Comparison: GC & HPLC

Partition in 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-attracts like or oil and water (similar

electrostatic properties)
Polar/polar & non-polar/non-polar
Molecules with dissimilar properties are not
attracted Polar retention forces
Hydrogen bonding
(permanent dipoles)
Dipole-Induced dipole

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+Cu
where u is the average velocity of the mobile phase.
A, B, and C are factors which contribute to band broadening.
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 time 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
Chromatographic grade gases (high purity)

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.

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.

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 IDs
Good resolution of early eluting compounds
Longer analysis times
Limited dynamic range
ID Effects - larger IDs
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

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

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

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