Glossary Of HPLC-LC Separation Terms – H

The listing should be helpful to those just starting in HPLC but it also can serve as a refresher for long-time users in the field.


h: Reduced plate height. Defined as HETP/d p, where HETP is the height equivalent to a theoretical plate and d p is the particle diameter. See also reduced plate height.

H: Same as HETP. See also efficiency.

η: See viscosity.

Head pressure (Δp): The difference in pressure between the inlet and outlet of a column measured in pounds per square inch. Governed by the following approximate equation for a column packed with spherical particles of typical internal porosity (0.5): Δp = 3000Lη/t M d p 2, where L is the column length in centimeters, η is the mobile-phase viscosity in centipoise, t M the column holdup time in minutes, and d p is the particle diameter in micrometers. Pressure can be expressed in pounds per square inch, bars, atmospheres, or pascals.

Heart cutting: Refers to collection of the center of the peak at which purity should be maximum in preparative LC. The term also is used in column switching.

H eff See effective plate height.

Helium sparging: See degassing. Helium has a very low solubility in most common liquids.

HETP: Height equivalent to a theoretical plate. A carryover from distillation theory; a measure of column efficiency; HETP = L/N, where L is column length and N is the number of theoretical plates. HETP should be approximately 2–3 d p for 5-μm particles with a typical well-packed HPLC column, HETP (or H) values usually are in the range of 0.01–0.03 mm. See also efficiency and h.

High performance CE: A technique in which small-diameter capillaries, buffered conducting solutions, and high voltages (as much as 30,000 V) separate ionic molecules based on their differential electrophoretic mobilities. Nonionic (neutral) molecules can be separated by MEKC.

High performance liquid chromatography (HPLC): The modern, fully instrumental form of liquid-phase chromatography technique that uses small particles and high pressures. Sometimes called high-pressure LC.

High pressure mixing: A configuration of a gradient HPLC system where the solvents are mixed on the high pressure side of multiple pumps (usually 2, binary); such a system offers a lower gradient delay volume than low pressure mixing systems where the solvents are mixed by proportioning valves before a single pump.

Holdup volume (V M ): The total volume of mobile phase in the column regardless of where it exists; V M = V e + V i, where V e is the interstitial volume and V i is the intraparticle volume. Also called the column void volume. IUPAC indicates that use of the term dead volume should be eliminated for this concept. The use of dead volume is limited to regions not swept by the flowing mobile phase system. Holdup volume is measured by injecting an unretained species that fits in all the pores. See also interstitial porosity and intraparticle porosity.

HPLC: See high performance liquid chromatography.

Hybrid silica: Silica gel comprising both organic and inorganic moieties with hybrid properties of polymeric packings and silica packings. Synthesized from silanes containing organic functionality. Different selectivity but better high-pH stability than bare or uncoated silica gel.

Hydrodynamic volume: The molecular volume defined by the effective diameter of a molecule in free solution at which the hydrodynamic sphere would be a sphere defined by the molecule as it revolves around its central axis in solution. Term used in SEC to define molecular shape and to explain why molecules with the same molecular weight often have different elution volumes. Measured by determining the Stokes radius.

Hydrophilic: Greek word for water loving. Refers to stationary phases that are fully compatible with water and to water-soluble molecules in general. Many columns used to separate proteins — such as ion-exchange, SEC, and affinity columns — are hydrophilic in nature and should not irreversibly sorb or denature protein in an aqueous environment.

Hydrophilic interaction chromatography: An alternative technique to reversed-phase HPLC for the separation of highly polar analytes that may be only slightly retained by reversed-phase chromatography, HILIC requires a high percentage of a nonpolar mobile phase and a polar stationary phase, similar to the requirements in normal phase chromatography. However, unlike normal-phase chromatography, which uses nonpolar solvents such as hexane and methylene chloride and tries to exclude water from the mobile phase, HILIC requires some water in the mobile phase to maintain a stagnant enriched water layer on the surface into which analytes may selectively partition. In addition, water-miscible organic solvents are used instead of the water-immiscible solvents used in normal-phase chromatography. With HILIC, sorbents such as bare silica, bonded diol, and polyhdroxyethylaspartamide are used. Polar analytes are well retained and are eluted in order of increasing hydrophilicity, just the inverse of reversed-phase LC.

Hydrophobic: Greek word for water fearing. Refers to stationary phases that are incompatible with water or to molecules that in general have little affinity for water. Hydrophobic molecules have few polar functional groups. Most have a high content of hydrocarbon (aliphatic and aromatic) functionality.

Hydrophobic interaction chromatography: A technique in which weakly polar (nonhydrocarboneous) packings are used to separate molecules by the interactions of their hydrophobic moieties and the hydrophobic sites on their packing surface. High concentrations of salt solutions are used in the mobile phases, and separations are generated by changing the salt concentration. The technique is analogous to salting-out molecules from solution. Gradients are run by decreasing the salt concentration. The technique often is used to separate proteins that are sensitive to denaturization by the organic solvents used in regular reversed-phase chromatography. Usually little or no organic solvent is used in the mobile phase in hydrophobic interaction chromatography.

Hydroxyapatite: A porous calcium hydroxy phosphate solid that chemically resembles bone and tooth. Used as a packing material in biochromatography for nucleic acid constituents, monoclonal antibodies, and proteins.

Hyphenated techniques: Refers to the family of techniques best known by their acronyms, including LC–mass spectrometry (MS), LC–Fourier transform IR spectroscopy (FTIR), and LC–MS–MS. See also multidimensional chromatography.


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