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High performance liquid chromatography
- 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Figure 1: HPLC instrument INTRODUCTION High-performance liquid chromatography (HPLC), is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column. HPLC has been used for manufacturing (e.g. during the production process of pharmaceutical and biological products), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and medical (e.g. detecting vitamin D levels in blood serum) purposes.
- 2. HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller sorbent particles (2–50 micrometer in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique. The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/VIS, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at. Figure 2: Principle of HPLC
- 3. TYPES OF HPLC Partition chromatography Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte molecules partition between a liquid stationary phase and the eluent. Just as in Hydrophilic Interaction Chromatography(HILIC; a sub-technique within HPLC), this method separates analytes based on differences in their polarity. HILIC most often uses a bonded polar stationary phase and a mobile phase made primarily of acetonitrile with water as the strong component. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences. HILIC bonded phases have the advantage of separating acidic, basic and neutral solutes in a single chromatographic run. The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time. The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups (e.g. hydroxyl-) and groups capable of hydrogen bonding inducing more retention. Coulombic (electrostatic) interactions can also increase retention. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times. Figure 3: Partition chromatography
- 4. NORMAL PHASE CHROMATOGRAPHY Also known as normal-phase HPLC (NP-HPLC) this method separates analytes based on their affinity for a polar stationary surface such as silica; hence it is based on analyte ability to engage in polar interactions (such as hydrogen bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. chloroform), and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on stearic factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers. Figure 4: Normal phase chromatography DISPLACEMENT CHROMATOGRAPHY The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities. There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution
- 5. chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentration. Figure 5: Displacement chromatography REVERSED PHASE CHROMATOGRAPHY Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is silica which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RP- HPLC to qualify drugs before their release. RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar water structure and plays the most important role in all processes in life science. RP-HPLC allows the measurement of these interactive forces. The binding of the
- 6. analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase. This solvophobic effect is dominated by the force of water for "cavity-reduction" around the analyte and the C18-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 7.3×10−6 J/cm², methanol: 2.2×10−6 J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis. Figure 6: Reverse phase chromatography SIZE EXCLUSION CHROMATOGRAPHY Size-exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography separates particles on the basis of molecular size (actually by a particle's Stokes radius). It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.
- 7. This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight heparins Figure 7: Size exclusion chromatography ION EXCHANGE CHROMATOGRAPHY In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on the column are excluded from binding, while solute ions of the opposite charge of the charged sites of the column are retained on the column. Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions (e.g. increasing the ion effect of the solvent system by increasing the salt concentration of the solution, increasing the column temperature, changing the pH of the solvent, etc.). Types of ion exchangers include: Polystyrene resins – These allow cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity. Cellulose and dextran ion exchangers (gels) – These possess larger pore sizes and low charge densities making them suitable for protein separation. Controlled-pore glass or porous silica In general, ion exchangers favor the binding of ions of higher charge and smaller radius.
- 8. An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. A decrease in pH reduces the retention time in cation exchange while an increase in pH reduces the retention time in anion exchange. By lowering the pH of the solvent in a cation exchange column, for instance, more hydrogen ions are available to compete for positions on the anionic stationary phase, thereby eluting weakly bound cations. This form of chromatography is widely used in the following applications: water purification, preconcentration of trace components, ligand-exchange chromatography, ion-exchange chromatography of proteins, high-pHanion-exchange chromatography of carbohydrates and oligosaccharides, and others. Figure 8: ion exchange chromatography BIOAFFINITY CHROMATOGRAPHY This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of these complexes involves the participation of common molecular forces such as the Vander waalsinteraction, electrostatic interaction, dipole- dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites. Figure 9: Bioaffinity chromatography
- 9. AQUEOUS NORMAL PHASE CHROMATOGRAPHY Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase solvents. Figure 10: Aqueous normal phase chromatography APPLICATIONS OF HPLC MANUFACTURING As briefly mentioned, HPLC has many applications in both laboratory and clinical science. It is a common technique used in pharmaceutical development as it is a dependable way to obtain and ensure product purity. While HPLC can produce extremely high quality (pure) products, it is not always the primary method used in the production of bulk drug materials. According to the European pharmacopoeia, HPLC is used in only 15.5% of syntheses. However, it plays a role in 44% of syntheses in the United States pharmacopoeia. This could possibly be due to differences in monetary and time constraints, as HPLC on a large scale can be an expensive technique. An increase in specificity, precision, and accuracy that occurs with HPLC unfortunately corresponds to an increase in cost. LEGAL This technique is also used for detection of illicit drugs in urine. The most common method of drug detection is an immunoassay. This method is much more convenient. However, convenience comes at the cost of specificity and coverage of a wide-range of drugs. As HPLC is a method of determining (and possibly increasing) purity, using HPLC alone in evaluating concentrations of drugs is somewhat insufficient. With this, HPLC in this context is often performed in conjunction with mass spectrometry. Using liquid chromatography instead of gas chromatography in conjunction with MS circumvents the necessity for derivitizing with
- 10. acetylating or alkylation agents, which can be a burdensome extra step. This technique has been used to detect a variety of agents like doping agents, drug metabolites, glucuronide conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis, and pesticides. Performing HPLC in conjunction with Mass spectrometry reduces the absolute need for standardizing HPLC experimental runs. RESEARCH Similar assays can be performed for research purposes, detecting concentrations of potential clinical candidates like anti-fungal and asthma drugs. This technique is obviously useful in observing multiple species in collected samples, as well, but requires the use of standard solutions when information about species identity is sought out. It is used as a method to confirm results of synthesis reactions, as purity is essential in this type of research. However, mass spectrometry is still the more reliable way to identify species. MEDICAL Medical use of HPLC can include drug analysis, but falls more closely under the category of nutrient analysis. While urine is the most common medium for analyzing drug concentrations, blood serum is the sample collected for most medical analyses with HPLC. Other methods of detection of molecules that are useful for clinical studies have been tested against HPLC, namely immunoassays. In one example of this, competitive protein binding assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin D. Useful for diagnosing vitamin D deficiencies in children, it was found that sensitivity and specificity of this CPBA reached only 40% and 60%, respectively, of the capacity of HPLC. While an expensive tool, the accuracy of HPLC is nearly unparalleled. BIBLIOGRAPHY www.wikipedia.com www.slideshare.com www.wikihow.com Images downloaded through Google image search. ABU SUFIYAN CHHIPA BPHARM VI SEM
- 12. .