Gas Chromatography
Let’s begin with an example problem: SPME head space analysis of
pesticides in tea and follow-up analysis by high speed GC.
Samples in 10mL sealed glass vials were placed in the MPS-2 autosampler for HS-
SPME extraction. Extracted compounds were thermally desorbed from the fiber
after its insertion into the hot (270 ◦C) splitless GC injection port. Desorption
time was 2min.
Anal Chim. Acta 611 (2008) 163
Gas Chromatography
Conventional HS-SPME
Fig. 3 – Comparison of conventional (a) and SPME-based (b) approach in analysis of
pesticide residues in tea samples. GC/TOF MS chromatograms of blank (non-spiked)
black (I) and fruit (II) teas; pesticide standard solution (200g kg−1) (III).
Malathion Heptachlor Anal Chim. Acta 611 (2008) 163
Gas Chromatography
Detection Figures of Merit (36 pesticides tested)
Pesticide MRL Linearity Dynamic LOQ RSD (%)
(μg/kg) (r2) Range (μg/kg) 50 μg/kg
(μg/kg) S/N = 12 N=5
HS-SPME
Malathion 500 0.9838 50-200 26 13
Heptaclor 20 0.9870 10-200 8 9
Ethyl Very time
acetate consuming !
extraction
Malathion 500 0.9977 10-200 8 11
Heptaclor 20 0.9983 1.25-200 1 5
MRL = maxiumum residue limit
Gas Chromatography - Instrument
A gas chromatograph (GC) is an analytical instrument that measures the content of
various volatile components in a sample. The analysis performed by a gas
chromatograph is called gas chromatography.
Principle of gas chromatography: The sample solution injected into the instrument
enters a gas stream which transports the sample into a separation tube known as the
"column." (Helium or nitrogen is used as the so-called carrier gas.) The various
components are separated inside the column. The detector measures the quantity of
the components that exit the column.
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Gas Chromatography
Column separation (gas-liquid, gas-solid) used for separating and
analyzing compounds that can be vaporized without decomposition.
Higher b.p Lower b.p
Separations generally based on
differences in boiling points!
Conditions
• Carrier gas (mobile phase) does NOTHING in GC but transport the
compounds. Not involved in separation mechanism (H2 and He common).
• Injection volume (0.1 – 10 μL generally). Temperature of injector is 50 oC
greater than least volatile (highest boiling point compound). All
compounds must be vaporized before transport onto column.
• Fixed temperature separation – average boiling point of all analytes is a
good starting point.
• Carrier gas is often dried by passage over molecular sieves as they strongly
retain water. Activated by heating to 300 oC in vacuum.
• Gaseous mobile phase carries gaseous compounds (analytes) through a
long column with a stationary phase.
Thermodynamics of Separations
The application of thermodynamics to a chromatographic separations
explains how the distribution coefficient (which itself determines the
magnitude of retention) is controlled by the standard energy of
distribution and the absolute temperature.
CStat
KD
CMobile
Amobile AStationary
GD
ln K D
RT
The Chromatogram
Average velocity
of moving
analyte zone
tr tm kb (last )
k
'
tm k a ( first )
Capacity Factor (2-10) Selectivity Factor (>1)
Plate Height – Equilibration Zones
Distillation theory
Height equivalent to a theoretical plate (H)
L(cm)
N ( platenumbe r )
H (cm / plate )
2
tr t r2
N 16 N 5.55 2
W W1/ 2
Why Do Bands Separate and Broaden with
Time on Column?
Longer on the column, longer the retention time (tr) and the broader the peaks.
Why Does Peak Width Matter?
Although the selectivity factor, α, describes the separation of band centers, it
does not take into account peak widths. Another measure of how well species
have been separated is provided by measurement of the resolution (R). The
resolution of two species, A and B, is defined as:
Rs
2 t rb t ra
wb wa
w = baseline peak width of two neighboring peaks.
Baseline resolution, R > 1.5
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 (unlike the plate model, which assumes that equilibration is infinitely fast).
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, we arrive at the Van Deemter
equation for plate height;
B
H A Cu
u
where u is the average velocity of the mobile phase. A, B, and C are factors which
contribute to band broadening.
Rate Theory of Chromatography
Van Deemter plot -
A plot of plate height (H) vs. average linear velocity (u) of mobile phase.
Rate Theory of Chromatography
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.
Efficiency of Separation (plate number)
H = L/N Small plate height = narrow peaks = better separation
Multiple Flow Path Term
One wants a small value for H! Plate numbers 100s-1000s for conventional
HPLC. Can be 106 for some high efficiency separations!!!!
Zone Broadening Terms
A term = Eddy Diffusion
Bterm = Longitudinal Diffusion
C term = Stationary Phase
Mass Transport
Why Do We Care About Flow Rate?
N (efficiency) = L/H N α Rs
Van Deemter Eq.
Flow Velocity (cm/s) = Flow Rate (cm3/s)/Cross sectional area (cm2)
Rate Theory of Chromatography
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Rate Theory of Chromatography
H is plate height
λ is particle shape (with regard to the packing)
dp is particle diameter
γ, ω, and R are constants (function of packing and k’)
Dm is the diffusion coefficient of the mobile phase
dc is the capillary diameter
df is the film thickness
Ds is the diffusion coefficient of the stationary phase.
u is the linear velocity
Goal: To achieve the fastest separation with the highest plate
number and resolution!!!
