chem 321 ch 33/lecture 15

ion-pair chromatography

An HPLC technique based on the partitioning of ionic species between a liquid mobile phase and a solid polymeric ionic exchanger; subset of reversed-phase chromatography in which easily ionizable species are separated on reversed-phase columns; the counterion forms an uncharged ion pair with a solute ion of opposite charge in the mobile phase. This ion pair then partitions into the nonpolar stationary phase giving differential retention of solutes based on the affinity of the ion pair for the two phases

all the characteristics of the ideal GC detector low internal volume (dead volume) to minimize extra-column band broadening should be small and compatible with liquid flow

The ideal detector for HPLC should have (3)

absorption of ultraviolet or visible radiation

The most widely used detectors for liquid chromatography are based on...

sampling loop

The most widely used method of sample introduction in liquid chromatography is based on a

1. the generation of pressures of up to 6000 psi (lb/in2) 2. pulse-free output 3. flow rates ranging from 0.1 to 10 mL/min 4. flow reproducibilities of 0.5% relative or better 5. resistance to corrosion by a variety of solvents

The requirements for liquid chromatographic pumps include (5)

affinity chromatography affinity ligand

a reagent (called what) is covalently bonded to a solid support. Typical ones are antibodies, enzyme inhibitors, or other molecules that reversibly and selectively bind to analyte molecules in the sam- ple. When the sample passes through the column, only the molecules that selectively bind to the the reagent are retained. Molecules that do not bind pass through the column with the mobile phase. After the undesired molecules are removed, the retained analytes can be eluted by changing the mobile phase conditions

Efficient, highly selective, widely applicable Only small sample required May be nondestructive of sample Readily adapted to quantitative analysis

characteristics of both HPLC and GC (6)

size-exclusion chromatography

fractionation is based on molecular size; silica or polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse. While in the pores, molecules are effectively trapped and removed from the flow of the mobile phase

Reciprocating types pumps

consists of a small cylindrical chamber that is filled and then emptied by the back- and-forth motion of a piston. The pumping motion produces a pulsed flow that must be subsequently damped because the pulses appear as baseline noise on the chromatogram

porous particle

consists of porous microparticles having diameters ranging from 3 to 10 mm; for a given size particle, a very narrow particle size distribution is desirable. The particles are composed of silica, alumina, the synthetic resin polystyrene-divinyl benzene, or an ion-exchange resin

Syringe-type pumps

produce a pulse-free delivery whose flow rate is easily controlled; relatively low capacity (,250 mL) and are inconvenient when solvents must be changed

Simple and inexpensive equipment Rapid Unparalleled resolution (with capillary columns) Easy to interface with mass spectrometry

4 advantages of GC

suppressor-based chromatography

A chromatographic technique involving a column or a membrane located between the analytical column and a conductivity detector; its purpose is to convert ions of the eluting solvent into nonconducting species while passing ions of the sample

dual pump heads or elliptical cams

Modern HPLC instruments use _______ to minimize such pulsations

the sample must dissolve in the mobile phase

Sample and mobile phase must be chemically similar because...

(1) partition, or liquid-liquid, chromatography; (2) adsorption, or liquid-solid, chromatography; (3) ion-exchange, or ion, chromatography; (4) size-exclusion chromatography; (5) affinity chromatography; and (6) chiral chromatography.

The types of high- performance liquid chromatography are often classified by the separation mechanism or by the type of stationary phase (6)

the screw-driven syringe type and the reciprocating pump

Two major types of pumps are used in HPLC instruments

pellicular and porous particle

Two types of packings are used in HPLC

single-column ion-exchange chromatography

analyte ions are separated on a low-capacity ion exchanger by means of a low-ionic strength eluent that does not interfere with the conductometric detection of analyte ions

reciprocating pumps

are used in almost all commercial instruments; advantages: small internal volume (35 to 400 mL), high output pressure (up to 10,000 psi), ready adaptability to gradient elution, and constant flow rates, which are largely independent of column back-pressure and solvent viscosity

guard column

between the injector and the column removes particulates and other solvent impurities; a short column packed with a similar stationary phase as the analytical column; prevents impurities, such as highly retained compounds and particulate matter, from reaching and contaminating the analytical column

scavenger column

between the mobile-phase container and the injector is used to condition the mobile phase; The solvent partially dissolves the silica packing and ensures that the mobile phase is saturated with silicic acid prior to entering the analytical column. This satu- ration minimizes losses of the stationary phase from the analytical column.

gradient elution

in HPLC is one in which the composition of the solvent is changed continuously or in a series of steps; two (and sometimes more) solvent systems that differ significantly in polarity are used and varied in composition during the separation

isocratic elution

in HPLC is one in which the solvent composition remains constant

chiral resolving agent

is a chiral mobile-phase additive or a chiral stationary phase that preferentially complexes one of the enantiomers.

sparging

is a process in which dissolved gases are swept out of a solvent by bubbles of an inert, insoluble gas

high-performance liquid chromatography (HPLC)

is a type of chromatography that combines a liquid mobile phase and a very finely divided stationary phase. In order to obtain satisfactory flow rates, the liquid must be pressurized to several hundred or more pounds per square inch; Pumping pressures of several hundred atmospheres are required to achieve reason- able flow rates with packings in the 3- to 10-um size range

gel filtration

is a type of size-exclusion chromatography in which the packing is hydrophilic. It is used to separate polar species

gel permeation

is a type of size-exclusion chromatography in which the packing is hydrophobic. It is used to separate nonpolar species.

silica particles for column packings

more rigid, which leads to easier packing and permits higher pressures to be used; more stable, allowing a great range of solvents to be used and exhibiting more rapid equilibration with new solvents

silica particles

most common packing in liquid chromatography; often coated with thin organic films, which are chemically or physically bonded to the surface

conductivity detector

well suited for ion chromatography; highly sensitive, for charged species, respond in a predictable way to concentration changes, simple to operate, inexpensive to construct and maintain, easy to miniaturize, and usually give prolonged, trouble-free service; The only limitation is the high electrolyte concentrations required to elute most analyte ions in a reasonable time. As a result, the conductivity from the mobile-phase components tends to swamp that from the analyte ions, greatly reducing the detector sensitivity

pellicular particle

spherical, nonporous, glass or polymer beads with typical di- ameters of 30 to 40 mm. A thin, porous layer of silica, alumina, a polystyrene-divinyl benzene synthetic resin, or an ion-exchange resin was deposited on the surface of these beads; separation of proteins and large biomolecules

suppressor-based ion chromatography

the ion-exchange column is followed by a suppressor column, or a suppressor membrane, that converts an ionic eluent into a nonionic species that does not interfere with the conductometric detection of analyte ions

column bleeding Chemically bonded phases

the mobile phase must be presaturated with the stationary phase or else what will occur what reduces this problem

partition chromatography

the stationary phase is a second liquid that is immiscible with the liquid mobile phase

liquid-liquid partition chromatography

the stationary phase is a solvent held in place by adsorption of the surface of the packing particles

liquid-bonded-phase chromatography

the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds

reversed-phase partition chromatography

the stationary phase is nonpolar and the mobile phase polar; the least polar analyte is eluted last.

normal-phase partition chromatography

the stationary phase is polar and the mobile phase nonpolar; the least polar analyte is eluted first; increasing the polarity of the mobile phase then decreases the elution time

Can accommodate nonvolatile and thermally unstable compounds Generally applicable to inorganic ions

two advantages of HPLC