CHAPTER 17 CHROMATOGRAPHIC METHODS AND HYPHENATED TECHNIQUES Introduction to Analytical Chemistry.

CHAPTER 17 CHROMATOGRAPHIC

METHODS AND HYPHENATED TECHNIQUES

Introduction toIntroduction toAnalytical ChemistryAnalytical Chemistry

Copyright © 2011 Cengage Learning17-2

17A Gas-liquid Chromatography

Two types of gas chromatography are encountered: gas-liquid chromatography (GLC) and gas-solid chromatography (GSC). Gas-liquid chromatography finds widespread use in where its name is usually shortened to gas chromatography(GC).


17A Gas-liquid Chromatography

Gas-liquid chromatography is based on partitioning of the analyte between a gaseous mobile phase and a liquid phase immobilized on the surface of an inert solid packing or on the walls of a capillary tubing.


17A-2 Carrier Gas System

Helium is the most common mobile phase, although argon, nitrogen, and hydrogen are also used.

Pressures at the column inlet usually range from 10 to 50 psi (lb/in. above room pressure) and provide flow rates of 25 to 50 mL/min.


Figure 17-1

Figure 17-1 Block diagram of a gas-chromatographic apparatus.


17A-3 Sample Injection System

The sample port (Figure 17-4) is ordinarily about 50°C above the boiling point of the least volatile component of sample sizes range from a few tenths of a microliter to 20 mL. Capillary columns require samples that are smaller by a factor of 100 or more.

Commercial gas chromatographs with capillary columns incorporate such splitters and also allow for sample injection without splitting when packed columns are used (split /splitless injection systems).


Figure 17-4

Figure 17-4 Cross-sectional view of a microflash vaporizer direct injector.


17A-4 Detectors

Flame-Ionization Detectors Most organic compounds, when pyrolyzed in a hot flame,

produce ionic intermediates that conduct electricity through the flame. Hydrogen is added to the carrier gas with this detector, and the eluent is mixed with oxygen and combusted in a burner equipped with a pair of electrodes.

Detection involves monitoring the current. FID exhibits a high sensitivity, a large linear response, and

low noise


17A-4 DetectorsThermal Conductivity Detectors

This device consists of an electrically heated source whose temperature at constant electric power depends on the thermal conductivity of the surrounding gas.

The chief limitation of the thermal conductivity detector is its relatively low sensitivity.


Figure 17-5

Figure 17-5 A typical flame ionization detector. (Courtesy of Hewlett-Packard Company.)


17A-4 Detectors

Electron-Capture Detectors The electron-capture detector (ECD) has become one of the

most widely used detectors for environmental samples because this detector selectively responds to halogen-containing organic compounds.

In this detector, the sample eluate from a column is passed over a radioactive β emitter.


17A-4 Detectors

Electron-Capture Detectors An electron from the emitter causes ionization of the carrier

gas and the production of a burst of electrons A constant standing current between a pair of electrodes

results from this ionization process The current decreases markedly, in the presence of organic

molecules containing electronegative functional groups that tend to capture electrons.


17A-4 Detectors

Electron-Capture Detectors Electron-capture detectors are highly sensitive and have The linear response of the detector, however, is limited to

about two orders of magnitude.


Figure 17-6

Figure 17-6 A typical thermal conductivity detector. (Courtesy of Varian Instrument Division, Palo Alto, CA.)


17A-4 Detectors

Hyphenated Methods of Detection Other important GC detectors With the thermionic detector, nitrogen- and phosphorous-

containing compounds produce increased currents in a flame in which an alkali metal salt is vaporized.

With the electrolytic conductivity detector, compounds containing halogens, sulfur, or nitrogen are mixed with a reaction gas in a small reactor tube. The products are then dissolved in a liquid, which produces a conductive solution.


17A-4 Detectors

Hyphenated Methods of Detection In the photoionization detector, molecules are photoionized

by UV radiation. The ions and electrons produced are then collected, and the resulting current is measured.

The effluent from chromatographic columns is often monitored continuously by the selective techniques of spectroscopy or electrochemistry.


17A-5 Gas Chromatographic Columns and Stationary Phases

Capillary, or Open Tubular, Columns Currently, the most widely used capillary columns are fused-

silica open tubular columns (FSOT columns). Their inside diameters are typically from 0.1 to 0.5 mm.


Figure 17-7

Figure 17-7 A 25-m fused-silica capillary column. (Courtesy of Varian, Inc., Walnut Creek, CA)



Packed Columns− Packed columns are fabricated from glass or metal tubing;

they are typically 2 to 3 m long and have inside diameters of 2 to 4 mm.

− The packing, or support, for a column holds the liquid stationary phase in place.

− The ideal solid packing consists of small, uniform, spherical particles with good mechanical strength and with a specific surface of at least 1 m²/g.



Packed Columns− the most widely used, packings for gas chromatography

were prepared from naturally occurring diatomaceous earth.

− The particle size of packings for gas chromatography typically fall in the range of 60 to 80 mesh (250 to 170 μm) or 80 to 100 mesh (170 to 149 μm).



Column Thermostating− Reproducible retention times require control of the column

temperature to within a few tenths of a degree.


17A-6 Liquid Phases for Gas-Liquid Chromatography

Desirable properties for the immobilized liquid phase include (1) low volatility (2) thermal stability (3) chemical inertness (4) solvent character-characteristics such that k and α values

for the solutes to be resolved fall within a suitable range.


Figure 17-9

Figure 17-9 Effect of temperature on gas chromatograms. (a) Isothermal at 45°C, (b) isothermal at 145°C, (c) programmed at 30°C to 180°C. [From W. E. Harris and H. W. Habgood, Programmed Temperature Gas Chromatography (New York: Wiley, 1966), p. 10. Reprinted with permission.]



Polar stationary phases contain functional groups such as － CO, － OH, unsaturation (double- or triple-bond), － NO₂, and － CN. Hydrocarbon-type stationary phases and dialkyl siloxanes are nonpolar.

Generally, the polarity of the stationary phase should match that of the sample components.



Polydimethyl siloxanes that have the general structure

The percentage description in each case gives the amount of substitution of the named group for methyl groups on the polysiloxane backbone


Table 17-2


17A-7 Applications of Gas-Liquid Chromatography

Qualitative Analysis A chromatogram provides but a single piece of information

about each species in a mixture (the retention time) Qualitative analysis of complex samples of unknown

composition is limited. Limitation has been largely overcome by linking

chromatographic columns directly with ultraviolet, infrared, and mass spectrometers.



Quantitative Analysis Quantitative GC is based on comparison of either the height

or the area of an analyte peak with that of one or more standards.

Peak area is independent of the broadening effects discussed earlier.

Therefore, area is a more satisfactory analytical parameter than peak height.



The Internal-Standard Method.The highest precision for quantitative GC is obtained

using internal standards because the uncertainties introduced by sample injection, flow rate, and variations in column conditions are minimized. In this procedure, a carefully measured quantity of an internal standard is introduced into each standard and sample, and the ratio of analyte peak area (or height) to internal-standard peak area (or height) is used as the analytical parameter.


17b High-performance LiquidChromatography

High-performance liquid chromatography (HPLC) is the most versatile and widely used type of elution chromatography.

Several types of high-performance liquid chromatography, include (1) partition, or liquid-liquid, chromatography;(2) adsorption, or liquid-solid, chromatography; (3) ion-exchange, or ion, chromatography; (4) size-exclusion chromatography; and (5) affinity chromatography.


Figure 17-12

Figure 17-12 Effect of particle size of packing and flow rate on plate height in liquid chromatography. (From R. E. Majors, J. Chromatogr. Sci., 1973, 11, 92, reprinted with permission.)


17B-1 Instruments for High-Performance Liquid

Chromatography

Mobile-Phase Reservoirs An elution with a single solvent of constant composition is

called isocratic. In gradient elution, two (and sometimes more) solvent systems that differ significantly in polarity are employed. The ratio of the two solvents is varied in a preprogrammed way, sometimes continuously and sometimes in a series of steps.


Figure 17-13

Figure 17-13 Block diagram showing components of a typical apparatus for HPLC. (Courtesy of Perkin-Elmer Corp., Norwalk, CT.)



Chromatography

Pumping Systems The requirements include (1) the generation of pressures of

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

Advantages of reciprocating pumps include small internal volume, high output pressure (up to 10,000 psi), ready adaptability to gradient elution, and constant flow rates.



Chromatography

Columns for High-Performance Liquid Chromatography

Most columns range in length from 10 to 30 cm and have inside diameters of 4 to 10 mm. Column packings typically have particle sizes of 5 or 10 μm. Columns of this type often contain 40,000 to 60,000 plates/m.

High-performance microcolumns with inside diameters of 1 to 4.6 mm and lengths of 3 to 7.5 cm have become available. These columns, which are packed with 3- or 5-μm particles, contain as many as 100,000 plates/m



Chromatography

Detectors Table 17-3 lists some of the common detectors and their

properties. The most widely used detectors for liquid chromatography

are based on absorption of ultraviolet or visible radiation. HPLC/MS systems can identify the analytes exiting from the

HPLC column.



Chromatography

Detectors Several electrochemical detectors have also been introduced

that are based on potentiometric, conductometric, and voltammetric measurements.


Figure 17-17

Figure 17-17 A UV detector for HPLC.


Figure 17-18

Figure 17-18 Amperometric thin-layer detector cell for HPLC.


17B-2 High-Performance Partition Chromatography

Column PackingTwo types of partition chromatography.Early work in liquid chromatography was based on

highly polar stationary phases such as triethylene glycol or water; a relatively nonpolar solvent such as hexane or i-propyl ether then served as the mobile phase. This type of chromatography is now called normal-phase chromatography.

In reversed-phase chromatography, the stationary phase is nonpolar, often a hydrocarbon, and the mobile phase is a relatively polar solvent.


17B-2 High-Performance Partition Chromatography

In normal-phase chromatography, the least polar component is eluted first; increasing the polarity of the mobile phase then decreases the elution time.

In the reversed-phase method, the most polar component elutes first, and increasing the mobile phase polarity, hence weakening its solvent strength, increases the elution time.


17B-3 High-Performance Adsorption Chromatography

All the pioneering work in chromatography was based on adsorption of analyte species on a solid surface.

Finely divided silica and alumina are the only stationary phases that find extensive use for adsorption chromatography.

A particular strength of adsorption chromatography not shared by other methods is its ability to resolve isomeric mixtures such as meta- and parasubstituted benzene derivatives.


17B-4 High-Performance Ion-Exchange Chromatography

Two types of ion chromatography are currently in use: suppressor based and single column.



Ion Chromatography Based on Suppressors Conductivity detectors have many of the properties of the

ideal detector.



Ion Chromatography Based on Suppressors The suppressor column is packed with a second ion-

exchange resin that effectively converts the ions of the eluting solvent to a molecular species of limited ionization without affecting the conductivity due to analyte ions.

For example, when cations are being separated and determined, hydrochloric acid is chosen as the eluting reagent, and the suppressor column is an anion-exchange resin in the hydroxide form.



Single-Column Ion Chromatography This approach depends on the small differences in

conductivity between sample ions and the prevailing eluent ions.


17B-5 High-Performance Size-Exclusion Chromatography

Size-exclusion, or gel, chromatography is a powerful technique that is particularly applicable to high-molecular-weight species.



Packings for size-exclusion chromatography consist of small (~10 μm) silica or polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse.

The average residence time of analyte molecules depends on their effective size. Molecules that are significantly larger than the average pore size of the packing are excluded and thus suffer no retention.



Chromatography based on the hydrophilic packings is sometimes called gel filtration, whereas techniques based on hydrophobic packings are called gel permeation.


Figure 17-23

Figure 17-23 Gel-permeation separation of components in an epoxy resin. (Courtesy of BTR Separations, a DuPont/ConAgra affiliate.)


Table 17-5


17D-1 Solid-Phase Extraction

As shown in Figure 17-24, the SPE consists of four steps—column conditioning,sample sorption, rinsing, and elution to desorb solutes.


Figure 17-24

Figure 17-24 (a), (b) CONDITIONING: The SPE column is conditioned with solvent to increaseretention reproducibility. (c), (d) SORPTION: Sample is loaded. (e) RINSING: Removes undesired matrix components. (f) ELUTION: Desired components are eluted; the strongly retained undesiredcomponents remain in the column.


17D-2 Solid Phase Microextraction

SPME uses a coated fused-silica fiber to draw organic compounds from the ambience by diffusion.

Alternatively, use of convection or agitation helps to speed up a deep sampling process. Headspace is another popular method, in which the SPME fiber laying in a confined space draws analytes from a sample of soil or water by diffusion.


Figure 17-25

Figure 17-25 Solid Phase Microextraction (Reprinted with permission from Bulletin 923: Solid Phase Microextraction: Theory and Optimization of Conditions, Figure A, available from: http://www.sigmaaldrich.com/Graphics/Supelco/objects/4600/4547.pdf, ©1998 Sigma Aldrich Co., St. Louis, MO.)


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CHAPTER 17 CHROMATOGRAPHIC METHODS AND HYPHENATED TECHNIQUES Introduction to Analytical Chemistry.

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