Glossary Of HPLC-LC Separation Terms – I

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.


IC: See ion chromatography.

Immobilized metal-affinity chromatography: See metal-affinity chromatography.

Immunoaffinity chromatography: A specific form of separation where an antibody is bonded or immobilized onto the surface of an HPLC support material. Based a molecular recognition mechanism, analytes that are specifically targetted by the antibody can be selectively retained via antibody–antigen interactions from a complex mixture. After interferences are washed away, retained analytes can be released by changing the mobile phase conditions such that the strong binding is disrupted.

Imprinted phases: Polymer and silica phases generated in the presence of a template or printing molecule. These phases have enhanced selectivity for the templating molecule.

Included volume: Also known as totally included volume. The volume at which a small molecule that explores the entire pore space of a column is eluted. See also size-exclusion chromatography.

Indirect detection: Used for non-UV absorbing or nonfluorescing analytes. A UV-absorbing or fluorescent compound added to the mobile phase maintains a high background signal; when a nonabsorbing or nonfluorescing analyte is eluted, the background is diluted and a negative peak is observed for that analyte. When an analyte acts to increase the concentration of the indicating species, it produces a positive peak. When a negative signal is detected, the detector signals are reversed to the output device.

Infinite diameter column effect: At a certain column length, a sample injected into center of a packed bed spreads by radial diffusion but never reaches column wall, where wall effects can cause band broadening. Phenomenon observed by John Knox, who showed that a sample peak collected in the exact center of the column exit displayed a higher efficiency than a sample peak collected near the wall. The infinite diameter effect depends on column length, internal diameter, particle size, and mobile-phase properties. Very seldom applied in HPLC.

Injection solvent: Solvent used to inject sample into an HPLC column; solvent should be of equal or lower strength than the mobile phase to prevent premature movement down the column due to the presence of a stronger solvent.

Inlet: The initial part of the column where the solvent and sample enter. An inlet frit usually holds the packing in place and, in some cases, protects the packed bed.

Inlet–outlet check valves: The check valves on an LC pump that allow mobile phase to flow in one direction but not in the reverse direction. Inlet check valves allow flow from the reservoir into the pump and outlet check valves allow mobile phase to flow to the column from the pump.

Inlet filter: Filtration devices attached to the inlet lines of the pump that removes particulate matter from the mobile phase before the solvent reaches the pump; reservoir filters are an inlet filter that resides in the solvent bottle.

In-line filter: A device that prevents particulate matter from damaging the column. Modern low-volume, in-line filters can be placed between the injector and the column without major contributions to band broadening. A filter in this position prevents sample particles from entering the packed bed or column inlet frit.

Interparticle porosity (´ e ): The interparticle volume of a packed column per unit column volume; ´e = V e/V c, where V e is the interstitial volume and V c is the total column volume. See also interstitial porosity.

Interparticle volume (V o ): The volume of mobile phase located outside the particles.

Interstitial porosity (´ e ): The fraction of the volume in the column located in the interparticle (interstitial) space; ´e = V e/V c.

Interstitial velocity (u e ): The actual velocity of the eluent as it moves through the column flowing around the particles; u e = F/A c´e. The interstitial velocity is the basis for computing the reduced velocity.

Interstitial volume (V e ): The volume between the particles. It does not include the volume in the pores of the particles. Also called the excluded volume (see SEC) and interparticle volume. Measured by injecting a molecule that does not permeate any pores and does not interact with the surface of the particles. In SEC, this volume is denoted V o.

Intraparticle porosity (´ i ): The fraction of the particle volume that is the pore volume; ´i = V pore /V particle .

Intraparticle volume (V i ): The volume inside the pores of the particles. Also called the internal and included volume. Can be measured by the BET method or mercury-intrusion porosimetry.

Ion chromatography (IC): An ion-exchange technique in which low concentrations of organic and inorganic anions or cations are determined using ion exchangers of low ion-exchange capacity with dilute buffers. Conductivity detectors often are used. IC is practiced in two forms: In suppressed IC, a second column or a membrane separator is used to remove the buffer counter ion from the analyte and simultaneously replace it with a hydrogen or hydroxide ion that concomitantly converts the buffer to an uncharged species thereby suppressing background and enhancing sensitivity. In nonsuppressed IC, low-concentration, weakly conducting buffers are carefully selected, the entire effluent is passed through the detector, and ions are detected above the background signal.

Ion-exchange capacity: The number of ionic sites on the packing that can participate in the exchange process. The exchange capacity is expressed in milliequivalents per gram. A typical styrene–divinylbenzene strong anion-exchange resin may have 3–5 mequiv/g capacity. Exchangers for IC have very low capacity. Capacity of weak anion and cation exchangers varies dramatically with pH.

Ion-exchange chromatography: A mode of chromatography in which ionic substances are separated on cationic or anionic sites of the packing. The sample ion, usually with a counterion, will exchange with ions already on the ionogenic group of the packing. Retention is based on the affinity of different ions for the site and other solution parameters such as pH, ionic strength, and counterion type. Ion chromatography basically is an ion-exchange technique.

Ion exclusion: The process in which ionized solutes can be separated from un-ionized or partially ionized solutes using ion-exchange resins. Separation results from Donnan potential in which ionic solutes exist at a higher concentration in solution than in the stationary phase, whereas nonionic solutes are evenly distributed between the mobile phase and resin. Therefore, ionic solutes will move faster down the column than nonionic solutes. Ion exclusion occurs in reversed-phase chromatography when anions are separated at pH values at which the silanol groups are ionized.

Ionic strength: Ionic strength is a characteristic of an electrolyte solution. It is typically expressed as the average electrostatic interactions among an electrolyte's ions. It is related to electrolyte concentration but the main difference between ionic strength and electrolyte concentration is that the former is higher if some of the ions are more highly charged. The higher the ionic strength of a mobile phase the more the mobile phase competes with the analyte for ionic or adsorptive sites.

Ion-moderated partitioning chromatography: A technique used for separating carbohydrates using strong cation-exchange packings that are in specific cationic form (for example, calcium, hydrogen, silver). The separation mechanism is complexation rather than ion exchange.

Ion-pair chromatography: Form of chromatography in which ions in solution can be paired or neutralized and separated as an ion pair on a reversed-phase column. Ion-pairing agents usually are ionic compounds that contain a hydrocarbon chain, which imparts a certain hydrophobicity so that the ion pair can be retained on a reversed-phase column. Retention is proportional to the length of the hydrophobic chain and the concentration of the ion-pair additive. Ion pairing also can occur in normal-phase chromatography when one part of the pair is dynamically loaded onto a sorbent, but this technique is not as popular as reversed-phase chromatography. Also known as ion-interaction chromatography or dynamic ion-exchange chromatography, which stresses that users sometimes do not know the precise mechanistic details of how the additive controls retention.

Ion retardation: Refers to using amphoteric ion-exchange resins, which retard ionic molecules and allow nonionic molecules or nonelectrolytes to be eluted preferentially.

Ion suppression: Buffering in an aqueous mobile phase at a particular pH to suppress solute ionization. For example, weak carboxylic acids can have their ionization suppressed by the adjustment of the pH below their pK a value. Useful for improving peak shape of weak acids and bases in reversed-phase chromatography.

Irregular packing: Refers to the shape of a column packing. Irregular packings are available in microparticulate sizes. The packings are obtained from grinding solid materials into small particles and sizing them into narrow fractions using classification machinery. Spherical packings are used more often than irregular packings in analytical HPLC, but the less-expensive, irregular packings are still widely used in preparative-scale LC.

Irreversible adsorption: When a compound with a very strong affinity for an adsorbent is injected onto a column, it can be adsorbed so strongly that it cannot be eluted from the column. A chemical reaction between the sample and the surface of the adsorbent is an example of irreversible adsorption. See also chemisorption.Isocratic: Using a time invariant–eluent composition in LC.

Isotherm: See adsorption isotherm.

Isothermal chromatography: Using conditions of constant temperature. The vast preponderance of all LC is performed under isothermal conditions.


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