GC Lecture Notes

Comparison: GC & HPLC

Partition in Chromatography Chromatography • Stationary phase, mobile phase, & analyte form a ternary system. • Each analyte is distributed between the two phases ( in equilibrium):  – Partition Coefficient K = CS/Cm  – CS: concentration of analyte on the stationary phase  – CM: concentration of analyte on the mobile phase

Factors Influencing Retention • Are those that influence distribution  – Stationary phase: type & properties  – Mobile phase: composition & properties  – Intermolecular forces between • Analyte & mobile phase • Analyte & stationary phase  – Temperature Intermolecular Forces • Based on electrostatic forces

 – “Like  – “Like--attracts like” or “oil and water” (similar  electrostatic properties) • Polar/polar & no n-polar/non-polar  –  Molecules  Molecules with dissimilar properties are not attracted • Polar retention forces  –  Hydrogen  Hydrogen bonding (permanent dipoles)  –  Dipole-Induced  Dipole-Induced dipole

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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.

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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

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Inert Helium Choice dictated by detector, cost, availability Pressure regulated for constant inlet pressure Flow controlled for constant flow rate Chromatographic grade gases (high purity)

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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. The pyrolysis products are swept directly onto the column.

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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 Is it worth the extra time and expense? Length effects - programmed analysis Retention more dependant on temperature Marginally increases analysis times Run conditions should be optimised • • •

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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. 

Thermal Conductivity Detector

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Flame Ionisation Detector

Advantages and Disadvantages of GC

Quantification in GC Response of detector varies with analyte Response factor to relate concentration to peak area Three methods: -Standard addition -Normalization peak area -Internal standard

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Quantification: Standard Addition

Quantification: Normalizing Peak Areas

Quantification: Internal Standard

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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 Ions are detected by electron multiplier which produces a signal proportional to ions detected

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