GC Lecture Notes

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Comparison: GC & HPLC Comparison: GC & HPLC

Partition in

Partition in ChromatograpChromatographyhy • Stationary phase, mobile phase, & analyte form

• Stationary phase, mobile phase, & analyte form aa ternary system. ternary system. • Each analyte is distributed between the two phases (

• Each analyte is distributed between the two phases ( in equilibrium):in equilibrium): –

– Partition Coefficient K = C Partition Coefficient K = CSS/C/Cmm –

– C CSS: concentration of analyte on the stationary phase: concentration of analyte on the stationary phase –

– C CMM: concentration of analyte on the mobile phase: concentration of analyte on the mobile phase

Factors Influencing Retention Factors Influencing Retention •

•Are those that influence distributionAre those that influence distribution –

– Stationary phase: type & properties Stationary phase: type & properties –

– Mobile phase: composition & properties Mobile phase: composition & properties –

– Intermolecular forces between Intermolecular forces between • Analyte & mobile phase • Analyte & mobile phase • Analyte & stationary phase • Analyte & stationary phase –

– Temperature Temperature

Intermolecular Forces Intermolecular Forces •

•

Based on electrostatic forces

–

– “Like

“Like--attracts like” or “oil and water” (similar

attracts like” or “oil and water” (similar

electrostatic properties)

• Polar/polar & no

• Polar/polar & no n-polar/non-polar

n-polar/non-polar

–

– Molecules with dissimilar properties are not

Molecules with dissimilar properties are not

attracted

attracted • Polar retention forces

• Polar retention forces

–

– Hydrogen bonding

Hydrogen bonding

(permanent dipoles)

–

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The Rate Theory of Chromatography

A more realistic description of the processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phase. The resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase.

If we consider the various mechanisms which contribute to band broadening = A + B / u + C u

where u is the average velocity of the mobile phase.

A, B, and C are factors which contribute to band broad ening.

A_{- Eddy diffusion}

The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.

B_{- Longitudinal diffusion}

The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening.

If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion.

C _{- Resistance to mass transfer}

The analyte takes a certain amount of tim e to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

Gas Chromatography – Overview

• Sample is vaporised and injected onto head of a chromatography column. • Elution is effected by the flow of an inert gaseous mobile phase.

• Separation is based upon the partition of the analyte between a gaseous mobile phase and a liquid phase immobilised on the surface of an inert solid (GLC) at a temperature above boiling point of analyte (multi-analyte: temperature programming).

• Mobile phase does not interact with molecules of the analyte.

• Eluted analyte detected by a detector and recorded by PC – Chemstation.

• GC columns are either packed (with silica particles coated in stationary) or capillary in nature. •

Carrier Gas  Inert

 Helium

 Choice dictated by detector, cost, availability  Pressure regulated for constant inlet pressure  Flow controlled for constant flow rate

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Sample Injection

•

GC column efficiency requires that the sample be of suitable size (to prevent column over loading) and be introduced as a plug of vapour.

•

Two common approaches include for introduction of 0.01 – 50 ml include: Microsyringe and valve loop.

•

The syringe technique is most common and can be used with both gas and low viscosity liquid samples by inserting the needle through a rubber septum to the column inlet port.

•

The region into which the needle projects must be heated in order to flash vaporise the sample.

•

However, overheating of the rubber septum must be avoided to prevent out gassing.

•

The most popular inlet for capillary GC is the split/splitless injector.

•

If this injector is operated in split mode, the amount of sample reaching the column is reduced (to prevent column overloading) and very narrow initial peak widths can be obtained.

•

For maximum sensitivity, the injector can be used in so-called splitless mode, then all of the injected sample will reach the column.

•

Injection may be manual or automated.

Split – Splitless Injection

• Septum purge outlet prevents components of previous injections from entering the column and minimizes the effect of septum bleed (low flow rate ~3 ml/min).

• The sample is injected into the liner region where it is completely vaporised. Mostly glass liners – zero dead volume

• The sample volume is then split between the column and the split outlet. Split injection is employed to dilute the sample and prevent column overloading. Typically 1:100 split ratios are employed with 99% of sample being vented to atmosphere.

• Method development: Some parameters of split/splitless injection that require optimisation, apart from instrumental design, are injector temperature, split ratio, split delay, injection volume, sample solvent and initial temperature of the column.

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Sample Valve Injection

• A version of reaction chromatography in which a sample is thermally decomposed to simpler fragments before entering the column. 1993, 65, 827

IUPAC Compendium of Chemical Terminology

• Many non-volatile solids can be decomposed thermally to produce characteristic gaseous products that can be chromatographed.

• Samples are placed directly on a small coil of Pt wire where it can be heated to several hundred degrees in a few milliseconds while the carrier gas is flowing over it.

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Column Configuration

Stationary Phases

 Choice of phase determines selectivity  Hundred of phases available

 Many phases give same separation

 Same phase may have multiple brand names

 Stationary phase selection for capillary columns much simpler  Like dissolves like

 Use polar phases for polar components

 Use non-polar phases for non-polar components Internal Diameter, Smaller ID’s

• Good resolution of early eluting compounds • Longer analysis times

• Limited dynamic range

ID Effects - larger ID’s

• Have less resolution of early eluting compounds • Shorter analysis times

• Insufficient resolution for complex mixtures

Length effects - isothermal analysis

• Retention more dependant on length

• Doubling column length doubles analysis times • Resolution a function of Square Root of Length • Gain 41% in resolution

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Characteristics of Ideal GC Detector • Good stability and reproducibility.

• Linear response to analytes that extends over several orders of magnitude. • Similarity in response toward all analytes.

• Temperature range from room temperature to 400C.

• A short response time that is independent of flow rate. • Non-destructive.

• High reliability and ease of use.

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Flame Ionisation Detector

Advantages and Disadvantages of GC

Quantification in GC

Response of detector varies with analyte

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Quantification: Standard Addition

Quantification: Normalizing Peak Areas

Quantification: Internal Standard

Basic GCMS Theory

• Sample injected onto column via injector • GC then separates sample molecules

• Effluent from GC passes through transfer line into the Ion Trap/Ion source • Molecules then undergo electron /chemical ionisation

• Ions are then analysed according to their mass to charge ratio