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GC and HPLC

Chem 223

Gas Chromatography (GC)

• Mobile phase is a carrier gas (usually He,N2, or H2)

• Analytes can be gases or volatile liquids(must be in gas phase to go throughcolumn)

• Columns may be packed or open– No multiple path broadening in open columns

A Schematic GC Instrument Open Tubular Column Types forGC

• Wall-coated open tubular (WCOT)• Support-coated open tubular (SCOT)• Porous-layer open tubular (PLOT)

Packed vs. Open

• Packed columns– Have greater capacity– Need bigger samples– Tend to produce

broader peaks• Open columns

– Have smaller capacity– Work with smaller

samples– Tend to have more

theoretical plates

What Does Ramping theTemperature Buy for You?

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Temperature Programming

• At low T, diffusion and equilibration are bothslow

• Raising the temperature– Decreases retention time– Sharpens peaks (all other things being equal)

• Typically, GC is done with a temperature program– Raising T drives less volatile material off the column

that otherwise would not elute– Usually this results in better separations

Typical GC Detectors

• Thermal conductivity (TCD)• Flame ionization (FID)• Electron capture (ECD)• Mass spectrometer (MS)

Thermal Conductivity Detector• Change in thermal

conductivityproduces signal

• General

• Low sensitivityoverall

Flame Ionization Detector• Hydrocarbons more easily

ionized than He; increasein ion current is signal

• High sensitivity forhydrocarbons

• Not general

Electron Capture Detector• Decrease in electron

current produces signal

• High sensitivity forhalogens

• Not general

Mass Spectrometer• Ionizes samples and

separates the ions bymass

• Peak patterncharacteristic ofcompound

• General

• Provides bothqualitative andquantitative analysis

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HPLC

• High performance (pressure) liquidchromatography

• One of the most heavily-used analyticaltechniques

• Uses a liquid mobile phase• Stationary phase consists of micron-sized

particles

A Typical HPLC Instrument

Influence of Particle Size

• Smaller particles yield higher resolution– Allow faster diffusion of solute between

stationary and mobile phases– Minimize multiple-path effects

• Smaller particles cause increased resistanceto solvent flow– Very high (70-400 bar) backing pressures are

required to compensate

Particle Size-Examples

Stationary Phases for HPLC• Normal-phase

– Polar stationary phase– Less polar mobile phase– Use a more polar solvent to increase elution strength

• Reversed-phase– Non-(or weakly)polar stationary phase– More polar solvent– Use a less polar solvent to increase elution strength

• Reversed-phase HPLC is more common– Eliminates tailing from polor compounds sticking to

polar column packing material– Less sensitive to polar impurities (like water) in the

solvent

Materials for Stationary Phases

• Microporous silica particles (3-10 mm diameter)– Adsorption of analyte occurs on particle surface

• Bonded stationary phase– React material with the silica surface to bond it– This allows modification of the surface to produce

tailor-made stationary phases– Octadecyl (C18) is the most common

• Would this be a reversed-phase or normal-phase material?

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An HPLC Column

• Guardcolumn isdisposable;protectsexpensivemaincolumnfrom stuffthat wouldfoul it

Solvent Programming in HPLC

• Solvent composition constant: isocraticelution

• Use of a changing mixture of solvents:gradient elution

Isocratic Elutions, Decreasing Eluent Strength

1) Benzyl alcohol, 2) phenol, 3) 3’,4’-dimethoxyacetophenone, 4) benzoin, 5) ethyl benzoate, 6) toluene, 7)2,6-dimethoxytoluene, 8) o-methoxybiphenyl. Reversed-phase column. A: aqueous buffer, B: acetonitrile

Gradient Elution (Same Sample)• Start with low

elution strengthand increase byadding moreacetonitrile

• Keepsseparationrelatively fastwhilemaintainingresolution

HPLC Detectors• UV detectors

– Measure UV absorbance, typically at 254 nm– Only work for things that absorb!

• Refractive index detectors– Deflection of light beam changes with changing

liquid composition– General– Insensitive

• Electrochemical– Uses a sensing electrode– Sensitive and specific

• Fluorescence– Very sensitive– Not general

• Mass spectrometer– Very sensitive and general– Expensive