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Transcript of HPLC
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY The “HP” portion of the acronym is sometimes assigned to the words high pressure (versus high performance), but it refers to the same analytical system. HPLC is used in drug analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics to name a few forensic applications. HPLC is based on selective partitioning of the molecules of interest between two different phases. Here, the mobile phase is a solvent or solvent mix that flows under high pressure over beads coated with the solid stationary phase.
While traveling through the column, molecules in the sample partition selectively between the mobile phase and the stationary phase.
Those that interact more with the stationary phase will lag behind those molecules that partition preferentially with the mobile phase. As a result, the sample introduced at the front of the column will emerge in separate bands (called peaks), with the bands emerging first being the components that interacted least with the stationary phase and as a result moved quicker through the column.
The components that emerge last will be the ones that interacted most with the stationary phase and thus moved the slowest through the column.
A detector is placed at the end of the column to identify the components that elute. Occasionally, the eluting solvent is collected at specific times correlating to specific components. This provides a pure or nearly pure sample of the component of interest. This technique is sometimes referred to as preparative chromatography. Most powerful of all chromatographic techniques.It can often achieve seperation and analyse that which would otherwise be difficult or impossible to infer. The development of open column methods like Paper Chromatography and Thin Layer Chromatography improved speed and resolution of Liquid chromatography greatly. Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC). In the HPLC technique, the sample is forced through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane by a liquid (mobile phase) at high pressure.
HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases.
Methods in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC)
and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is termed reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more. Types Partition Chromatography
Partition chromatography was the first kind of chromatography that chemists developed.
The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids.
Partition chromatography uses a retained solvent, on the surface or within the grains or fibres of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of somecoulombic and/or hydrogen donor interaction with the solid support.
Molecules equilibrate (partition) between a liquid stationary phase and the eluent.
This method separates analytes based on polar differences, often using a bonded polar stationary phase and a non-polar, water miscible, mobile phase. Normal-phase chromatography
Also known as normal-phase HPLC (NP-HPLC), or adsorption chromatography, this method separates analytes based on adsorption to a stationary surface chemistry and by polarity.
It was one of the first kinds of HPLC that chemists developed.
NP-HPLC uses a polar stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents.
The analyte associates with and is retained by the polar stationary phase.
The interaction strength depends not only on the functional groups in the analyte molecule, but also on steric factors.
The effect of sterics on interaction strength allows this method to resolve (separate) structural isomers.
Partition and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HILIC bonded phases which improve reproducibility. 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.
Operating parameters are adjusted to maximize the effect of this difference.
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 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 concentrations. Reversed phase HPLC
Non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With these stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily.
RPC operates on the principle of hydrophobic forces, which originate from the high symmetry in the dipolar water structure and play the most important role in all processes in life science. RPC allows the measurement of these interactive forces. The binding of the 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 in the aqueous eluent. 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. Size-exclusion chromatography
Also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of size.
It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a 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.
This technique is widely used for the molecular weight determination of polysaccharides. Ion-exchange chromatography
Retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded.
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.
An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. An increase in pH reduces the retention time in cation exchange while a decrease in pH reduces the retention time in anion exchange.
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-pH anion-exchange chromatography of carbohydrates and oligosaccharides, and others. 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 Van der Waals interaction, 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. 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 reverse-phase solvents. Parameters
The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.
Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity.
Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector.
Narrow-bore columns (1–2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors,fluorescence detection or with other detection methods like liquid chromatography-mass spectrometry
Capillary columns (under 0.3 mm) are used almost exclusively with alternative detection means such as mass spectrometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ. Particle size
Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 μm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.
This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (column diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase extraction. Pore size
Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.
Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressuremay reach as high as 40 MPa (6000 lbf/in2), or about 400 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns.These "Ultra High Performance Liquid Chromatography" systems or RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in²), or about 1000 atmospheres. The term "UPLC" is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique. Introduction Principle HPLC is a technique that has arisen from the application of theories and instruments originally developed for gas chromatography. It was known from Gas Chromatography theories that efficiency would be improved if particle size of the materials used in Liquid Chromatography could be reduced.
As HPLC developed, particle size reduced.
Present norms require usage of microparticulate column packing- silica particles of sizes 10,5 or 3 micrometres. As in other forms of chromatography, the time taken for the solute to pass through the chromatographic system is a characteristic of the solute. GC vs HPLC For GC, the mixture to be examined must be in vapour state. However, only 20% of chemical compounds are suitable for GC.The rest are either thermally unstable or non volatile.
Moreover, polarity in a compound results in poor chromatographic behaviour. In case of HPLC, the only restriction is that we must be able to dissolve our sample in a solvent. Thus HPLC is a preffered method fro macromolecules, labile products, drugs and other biochemicals. Reversed phase HPLC
Structural properties of the analyte molecule play an important role in its retention characteristics.
In general, an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) results in a longer retention time because it increases the molecule's non-polar surface area, which is non-interacting with the water structure.
On the other hand, polar groups, such as -OH, -NH2, COO– or -NH3+ reduce retention as they are well integrated into water. Very large molecules, however, can result in an incomplete interaction between the large analyte surface and the ligand's alkyl chains and can have problems entering the pores of the stationary phase.
Another important component is the influence of the pH since this can change the hydrophobicity of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. Chromatography is excellent for seperation, but it does not provide details necessary for identification. Such details are provided by spectrometric techniques.
However, today HPLC has been combined with such techniques to give us that benefit.