Glossary of HPLC Terms

Adsorption

The process of interaction between the solute and the surface of an adsorbent. The forces involved can be strong such as hydrogen bonds, or weak such as 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.

Backflushing

A column switching technique in which a four-way valve placed between the injector and the column allows the mobile phase to flow in either direction. Backflushing is used to elute strongly held compounds at the head of the column. It can be used to analyze these compounds or merely to remove them from the column.

Baseline

The baseline is the line drawn by the data system when the only signal from the detector is from the mobile phase.

Carrier

A term used most often in affinity chromatography, which refers to the support that is used to carry the active ligand, usually by a covalent bond. It can also refer to the support in other chromatographic modes.

Chromatography

A chemical separation technique based on the differential distribution of the constituents of a mixture between two phases, one of which moves relative to the other.

Chromatographic methods

A record of the parameters used in a separation that yields a particular result. It allows another analyst following the method and conditions to reproduce the separation, and achieve the same results.

Column

A tube which contains the stationary phase. The stationary phase differentially interacts with the sample’s constituent compounds as they are carried along in the mobile phase.

Column chromatography

Any form of chromatography that uses a column to hold the stationary phase. Open column chromatography, HPLC and open tubular capillary chromatography are all examples.

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. Degassing is carried out by: heating the solvent, by vacuum ( in a vacuum flask), on-line using evacuation of a tube made from a gas permeable substance such as PTFE, or by helium sparging.

Detector

An electronic device that quantitatively discerns the presence of the separated components as they elute. There are different types of detectors. Some of the common detector types are: UV/Visible light absorbance, differential refractive index, electrochemical, conductivity, and fluorescence.

Detectors in HPLC

• Monochromatic UV detector
• Variable UV-VIS detector
• Diode or Photodiode array detector
• ESI-MSD
• ESI-MSⁿ
• Refractive index detector – (RID) detects all solutes
• Coulometric detector
• Amperometric detector
• Fluorescence detector – detects solutes with fluorophores with very high sensitivity
• Radioactive detector

Detector Sensitivity

The sensitivity setting is the line between normal background noise and a true peak. Perturbations from the baseline that fall below the sensitivity setting are considered noise and are filtered out. Setting the sensitivity too high may result in missing small peaks, while setting it too low may result in a lot of spurious raw data as the software tries to integrate peaks out of the noise.

Flow rate (F)

The volumetric rate of flow of mobile phase through an LC column. For a conventional HPLC column of 4.6 mm i.d., typical flow rates are 1 to 2 mL/min.

Fluorescence detectors

Fluorescence detectors project a specified wavelength of light into the sample, causing the component of interest to fluoresce and the emitted light is detected. Fluorescence detectors are commonly used with derivatization methods. Fluorescence detectors are very selective and very sensitive.

Frit

The porous component 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 endfitting. Frits are made from stainless steel or other inert metal or plastic, such as porous PTFE or polypropylene.

Guard column

A small column placed between the injector and the analytical column. It protects the analytical column against contamination by sample particulates, and by strongly retained species. The guard column is usually packed with the same material as the analytical column and is often of the same i.d. It is much shorter, costs less, and is usually discarded when it becomes contaminated.

Injector

A mechanism for accurately injecting a predetermined amount of sample into the mobile phase stream. The injector can be a simple manual device, or a sophisticated autosampler that permits automated injections of many different samples for unattended operation.

Inlet

The initial part of the column where the solvent and sample enter. There is usually an inlet frit that holds the packing in place and, in some cases, protects the packed bed.

Ion chromatography (IC)

An ion-exchange technique in which low concentrations of anions or cations are determined using low capacity ion exchangers with weak buffers. Conductivity detectors are often used. Ion chromatography is practiced in two forms, suppressed IC and non-suppressed IC.

Isocratic

A constant composition mobile phase used in liquid chromatography.

k or k'

The capacity factor. It can be calculated from the equation, where t_R is retention time for the sample peak, and t_o is the retention time for an unretained peak.

Linearity

A measurement process which ensures accurate quantitation. In chromatography it is important that the detector provide a linear response over the range of sample concentrations it encounters.

Microbore

Columns with smaller than usual internal diameters ( < 2 mm) used in HPLC.

Mobile phase

The solvent that moves the solute through the column.

N

The number of theoretical plates. A measure of the efficiency of a column, where t_R is retention time, and w_b is the base width of the peak. Sometimes it is measured as, where w_l/2 is the peak width at half height.

Narrow-bore column

Columns of < 0.5 mm i.d. used in HPLC.

Normal-phase chromatography

A mode of chromatography carried out with a polar stationary phase and a nonpolar mobile phase. Adsorption on silica normal phase system. It also refers to the use of polar bonded phases, such as CN or NH₂. It is sometimes referred to as straight phase chromatography.

Particle size (d_p)

The average particle size of the packing in an LC column.

Peak

When the detector registers the presence of a compound, the normal baseline signal it sends to the data system changes, resulting in a deflection from the baseline called a peak. Well resolved peaks are symmetrical, touch the baseline, and do not interfere with other peaks.

Peak Identification

Peak identification is usually performed by comparing the sample chromatogram to a chromatogram of a standard solution separated under the same conditions. Peaks that appear at the same elution time as peaks in the standard are identified as the same component.

Peak shape

The profile of a chromatographic peak. Theory assumes a Gaussian peak shape (perfectly symmetrical). A peak asymmetry factor is used to describe a shape as a ratio.

Photodiode Array (PDA)

PDA detectors are UV/Vis detectors that record the absorbance of light at many different wavelengths simultaneously.

Physical factors

Variables such as particle size, particle surface area, column dimensions, leaks in the fluid path, system bandspreading, and detector cell design.

Preparative chromatography

The process of using liquid chromatography to isolate a sufficient amount of material for other experimental or functional purposes. For pharmaceutical or biotechnological purifications, columns several feet in diameter can be used for multiple grams of material. For isolating just a few micrograms of a valuable natural product, an analytical column can be used. Both are preparative chromatographic approaches.

Reversed-phase chromatography (RPC)

The most common HPLC mode. Uses hydrophobic packings such as octadecyl or octysilane phases bonded to silica or neutral polymeric beads. The mobile phase used 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. For example, in the RPC of anions, the addition of a buffer and tetraalkylammonium salt would allow ion pairing to occur, resulting in separations that rival ion-exchange chromatography.

Sample capacity

The amount of sample that can be injected onto an LC column without overload. It is often expressed as grams of sample per gram of packing. Overload is defined as the sample mass injected at which the column efficiency falls to 90 percent of its normal value.

Sampling rate

The frequency with which the detector checks the flow cell. Sampling rate is often called time constant. If it is set too fast, too much raw data will be generated. If it is set too slow, a narrow peak could be missed.

Semipreparative chromatography

Preparative liquid chromatography carried out on an analytical size (4 to 5 mm i.d.) or slightly larger (6 to 10 mm i.d.) column. Normal injection size is in the milligram to low gram range.

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 but the principle is different from HPLC. It is sometimes referred to as digital chromatography. This processs, as it is 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.

Support

The solid particles in a column. The support can be naked, coated, or can have a chemically bonded phase in HPLC.

Tailing

A peak with an asymmetrical factor of >1. An asymmetrical peak is the result of a component that is excessively retarded in eluting. Tailing is caused by sites on the packing that have a stronger than normal retention for the solute. A typical example of a tailing phenomenon is the strong adsorption of amines on the residual silanol groups of a low coverage reversed-phase packing.

Thin Layer Chromatography

The chromatographic technique for separating and analyzing mixtures of substances, using a thin layer of stationary phase attached to a glass plate and using the passage of liquid up the plate by capillary action as the mobile phase.

Ultraviolet/Visible Light (UV/Vis)

The tunable or variable wavelength UV/Vis detector is the most popular form of detector. For methods involving organic compounds in aqueous mobile phases, the UV/Vis detector takes advantage of compounds’ varying absorptivities of ultraviolet and visible light.

X-axis

The X-axis of the chromatogram records the time or volume of mobile phase that passes though the detector.

Y-axis

The Y-axis of the chromatogram records the strength of the detector signal, which is usually proportional to the concentration of sample in the eluent passing through the detector. The units depend on the type of detector being used.