Glossary Of HPLC-LC Separation Terms – C

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.

C

C term: The interphase mass transfer term of the van Deemter equation. See also mass transfer and van Deemter equation.

C8: See octylsilane.

C18: See octadecylsilane.

C 4 , C 8 , C 18 , etc.: Refer to the alkyl-chain length of a reversed bonded phase.

C S See Langmuir isotherm.

Capacity: See sample capacity.

Capacity factor (k): Old term for a chromatographic parameter that measures the degree of retention. Now defined as the retention factor (k)by the International Union of Pure and Applied Chemistry (IUPAC). See also retention factor for method of calculation.

Capillary column: Refers to columns with inner diameters less than 0.5 mm.

Capillary electrochromatography (CEC): A hybrid technique in which capillary columns are packed with chromatographic sorbents and electroosmotic flow rather than pressure moves mobile phase through the column; technique has the surface-mediated selectivity potential of HPLC and the high efficiency of capillary electrophoresis (CE).

Capillary LC: Generally refers to HPLC performed in a fused-silica or other type of capillary column; the inner diameters typically are less than 0.5 mm; has also been called micro-LC.

Capillary micellar electrochromatography: The CEC version of micellar electrokinetic capillary chromatography (MEKC).

Capillary tubing: Tubing to connect various parts of a chromatograph and direct flow to the proper places. Most capillary tubing used in HPLC is less than 0.020 in. in inner diameter. The smallest useful inner diameter is approximately 0.004 in.Capping: Same as endcapping.

Carbon Load: For a bonded phase silica, term usually used to describe the surface coverage or the degree to which the available silanols on the column packing’s surface have reacted and been replaced with the bonded phase; the higher the carbon load, the lower number of residual silanols. The carbon load is normally expressed as a % carbon (e.g. 12% carbon). In reversed-phase LC, the higher the carbon load, the greater the analyte retention.

Carrier: A term most often used in affinity chromatography; refers to the support that binds the active ligand, usually by a covalent bond; can also refer to the support in other chromatography modes such as liquid–liquid chromatography.

Carrier gas: The mobile phase in gas chromatography (GC).

Cartridge column: A column type that has no endfittings and is held in a cartridge holder. The column comprises a tube and packing contained by frits in each end of the tube. Cartridges are easy to change and are less expensive and more convenient than conventional columns with endfittings.

Cation-exchange chromatography: The form of ion-exchange chromatography that uses resins or packings with functional groups that can separate cations. An example of a strong cation functional group would be a sulfonic acid; a weak cation-exchange functional group would be a carboxylic acid.

CE: Capillary electrophoresis.

CEC: See capillary electrochromatography.

CGE: See capillary gel electrophoresis.

CZE: See capillary zone electrophoresis.

Chain length: The length of carbon chain in the hydrocarbon portion of a reversed-phase packing. It is expressed as the number of carbon atoms (C8, C18, and so forth). It specifically excludes the short chains — typically methyl, isopropyl, and sec-butyl groups — that also are attached to the silane.

Channeling: Occurs when voids created in the packing material cause mobile phase and accompanying solutes to move more rapidly than the average flow velocity, which in turn allows band broadening to occur. The voids are created by poor packing or erosion of the packed bed.

Check valve: A device inserted into a moving liquid stream that allows flow of the stream in only one direction; most often used on the inlet and outlet sides of an HPLC pump.

Chemisorption: Sorption caused by a chemical reaction with the packing. Most of these interactions are irreversible and usually occur on packings with reactive functional groups such as silanol or bonded amino phases. Chemisorption is common with metal oxide phases that have strong Lewis acid sites.

Chiral recognition: The ability of a chiral stationary phase to interact differently with two enantiomers leading to their HPLC separation.

Chiral stationary phases: Stationary phases that are designed to separate enantiomeric mixtures. The phases can be coated or bonded to solid supports, created in situ on the surface of the solid support, or exist as surface cavities that allow specific interactions with one enantiomeric form.

Chlorosilane: A chemical reagent used to prepare siloxane bonded phases; reactivity changes from a monochlorosilane < dichlorosilane < trichlorosilane; the alkyl portion (octadecyl, octyl, etc.) will dictate the hydrophobicity of the resulting bonded phase; alkoxysilanes can be used but are less reactive.

Chromatogram: A plot of detector signal output or sample concentration versus time or elution volume during the chromatographic process.Chromatograph: As a noun: a device used to implement a chromatographic separation. As a verb (IUPAC): the act of separating by elution through a chromatographic bed.

Chromatographic conditions: Those chromatographic method experimental parameters that describe how an analysis was performed. Sufficient information must be presented so that the analysis can be duplicated for verification purposes.

Classification: The process of sizing column packing particles; generally in HPLC, small particle-size distribution provides better efficiency and a greater permeability because of the absence of fines. Classification can be performed by sedimentation, elutriation, and centrifugal air techniques.

Column back pressure: See head pressure.

Column chromatography: Any form of chromatography that uses a column or tube to hold the stationary phase. Open-column chromatography, HPLC, and open-tubular capillary chromatography all are forms of column chromatography. Most often refers to open-column chromatography used for preparative-scale work.

Column dead time: The time associated with the dead volume; determined by the dead volume divided by the flow rate; in reversed-phase LC, uracil is often used to measure dead volume and dead times.

Column length (L): The length of chromatography column in HPLC or capillary in CE used to perform the liquid-phase separation.

Column packing: The solid material, usually a porous solid with or without a chemically interactive surface, placed inside of the column used to differentially retain analytes; referred to as the stationary phase; common packings are unbonded and bonded silica, resins, inorganic-organic hybrids, and graphtized carbon.

Column performance (N): Refers to the efficiency of a column; the number of theoretical plates for a given test compound.

Column plate number (N): Denotes the column efficiency; the larger the plate number, the more theoretical plates the column possesses; a typical well-packed column with a 5-μm d p porous packing in a 15 cm × 4.6 mm column should provide 10,000–12,000 plates.

Column switching: Using multiple columns connected by switching valves for better chromatographic separations or sample cleanup. Fractions from a primary column can be switched to two or more secondary columns, which in turn can be further diverted to additional columns or to detectors; sometimes called multidimensional chromatography.

Column volume (V c ): The volume of the unpacked column; V c = A c L, where A c and L are the cross-sectional area of the tube and the tube length, respectively.

Competing base: Adding a small basic compound such as triethylamine or dimethyloctylamine at 10–50 mM concentration to the mobile phase in reversed-phase chromatography to inhibit basic analytes from interacting with residual silanols; works by the law of mass action because concentration of competing base is much greater than analyte. See also additive.

Comprehensive two-dimensional chromatography: Two Dimensional Chromatography applied to every fraction. See Two Dimensional Chromatography. For complete series of definitions around the topic of 2D chromatography see P.Schoenmakers, P. Marriott, and J. Beens, LCGC Europe 16(6) 335–339 (2003).

Controlled surface porosity support: Same as porous-layer bead and pellicular packing.Counterion: The ion in solution used to displace the ion of interest from the ionic site in an ion-exchange process. In ion pairing, it is the ion of opposite charge added to the mobile phase to form a neutral ion pair in solution.

Coupled columns: A form of column switching that uses a primary column connected to two secondary columns by a selector valve. Fractions from the first column can be selectively transferred to the second and third columns for additional separations. This term also is used to describe two or more columns connected in series to provide an increased number of plates.

Coverage: Refers to the amount of bonded phase on a silica support in bonded-phase chromatography. Coverage usually is described in micromoles per square meter or in terms of percentage carbon (w/w).

Critical micelle concentration: The concentration of an ionic surfactant above which a micelle is formed by aggregation; micelles added to a mobile phase improve the separation of nonionic substances in HPLC and CE (MEKC) by a partitioning mechanism.

Cross-linking: During the process of copolymerization of resins to form a three-dimensional matrix, a difunctional monomer is added to form cross-linkages between adjacent polymer chains. The degree of cross-linking is determined by the amount of the monomer added to the reaction. For example, divinylbenzene is a typical cross-linking agent for the production of polystyrene ion-exchange resins. The swelling and diffusion characteristics of a resin are governed by its degree of cross-linking.

Cyano phase: A chemically bonded phase that terminates with the CN functional group; it can be used in normal phase as a moderate polarity sorbent and in reversed phase as a short chain bonded phase.

Cyclodextrins: Cyclic oligomers of several D-(+)-glucopyranose units used in chiral HPLC and CE separations; popular ones are named α-, β-, and γ-cyclodextrins; they have a truncated cone shape, a relatively hydrophobic cavity, and primary and secondary hydroxyl groups at their ends; they separate on the basis of differential inclusion of enantiomers; modified cyclodextrins with derivatized hydroxyl groups also are used for selectivity modification.