A B C D E F G H I J K L M N O P Q R S T U V W X Y Z

HPLC :

  • A

    • Adsorption Chromatography—Separation mode resulting from compounds that have different adhesion rates for the packing surface. (See Normal- Phase Chromatography.)

    • Alpha (a)—(Separation or chemistry factor). A measure of separation between two peak maxima. Ratio of their k′ values.

    • Attenuation—Measure of detector sensitivity. A larger value means less sensitivity.

    • Autoinjector—An injection device for automated methods development in which the sample loop is repeatedly filled from a large sample reservoir rather than a sample vial carousel.

    • Autosampler—A multiple sample injector, usually with a rack or carousel to hold sample vials or a sample well plate, designed for unattended pro- grammed operation in which a sample is loaded by either pushing or pulling sample into the loop injection loop with air or hydraulic pressure.

    • Autozero—Detector, integrator, or computer function capable of setting detector signal value (baseline?) to zero.

  • B

    • Band—The disk of resolved compound moving down the column. Band spreading cause by diffusion tends to remix already separated bands.

    • Baseline—Detector signal versus time if no peaks are present. Good indica- tor of pulsing, air bubles, electrical noisse, or impurities.

    • Baseline Resolution—Chromatographic goal of methods development in which all valleys between adjacent peaks touch the baseline, indicating complete resolution of peaks.

    • Buffer—Mobile phase modifier used to control pH. Usually salts of weak acids or bases, most effective at their pK a , where concentrations of ionized and unionized form are equal.

  • C

    • C 8 (Octyl)—Nonpolar column or packing with 8 carbon hydrophobic hydro- carbon chain bound to silica.

    • C 18 (Octyldecyl)—Nonpolar column or packing with an 18 carbon hydrophilic hydrocarbon chain bound to silica. Used for reversed phase separations. (See ODS.)

    • Cartridge Column—Disposable off-line tube packed with >1 g of packing used for sample and solvent preparation. (See SFE and Windowing.)

    • CAD (Charged Aerosol Detector)—A universal, mass detector that evapo- rates column effluent using a gas nebulizer in the presence of a caronal dis- charge needle that ionizes compound droplets so they can be detected on an electrometer.

    • Check Valve—Mechanism in the pump head inlet and outlet to ensure one- way solvent flow; usually a sapphire ball in a stainless steel cone. Major point of buffer precipitation and pump pressure loss.

    • Chip HPLC—Nano-flow, micro-sample HPLC system in which the packed column resides within the body of the injector. Originally touted as the HPLC of the future for nano-level LC/MS, but its potential has been slow to materialize.

    • Chromatography—A separation technique producing a qualitative record of the relative amounts of components, a chromatogram. HPLC modes include partition and adsorption (polarity), GPC or SEC (size), ion exchange (charge), or affinity (compound-specific retention).

    • Column—A metal tube in which the HPLC separation occurs, packed with porous packing held in place at each end by a fritted filter in an end-cap. End-caps are secured to the column with ferrules and can be opened for frit cleaning.

    • Column Blank—A length of tubing, fitted with compression fittings simulat- ing column ends, used to replaced the column for system cleaning and diagnosis.

    • Column Heaters—Heaters designed to allow elevated temperature operation by jacketing the column, injector, and tubing lines. Especially useful for shortening run times and inducing a changing when using temperature- resistant zirconium columns. Best systems use fast response Peltier healing/cooling.

    • Compression Fitting—A device for connecting tubing to other system parts. Usually made up of a ferrule and a threaded screw or cap, which slide over the tubing. Tightening the screw cap forces the ferrule into a conical hole, squeezing (swaging) it permanently onto the tubing.

  • D

    • Dead Volume—Unnecessary volume in a system that can remix separated bands of compounds, usually in tubing or fittings, especially from the injec- tor to the column and from the column to the detector.

    • Deoxygenation—Removing oxygen from a solvent by vacuum replacement with nitrogen or helium gas to prevent oxidation of sensitive compounds or columns (such as the amino columns).

  • E

    • Efficiency (N)—A measure of the narrowness of elution bands, the sharpness of peaks, and the performance of a column. Results are in theoretical plates. The Huber equation calculates efficiency versus flow rate, which is plotted on as a Van Deampter plot, which compares column efficiency with flow rate.

    • ELSD (Evaporative Light Scattering Detector)—A universal, mass evapo- rated detector that measures the amount of compounds present by the amount their droplets deflect an incident light beam.

    • Elution—Washing bands of separated bands out of the column with mobile phase. The liquid output of a column is the eluant; the amount of solvent needed to reach a peak’s maximum is its elution volume.

    • Elutotropic Series—Solvents ranked in order of polarity or eluting strength. The strongest solvent is the one most like the packing material in polarity.

    • End Absorption—UV absorption, from 210 nm down, going nonlinear at 180 nm due to dissolved oxygen. Most carbon-oxygen containing com- pounds absorb in this area.

    • End-capping—After silylation, reaction of bonded-phase packing with a reactive small molecule to tie up unreacted silanols on the silica surface. Sharpens peaks from basic compounds.

    • Exclusion Volume—In size-exclusion chromatography, Vo, the volume of solvent necessary to washout unretarded compounds too large to penetrate the pores of a size-separation column. The inclusion volume, 2Vo, is the elution volume needed to elute all compounds small enough to fully pene- trate the pores.

  • F

    • Fines—Small particles of packing material in a column that tend to migrate and plug the outlet frit, raising column back-pressure. Commonly seem with irregular packings that have microfractures that break off small pieces of packing material under pressure changes.

    • Flow Cell—Low volume (8–20 mL) detector cell designed to accept eluant output from an HPLC or an ion chromatography.

    • Frits—Porous stainless steel filters at either end of the column that serve as bed supports and filter the sample coming in from the injector.

  • G

    • GPC (Gel Permeation Chromatography)—Separation mode based on the molecular sizes of the compounds. [See SEC (size exclusion chromatography).]

    • Gradient—A reproducible change in a separation parameter that can be used to speed a separation. In a binary solvent gradient, % solvent B increases while %A decreases, causing late-eluting peaks to come off faster and sharper.

    • Guard Columns—Short, protective columns placed in-line between the injec- tor and the main column.

  • H

    • Helium Sparging—A solvent degassing technique in which helium gas is bubbled through solvents to displace dissolved gases before solvent mixing, compression, and pumping.

  • I

    • Interface—An ionizing, evaporative device designed to take effluent from the HPLC and prepare it for injection directly into the source of a mass spectrometer.

    • Ion Displacement—Use of strong salt solutions to displace compounds bound to ion-exchange columns.

    • Ion Exchange Chromatography—Separation mode for ionized compounds on charged columns. Anion-exchange columns attract and separate anions; cation-exchange columns separate cations.

    • Isocratic—Constant mobile phase composition. The opposite is a gradient in which the mobile phase composition is altered during the run. Isocratic con- ditions are not restricted to single solvents or solvent mixtures, but can include multiple components in the mobile phase.

  • K

    • k¢ (Retention Factor)—A measure of the relative solvent volume needed to wash a compound off a column at a given solvent polarity. Normalized with the void volume of the column to make it independent of column length.

  • L

    • Lamps—Light source for a detector. A deuterium lamp is fully variable from 190 nm to 400 nm; a tungsten lamp from 370 to 700 nm. Other lamps show discrete bands; mercury, 254 and 436 nm; cadmium, 228 nm; zinc, 214 nm.

    • LC/MS (Liquid Chromatography/Mass Spectrometry)—Chromatography system in which an HPLC is married to a mass spectrometric detector through an evaporated, ionizing interface. A variety of mass spectrometers are used to produce various LC/MS and LC/MS/MS configurations. MS detectors are universal, mass detectors that provide molecular weight infor- mation and can give a definitive identification of separated compounds.

    • Loop and Valve Injector—Device for placing sample onto the column head. Modern design consists of a loop, partially or overfilled at atmospheric pres- sure, that is rotated into the flowing stream from pump to column. Sample is back-filled from the end of the loop closest to the column, described as “last in, first out” filling.

  • M

    • Microporous Packing—Modern, fully porous, high-resolution separations packing with average particle diameters of 3–10 mm.

    • Mobile Phase—The solvent mixture pumped through the column carrying the injected sample; the liquid phase of the solid-liquid equilibration.

    • Monolith Column—Porous silica column prepared in situ to completely fill the column tube with a fully porous silica foam skelton. After the organic polymer support is heated off, the silica surface is silylated in place to product bonded-phase surface. Column is high resolution and can be used at high flow rates with relatively low back-pressure (see Chapter 16).

    • Multichannel HPLC—HPLC system designed to run parallel HPLC columns into a multi-flow cell UV or fluorescent detector. Designed for production laboratories to speed QA/QC monitoring (see Chapter 16).

  • N

    • Nanoflow HPLC—HPLC system with accurately controlled reciprocating and syringe pumps designed to use capillary and small diameter, high- resolution columns as front ends for electrospray and nanospray mass spec- trometer interfaces.

    • Needle Port Seal—Teflon R throat seal in injector needle port that prevents flow back of injected sample solution.

    • Normal Phase Chromatography—Separations mode run on nonbonded, anhydrous porous silica using a nonpolar mobile phase. (See Adsorption Chromatography.)

  • O

    • ODS—Octadecylsilyl bonded phase material or column in which the material bound to silica is an 18-carbon saturated hydrocarbon chain. (See C 18 .)

  • P

    • Pacification—Treatment of a column-bridged HPLC system with 20% (6 N) nitric acid to remove buffer and organic deposits and protect metal surfaces from corrosion. The column must be removed before acid treatment. Overnight water wash is needed to remove the last traces of acid.

    • Peak Areas versus Peak Heights—Integration and quantitization can be based on either the height or area of the peak. With well-resolved peaks seen in research labs, areas give more accurate results; with less well-resolved peaks or shoulders seen in clinical or biomatrix separations, peak heights give best results.

    • Pellicular Packing—First analytical packing; it had a solid core and a crust of porous silica. Now used primarily for packing guard columns and columns for separating very large molecular weight compounds (i.e., DNA, RNA).

    • Plate Count—A measure of column efficiency derived by comparing peak width to retention time. A higher number indicates a more efficient sepa- ration. Theoretical plates are an arbitrary unit assigned to the efficiency value, in analogy to efficiency units in open column distillations.

    • Plunger—A piston, usually of sapphire, driven by the pump motor into the pumping chamber to pressurize and displace solvent through the outlet check valve. The rear of the chamber is sealed by the plunger seal, made of hardened Teflon R that fits tightly around the plunger.

    • Polarity—A measure of a solvent’s, column’s, or compound’s ability to attract similar molecules. Polar compounds have large dipole moments, large dielectric constants, and usually form hydrogen bonds (e.g., water). Non- polar compounds such as hexane are on the opposite end of the polarity scale. (See Elutotropic Series.)

    • Pulse Dampeners—Device used to control pump pulsing. Usually a tight coil of metal tubing in a metal container that acts as a baffle and counters pulsing by a spring recoil effect.

  • R

    • Reciprocating Pumps—Single- and dual-headed pumps that use a piston and check valves to pump solvent from a reservoir into the system.

    • Resolution (R)—A measure of the completeness of a separation. Influenced by k′ (solvent polarity), N (column efficiency), and a (system chemistry).

    • Retention Time—The time or mobile phase volume need to elute and detect a component of the mixture in a detector.

    • Reverse-Phase Chromatography—Separation mode on bonded phase columns in which the solvent/column polarities are the opposite of normal- phase separations. Polar compounds elute before nonpolar compounds, Nonpolar columns require polar solvents.

    • RP 18 —Reverse phase, bonded packing with 18-carbon side chain. (See C 18 , ODS.)

    • Rotor Seal—Teflon R surface that seals the injector and separates the flowing mobile phase from the sample loop until an injection is completed.

  • S

    • Sample Clarification—Removal of particulates from the injection sample by either filtration or centrifugation.

    • Saturation Column—Sacrificial column placed before the injector to protect the main column from pH degradation.

    • Seal—Wear surface that both lubricates and separates moving parts in the HPLC. (See Plunger Seal, Rotor Seal, and Needle Port Seal.)

    • SEC (Size Exclusion Chromatography)—A separation mode employing control pore size packing to achieve resolution of molecules based on size and shape. (See GPC.)

    • Separation Factor (a)—A measure of peak separation between peaks. Product of dividing one peak k′ by the other. Also called the chemistry factor because it is controlled by changes in the chemistry of the column, mobile phase, and the sample.

    • SFE—Separation and filtration cartridge column. Also referred to as a SPE (solid phase extraction column). (See windowing in Chapter 12.)

    • Silica—Particles or spheres of crystalline silicic acid used in chromatography. Its surface is polar, acidic, and tends to attract water of hydration and polar compounds.

    • Silylation—The first step in forming bonded-phase packings from dried silica and chlorodialkylchlorosilanes.

    • Stationary Phase—A term used to describe the column packing, indicating that it is part of a two-phase equilibrium with the mobile phase or column solvent.

    • Syringe Pump—A pulseless pump made up of a motor-driven piston or plunger in a solvent-filled cylinder. Useful only when small solvent volumes are to be pumped; often used in micro-flow or nano-flow HPLC systems.

  • T

    • Tailing—Unsymmetrical peak formation in which the side of the peak away from the injection returns very slowly to the baseline. Usually due to an unresolved equilibration and incomplete separation.

  • U

    • Ultra-fast HPLC—An HPLC system designed to use 2-mm spherical packing at high flow rate and pressure (∼12,000 psi) to produce very rapid, high- resolution separates. This system is designed for interfacing into an LC/MS system or to increase separation speed.

  • V

    • Voids—Spaces or openings in the column bed leading to poor chromatogra- phy. End voids are directly under the inlet frit. Center voids are channels through the center of the packing bed.

    • Void Volume—The solvent volume inside the packed column. It usually can be measured as an early refractive index baseline upset when injecting a sample dissolved in a solvent even slightly different from mobile phase.

  • W

    • Windowing—A technique using cartridge columns (SFE) to speed chro- matography by first removing polar and nonpolar impurities, leaving only a solvent fraction containing the compounds of interest.

  • Z

    • Zero Dead Volume—Fittings designed to leave no extra column volumes that might cause band spreading or remixing of peaks.