Glossary of HPLC-LC Separation Terms - A

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.


α: See separation factor.

A solvent: Usually the weaker solvent in a binary eluent or gradient elution separation. In reversed-phase liquid chromatography (LC), the A solvent typically is water or a water-rich mixture.

A term: The first term in the van Deemter equation. See eddy dispersion term and van Deemter equation.

Absorption: The process of retention in which the solute partitions into a liquid-like coating.

Activity: The relative strength of the surface of the packing in adsorption chromatography. For silica gel, the more available the silanol groups, the more active the surface. Activity can be controlled by adding water or other polar modifier that hydrogen bonds to the active sites, thereby reducing the surface activity.

Adapter: A union with different threads on each end; generally used to connect two different types of tubing together.

Additive: A substance added to the mobile phase to improve the separation or detection characteristics; for example, a competing base to negate the effects of silanols, a chelating agent to block metal sites, or a UV-absorbing compound to perform indirect photometric detection.

Adjusted retention time (t R '): A measure of the retention time adjusted for the holdup time; t R' = t R – t M, where t R is the retention time and t M is the holdup time (the time it takes for a small, unretained compound that completely permeates the pores to be eluted from the chromatographic column).

Adjusted retention volume (V R '): Adjusts the retention volume for the holdup volume; V R' = V R – V M, where V R is the retention volume of the peak of interest and V M is the holdup volume (the volume corresponding to the total volume of mobile phase in the column). See also dead volume and holdup volume.

Adsorbent: Packing used in adsorption chromatography. Silica gel and alumina are the most frequently used adsorbents in high performance liquid chromatography (HPLC).Adsorption: The process of retention in which the interactions between the solute and the surface of an adsorbent dominate. The forces can be strong forces (hydrogen bonds) or weak (van der Waals forces). For silica gel, the silanol group is the driving force for adsorption, and any solute functional group that can interact with this group can be retained on silica. The term adsorption places emphasis on the surface versus penetration or embedding in the stationary phase coated or bonded to a surface.

Adsorption chromatography: One of the basic LC modes that relies upon adsorption to the surface of an active solid to effect the separation. Silica gel and alumina are the most frequently used normal-phase adsorbents, and molecules are retained by the interaction of their polar function groups with the surface functional groups; for example, silanols of silica. Carbon also is used as an adsorbent in a reversed-phase mode.

Adsorption isotherm: A plot of the equilibrium concentration of sample in the mobile phase per unit volume versus the concentration in the stationary phase per unit weight in adsorption chromatography. The shape of the adsorption isotherm can determine the chromatographic behavior of the solute; for example, peak tailing, peak fronting, and column overload.

Aerogel: A packing prepared when the dispersing agent is removed from a gel system without collapsing the gel structure. Silica gels and glass beads used for size-exclusion chromatography (SEC) are examples of aerogels that can retain their structures even at the high pressures used in HPLC. See also xerogels.

Affinity chromatography: A technique in which a biospecific adsorbent is prepared by coupling a specific ligand — such as an enzyme, antigen, or hormone — for the macromolecule of interest to a solid support (or carrier). This immobilized ligand will interact only with molecules that can selectively bind to it. Molecules that will not bind will be eluted unretained. The retained compound later can be released in a purified state. Affinity chromatography is normally practiced as an on–off separation technique.

Agarose: High molecular weight polysaccharide used as a separation medium in biochromatography. It is used in bead form, often in gel-filtration chromatography, with aqueous mobile phases.

Alkoxysilane: A reactant used for the preparation of chemically bonded phases. It will react with silica gel as follows: R3SiOR + ≡SiOH → ≡Si–OSiR3 + ROH, where R is an alkyl group.

Alumina: A normal-phase adsorbent used in adsorption chromatography. Aluminum oxide is a porous adsorbent that is available with a slightly basic surface; neutral and acidic modifications also can be made. Basic alumina can have advantages over silica, which is considered to have an acidic surface.

Amino phase: A propylamino phase used in normal bonded-phase chromatography. It is somewhat reactive for solute molecules such as aldehydes or mobile-phase additives that can react with amines. The amino phase has found some applications as a weak anion exchanger, and it also is used for the separation of carbohydrates with a water–acetonitrile mobile phase. It is a relatively unstable phase.Amphoteric ion-exchange resin: Ion-exchange resins that have both positive and negative ionic groups. These resins are most useful for ion retardation in which all ionic materials can be removed from solution because the anionic and cationic functionalities coexist on the same material.

Analyte: The compound of interest to be analyzed by injection into and elution from an HPLC column.

Analytical column: An HPLC column used for qualitative and quantitiative analysis; a typical analytical column will be 50–250 mm × 4.6 mm but columns with smaller diameters (down to 0.05 mm i.d.) can also be considered as analytical columns; can be constructed of stainless steel, glass, glass-lined stainless steel, PEEK, and other metallic and nonmetallic materials.

Anion exchange: The ion-exchange procedure used for the separation of anions. Synthetic resins, bonded-phase silicas, and other metal oxides can be analyzed in this mode. A typical anion-exchange functional group is the tetraalkylammonium, which makes a strong anion exchanger. An amino group on a bonded stationary phase is an example of a weak anion exchanger.


Asymmetry: Factor describing the shape of a chromatographic peak. Chromatographic theory assumes a Gaussian shape and that peaks are symmetrical. A quantitative measure is the peak asymmetry factor, which is the ratio of the distance from the peak apex to the back side of the chromatography curve over the distance from the peak apex to the front side of the chromatography curve at 10% of the peak height. Other measures of asymmetry are commonly used, especially the U.S. Pharmacopeia (USP) method. (Fig.1). See also Foley–Dorsey equation.

Asymmetry factor: A factor that denotes band shape. The asymmetry factor is calculated from the chromatographic peak by dropping a perpendicular at the peak apex and a horizontal line at 10% of the peak height; at the intersection, the distance to the tail of the peak along the horizontal line (distance B) divided by the distance along the horizontal line to the front of the peak (distance A) produces a ratio called the peak asymmetry factor (see Figure 1). The ratio is 1 for a symmetrical peak, less than 1 for a fronting peak, and greater than 1 for a tailing peak. The higher the value, the less symmetrical the peak; values greater than 2 are unacceptable.

Atmosphere (atm): A measure of the pressure drop across an HPLC column; 1 atm = 14.7 lb/in.2 (psi). See also bar and pascals.


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