Adsorption
The process of interaction between the solute and the surface of an adsorbent. The forces involved can be strong (for example, 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 by liquid-solid chromatography on silica.
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 elute unretained. The retained compound can later be released in a purified state. Affinity chromatography is not a chromatographic technique as such, but is actually selective filtration.
Anion Exchange Chromatography
The ion exchange procedure used for the separation of anions. Both resins and bonded phases are available for this mode. The tetraalkylammonium group is a typical strong anion exchange functional group. An amino group on a bonded or coated stationary phase would be an example of a weak anion exchanger.
Asymmetry
Factor describing the shape of a chromatographic peak. Theory assumes a Gaussian shape and that peaks are symmetrical. The peak asymmetry factor is the ratio (at 10% of the peak height) of the distance between the peak apex and the back side of the chromatographic curve to the distance between the peak apex and the front side of the chromatographic curve. A value > 1 is a tailing peak, while a value < 1 is a fronting peak.
Backpressure
The pressure required to pump the mobile phase through the column. It is related to mobile phase viscosity (h), flow rate (F), column length (L) and diameter (dc), and particle size (dp) by the following equation:
Band Broadening
The dilution of the chromatographic band as it moves down the column. The measure of band broadening is band width, tw, or more correctly, the number of theoretical plates in the column, N.
Band Width (tw)
The width of the chromatographic band during elution from the column. It is usually measured at the baseline by drawing tangents to the sides of the Gaussian curve representing the peak. Small band widths usually represent efficient separations. Also referred to as peak width.
Capacity Factor (k)
Expression that measures the degree of retention of an analyte relative to an unretained peak, where tR is the retention time for the sample peak and to is the retention time for an unretained peak. A measurement of capacity will help determine whether retention shifts are due to the column (capacity factor is changing with retention time changes) or the system (capacity factor remains constant with retention time changes).
Cation Exchange Chromatography
The ion exchange procedure used for the separation of cations. Both resins and bonded phases are available for this mode. The sulfonic acid group is a typical strong cation exchange functional group. A carboxylic acid group on a bonded or coated stationary phase would be an example of a weak cation exchanger.
Chain Length
The length of carbon chain bonded to a reversed phase packing. It is expressed as the number of carbon atoms (e.g., C8, C18).
Chromatogram
A plot of detector signal output versus time or elution volume during the chromatographic process.
Counterion
In an ion exchange process, the ion in solution used to displace the ion of interest from the ionic site. In ion pairing, it is the ion of opposite charge added to the mobile phase to form a neutral ion pair in solution.
Coverage
Refers to the amount of bonded phase on a silica support. Coverage is usually described in µmol/m2 or % Carbon.
Dead Volume (Vd)
The volume outside of the column packing itself. The interstitial volume (intraparticle volume + interparticle volume) plus extra column volume (contributed by injector, detector, connecting tubing, and end fittings) all combine to create the dead volume. This volume can be determined by injecting an unretained compound (i.e. a compound that does not interact with the column packing). Also abbreviated Vo or Vm.
Degassing
The process of removing dissolved gas from the mobile phase before or during use. Dissolved gas may come out of solution in the detector cell and cause baseline spikes and noise. Dissolved air can affect electrochemical detectors (by reaction) or fluorescence detectors (by quenching).
Efficiency (N)
Also number of theoretical plates. A measure of peak band spreading determined by various methods, some of which are sensitive to peak asymmetry. The most common are shown here, with the ones most sensitive to peak shape shown first:
Eluate
Combination of mobile phase and solute exiting column; also called effluent.
Eluent
Mobile phase used to carry out a separation.
Eluotropic Series
A series of solvents with an increasing or decreasing degree of polarity, generally used to explain solvent strength in liquid-solid or adsorption chromatography. A nonpolar solvent such as pentane would be at one end of the scale; dichloromethane would be an intermediate solvent; a strongly polar solvent, such as water, would be at the other end of the scale. Thus, when developing a method or running a gradient, an eluotropic series is useful for selecting solvents.
Elution Volume (VR)
Refers to the volume of mobile phase required to elute a solute from the column at maximum concentration (apex). VR = F o tR, where F is flow rate in volume/time and tR is the retention time for the peak of interest.
Exclusion Limit
In SEC, the upper limit of molecular weight (or size) beyond which molecules will elute at the same retention volume, called the exclusion volume. Many SEC packings are referred to by their exclusion limit.
Frit
The porous element at either end of a column that serves to contain the column packing. It is placed at the very ends of the column tube or, more commonly, in the end fitting. Frits are made from stainless steel or other inert metal or plastic, such as porous PTFE or polypropylene.
Gaussian Curve
A standard error curve, based on a mathematical function, which is a symmetrical, bell shaped band or peak. Most chromatographic theory assumes a Gaussian peak.
Gel Filtration Chromatography (GFC)
Size exclusion chromatography carried out with aqueous mobile phases. Generally refers to separations carried out on soft gels such as polydextrans. Most gel filtrationseparations involve biopolymers.
Gel Permeation Chromatography (GPC)
Size exclusion chromatography carried out with organic mobile phases. Used for the separation and characterization of polymers. SEC with aqueous mobile phases is referred to as aqueous GPC, or GFC.
Gradient Elution
Technique for decreasing separation time by increasing mobile phase strength over time during the chromatographic separation. Other gradients include temperature, pH, salt, and flow rates.
HETP
Height equivalent to a theoretical plate. A carryover from distillation theory: a measure of a column's efficiency. For a typical well-packed HPLC column with 5 µm particles, HETP (or H) values are usually between 0.01 and 0.03 mm.
where L is column length in millimeters and N is the number of theoretical plates.
Hydrophilic
"Water loving": refers both to stationary phases that are compatible with water and to water soluble molecules in general.
Hydrophobic
"Water hating": refers both to stationary phases that are not compatible with water and to molecules in general that have little affinity for water. Hydrophobic molecules have few polar functional groups: most are hydrocarbons or have high hydrocarbon content.
Ion Exchange Chromatography (IEC)
A mode of chromatography in which ionic substances are separated on cationic or anionic sites of the packing. The sample ion (and usually 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 on a number of other solution parameters (pH, ionic strength, counterion type, etc).
Ion Exchange Capacity
The number of ionic sites on the packing that can take part in the exchange process. Exchange capacity is expressed in mEq/g.
Isocratic
Use of a constant composition mobile phase in liquid chromatography.
Linear Velocity
The flow rate normalized by the column cross section. This effects column performance and is directly related to column pressure. Linear velocity is given by the following equation where L is column length and to is the breakthrough time of an unretained peak:
Mass Transfer
The process of solute movement into and out of the stationary phase or mobile phase. The C-term of the van Deemter equation is referred to as the mass transfer term. The faster the process of mass transfer, the better the efficiency of the column. In HPLC, mass transfer is the most important factor affecting column efficiency. It is increased by the use of small particle packings, thin layers of stationary phase, low viscosity mobile phases, and high temperatures.
Mobile Phase
The solvent that moves the solute through the column.
Modifier
Additive that changes the character of the mobile phase. For example, in reversed phase, water is the weak solvent; methanol, the strong solvent, is sometimes called the modifier.
N (Number of Theoretical Plates)
See Efficiency.
Octadecylsilane (ODS or C18)
The most popular reversed phase packing in HPLC. Octadecylsilane phases are bonded to silica or polymeric supports. Both monomeric and polymeric phases are available.
Overload
The mass of sample injected onto the column at which efficiency and resolution begin to be affected if the sample size is further increased.
Partition Coefficient (K)
The amount of solute in the stationary phase relative to the amount of solute in the mobile phase.
Peak Shape
Describes the profile of a chromatographic peak. Theory assumes a Gaussian peak shape (perfectly symmetrical); peak asymmetry factor describes shape as a ratio. See Asymmetry.
Pore Volume
The total volume of the pores in a porous packing; usually expressed in mL/g.
Porosity
For a porous adsorbent, the ratio of the volume of the interstices to the volume of the solid particles. The pore volume is also used as a measure of porosity.
Recovery
The amount of solute (sample) that elutes from a column relative to the amount injected. Most often used with protein separations in which proteins irreversibly bind to active sites on the packing in certain columns.
Residual Silanols
The silanol (-Si-OH) groups that remain on the surface of a packing after a phase is chemically bonded onto its surface. These silanol groups may not be accessible to the reacting bulky organosilane (e.g., octadecyl-dimethylchlorosilane) but may be accessible to small compounds. Often they are removed by end-capping with a small organosilane such as trimethylchlorosilane.
Resolution (Rs)
Ability of a column to separate chromatographic peaks. Resolution can be improved by increasing column length, decreasing particle size, increasing temperature, changing the eluent or stationary phase. It can also be expressed in terms of the separation of the apex of two peaks divided by the tangental width average of the peaks:
Retention Time (tR)
The time between injection and the appearance of the peak maximum.
Retention Volume (VR)
The volume of mobile phase required to elute a substance from the column: VR = F o tR.
Reversed Phase Chromatography (RPC)
The most common HPLC mode. Uses hydrophobic packings such as octadecyl- or octylsilane phases bonded to silica or neutral polymeric beads. Mobile phase is usually water and a water-miscible organic solvent such as methanol or acetonitrile. There are many variations of RPC in which various mobile phase additives are used to impart a different selectivity.
SAX
Strong anion exchanger.
SCX
Strong cation exchanger.
Selectivity (alpha)
A thermodynamic factor that is a measure of relative retention of two substances, fixed by a certain stationary phase and mobile phase composition. Where k1 and k2 are the respective capacity factors.
Silanol
The Si-OH group found on the surface of silica gel. There are different strengths of silanols, depending on their location and relationship to each other. The strongest silanols are acidic and often lead to undesirable interactions with basic compounds during chromatography.
Siloxane
The Si-O-Si bond. A principal bond found in silica gel or for attachment of a silylated compound or bonded phase. Stable except at high pH value.
Size Exclusion Chromatography (SEC)
A noninteractive technique which separates solutes according to their molecular size in solution.
Solid Phase Extraction (SPE)
A sample preparation technique that uses a solid phase packing contained in a small plastic cartridge. The solid stationary phases are the same as HPLC packings; however, the principle is different from HPLC. The process, as most often practiced, requires four steps: conditioning the sorbent, adding the sample, washing away the impurities, and eluting the sample in as small a volume as possible with a strong solvent.
Solute
The dissolved component of a mixture that is to be separated in the chromatographic column.
Solvent Strength
Refers to the ability of a solvent to elute a particular solute or compound from a column. See eluotropic series.
Stationary Phase
The immobile phase involved in the chromatographic process.
Tailing
The phenomenon in which the normal Gaussian peak has an asymmetry factor > 1. Tailing is most often caused by sites on the packing that have a stronger than normal retention for the solute.
Tailing Factor (T)
A measure of the symmetry of a peak, given by the following equation where W0.05 is the peak width at 5% height and f is the distance from peak front to apex point at 5% height. Ideally, peaks should be Gaussian in shape or totally symmetrical.
T = W0.05/2f
Theoretical Plate
Relates chromatographic separation to the theory of distillation. Measure of column efficiency. Length of column relating to this concept is called height equivalent to a theoretical plate (HETP). See HETP.
van Deemter Equation
An equation used to explain band broadening in chromatography. The equation represents the height equivalent of a theoretical plate and has three terms. The A term is used to describe eddy diffusion, which allows for the different paths a solute may follow when passing over particles of different sizes. The B term is for the contribution caused by molecular diffusion or longitudinal diffusion of the solute while passing through the column. The C term is the contribution of mass transfer and allows for the finite rate of transfer of the solute between the stationary phase and mobile phase. n is the reduced velocity of the mobile phase as it passes through the column.
Void
The formation of a space, usually at the head of the column, caused by a settling or dissolution of the packing. A void in the
column leads to decreased efficiency and loss of resolution.
Void Time (tm or t0)
The time for elution of an unretained peak.
Void Volume (V0)
The total volume of mobile phase in the column: the remainder of the column is taken up by packing material. Can be determined by injecting an unretained substance that measures void volume plus extra column volume.
WAX
Weak anion exchanger.
WCX
Weak cation exchanger
