precision, selectivity, precision and cost, time and the necessary equipment. However, the injection volumes in HPLC are usually higher compared to GC due to the larger volume of HPLC columns. Precision in HPLC is higher due to the use of loop injectors. HPLC is used to analyze a range of analytes because GC is limited to volatile analytes. On the other hand, the theoretical plates are more for GC giving a higher resolution compared to HPLC for complex mixtures (Harvey 2000, p. 589). Despite chromatography being useful in separating complex mixtures, many uncommon components of a sample cannot be readily identified from a chromatogram alone. Mass spectroscopy on the other hand can be used to identify enumerable components but it becomes difficult to discern them from a complex mixture. As such, the physical coupling of HPLC to MS and GC to MS allows for these weaknesses to be compensated for (Wren 2009). Using the mass spectrometer as the detectors improves detection limit to between 25 femtograms and 100 picograms. References Allman, R 2010, Analytical techniques: Chromatography, Wave signal, accessed on 20 March 2013, Clark, J 2007, Gas-Liquid Chromatography, Chemguide, accessed on 20 March 2013, Clark, J 2007, High Performance Liquid Chromatography, Chemguide, accessed on 20 March 2013, Harvey, D 2000, Modern Analytical Chemistry, e – book, accessed on 21 March 2013, http://www.chemmsu.ru/download/2kurs/analitika/ModernAnalyticChemistry.pdf Sheffield Hallam University, n.d., Chromatography, Sheffield Hallam University, accessed on 20 March 2013, Wren, C 2009, High Performance Liquid Chromatography and Gas Chromatography Coupling with Mass Spectrometry, Metro State Atheists, accessed 20 March 2013, Read More