Advancements to allow fast HPLC - Essay Example

Advancements to Allow Fast HPLC Contents Introduction Advancements: Fast HPLC Monolithic Columns High Pressure Operation HTLC Column Classification Conclusion References Introduction One of the major domains of chemistry that promises lots of advancements due to extensive research is Analytical Chemistry. Analytical Chemistry is a branch of chemistry that deals with the analysis of the chemical nature of matter. Analysis of chemical composition of matter is the supporting pillar to the development of any new product or structure. The collective term used for the various laboratory procedures available to separate matter into its constituent compositions to analyze their chemical nature is known as Chromatography. High Performance Liquid Chromatography (HPLC) is a type of Column Chromatography, which is used to separate a mixture into its constituent components with the use of chemical interactions. A series of chemical interactions are used in between the substance of subject and the chromatography column. Although the concept of Liquid Chromatography dates back to 1940s, the path breaking research, in the field of HPLC is being carried out on a large scale even today. More samples of matter are being analyzed for their chemical behavior than ever before in the recent times. Chemists, researchers and pharmaceutical companies need to perform chromatography on large quantities of samples. The call of the day is to speed up the process of HPLC while at the same time not forsake its accuracy. The solution is to speed up the process of HPLC by a magnitude. This process known as Fast HPLC is a boon to all people and organizations concerned. The most common principle behind Fast HPLC is rapid analysis using short columns. The particles filled in the columns are also short. Various developments are taking place in the HPLC technology to aid Fast HPLC and in all cases it is observed that Fast HPLC operate nearly five times faster while at the same time not sacrificing performance, reliability and simplicity. This report aims to enumerate the various developments that are being carried out in the field of HPLC that aid Fast HPLC. The HPLC process is described with relevant terms. Several research works being carried out are explained with the intended purpose of the research. Particular topics covered in this report include advancements in monolithic columns, small particle columns and high pressure and high temperature modes and relevant developments in other methods. The report aims at understanding the advantages brought about by these advancements in the field of HPLC. Specific concentration of the report is the developments in the field of columns used in HPLC. An analysis is made as to how, advances in the columns speeds up the process of chromatography. The applications of these developments are enumerated and an analysis is provided as to how the modern developments have changed the way the process is conducted. [1][2][3] Etc. The Process The performance of HPLC is affected by a number of factors. The most important among the factors are 'Column Efficiency', 'Performance of Mobile Phase', 'Performance of Stationary Phase', 'Injectors', 'Pumps', 'Detectors', 'Columns and Column Packing' and 'Automation'. Advancements in several of these factors have led to the development of Fast HPLC. Advancements: Fast HPLC Monolithic columns: A method popularly used as a separation media for liquid chromatography has been that of monolithic stationary phases and columns. These have not taken much time to become popular however some of their features have yet to be clearly studied. Researchers comment that chromatographic behavior of these columns should be studied along with their physico-chemical and structural properties to attain progress in their design and production. Recently a technique known as fast high performance LC is becoming popular for laboratory. Here shorter columns that have higher flow rates are used thereby analysis time is considerably reduced. An implied result of this had been the fact that in order to maintain efficiency, shorter columns require smaller particles. An issue here is that smaller particles increase backpressure. This fact puts a restriction on the allowable column length and flow rates for LC pumps. Monolithic LC columns have eliminated this problem. These columns consist of rod shaped porous network along with a bi-modal pore distribution. The format of HPLC column typically includes dimensional and morphological properties of separation tube and stationary phase with in. As cylindrical geometry simplifies models and calculations the tubes are always cylindrical. An added advantage to this is that a wide range of cylindrical tubing from different materials is readily available. However formats are taking change and rectangular, square and other perimeter shape models are expected to become widely used. Initially there was a controversy regarding irregularly shaped or spherical particles. This resulted in change of morphology of stationary phases. Nowadays chromatographers use spherical particles that are characterized by its diameter, pore size, volume and surface area. Experiments have been successful by reducing the particle diameter from around 50-60 micrometers to 3-3.2micrometers. A recent bookmark has been use of still smaller particles (that is sub-2-micrometers). A new kind of monolithic columns known as silica and organic polymer based monolithic HPLC columns have come up commercially and they made the term particle size obsolete. Below we find the tabular representation for HPLC columns in cylindrical formats [6] Description Dimensions (internal diameter) Typical flow rate Velocity 1-10mm/sec Open tabular LC < 25 micro meters 100mm >20ml/min A major factor for contributing advancements in this field has been development of LC-MS in drug discovery. This field needs mass analyzers that can provide more structural information at high sensitivity. It requires that maximum information be achieved from sample amounts that are taken. In proteomics, minute protein samples are available which have a reduction in column dimensions into sub-mm internal diameter. Manufacturers have taken technical issues like sub micro liter/min flow rates and sub-micro liter volume sample as challenge and came up with expected results by using very narrow Internal diameter connection capillaries that minimize extra column band spreading. Five compounds namely acetophenone, methylbenzoate, hexyl paraben, heptyl paraben and antracene are subjected to study their H-U curve characteristics. It was found that antracene showed a smooth and sharp hyperbola having a " k' " value of 0.84 and rest others had comparatively low HETP (mm) for smaller values of linear velocity. Heptyl paraben k'=0.65 showed an increase in HETP (mm) [6]. Others including acetophenone, methylbenzoate and hexyl paraben had k' values of 0.28,0.38 and 0.55 respectively. Changes are observed in the patterns of descriptive dimensions of columns, geometry and morphology. This is demanding a clean description about the new formats. Researchers anticipate that as the trend is for having diameter reduction, this monolithic column technology will dominate the stationary phases. As there might be a problem to manipulate and control particles in narrow Internal diameter capillaries, alternate solvent drive methods are likely to become important (Electric field assistance). Scalability based on operational demands is the factor that makes HPLC such a strong separation technique. In a range from micrometer internal diameter to "cm" Internal diameter, HPLC provides a uniform mechanism for easy adoption of parameters to separation problems. It requires much less effort than other techniques. In the year 2001 the commercial existence of monolithic reversed-phase (RP) silica columns has grabbed the crucial role of being the faster analysis mediums. This was carried out by higher flow rates with the help of typical packed columns. These monolithic columns are made up of monomers that are polymerized. This process results in a continuous porous rod instead of a packed bed built up of individual particles. This particular porous rod provides macropores (2 m) that flow out easily with least resistance. On the other hand, mesopores (13 nm) present excellent chromatographic performance. The various aspects of monolithic column preparation and characterization [1] and [2] have been dealt in detail by Tanaka et al. the most important task of reviewing the characterization and fundamental studies of the chromatographic behavior of monolithic columns used for RP-LC [3]has been carried out by Miyabe and Guiochon. Similarly the various tasks such as development, characterization, and several applications of silica-based monolithic columns [4] have been reviewed by Cabera. A report obtained by numerous authors over the efficiency in terms of plate height regarding the monolithic columns is that the columns are relatively flat with increasing linear velocity. But 5-m-particle columns unfortunately showed reduced efficiency. The increases in pressures were meek for monolithic columns. They also succeeded to fit in the ranges of operation for conventional pumps, though the flow rates were at 9 mL/min. As the back pressures were quite low they allowed the monolithic columns to be coupled in series as well as support higher range for flow rate programming. Certain issues due to the individual nature of monolithic column formation and the process to bind the outer polymer cladding have come up. The major concerns are reproducibility and robustness. These issues have been explored by several authors. Wu et al. found that the monoliths were far less retentive when compared to packed columns. Furthermore lower solvent strengths were required for comparable retention [5]. The experiments also proved that the reproducibility of columns was good. Nederkassel et al. described the transfer and robustness of pharmacopeial methods on Chromolith columns [6]. Gerber et al. also reported good separations with decreased analysis times. His experiments also proved good column stability [7]. In general all the researchers noted the loss of efficiency at high flow rates for larger molecules. This phenomenon was due to mass-transfer effects. There have been many studies regarding the characterization of monolithic columns. Tanaka [6] [7] gives a detailed discussion about the characterization of the monolithic columns along with its preparation details. The chromatographic behavior monolithic columns and its wide usage in RP-LC is well explained by Miyabi and Guichon [8] Cabera [9] gives an account of developments in characterization and its applications. In all the studies it was found that efficiency, which is measured in terms of plate height for monolithic columns, was a flatter curve when compared to 5-micrometer particle columns. Pressure changes for monolithic columns were in accordance to the acceptable ranges and supported flow rates up to 9ml/min. Few applications of monolithic silica are mentioned below: 1) Columns used: Monolithic silica columns with chemically bonded tert butyl carbamoylquinine. Stationary phase: Chiral anion exchanger selector. Purpose: Enantiomer separations. [9] Separation of suprofen was performed using the enatioselective silica rod type chiral phase and it took less than 10 minutes of total time for baseline separation. This is an indication of the sensitivity and reduced separation time by the monolithic silica material. In this process a comparative study of the chromatographic behavior of tertbutyl carbamoylquinine silica rod is done and the spectrum of chiral test components like N-derivative amino acids (DNB, AC, DNZ, Z amino acids) and for suprofen were obtained. 2) Simple and Comprehensive Two Dimensional (2D) Reversed-Phase HPLC Using Monolithic Silica Column [10] 2D separation using monolithic silica columns in reversed phase is done in three phases. 1) 1D effluent was directly injected into 2D HPLC for 28 sec and later 2 sec injection time was given. 2) Effluent of 1D was held by two six-port valves in an alternate fashion for about 30sec for a single 2D column for effective 2D HPLC without having to lose 1D effluent. 3) Two monolithic silica columns with a switching valve along with two sets of chromatographs were used to alternately work and to separate each fraction of 1D effluent with the 2D columns. Different results were obtained from these stationary phases that ultimately made 2D separation possible. These 2D HPLC uses the high efficiency of monolithic silica columns at high linear velocities. 3) Evaluation of a monolithic silica column operated in the hydrophilic interaction chromatography mode with evaporative light scattering detection for the separation and detection of counter-ions [11] In this process a monolithic silica column is operated in the hydrophilic interaction chromatography (HILIC) mode along with an evaporative light scattering detector (ELSD). The metals Lithium, sodium and potassium were the test counter-ions here. Chromatographic properties of this column were determined by changing various key parameters, such as pH, flow rate, buffer strength, acid and organic modifier. Upon increasing the organic content was from 60 to 90% (acetonitrile) for these cations, retention time went up on approximately seven fold. Buffer concentration and pH had no significant effect as the effect that was noticed with organic content change. Flow rates around to 5mL/min were used to perform counter-ion separations, which took less than 3min. The changes in retention, resolution, and peak shape were observed and an optimized method was found and it was later evaluated for linearity, reproducibility, and limit of detection (LOD) for sodium. Linearity, with an R2 value of 0.999 across the working-standard range was acceptable and a LOD of 0.1g/mL was computed. The accuracy of predicting the counter-ion was roughly 3% when salt content was corrected for potency. 4) New possibilities in ion chromatography using porous monolithic stationary-phase media [12] Porous monolithic stationary phase media has become widespread for HP separations of organic and inorganic ions. For cation and anion separations, commercially, silica monoliths and polymer-based monoliths are available. They are produced with coated Ionic sites. These have given way to a number of new approaches for Ion separation that include: 1) Low and medium pressure Ion chromatography (IC) 2) Ultra fast IC 3) Capillary IC 4) Multi column and Multi dimensional IC 5) Double gradients IC Small-particle columns with High temperature and high pressure: One of the areas in order to improve the overall productivity is particle size reduction. Researchers comment that use of small particles results in more efficient columns. It is also possible that smaller irregular particles would result in performance enhancement. Small particles were developed for HPLC based on the results of research work. 5 micrometer was for sometime standard particle diameter. After that high performance 3-micrometer particles were introduced. Recently Zorbex Rapid Resolution HT columns introduced the sub 2 micrometer. Very small particles have an advantage that keeping the plate count it can shorten the column. As separation time is proportional to column length, a faster separation is possible with smaller particles. Lab experiments prove that reduction in particle size results in flatter deemter curves at higher linear velocities. For instance 2-micrometer particle gives a flatter curve than a 5-micrometer column at high velocity. [19] Sub-2-m-particle columns have been tested for pharmaceutical analyses at pressures accessible by conventional instrumentation [13] Here a comparison of performance of a wide range of analytical columns in pharma domain is given: Zorbax Eclipse XDB-C18 (75 mm - 4.6 mm i.d., 3.5 m) and Zorbax Eclipse XDB-C18 (50 mm - 4.6 mm i.d., 1.8 m) that are known to be improved types of analytical columns have been tested to determine the presence of estradiol, which is the active substance, methylparaben, propylparaben which are preservatives and estrone which is the degradation product and those results are contrasted with the conventional C18 columns (250 mm - 3.0 mm i.d., 5.0 m). Particle size, column length and Octadecylsilica type are the parameters in which these Zorbax columns differ. Flow-rates up to about 2.5 ml min-1 that are considerably higher could be applied without having to worry much about the backpressure. Analysis time run was reduced to 3.5 min compared to 12 min obtained for the conventional C18 column. These data for Zorbax columns highlights the advantages of these columns for their practical use in pharmaceuticals, especially considering Analysis time and solvent consumption. High-pressure operation To better know the reason for opting high-pressure operation a comparison of high pressure and low-pressure operation is given below: High-pressure vs. low pressure [8] I. Low pressure a. First LC experiments done this way b. Large diameter column (>1 cm) c. Large particles (> 37 m) d. Mobile phase uses gravity feed or low-pressure pumping e. Low pressure f. Low resolution g. Mainly used for preparative scale separations today II. High pressure a. Small particles ( 10 m) b. Small diameters ( 4 mm) c. High pressures ( 5000 psi) d. High resolution or "high performance" e. Called high-performance liquid chromatography or HPLC f. Mainly used for analytical separations today According to the equation, [9] Where is the flow resistance factor, is the viscosity of the solvent, L is the length of the column, and dp is the diameter of the particles. It is clear that backpressure is inversely proportional to square of the particle diameter. As the recent developments tend to decrease the particle diameter in order to attain reduced analysis time, it should even consider the increased pressure that is resulted. Until recently there was a practical limitation of pressure that was around 600psi. However commercial systems come up with LC systems that can operate with pressure more than 10,000 psi. These also allow optimal column lengths to attain high efficiencies for chromatographic separations. These high-pressure systems are used for drug substance analysis. Lab experiments prove that this combination of reduced particle diameter, increased pressure and optimal column length would help achieve analysis time that is reduced in comparison to the same with large particles. This technique is nowadays widely used in pharmaceutical impurity analysis. The ultimate aim is to reduce the separation time for analysis. An application worth mentioning here is the study of Peak capacity in Ultra performance liquid chromatography (UPLC) Peak capacity in ultra performance liquid chromatography (UPLC) is measured using a commercial instrument with the specifications 2.1 mm Internal diameter and columns packed with1.7 m porous particles. Peak capacity, for a small molecule was measured as a function of gradient time, mobile phase flow rate, and column length. It was observed that, the highest peak capacity is obtained from a short column operating at high linear velocities, if fast analysis was considered. If there is a situation where an even higher peak capacity is required, then there is only one option left that is to opt for longer analysis time. This ultimately reaches to a point where switching to a longer column becomes the best approach. (Elsevier B.V, 2005) High-temperature liquid chromatography HTLC is performed at high temperatures. The advantage of high temperature is that diffusivity or mobility of molecules increases with increase in temperature. Also high temperature decreases viscosity and thereby backpressure and hence allows high flow rates. [11] Lab experiments proved that increase in temperature increases analyte diffusivity thereby reducing interface mass transfer, which reduce the analysis time by 10-20 folds. [9] However few problems concern this HTLC that include stationary phase stability at temperature higher than 150C and analyte instability at higher temperature. Commercial manufacturers came up with solutions like having thermally stable stationary phases. Even these approaches have a few side effects like thermal mismatch when the variation in temperature between effluent and stationary phase leads to peak broadening [5] and analyte degradation [6] where analyte gets spoiled. These issues are yet to be addressed however it offers many advantages in a few cases. Column Classification: There are two purposes for classifying the columns 1) Separation of an equivalent column is necessary as the conventional one is not available. 2) In order to attain semi-orthogonal conditions, a column having different selectivity is needed. In order to reduce time in the process of identifying desired columns, these characterization techniques are very useful. Systematic and Automated Method Development: Impurity separation in the pharmaceuticals Industry requires great deal of time and effort. In 1990's [23] high purity silica opened options to facilitate study of wide ranges of pH. As faster and robust methods for development were necessary, this led researchers to go for automated methods to study pH. Multiple solvents and Detectors on a single LC system were present here. Organic solvent like methanol and acetonitrile, Detectors like ELCD, MS and PDA were all on one LC system[24]. Conclusion: Advancements in HPLC are heading towards newer and quicker separations along with improved separation capability. The monolithic column, reduced particle diameter, high pressure-high temperature working operations, newer manufacturing products etc are the evidences for it. HTLC is not widely used but research is going on to eliminate the side effects and hence have the advantage of reduced analysis time. In the field of determination of drug impurities, a major trend in Liquid Chromatography is toward faster separations. The trend also focuses towards improved separation capability. This is indicated by applications of monolithic columns and short, sub-2-m columns that may be operated at high pressures. Much more efficient method development and exploration of a wide range of mobile phase pH and stationary phase in an automated fashion is possible with faster separation methods. The bottom line is that these advances in the field of HPLC will definitely lead to improved capability and quicker drug impurity profiling. These are definite bookmarks in the field of liquid chromatography. References (1) "HPLC: A Users Guide" www.pharm.uky.com Advances in hplc techno (2)Toshihiko Hanai, Yokohama "HPLC: Practical Guide" Health Research Foundation. [3] Willard, H.; Merritt, L.; Dean, J.A.; Settle Jr., F.A. Instrumental Methods of Analysis, 7th ed., Wadsworth Publishing Co., 1988 [4] Monolithic Silica Columns, James.D, Loyd, University of Alabama, Literature Seminar, www.bama.ua.edu/chem/seminars/student_seminars/spring03/papers-s03/Loyd-s03.pdf [5] Trends in HPLC Column formats.(Micro bore ,Nano bore, Smaller. Journal of Separation Science, Volume 27, Issue 10-11,July 2004,pages 853-873 [6] Preparation and application of monolithic beds in the separation of selected natural biologically important compounds, Journal of separation science 30 (1), pp 55-66 [7] Monolithic silica columns for high-efficiency chromatographic separations. Tanaka, N, kobayashi, H,Ishizuka,N,Minakuchi,H,Nakanishi,k,Hosoya,K,Ikegami. Journal of chromatography A, Volume 965, Issue 1-2, 2 August 2002,Pages 35-49. [8] Characterization of monolithic columns for HPLC, Miyabe, K,Guiochon, G. [9] Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations. Lubba, D, LINDER, w, Journal of chromatography A, VOLUME 1036,ISSUE 2, 21 MAY 2004, PAGES 135-143 ( D. Lubda and W. Lindner, J. Chromatogr., A 1036 (2004), p. 135. [10] Simple and Comprehensive Two Dimensional Reversed-Phase HPLC Using Monolithic Silica Column, Tanaka,N,Kimura,H,Tokuda,D, Volume 76,Issue 5, 1 March 2004,Pages 1273-1281. [11] Evaluation of a monolithic silica column operated in the hydrophilic interaction chromatography mode with evaporative light scattering detection for the separation and detection of counter-ions, Pack, B.W, Risley, D.S, JOURNAL OF CHROMATOGRAPHY, Volume 1073, Issue 1-2, 6 May 2005,pages 269-275 [12] New possibilities in ion chromatography using porous monolithic stationary-phase media, Paull,B,Nesterenko,P.N, Trac- Trends in Analytical chemistry, Volume 24,issue 4, aprill 2005,apges 295-303. [13] A comparison of performance of various analytical columns in pharmaceutical analysis, 2005,journal of chromatography A, 1088(1-2),pp.24-31 [14] A comparison of performance of various analytical columns in pharmaceutical analysis: Conventional C18 and high throughput C18 Zorbax columns, Noyao kova,L,solich,p, Journal of chromatography A, Volume 1088,Issue 1-2, 23 september 2005,pages 24-31. [15] Ultrahigh-Pressure Reversed-Phase Liquid Chromatography in Packed Capillary Columns, MacNair,J.E.Lewis,K.C.Jorgenson,J.W., Analytical chemistry, Volume 69,Issue 6,1997,Pages 983-989. [16] Recent developments in columns for UPLCa,,c:additional UPLCa,,c chemistries provide flexibility for methods development, Grumbach, E.S, wheat, T.E, MAZZEO, J.R. Volume 18, Issue 9 SPEC. ISS., September 2005, Pages 44+46 [17] Peak capacity in gradient ultra performance liquid chromatography(UPLC), Wren,S Volume 38, Issue 2, 15 June 2005, Pages 337-343.A.C. [18] A.D. Jerkovich, R. LoBrutto, R.V. Vivilecchia, LC-GC North America, Supplement, May 2005, 15. [19] J.D. Thompson and P.W. Carr, Anal. Chem. 72 (2000), p. 1253. [20] B. Yan, J. Zhao, J.S. Brown, J. Blackwell and P.W. Carr, Anal. Chem. 74 (2002), p. 1017. [21] Effect of tempreture in reversed phase liquid chromatography, Guillarme,D.Heinisch,S,Rocca,J.L, Volume 1052, Issue 1-2,15 October 2004,pages 39-51. [22] U.D. Neue, E.S. Grumbach, J.R. Mazzeo, K. VanTran and D.M. Wagrowski-Diehl In: I.D. Wilson, Editor, Bioanalytical Separations, Handbook of Analytical Separations Vol. 4, Elsevier, Amsterdam, The Netherlands (2003), p. 185. [23] B.D. Karcher, M.L. Davies, J.J. Venit and E.J. Delaney, Am. Pharm. Rev. 7 (2004), p. 62. [24] www.lcgceurope.com/lcgceurope/HPLC Read More