A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as gradient elution. A normal gradient for reversed phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol (which is hydrophobic); whereas the more hydrophilic compounds will elute under conditions of relatively low methanol/high water. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the HPLC method which gives the best separation of peaks.
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. Very polar solvents in a mixture tend to deactivate the column by occupying the stationary phase surface. This is somewhat particular to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather than a soft layer on a surface).
NP-HPLC had fallen 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.
Reversed phase HPLC (RP-HPLC or RPC) has a 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 more non-polar, while polar molecules elute more readily. An investigator can also increase retention time by adding a polar solvent to the mobile phase, or decrease retention time by adding a more hydrophobic solvent. RPC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RPC to qualify drugs before their release.
RPC operates on the principle of hydrophobic interactions, which result from repulsive forces between a polar eluent, the relatively non-polar analyte, and the non-polar stationary phase. 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. Gradient elution uses this effect by automatically changing the polarity of the mobile phase during the course of the analysis.
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.
Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond.
Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5 × 10-7 J/cm² per Mol for NaCl, 2.5 × 10-7 J/cm² per Mol for (NH4)2SO4), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).
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. Ammonium formate is commonly added in mass spectrometry to improve detection of cerain analytes by the formation of ammonium adducts. A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is one of the strongest organic acids. The effects of acids and buffers vary by application but generally improve the chromatography.
Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine. Because the 2,2'-bipy can chelate the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) when metal ions are present on the surface of the silica...
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.
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.
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.
A composition of the mobile phase that remains constant throughout the procedure is termed isocratic.
In contrast to this is the so called "gradient elution", which is a separation where the mobile phase changes its composition during a separation process. One example is a gradient in 20 min starting from 10% methanol and ending up with 30% methanol. Such a gradient can be increasing or decreasing. The benefit of gradient elution is that it helps speed up elution by allowing components that elute more quickly to come off the column under different conditions than components which are more readily retained by the column. By changing the composition of the solvent, components that are to be resolved can be selectively more or less associated with the mobile phase. As a result, at equilibrium they spend more time in the solvent and less time in the stationary phase, and therefore they elute faster.
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.
WIPO PUBLISHES PATENT OF F. HOFFMANN-LA ROCHE FOR "HYDROPHOBIC INTERACTION CHROMATOGRAPHY METHOD" (GERMAN INVENTORS)
Nov 26, 2011; GENEVA, Nov. 25 -- Publication No. WO/2011/144606 was published on Nov. 24. Title of the invention: "HYDROPHOBIC INTERACTION...
WIPO ASSIGNS PATENT TO AVANTOR PERFORMANCE MATERIALS FOR "PROCESS FOR TRYPSIN AND CHYMOTRYPSIN PURIFICATION UTILIZING HYDROPHOBIC INTERACTION CHROMATOGRAPHY" (AMERICAN INVENTORS)
Oct 27, 2011; GENEVA, Oct. 27 -- Publication No. WO/2011/126957 was published on Oct. 13. Title of the invention: "PROCESS FOR TRYPSIN AND...
WIPO ASSIGNS PATENT TO NATRIX SEPARATIONS FOR "HYDROPHOBIC INTERACTION CHROMATOGRAPHY MEMBRANES, AND METHODS OF USE THEREOF" (CANADIAN INVENTORS)
May 23, 2011; GENEVA, May 23 -- Publication No. WO/2011/058439 was published on May 19. Title of the invention: "HYDROPHOBIC INTERACTION...