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چهارشنبه 2 بهمن‌ماه سال 1387 ساعت 15:39
                                        Glossary of HPLC Terms


Adsorption Chromatography–Separation mode resulting from compounds that have different adhesion rates for the packing surface
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 that 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 programmed 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
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 indicator 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 pKa, where concentrations of ionized and unionized form are equal
C8 (Octyl)–Nonpolar column or packing with 8 carbon hydrophobic hydrocarbon chain bound to silica
C18–Octyldecyl–Non-polar column or packing with an 18 carbon hydrophilic hydrocarbon chain bound to silica. Used for reversed phase separations
Cartridge Column–Disposable off-line tube packed with >1 gm of packing used for sample and solvent preparation
CAD (charged aerosol detector)–A universal, mass detector that evaporates column effluent using a gas nebulizer in the presence of a caronal discharge 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 with in 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
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 endcap. 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 simulating 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 column. 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
Dead Volume–Unnecessary volume in a system that can remix separated bands of compounds, usually in tubing or fittings, especially from the injector 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)
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 to flow rate
ELSD (evaporative light scattering detector)–A universal, mass evaporated 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 non-linear at 180 nm due to dissolved oxygen. Most carbon-oxygen containing compounds absorb in this area
Endcapping–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 penetrate the pores
Fines–Small particles of packing material in a column which tend to migrate and plug the outlet frit raising column backpressure. Commonly seem with irregular packing that have micro fractures 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 serves as bed supports and filter the sample coming in from the injector
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 place in-line between injector and the main column
Helium Sparging–A solvent degassing technique in which helium gas is bubbled through solvents to displace dissolved gases before solvent mixing, compression and pumping
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 conditions are not restricted to single solvents or solvent mixtures, but can include multiple components in the mobile phase.
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
Lamps–Light source for a detector. A deuterium lamp is fully variable from 190 nm to 400 nm; 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 provides molecular weight information 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 pressure, 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
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 Columns–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 rate with relatively low backpressure
Multichannel HPLC–HPLC system designed to run parallel HPLC columns into a multi-flow cell UV or fluorescent detector. Designed for production laboratory to speed QA/QC monitoring
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 spectrometer interfaces
Needle Port Seal–TeflonR 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
ODS–Octadecylsilyl bonded phase material or column in which the material bound to silica is an 18-carbon saturated hydrocarbon chain
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 column 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 separation. 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
Polarity–A measure of a solvent, column 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
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
Reciprocating Pumps–Single and dual headed pumps which 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 nonpolars compounds, Nonpolar columns require polar solvents.
RP18–Reverse phase, bonded packing with 18-carbon side chain (see C18, ODS)
Rotor Seal–TeflonR surface that seals the injector and separates the flowing mobile phase from the sample loop until an injection is completed
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 base on size and shape
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
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 compound
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
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
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,
Voids–Spaces or openings in the column bed leading to poor chromatography. 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
Windowing–A technique using cartridge columns ( SFE) to speed chromatography by first removing polar and nonpolar impurities leaving only a solvent fraction containing the compounds of interest
Zero Dead Volume–Fittings design to leave no extra column volumes that might cause band spreading or remixing of peaks


Dr. Marvin C. McMaster
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