High Pressure Liquid Chromatography (HPLC)

High Pressure Liquid Chromatography (HPLC) (Sometimes 'High Performance')Research with thin-layer and column chromatography showed that separations are much more effective when the stationary phase is a very thin layer on the surface of very small and very uniform spherical beads. However, resistance to flow of the mobile phase is very much higher, and in order to get a useful flow of a liquid mobile phase, e. g., 1 - 3 milliliters/minute, pressures of around 15 Mpa (about 2,000 psi) must be applied to the mobile phase. It is possible to apply such pressure from a cylinder of compressed gas, but most systems use a reciprocating piston pump or diaphragm pump with some means of damping the pressure fluctuations from the piston. The sample is usually dissolved in the mobile phase before injection. Columns are typically 4.6 mm ID (6 mm OD) stainless steel tubing 250 mm long. A typical packing will have octadecylsilyl (C18-Si-) (ODS) groups bonded to 5 µm silica beads. The packing is held inside the column by “frits”, discs with pores about 0.5 µm in diameter.
Liquid-liquid chromatography began with samples dissolved in organic solvents and a stationary phase of water adsorbed on particles or fibers of the solid support. More generally, the stationary phase was more “polar” than the mobile phase. That is the so-called "normal phase" chromatography. But stationary phases such as ODS have been particularly useful for separating samples dissolved in water (and most HPLC is now done with bonded phases). Liquid chromatography with the stationary phase less polar than the mobile phase is called “reverse phase”, but is now the common situation. The mobile phase is very often not just water but a mixture of water with methanol (CH3OH) or acetonitrile (CH3CN). “Solvent programming”, a stepwise or continuous change (gradient elution) of the mobile phase composition, is used to speed up separations, like temperature programming in gas chromatography.
“Chiral”columns have been developed relatively recently to separate optical isomers. This separation is important because many pharmaceuticals are active in only one chiral form. For instance, natural Vitamin E is D-a-tocopherol, while half of synthetic Vitamin E is the less active L- isomer.
As in gas chromatography, a few microliters of the solution are measured into a “sample loop” on an “injector”. When the injector is operated, the sample loop is suddenly switched into the flow of mobile phase just before it reaches the column. The mobile phase leaving the column passes immediately into a “detector” which is used to determine the presence and concentration of a solute.
Because the pressure on the mobile phase drops rapidly as it leaves the column, bubbles may form from dissolved gases. Chromatographers “degas” the mobile phase before use by boiling it, ultrasonic treatment, bubbling helium through it to flush out other gases, or applying a vacuum.
There are no relatively inexpensive HPLC detectors as sensitive and broadly useful as the flame ionization and electron capture detectors used in gas chromatography. A refractive index (RI) detector responds to most components, but is not very sensitive. An ultraviolet (UV) detector is quite sensitive for molecules which absorb ultraviolet light, and a variable wavelength UV detector can be set to the absorption maximum for a particular molecule of interest, or to a short wavelength where most molecules absorb. A diode array detector (DAD) disperses the transmitted light into a spectrum, providing an absorption spectrum of each component that absorbs ultraviolet light. Still more sensitive, and still less general, is the fluorescence detector which measures fluorescence emitted from components which have absorbed ultraviolet light. There are various special-purpose detectors. Amperometric systems measure electron flow which oxidizes or reduces certain components (sugars, for instance), and polarimetric detectors, generally not very sensitive, detect components are optically active. Mass spectrometric detectors are now used, but were late to arrive because it was difficult to separate the mobile phase molecules so as to maintain adequate vacuum in the mass spectrometer.
Special case: Supercritical fluid chromatography (SFC). Liquids can vaporize, and gases can condense to the liquid phase. Both changes depend on the pressure and temperature. Every gas has a critical temperature above which it cannot be condensed at any pressure. The critical pressure is the pressure required at the critical temperature. If a liquid (such as a condensed gas) is warmed, under high pressure, to the critical temperature and beyond, strange things happen. The boundary between gas and liquid vanishes, and it is best not to ask physical chemists too many questions about the remaining phase, the “supercritical fluid”. Whatever their exact nature, supercritical fluids can be very useful solvents for HPLC, which runs under high pressure anyway. The most commonly used of these fluids is carbon dioxide. (Supercritical fluids are also useful for extracting things from solids, and, when the solids are clothing, for dry cleaning.)
Reports and procedures will specify the mobile phase composition, flow rate and perhaps the pressure; the column dimensions; the column packing (particle type and size; coating); the detector and its operating conditions (e. g., wavelengths for UV and fluorescence detectors), integrator settings, and, somewhere, retention times.
There are some kinds of liquid chromatography that do not fit the partition or adsorption models very well. They are often done in simple vertical columns.
Gel chromatography: The mobile phase carries sample through a gel, such as agar or polyacrylamide. Small molecules pass through quickly. The larger molecules cannot get through as quickly, and are separated by size. Various kinds of gels are used for polar (aqueous) or nonpolar mobile phases. Sometimes these gels are called “molecular sieves”, but they are not the Molecular Sieves (tradename) used in gas-solid chromatography to separate gases.
Exclusion chromatography: The packing is made of porous beads. Small molecules diffuse into the pores and so do not move as fast as the mobile phase. The largest molecules cannot fit into the pores, and move with the solvent flow. If the beads are charged, ions of the same charge are repelled while those of opposite charge are attracted in ion exclusion chromatography.
Ion-pair chromatography: In liquid chromatography, strongly ionized sample components which would not partition into the liquid phase can be combined with large ions having the opposite charge [such as an alkyl sulfonate (negative) or a tetrabutylammonium (positive)] to produce “ion pairs” that act like a compound and will partition.
Affinity chromatography: This uses a specific “ligand” (such as an antigen) bonded to the stationary phase to separate a specific substance such as the corresponding antibody.
There are some special procedures and terms:
Headspace analysis: The sample is held for a time in a closed container, with a gas phase. Volatile molecules move from the sample into the gas phase, depending on their volatility. When the system has reached equilibrium, a sample of the gas phase is injected into a gas chromatograph, and the concentration in the gas phase (headspace) is used to determine the concentration in the liquid phase. Purge and trap: A relatively large volume of the liquid sample (perhaps a liter) is held in a container. A gas (e. g., helium) is bubbled through to sweep out the volatile molecules, and then passed through a short trap column containing some of the column packing (“solid phase”) which collects and concentrates the volatiles in a much smaller volume. Then the trap is heated rapidly and the volatiles are driven into the gas chromatograph.
Solid phase extraction (SPE): A relatively large volume of liquid sample is exposed to a small volume of a stationary phase that extracts and concentrates material from the sample. That material can then be redissolved and chromatographed.
Response factor: Detectors typically respond more strongly to some molecules than to others, so that peak size alone is not the best means of measuring quantities of individual components. Once the components have been identified, the analyst can chromatograph known amounts of individual materials to relate the peak size to quantity. The relationship is the “response factor”.
Area percent: The area of a specific peak divided by the total area of all the peaks. It is commonly used to report analysis results. Users should remember that response factors differ, so that area percent is not the same as weight percent, and that some components may not be detected at all by the detector used.