Aastha Ahuja et al. /International Journal of Chemical and Analytical Science 2010, 1(9),205-207 Review Article Available online through ISSN: 0976-1209 www.ijcas.info Advances In HPLC Column Packing *Aastha Ahuja, Rahul Chib, Mukul Sonker, Geet Sethi, Shweta Gupta , Anju Hooda Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), University of Delhi Pushp vihar, sector-3, New Delhi-110017

Received on: 20-05-2010; Revised on: 16-06-2010; Accepted on:15-07-2010 ABSTRACT High Performance Liquid Chromatography (HPLC) is a highly improved form of column chromatography in which instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres(500-5000 p.s.i). The sensitivity and range of the technique depends on the choice of column and on the efficiency of the overall system. Column technology has seen great developments over the years. The transition from large porous particles and pellicular materials to small porous particles occurred in the early 1970s, when micro particulate silica gel (10 -mm dp) came into light and appropriate packing methods were developed. Monolithic columns are a promising alternative to packed columns in the future but much of the research is yet to be done. The use of CAPILLARY COLUMNS (4 mm) has increased in recent years, in part because small-diameter columns use much less solvent and provide higher sensitivity. While speaking of the future of packaging development, silica gel with chemically bonded phases will be around for a long time. The field is a promising one with great scope for research and development.

Keywords: HPLC, column packing, Perfusion chromatography,Monoliths.

INTRODUCTION HPLC was developed in the late 1960s and early 1970s. Today, it is widely not adsorbed elutes faster. Thus, the adsorption decides the retention time of the applied for separations and purifications in a variety of areas including phar- molecule. After passing through the column the mobile phase enters into the maceuticals, biotechnology, environmental, polymer and food industries. [1] HPLC detector attached to the printer, where the molecule is detected and print is has over the past decade become the method of choice for the analysis of a wide taken for graphical separation of the molecule. Column technology has devel- variety of compounds. The main advantage of HPLC over GC is that the oped alongside the development of HPLC instrumentation the new “HPLC” analytes need not necessarily be volatile, so macromolecules are suitable for instruments could develop up to 6,000psi (400 bar) of pressure, and included HPLC analysis. [2] improved detectors and columns. With continued advances in performance, the name was changed to HPLC from liquid chromatography (LC). Today, trace Principle: concentrations of compounds, as low as “parts per trillion” (ppt), are easily In isocratic HPLC the analyte is forced through a column of the stationary obtained. [4] phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the History Of HPLC column. The sample to be analyzed is introduced in a small volume to the stream Very few reliable chromatographic methods were available to the scientists, of mobile phase and is retarded by specific chemical or physical interactions before the 1970’s. During 1970’s, a number of techniques including open-col- with the stationary phase as it traverses the length of the column. Nature of the umn chromatography, paper chromatography, and thin-layer chromatography, analyte, stationary phase and mobile phase composition accounts for the retar- were used to carry out chemical separations. But these were however inadequate dation. The time at which a specific analyte elutes (comes out of the end of the for quantifying compounds and for resolution between similar compounds. Be- column) is called the retention time and is considered a reasonably unique fore the advent of HPLC, low-pressure, gravity-fed columns with large, irregu- identifying characteristic of a given analyte. Pressure increases the linear veloc- larly shaped porous particles such as silica gel or alumina were frequently used. ity (speed) of the component providing it less time to diffuse within the column, And, because of their high surface area, these porous particles had excellent which leads to improved resolution in the resulting chromatogram. Any miscible sample capacity. However, these large particle sizes gave poor column effi- combinations of water or various organic liquids (the most common are metha- ciency. Thus, Pressure liquid chromatography began to be used, during this time, nol and acetonitrile) serves as the solvent system. Water may contain buffers or to decrease flow through time, thereby reducing purification time of compounds salts to assist in the separation of the analyte components, or compounds such being isolated by column chromatography. as Trifluoroacetic acid which acts as an ion pairing agent In spite of being considered somewhat mature, new developments in HPLC still continue. There Flow rates still continued to remain inconsistent. HPLC was developed in the have been improvements in column construction, packing material design, mid- 1970’s and made better with the development of column packing materials bonded phase chemistry and formats. In addition to this, new phases have and the convenience of on-line detectors. In late 1970’s, new methods including extended the pH range (high and low), providing it more versatility. [3] reverse phase liquid chromatography became popular. It made separation be- tween very similar compounds easier. HPLC was frequently used for the separa- Instrumentation: tion of chemical compounds, by the 1980’s. The new techniques made separa- The heart of a HPLC system is the column. The column contains the particle tion, identification, purification and quantification much easier, better and re- that contains the Stationary phase. Pump forces the mobile phase to pass producible. Computers and automation further added to the convenience of through the column and injector injects the mixture to be separated into the HPLC. flowing mobile phase. Molecules that are adsorbed maximum by stationary phase are eluted slowest through the column, whereas the molecules which are The last decade saw vast improvements in the form of development of micro- columns, and other specialized columns. The dimensions of the typical HPLC column are: 250 mm in length with an internal diameter between 3-5 mm and *Corresponding author. that of micro-columns, or capillary columns, ranges from 3 µm to 200 µm. Fast Aastha Ahuja HPLC makes use of a column that is shorter than the characteristic column, Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), with a length of about 3 mm, and they are packed with smaller particles. [5, 6] University of Delhi,Pushp vihar, sector-3, New Delhi-110017 Tel.: + 91-9899430775 E-mail:[email protected]

International Journal of Chemical and Analytical Science Vol.1.Issue 9.September 2010 205-207 Aastha Ahuja et al. /International Journal of Chemical and Analytical Science 2010, 1(9),205-207 Development In Porous Packings Comparison of current packing types in HPLC Porous packings have been in favor throughout the history of HPLC. The transition from large porous particles and pellicular materials to small porous c d particles occurred in the early 1970s, when micro particulate silica gel (10 -mm dp) came in the scene and appropriate packing methods were developed. Until Diffusive pore spherical materials were developed and perfected, till the 1970s irregularly shaped micro particulate packings were in vogue. The spherical packings had Through pore several advantages over their predecessors, such as; these could be packed more homogeneously, gave better efficiencies, and could be manufactured in higher purity. [7] Totally porous particle Perfusion particle The major surface area of the particle is contained within the diffusive pores Comparison of current packing types in HPLC which dominate the typical porous packing (Figure-1). In a porous particle, solutes get transferred from the moving mobile phase, outside of the particles, Thin porous e Porous f into the stagnant mobile phase within the pores, to interact with the stationary Shell phase. Particle size reduction improves both the interparticle mass transfer as layer well as the intraparticle mass transfer. After this interaction, the solute molecule must diffuse out of the particle and continue its journey down the column. Such a mass transfer takes place many thousands of times as the differential separa- tion process advances and the solute is eluted from the column. The mobile phase in which the solute was originally present, moves down the column ahead Poroshell particle of the solute, while the solute spends its time in the diffusive pores. This slow Non -porous silica (NPS) rate of mass transfer into and out of the porous particle is a major source of band or nonporous resin (NPR) broadening in HPLC. The advantage of using smaller particles is that they Fig.1. Pictorial comparison of liquid chromatorgraphy column packing shorten the path length of this diffusion process, improve mass transfer, and provide better efficiency. But however, congruent with the improvement in a. Large, totally, porous particles (100 +m m) (normally irregular shaped) efficiency was the decrease in column permeability; that is, an increase in b. Superficially porous particle (50 m m) ( also called as pellicular or porous layer bead column backpressure. The increase in pressure is proportional to the inverse of c. Microparticulate totally porous particle (5 m m) the square of the diameter of the particle. Thus, halving particle diameter will d. Perfusion particle (12 m m) increase the column head pressure by a factor of four. A current trend in HPLC e. Non-porous silica (NPS) or nonporous resin (NPR) for high-throughput separations is to use shorter columns with smaller particles f. Poroshell particle (5 m m) (3- or 3.5-mm particles in a 50 or 20 mm x 3 4.6 mm column) rather than Nonporous and Superficially Porous Packing longer columns with larger particles (5-mm particles in a 150–250 mm x 3 4.6 The rates of mass transfer can be improved by the use of nonporous packing. mm column).Separation time is directly proportional to length of column, thus There are two types of nonporous packing-1) nonporous silica 2) nonporous shortening the column results in faster separations. [8] resin. The nonporous packing reminds of the older pellicular or porous layer beads. However, they are of much smaller particle size. Faster rates of mass Perfusion Chromatography transfer and separations of only a few minutes can be achieved for both large Perfusion chromatography is a technique arised to overcome the problem asso- and small molecules using the nonporous layer. The thin layer of stationary ciated with mass transfer in the separation of large molecules such as proteins by phase limits the capacity of the packing and makes the nonporous silica and HPLC. Perfusion chromatography enables the assessment of protein composi- nonporous resin unsuitable for preparative separations. The backpressure from tion of a foodstuff at sufficient speed and low cost to be suitable in routine the nonporous silica columns are generally much greater than those experi- analysis. [9] Perfusion media are available in different chromatographic modes: enced with micro particulate HPLC porous packing of popular particle sizes reversed-phase, ion-exchange, hydrophobic interaction, and affinity. [10] (that is, 5- and 3-mm) as a result of their small particulate sizes and hence are not commonly used. Superficially porous packing is similar to the nonporous silica particles except for the fact that the particle size is larger, about 5 mm Perfusion media are constituted by two set of pores: in diameter, providing a much lower pressure drop. They provide increased A) Through pores (6000-8000). The through - pores allow mobile phase to sample capacity as the surface area is larger (4–6 micro metre/g) than the pass through the packing itself, thereby increasing the rate of mass transfer in nonporous silica. These Poroshell particles are recommended for larger the mobile phase. Instead of predominantly flowing around the particle, a biomolecules that diffuse slowly into porous packing. The biomolecule peaks portion of the mobile phase flows through the particle, there by allowing the broaden due to slow diffusion into and out of the pores when flow rates are solute to spend less time undergoing the mass transfer process and giving nar- increased with porous packing. The thin layer of stationary phase is derivatized rower peaks. The process is actually a combination of diffusion and convection with alkyl bonded moieties such as C3, C8, and C18, providing separations of proteins by reversed - phase chromatography. The Poroshell type packings B) Diffusive pores (800-1500 A) which enable better access of macromol- provide good recovery of biomolecules. [11] ecules to the inside of the particle by the combination of convective and diffusive flow. The diffusive pores are the same type present in the porous Monolithic Columns particles and provide the sorption capacity A monolith can be described as a single large “particle” without interparticular voids. Monolithic columns are a promising alternative to packed columns in the future. The rapid emergence of monolithic-type HPLC columns, both in Comparison of Early packing types in LC normal-bore and capillary - bore sizes, is changing the dimension format of a b stationary phases. [12]

All the mobile phase flows through the stationary phase greatly accelerating the rate of mass transfer. In contrast to diffusion, which is the typical driving force for mass transfer within the pores of particulate stationary phases, this convective flow through the pores enables a substantial increase in the separa- tion speed of large molecules such as proteins. Monolithic columns are a relatively recent format of stationary phase for HPLC. Their unique proper- 50mg ties, like the ease of their preparation, the tolerance to high flow-rates, and Totally porous particle Superficially porous particle the rapid speed of chromatographic separations that can be achieved at ac-

International Journal of Chemical and Analytical Science Vol.1.Issue 9.September 2010 205-207 Aastha Ahuja et al. /International Journal of Chemical and Analytical Science 2010, 1(9),205-207 ceptable back pressures, make the monolithic column format superior in some columns (.1 mm i.d.) are also used with conventional electrospray operating at applications to the more common columns packed with beads. As monoliths flow rates of a few microliters per minute The primary benefits of using are rather “young,” the number of different stationary phases, separation capillary columns in proteomic analysis is sensitivity – the ability to detect mechanisms and methods developed with them remains much smaller than extremely low levels of proteins. Because of the complexity of proteomic that available for packed columns. It is only a question of time until the range samples, often containing thousands of peptides, high resolution is required covered by monolithic technology will be extended and successfully compete together with very high sensitivity. [17] with all other well-established separation technologies. [13] Future Direction In Packing Development Advantages and Disadvantages of LC Columns Recent researches in packaging development have made it clear that silica gel with chemically bonded phases possess great potential and scope for develop- Columns packed with silica-based particles smaller than 2 µm: [14] ment in the near future. Silica forms the macromolecular substances which form the building materials of the matrices of most sorbents. They also Advantages consist of carbon and numerous organic natural or synthetic polymers (eg. · Columns of different length and internal diameter are avail- dextran. cellulose, polystyrene, polyvinylacetate, polyvinyl alcohol). They able. have excellent efficiency, rigidity, lower cost than alternatives and ability to · Short columns packed with 1.8-µm particles show excellent be functionalized to fit just about any HPLC mode. The use of smaller porous resolution and high plate numbers even for ultrafast separa- particles packed into short columns for simple sample mixtures is now a tions. common practice and shall continue to take place in the near future. The · Available column internal diameters and chemistries make them shorter column with smaller particles can provide the same resolution as that compatible With LC-MS. of a longer column with larger particles. Extra column effect, dwell volumes, and injection and detection volumes must match the narrow peak widths that Disadvantages are encountered so that band spreading does not occur are essential for opti- · Higher back pressure at increased flow rates. An appropriate par- mized results. [18, 19] ticle size distribution can contribute to reducing this effect. Columns packed with polymeric or silica-based monolithic station- REFERENCES ary Phases: [15] 1. R.B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149. 2. Y. Liu, M.L. Lee. “Ultrahigh pressure liquid chromatography using elevated temperature.” Journal of Chromatography. 2006; 1104 (1-2): 198–202. 3. K.K. Unger and E. Weber. “A Guide to Practical HPLC.” Git Verlag GMBH, Darmstadt, Advantages 1999, p.45. · Fast and ultrafast LC at high flow rates and low back pressures. 4. J.J. Bergh, J.C. Breytenbach. “Stability-indicating High-performance Liquid- chromato- · Long columns (~ 100 mm) can be used due to lower back pressure. graphic Analysis of Trimethoprim in Pharmaceuticals.” J. Chromatogr. 1987; 387: 528-531. 5. P.B. Hamilton, Anal. Chem., 1960, 32(13), 1779-1781. · Comparable efficiency, resolution, and precision are achieved, as 6. J.J. DeStefano, T.J. Langlois, and J.J. Kirkland, J. Chrom. 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International Journal of Chemical and Analytical Science Vol.1.Issue 9.September 2010 205-207