Analytical Technique on High Performance Liquid Chromatography - Research Paper Example

Analytical Тесhniquе on High Реrfоrmаnсе Liquid Chromatography (НРLС) Your name Class name Name of University Abstract: High Performance Liquid Chromatography (HPLC) is a separation technique achieved by injecting a liquid sample into a solvent stream pumped through a chromatographic column (stationary phase). With the aim of providing auxiliary information in the discipline of pharmacology concerning the use of HPLC, this article reviews various aspects of HPLC such as principles, applications in pharmaceutical analysis, instrumentation, configuration, analytical methods such as such as isocratic, gradient, normal phase LC and reversed phase LC. It further explains the concept of efficiency and peak integration, and highlights the need of troubleshooting in the hardware mechanics and separation fundamentals so as to obtain reliable chromatographic data. Objectives: After reading this article review, the reader should be able to: i. Discuss the theory behind High Performance Liquid Chromatography (HPLC) separation. ii. Discuss the system components of HPLC. iii. Discuss the applications of HPLC in pharmaceutical analysis. iv. Discuss various analytical methods for HPLC. v. Discuss the concept of troubleshooting in the HPLC system. Methods: High Performance Liquid Chromatography (HPLC) technique depends on the degree of retention of various components in the column. Key Findings: HPLC is an important qualitative and quantitative tool in pharmaceutical analysis. Summary: High Performance Liquid Chromatography (HPLC) is the most versatile, fastest, safest, and dependable chromatographic technique for pharmaceutical quality control. Keywords: High-Perfomance Liquid Chromatography (HPLC); applications; system components; elution. INTRODUCTION High Performance Liquid Chromatography (also referred to as high pressure liquid chromatography, HPLC) involves the injection of a small sample dissolved in a solvent into a stream of solvent (mobile phase) pumped into a column packed with solid particles (stationary phase). It is generally used in biochemistry to identify, separate and quantify individual components of active compounds. [1] At the chromatographic column, the individual components of the sample in solution are moved down the column as the liquid streams through under pressure delivered by a pump. [2] The detector indicates the retention time (the time taken for the analyte to come out of the column) of the analyte. The retention time varies depending on the interactions between the solvents, analyte and the stationary phase. History of HPLC The use of chromatography to elicit separation can be traced back to a period between 1903 with its pioneer being a Russian botanist Michael Tswett. The technique was rediscovered by Richard Kuhn in 1930s. In 1941, Martin and Synge discovered liquid-liquid partition chromatography and subsequent paper chromatography by Consden in 1944 which revolutionized approaches to biochemical analysis.[3] According to Kirkland S and Glajch [4], Martin & Synge laid foundation for HPLC by pointing out that improved performance of liquid chromatography could be achieved by use of smaller particles and higher pressures. Major technical advances such as quality of analytical columns and the mobile phase have resulted in much reduced elution time saving both time and solvent and have perhaps contributed to the popularization of HPLC. Pump technology has advanced and can deliver the solvent accurately over a broad range of flow rates. [2] Such advances among others have allowed HPLC to become more accessible as an analytical tool in chromatographic analysis. Applications of HPLC in Pharmaceutical Analysis There are a number of applications in pharmaceutical analysis for the creation of a new drug. This ranges from initial drug discovery to the manufacture of products that will be administered to patients. [5] The first step (drug discovery) involves the selection of the disease which the drug will treat. The decision will be based on financial considerations and unmet medical needs. The second step is the identification of a suitable drug target. [2] Drug targets are the areas in body cells such as proteins, nucleic acids, carbohydrates and lipids macromolecules where the drug attaches itself. Once the potential drug target has been identified the challenge remaining is to find a chemical which will interact with the target. A bioassay is required in evaluating the effect of chemicals on the target. [6] This test checks whether the desired interaction between the drug and the target takes place. Vistro tests, on the other hand, involve special cell, enzymes or tissues. The tests are performed and designed in a way that produces an easily measurable effect when interaction occurs. [4] This can either be an enzyme catalyzed reaction that results in a color change or a cell growth. High throughput screening (HTS) refers to the automated testing of large compounds over a large number of targets. For instance, several thousand compounds can be tested in 30-50 biochemical tests at once. [7] Compounds screened in this method might be from an existing compound library (i.e. a collection of compounds synthesized by a pharmaceutical company over a number of years of research). Alternatively, they might be sourced from combinatorial synthesis. This involves a method of producing a large number of compounds in a short period of time by using a defined reaction rate and a large variety of starting reagents and materials. [4, 8] Usually the process is performed on a very small scale so as to allow for automation of the process. The screening results identify the hits (compounds active in the screens with the potential of being turned into drugs). These compounds are further analyzed and screened to reduce their number and end up with lead compounds (a structure that indicates a pharmacological activity that serves a useful starting point in the drug design). HPLC isolates and purifies the hits and lead compounds during a combinatorial synthesis. [2] Finally, the drug design involves the studying of the structure-activity relationships (SARs) through biological testing, once a lead compound is known. Drug design defines the regions of the lead compound of functional groups which are important to its biological activity. [9] The drug target identification is optimized by synthesizing and testing a range of products similar to the lead compounds in a process referred to as pharmacodynamics. METHODS HPLC System Components A typical configuration of a HPLC system consists of a mobile phase reservoirs, degasser, a pump (solvent management system), injector (sample management system), column compartment, detector, data processor and waste. [2] The diagram below describes the system. A- Degasser B- Pump C- Injector D- Column compartment E- Detector F- Data processor G- Waste H- Mobile phase reservoir Fig 1. Configuration of a typical HPLC system. RETRIEVED from Mc Poolon 2009, An Introduction to HPLC for Pharmaceutical Analysis. Mobile Phase reservoir This consists of plastic coated glass containers of an appropriate size to contain enough mobile phase for the analysis being carried out. Fitted with a filter at the point of contact with the mobile phase, PTFE tubing connects the contents of the reservoir with the HPLC system. [10] The purpose of the filter is to remove any particulate matter or un-dissolved material and as well as act as a ‘sinker’ to hold the tubing at the bottom. For HPLC, the mobile phase consists of a stream of liquid that is forced through the chromatographic column (stationary phase) and carries through with it the analyte. Tayler and Francis [11] argue that what the mobile phase consists of depends on both the nature of the compounds being analyzed and the stationary phase. Degasser This component is necessary in HPLC systems to help eliminate gases dissolved in solvents. Solvents might have some quantities of oxygen dissolved in them and this oxygen can be discharged in the form of very tiny bubbles in the system with a drop of pressure between the detector and the column or when a solvent with high solubility for oxygen is dissolved in another solvent with low oxygen solubility [2, 11] Pressure fluctuations due to these gas bubbles might be realized when the mixing is done before the pump. Dissolved gases in the mobile phase can influence both the injected volume when small sample volumes are injected and also perturb the reading of detectors. [12] This can be curbed by use of degasser apparatus. The solvent is forced down a piece of special polymeric tubing that is placed in a vacuum chamber (chromatographic column). With its selective permeability, the tubing material (membrane), the vacuum created by a pump reduces the content of the gases from the solvent. Pump The pump delivers a continual solvent stream through the injector, column and detector. The pressure pumps generate high pressure that is needed to move the mobile phase through the chromatographic column by overcoming its resistance. [10, 12] The flow of the solvent is characterized by the volumetric flow rate, u. The pumping parameters can, however, vary depending on the purpose of the analysis but the volumetric flow rate, u must be constant or with very small fluctuations. Injector In this HPLC system, the role of the injector includes the addition of small, precisely measured volume of a solution that contains the sample through the mobile phase. Moldoveanu and David [13] argue that the injection must be done accurately and reproducibly. The nature of the solvent for the sample and the injection volume are the two significant parameters which must be considered by the operator in regards to injection. The solvent choice should be one that completely dissolves the sample and as well be soluble in the mobile phase. [13] Injector systems can be automated (computer-controlled auto-samples) to the extent of selecting the desired sample from a ray, and repeating the injection or upon receiving an electrical signal from the computer or at a specified time interval. [14] Column compartment With the design to perform separations in HPLC, the chromatographic columns are comprised of a tube made of stainless steel metal or PEEK plastic. The tube is filled with a stationary phase and for the stationary phase to be kept from moving, special frits are placed at the ends inside the column to perform the function. [7-9] The temperature of the column and the mobile phase should be maintained steady during an analysis. However, to achieve better efficiency, most separations are done at ambient temperature but the chromatographic column is heated using a heating block, a water bath or a column oven. Detector In HPLC, detectors are used to measure the concentration of eluting analytes. This is done through the monitoring of one of their inherent behavior for instance UV absorbance. [8] Detectors can either be specific to particular classes of analytes or universal to all analytes. The Ultraviolet/visible (UV/vis) absorbance detector is used in pharmaceutical analysis to monitor the absorption of visible or UV light in the HPLC eluent. In some cases electrochemical detectors, evaporative light-scattering detectors (ELSD), differential refractometers (RI), fluorescence spectrophotometers, mass spectrometers (MS), charged aerosol detectors (CAD), or other special detectors may be used. [15] The absorbance detector for ultraviolet/visible (UV/vis) has a monochromater (deuterium lamp and a small flow cell. The monochromater selects specific wavelengths that can pass through the exit slit. [7, 13] The principle for this detection is Beer’s Law which states that: Absorbance (A) =Molar absorptivity (e) *path length (b) * concentration Meyer [16] defines absorbance as “the negative logarithm of transmittance, which is equal to the ratio of transmitted light intensity and the incident light intensity.” Data processor The data acquisition computer receives signals from the detector and records them on electronic integrators or strip chart recorders. These strip chart recorders and the electronic integrators vary in their complexity and in the ability to store, process, and reprocess chromatographic data. Data acquisition computer not only controls the HPLC instrument modules, but also it uses the signal received to determine retention time (time of elution), for qualitative analysis (sample components) and quantitative analysis (amount of sample) [4]. Basically, it converts data from the detector into meaningful results. Waste collection This involves the collection of the liquid waste. Separation mode HPLC has not only a variety of configurations but also separation modes. Depending on the choice of operation, the HPLC system can either be operated in isocratic mode or gradient elution (GE). [16] Isocratic mode- this involves the use of isocratic pump that constantly delivers the composition of the mobile phase down the chromatographic run. [9, 17] This mode of separation best suits a routine work that involves the analysis of few components. This allows both the quantification and identification of each component in the analyte. In this case, the solvents have to be pre-mixed. Compared to Gradient elution, isocratic mode is generally preferred due to its simplicity and better precision. Gradient elution (GE), on the other hand, involves a gradient pump that delivers a variable composition of the mobile phase within the chromatographic run time [5, 9}. Gradient elution involves the use of a weak eluting solvent at the start of the separation and adding an increasing amount of a strong eluting solvent during the chromatographic run time. GE-HPLC improves separation efficiency and is preferred in separating complex mixtures whose polarity varies. It can also be used as an experiment in finding an appropriate mobile phase to use in isocratic separation DISCUSSION The column and the solvent Based on the polarity of the liquid chromatographic phases (mobile and stationary), there are two other LC techniques which include Normal phase (NP) and reversed phase (RP) liquid chromatography. [10, 18] Both normal phase and reversed phase liquid chromatography analysis can utilize gradient eluent of isocratic separation modes. Separation using one of the four techniques depend on a number of variables especially the scope of analysis, the chemical structure and number of compounds to be separated. For normal phase HPLC, the chromatographic column is packed with tiny silica particles. The solvent used in this case is non-polar e.g. non polar-hexane. [10] Polar compounds contained in the mixture will stick longer to the polar silica when passed through the column than non-polar compounds will. Therefore, non-polar compounds in the mixture will pass more quickly through the column than the polar ones. On the other hand, the silica in the column for reversed phase HPLC is modified to make it non-polar. This is made possible by attaching a long hydrocarbon chains to their surface. In this case, a polar solvent such as methanol (a mixture of water and alcohol) is used. [10] The polar solvent will strongly attract polar molecules in the sample dissolved in the solvent that is being forced down the column. Consequently, there will be no much attraction between the polar molecules in the sample solution and the stationary phase (the hydrocarbon chains attached to the silica). Therefore, polar molecules in the mixture will take more time moving with the solvent. On the contrary, non-polar molecules will attract with the hydrocarbon groups due to the van der waals forces of dispersion. [13] Non-polar molecules will take less time in the liquid dissolved in the solvent with the low solubility since there is a need to break hydrogen (H-H) bonds in attempts to squeeze in between the polar molecules. [24] Therefore, the polar molecules will travel more quickly through the column. Solvent and modifier type Most separations in reversed phase (RP) liquid chromatography use chemically modified silica as the partition mechanisms in the stationary phase. The surface of the support is reacted with a variety of reagents in order to produce bonded derivatives that carry active sites on the surface. [19] The nature of this bonded phase determines the separation properties of the chromatographic analysis. The table below shows the commonly bonded stationary phases. Common bonded phases octyl Si-(CH2)7-CH3 C8 cyanopropyl Si-(CH2)3-CN CN phenyl Si-(CH2)3-C6H5 C6H5 octadecyl Si-(CH2)17-CH3 C18 aminopropyl Si-(CH2)3-NH2 NH2 diol Si-(CH2)3-OCH(OH)-CH2-OH   Efficiency (N) Winder [20] defines efficiency of a chromatographic column as the number of theoretical plates (N), mathematically expressed as where tR is the retention time of the solute, Wh is the width of the peak determined at half peak height. Both tR and Wh are measured in the same units. Efficiency can also be expressed per metre (N') i.e. , where l is the length of column in metres. A column is efficient if the lateral diffusion of solute bands is restricted during their column residence time. [22, 23] Basically, the narrower the peaks are, the better the separation from the neighboring peaks and the more accurately they can be measured. [10] Measuring the column performance in terms of peak skewness or asymmetry, efficiency, and peak resolution are key system suitability tests. However, it should be noted that efficiency should never be used as a system suitability criterion in gradient elution. Peak Integration Peak intensity in MR spectra can be measured using peak integration, peak fitting and basis spectrum fitting. Stagg and Rothman [21] define peak integration as method used to obtain precise values for the concentration of sample components. It is normally performed in the frequency domain (the domain in which the spectral peaks are observed). The strength of peak integration is its ease of implementation and simplicity. However, it has drawbacks such as failure to effectively separate the contributions from overlapping peaks especially in vivo MRS [2, 7-9, 21]. Further, peak integration measurements are influenced by errors in the phase of spectrum and the choice of the frequency range over which the peaks are being integrated. According to Fourier transformation [26], the area of a signal in the frequency domain is exactly equal to the amplitude of the first point of the same signal in the time domain. In a case of a spectrum containing multiple peaks, the amplitude of the first point in the free induction decay is equivalent to the sum of the peak areas in the spectrum and thus cannot be used to estimate the area under individual peaks. [3, 5] HPLC troubleshooting Often, desirable results might not be obtained in a HPLC analysis maybe due to the instrument (i.e. hardware used such as pumps, auto-samplers, detectors and columns) or separation procedures. Understanding the hardware mechanics and separation fundamentals, therefore, becomes crucial for a reliable chromatographic data. [11, 25] Introducing the concept of resolution in HPLC is another key concept that ensures reliability of the data. A resolution between two peaks requires proper retention factor (a function of the mobile phase strength, enough efficiency (a function of the column and packing dimensions), the correct selectivity (chemical interaction provided by the mobile phase and the stationary phase). [23] CONCLUSION It can be concluded that HPLC is the most widely used analytical method in the pharmaceutical industry, both quantitatively and qualitatively. In the pharmaceutical analysis laboratories, HPLC is used for checking the purity of new drug candidates, quality control of final drug products, identification of active components of dosage forms, scaling up of synthetic procedures, monitoring changes, and in-process testing for developing new formulation. It identifies, separates and quantifies individual components of active compounds. This is done at the chromatographic column where the individual components of the sample in solution are moved down the column as the liquid streams through under pressure delivered by a pump. The retention time for different analytes recorded by the detector is used for various analyses. With improved technology, the modern computer based data stations with large storage capacity collects, processes and can even store data for possible future processing. This entire review shows how versatile, reproducible HPLC chromatographic technique is in the estimation of drug products and their components. REFERENCES 1Malviya R, Bansal V, Pal OP and Sharma PK. High Performance Liquid Chromatography: A Short Review. Journal of Global Pharma Technology. 2010; 2(5): 22-26. 2McPolin O. An Introduction to HPLC for Pharmaceutical Analysis. Northern Ireland: Mourne Training Services; 2009. 3Jandera P. 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International Journal of Pharmaceutical Compounding. 2004 May/June; 8(3): 223-227. 24 Moffat AC, Osselton MD, Widdop B. Clarke’s Analysis of Drugs and Poisons. London: Pharmaceutical Press; 2004. 25Pungor E. A Practical Guide to Instrumental Analysis. Boca Raton: CRC Press; 1995. 26Simpson CF. Practical High-Performance Liquid Chromatography. London: Heyden and Son; 1976. Read More