Terminology Used in Chromatography

 

Introduction:

There are many number of terms used in the chromatographic analysis. we have listed few of them which are routinely used in everyday analysis. 

1.Chromatography:

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.

2.Chromatogram:

A plot of detector signal output or sample concentration versus time or elution volume during the chromatographic process.

3.Chromatograph:

Instrument for carrying out chromatography

4.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.

5.Stationary Phase:

The stationary phase is one of the two phases forming a chromatographic system. It may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid. This solid may or may not contribute to the separation process. The liquid may also be chemically bonded to the solid (Bonded Phase) or immobilized onto it (Immobilized Phase).

6.Mobile Phase:

A fluid which percolates through or along the stationary bed, in a definite direction. It may be a liquid (Liquid Chromatography) or a gas (Gas Chromatography) or a supercritical fluid (Supercritical-Fluid Chromatography). In gas chromatography the expression Carrier Gas may be used for the mobile phase. In elution chromatography the expression Eluent is also used for the mobile phase.

The expression Chromatographic Bed or Sorbent may be used as a general term to denote any of the different forms in which the stationary phase is used.

Note: Particularly in gas chromatography where the stationary phase is most often a liquid, the term Liquid Phase is used for it as compared to the Gas Phase, i.e., the mobile phase. However, particularly in the early development of liquid chromatography, the term "liquid phase" had also been used to characterize the mobile phase as compared to the "solid phase" i.e., the stationary phase

7.Solvent:

A term sometimes referring to the liquid stationary phase in partition chromatography

8.Absorption:

The process of retention in which the solute partitions into a liquid like coating

9.Adsorbent:

Packing used in adsorption chromatography. Silica gel and alumina are the most frequently used adsorbents in high performance liquid chromatography (HPLC).

10.Analyte:

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

11.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.

12.Buffer:

A solution that maintains constant pH by resisting changes in pH from dilution or addition of small amounts of acids and bases.

13.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.

14.Capillary Column:

Refers to columns with inner diameters less than 0.5 mm.

15.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).

16.Capillary Gel Electrophoresis (CGE):

A technique in which a capillary is filled with, or the walls coated or covalently bonded with, cross-linked polyacrylamide to simulate slab gel electrophoresis; this polymer network uses a sieving mechanism; used for protein, carbohydrate, and DNA separations such as fingerprinting and sequencing.

17.Column Performance (N):

Refers to the efficiency of a column; the number of theoretical plates for a given test compound.

18.Carrier Gas:

The mobile phase in gas chromatography (GC).

19.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.

20.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.

21.Column Length (L):

The length of chromatography column in HPLC or capillary in CE used to perform the liquid-phase separation.

22.Dwell Time:

The time equivalent to dwell volume; determined by the product of flow rate and the dwell volume.

23.Dwell Volume:

The volume between the point of mixing of solvents (usually in the mixing chamber or at the proportioning valves in the liquid chromatograph) and the head of an LC column. Important in gradient elution or in isocratic elution situations when changes in solvent composition are made so that the column experiences the composition change in the shortest possible time. Low-pressure mixing systems generally have larger dwell volumes than high-pressure mixing systems.

24.Dead Volume (VM):

The column dead volume comprises the entire space accessible to a small molecule that can fully permeate all pores of a packing material. It includes the interstitial volume and the unoccupied pore volume. It is denoted as VM. The system dead volume includes the additional volume in the tubing that connects the injector and detector to the column. The system dead volume usually is approximated by injecting a small, essentially unretained species. Uracil, acetone and thiourea are most commonly used species in reversed-phase chromatography. See also adjusted retention volume, holdup volume, and void volume.

25.Electrophoresis:

The movement of sample ions under the influence of an applied voltage.

26.Eluent:

The mobile phase used to perform a separation.

27.Elution:

The process of passing mobile phase through the column to transport solutes down a column

28.Flow Rate (F):

The volumetric rate of flow of a mobile phase through an LC column. Typical flow rates are 1–2 mL/min for a conventional 4.6-mm i.d. HPLC column.

29.Fronting:

Peak shape in which the front part of the peak (before the apex) in a chromatogram tapers in advance of the remainder of the peak; that is, the front is less steep than the rear. The peak has an asymmetric distribution with a leading edge. The asymmetry factor for a fronting peak has a value of less than one. Tailing is the opposite effect. Fronting can result at high sample loads because of positive curvature in the isotherm and from using poorly packed columns.

30.Gradient:

A process to change solvent strength as a function of time (normally solvent strength increases) thereby eluting progressively more highly retained analytes. Typically, gradients can be binary, ternary, and quaternary solvent mixtures in which solvents are blended to achieve the proper strength.

31.Gradient Elution:

Technique for decreasing separation time by increasing the mobile-phase strength over time during the chromatographic separation. Also known as solvent programming. Gradients can be continuous or stepwise. Binary, ternary, and quaternary solvent gradients have been used routinely in HPLC.

32.Hydrophilic:

Greek word for water loving. Refers to stationary phases that are fully compatible with water and to water soluble molecules in general.

33.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.

34.Guard Column:

A small column placed between the injector and the analytical column. It protects the analytical column from contamination by sample particulates and strongly retained species. The guard column usually is packed with the same material as that in the analytical column and is often of the same inner diameter. It is much shorter, costs less, and usually are discarded when it becomes contaminated. Integrated guard–analytical column systems often are preferred to minimize extra column effects caused by connecting tubing with separate guard and analytical columns.

35.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.

36.Interstitial Volume (Ve):

 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 Vo

37.Linear Velocity (u):

The velocity of the mobile phase moving through the column. Expressed in centi-meters per second. Related to flow rate by the cross-sectional area of the column. Determined by dividing the column length (L) by the retention time of an unretained compound.

38.Liquid Chromatography (LC):

A separation technique in which the mobile phase is a liquid. Most often performed in a column.

39.Method Development:

A process for optimizing the separation, including the sample pre-treatment, to obtain a reproducible and robust separation. Usually, it emphasizes the search for the stationary phase, eluent, and column temperature combination that provides an adequate, if not optimum, separation.

40.Method Validation:

A process of testing a method to show that it performs to the desired limits of precision and accuracy in retention, resolution, and quantitation of the sample components of interest.

41.Octadecylsilane:

The most popular reversed phase in HPLC. Octadecylsilane phases are bonded to silica or polymeric packings. Both monomeric and polymeric phases are available. Abbreviated in column names as C18 and ODS.

42.Octylsilane:

A popular stationary phase in reversed-phase chromatography. Usually provides slightly less retention than the more popular C18. Both monomeric and polymeric phases are available. Abbreviated in column names as C8.

43.Peak Shape:

Describes the profile of a chromatography peak.

44.Planar Chromatography:

A separation technique in which the stationary phase is present as or on a plane (IUPAC). Typical forms are paper and thin-layer chromatography

45.Recovery:

The amount of solute or sample that is eluted from a column relative to the amount injected.

46.Retention Time (tR):

Also called the total retention time. The time between injection and the appearance of the peak maximum.

47.Tailing Factor:

U.S. Pharmacopeia measure of peak asymmetry defined as the ratio of the peak width at 5% of the apex to twofold the distance from the apex to the 5% height on the short time side of the peak. Greater than unity for tailed peaks.

48.Theoretical Plate (N):

A concept described by Martin and Synge. Relates chromatographic separation to the theory of distillation. Length of column relating to this concept is called height equivalent to a theoretical plate.

49.Void Volume (VM):

The total volume of mobile phase in the column; the remainder of the column is taken up by packing material. This volume can be determined by injecting an unretained substance. Also called dead volume. The symbol V0 is often used to denote the void volume. This is valid only for a column packed with nonporous particles. V0 is valid when used to denote the excluded volume (Ve) in SEC.

50.Column back pressure:

The difference in pressure between the column inlet and outlet.