Chương 11: Phương pháp

sắc ký khí (GC)

 GC:
Figure1: The diagram of separation
- Mobile phase: inert gas process.
- Stationary phase: solid or
liquid.
- Sample is injected into the
gas phase (carrier gas).
- Gas + sample moving into
separation column.
- Solutes are separated Figure 2: The schematic diagram of GC instrument
based on the different
partitioning of the solutes in
the mobile phase and the
stationary phase.
- After column, solutes are
moving to the detector
producing signal.
Separation column
 How to select the stationary phase?

-Rule: similar compounds will be dissolved

in each other.
-Polar substances: polar stationary phase
-Non-polar substances: non-polar
stationary phase
-Non-volatile, non-degradable at working
temperature of the column.
-No irreversible reactions between the
solutes and the stationary phase and the
carrier gas.
 Stationary phase
Materials for the stationary phase :
Stationary phase polarity Trade Temperature Representative
name limit applications

Squalane Non Squalane 150 Low-boiling aliphatics

hydrocarbons

Apezion L Non Apezion L 300 Amides, esters, Fatty acid

Polydimethyl siloxane Slightly SE-30 300-350 Amino acids, drugs,

pesticide, phenols

Phenylmethyl moderately OV-17 375 Drugs, pesticide,

polysiloxane polyaromatic,

Triflouzopropylmethyl moderately OV-210 275 Amino aicds, drugs,

polysiloxane ketones, halogenated
compounds
 GC Instrumentation
Columns
• Stationary Phases discussed previously
• OT columns
– Best resolution
– More robust
– High resolution (thin film 0.25 mm diameter) vs.
higher capacity (thick film 0.53 mm diameter)
– More expensive, difficult to make in lab
• Packed columns
– More capacity
– Better with less (mass) sensitive detectors (thermal
conductivity detector)
 Mobile phase
Inert gas: H2, N2, Ar...
Mobile phase rate:
for packed column: 25-150 mL/min
for capillary column: 1-25 mL/min
 (Mobile Phase)
• Since there is no mobile phase –
analyte interaction in GC, why
does the mobile phase matter?
– Affects diffusion
• Smallest MW gases diffuse
faster
• van Deemter B term at low
flow rates (fast is worse)
and C term at higher flow
rates (fast is better)
• Hmin not affected much, but
umin affected by gas chosen
• Smallest MW allows fastest
runs at min. H
– Detector requirements
– He is most common (inert, safe CO2 min H2 min
gas with high diffusivity for
better efficiency at high flow
rate)
– H2 also can be used with even
better efficiency, but is less safe
 Elution order in GC
Depending 2 factors:
- Boiling temperature of solutes: solute with
lower boiling temperature eluted first, solute
with higher boiling temperature  eluted later.
-Interaction in GC: partition between the solutes
and the stationary phase. Solute well dissolved
in the stationary phase eluted first.
+ Non-polar stationary phase  more polar
solute eluted first, less polar solute eluted later.
+ Polar stationary phase: less polar solute
eluted first, more polar solute eluted later.
 GC Instrumentation
Flow Control
• Flow can be controlled by Pressure
regulating inlet pressure (either Transducer
constant pressure or Solenoid valve
compensation for constant linear
velocity).
• Equipment consists of valves for
regulating pressure (constant
pressure) in older instruments or
electronic pressure control
(solenoid valve opens or closes
in response to pressure). Soap film
• Flow rate is typically checked at
detector using bubble meter.
soap
 GC Instrumentation
Sample Injection
• Several types of injectors are available and choice of
injector depends on sample phase, analyte
concentration, and other sample properties
• The most common injectors are designed for liquids (but
can be used for gases)
• Injectors for gases only can be used for gases
• Liquids require much smaller volumes (1 μL, a typical
liquid injection volume, is equivalent to ~ 1 mL after
evaporation) and column overloading is common
• Column overloading is most common with narrow
diameter OT columns and least common with packed
columns
• Most injectors are heated (except on-column)
Sample volume
For packed column: directly inject
sample volume from 1 to 20µ L.
For capillary column: to prevent the
overload of column, sample volume
must be reduced by splitting the initial
volume to a smaller fraction that goes
through the column, about 0,02 -
0,002µL (10 - 100ng).
 GC Instrumentation
Sample Injection – Gas Samples
6 port valve
• Fixed Loop Injectors
He in
– A loop of fixed volume is
To GC column
filled with a gas
– The injection valve is
twisted so that the mobile
phase pushes the gases in
the loop into the column Waste
Gas
– Very similar to most sample in
common injections in
HPLC (Covered later) INJECT POSITION
LOAD POSITION
– Very reproducible injection
 GC Instrumentation
Sample Injection – Gas Samples
• Specialized Injectors (Fixed loop injectors with trapping
capability)
– Best for trace analysis
– In place of loop is a trap (adsorbant or cold trap) so that all gas
sent into loop gets trapped, then injected
– These allow injection of greater volumes but may require
removal of interferents (oxygen, water) and require better
quantitative control of gases (careful volume or pressure
monitoring)
• Other ways to inject gas samples (using injectors
designed for liquids)
– Direct syringe injection (samples at higher concentrations)
– Solid phase microextraction (SPME with fibers exposed to gas
samples)
 GC Instrumentation
Sample Injection – Liquid Samples
• Split/Splitless Injectors
– Injectors capable of running in Syringe port
two modes: split and splitless outside
– Split injections used to avoid Septum
overloading columns Split vent
• Injection Process
– Syringe pierces septum and
liner Inside oven
depressing plunger deposits
liquid
– Analyte volatilizes He in
Split valve
– Part injected (usually smaller
fraction)
– Part passed to vent
– Fraction vented depends on To
split valve Column
 GC Instrumentation
Sample Injection – Liquid Samples
• Split injection is used for:
– Higher concentrations
– Smaller diameter (OT) columns
– More volatile components/less volatile solvents
• In split injection, solvent overload is less problematic
• Split ratios can vary from 3:1 (to vent: to column) to
1000:1
• Splitless injection is used for trace analysis (~50% of
injected sample put on column)
• Splitless injection usually requires temperature
programming where solvent passes through cool column
while analytes are trapped
• Split/Splitless injectors also used with SPME
 GC
Additional Information
• More Details on Split vs. Splitless Injection
– Split liner contains baffles to help mix sample so that
fraction to column is more consistent (but this
fraction still varies to make split less quantitative)
– Because of greater total flow in split mode, the liner is
flushed out more quickly
– In splitless injection, it takes time to purge vapors
from liner, so peak would be wide without
temperature ramping
– In split injection, the split vent is closed later along
with a decrease in He flow
– In splitless injection, the split vent is opened later to
flush out residual gases.
 GC Instrumentation
Sample Injection – Liquid Samples
• Other injectors:
– Direct injection (liner to column)
– On-column injection (needle goes into
column)
– First two types work with packed columns and
wide-bore OT columns
– Cold on-column (used with temperature
programming on inlet)
 GC Instrumentation
Sample Injection – Syringes
• For gas samples, gas-tight syringes needed
• For liquid samples, manual injection results in
variability in injected volume. An internal
standard normally is required for quantitative
work (to adjust for variation in amount injected).
• Use of autosamplers allows accurate injection of
liquids.
 GC Instrumentation
Ovens/Heating
• The columns are housed in thermostated ovens
• Most GCs have temperature programming
capability
• Most GC ovens have minimum temperatures of
around 35 to 40°C
• The maximum temperature is usually based on
avoiding damage to the column
• Operation at sub-ambient temperatures possible
with cryogenic cooling (mostly for permanent
gases/highly volatile liquids)
 Temperature control

- Very important in GC
-Column is housed in the oven, where there is a temperature
control oven. ̣
- Isothermal separation: constant temperature and a bit lower
than the boiling temperature of solutes.
-Temperature program separation: when the boiling
temperatures of the solutes are too much different. At first,
temperature is set constant for solutes with lower boiling, then
gradually raised to reach the boiling temperature of the solute
with higher boiling temperatures.
 GC Instrumentation
Detectors - Classes
• Universal Detectors
– Should detect wide range of compounds
– Response should be based on mass or moles of
analyte
– Used for comprehensive understanding of samples
• Selective Detectors
– Should detect only specific types of compounds
– Best for selected analytes in complex samples
• Multi-dimensional Detectors (selective and
universal)
 GC Instrumentation
Detectors
• Performance criteria
– Minimum detectable amounts or concentrations
(amount or concentration for peak with signal/noise =
3)
– Linear range (or useful range for non-linear
detectors)
– Destructive vs. non-destructive
– Concentration type vs. mass flow rate type
– Other (cost, speed, size, precision, etc.)
• Most GC detectors perform well for cost
vs. HPLC
 GC Instrumentation
Universal Detectors
• Thermal Conductivity Detector (TCD)
– Principle:
• heat conducted by gas from heated filament
• Filament resistance is temperature dependent
• Heat conduction depends on gas MW
– All gases (w/ MW ≠ carrier MW) detected, but
variable sensitivity.
– Simple, but not very sensitive
 GC Instrumentation
Universal Detectors
• Flame Ionization Detector (FID)
– Principle
• GC effluent “burned” in hydrogen flame
• Ions produced in combustion and collected in electrode
• Current proportional to mass combusted
– Good sensitivity
– Hydrocarbons are detected; similar response for
hydrocarbons, but decreased response with functional
groups
– Sample consumed in flame
– One of the most common because quantitation is
good and possible without standards
 Detectors
- Principle:
FID
- The combustion of organic
(Flame ionization detector )
compounds in a flame results in a
formation of cations and
electrons.
- Apply a large enough potential 
generate a current, proportional to
the amount of analytes.
- Advantage:
+ Apply for most of organic
compounds.
+ Relatively low limit of detection
(LOD)
+ Popular, inexpensive
 GC Instrumentation
Selective Detectors
• Based on Gas Ionization
– Electron Capture Detector (ECD)
• Uses β emitter to produce electrons that cause current
• Compounds with electronegative elements (e.g. halogens)
“capture” electrons reducing current
• One of the most sensitive detectors available
– Photoionization Detector (PID)
• Uses UV light to photoionize compounds (M + hν → M+ + e-)
• Sensitive to unsaturated compounds
 ECD ( Electron captured
detector )
• Radioactive decay-based detector
• Selective for compounds containing
electronegative atoms, such as halogens,
peroxides, quinones, nitro groups.
1. Structure:
 2. Principle

• Based on the capture of electrons by electronegative

atoms in a molecule
• Electrons are produced by ionization of the carrier gas with
a radioactive source such as 63Ni ( emitter)
N2 + β = N2+ +2e, electrons transfer to anode.
• No electrophilic compounds go to detector:
 N2+ transfers to cathode
 electrons transfer to anode electric current.
• Electrophilic compounds go to detector:
 β+X X- ; X- + N2+ X + N2
 3. Advantages and
disadvantages:
Advantages Disadvantages
- Useful for environmental testing: - Very sensitive when change speed of gas,
+ detection of chlorinate pesticides or herbicides temperature.
+ detection of polynuclear aromatic carcinogens - Sensibility and linear dynamic significantly
+ detection of organometallic compounds change with different compounds.
- Very sensitive for halogen (I-, Br-, Cl-, F-), - Carrier gas : high purity
- -
nitro , sulfur containing compounds.
- Low LOD 10-12 – 10-13 g (0.1ppb)
- Minimal dead volume -> cleaned by gas ->
increasing repeat + thermal stability for
detector.
 GC Instrumentation
Selective Detectors
• Element Selective Detectors
– Atomic plasma emission (expensive)
– Specific detectors for S, N, P containing compounds
based on ionization (NPD) or emission of light (flame
photometric detector and sulfur chemiluminescence
detector)
• Multiple detectors
– Multiple dectors can be used provided 1st is non-
destructive if in series (e.g. PID/FID gives
aromatic/unsaturated fraction of hydrocarbon
samples)
 GC Instrumentation
Mass Spectrometer Detectors
• Very Popular Detector
• Both selective (single ion mode) and
universal (total ion mode); plus allows
structural information
• Will cover in more detail in separate
section later
 GC Instrumentation
Some Questions
1. The mobile phase in GC is switched from He to N2.
Why would you want to decrease the flow rate?
2. How is the retention of polar compounds affected by
switching from He to H2 as a carrier gas?
3. List two ways to inject gas samples on GCs set up for
analysis of liquids.
4. For what type of columns can direct injection be used?
5. A student is analyzing reaction products present in the
parts per thousand level and is using a GC with a 0.25
mm OT column and a split/splitless injector. What
type of injection should be used?
 GC Instrumentation
Some Questions
6. Which chromatogram to the 20

18

right is from a split injection? 16

14

Which is from a splitless 12

Response
10

injection? 8

7. A food sample is being 4

analyzed by GC for P 0
0 0.5 1 1.5 2 2.5
Time (min)
3 3.5 4 4.5 5

containing pesticides. Even

after extraction, there are
hundreds of compounds. 2
1.8

What injector, column

1.6

1.4

Response 1.2

(dimensions), and detector 1

0.8

should be chosen? 0.6

0.4
0.2
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Time (min)
 GC – Role of Temperature
• Temperature is the
most common way of Plot for each compound
Different
changing retention selectivity
possible for other
(high T, less compounds

retention) Log K

• Relationship between
temperature and
Homologous
distribution constant: series

H
log K    const . 1/T Low T
2.3RT

Isothermal = single T
 GC – Role of Temperature
Temperature is good for early
eluting compounds
• For isothermal GC, peak gaps
and widths tend to increase Wide separation of
late eluting
with increasing retention time compounds
(for constant logK)
• When wide separation for later
eluting compounds, an
increase in k is desired, but
will result in unresolved early
Later eluting
eluting peaks compounds now elute
• The solution is temperature faster

programming where T
increases with time so that k
decreases with time
Peak width stays narrower
 GC – Role of Temperature
• Advantages of Gradient Separations
– Compounds of a wide range of volatilities can be
separated in a single run
– Because compounds are “trapped” at the head of the
column until the temperature is high enough that
they are volatile, narrow peaks result (even when not
injected as narrow plug)
– Signal to noise is improved in late eluting peaks
– Quicker and more efficient elution occurs
– Less overlap of compounds with solvent peak
 GC – Role of Temperature
• Disadvantages of Gradient Separations
– Must include time for oven to cool and re-establish
equilibrium in total analysis time (so decrease in time
is not as large as chromatogram may indicate)
– Baseline tends to be less stable (some detectors’
response depends on T and increasing column bleed
can be a problem for trace analysis)
 GC – Other Topics
• Solid Phase MicroExtraction (SPME)
– Mentioned as advanced simple separation
– Simplifies many sample extraction steps (time
savings)
– Can be used with both liquid and gas samples
– Selectivity can be changed by changing fiber type
(polar vs. non-polar)
– Main disadvantage is quantitation is more difficult
(best to have standard in matrix like sample)
– Longer fiber exposure allows greater sensitivity
 GC – Other Topics
1 st Separation
Then Separationin
in 1
2nd dimension
dimension
• Comprehensive 2 Dimensional GC
(or GC x GC)
– See Chapter 15, section 1
– Also materials from Crimi and
Snow, LCGC North America (2008),
26, 62-70.
– Another recent review (focused on
applications) is Adahchour et al., J.
Chromatogr. (2008), 1186, 67-108.
– a simple TLC example of a 2 High alkane affinity
dimensional separation:
• Separation first in 1 dimension (e.g. Resultant Plate
hexane) High aromatic
• Then separation in 2nd dimension affinity
(using different solvent – say
benzene) In hexane
– GC x GC is most common multi-
dimensional instrumental method Inseparable
in hexane

In benzene
 GCxGC – Added Slide
Effective Use of 2 dimensions
• Effectiveness of Method
– Useful when columns and

2nd Dimension
samples chosen so 2nd
dimension gives additional
separations
– Not that valuable if 2nd
dimension does not help much
1st Dimension
– Separation principles in the two
dimensional should be Less Effective Separation
“orthoganol” (based on different

2nd Dimension
principles)

1st Dimension
 GC
GC x GC
• GC x GC’s main advantages are much
greater peak capacity and alternative
selectivities
• GC x GC is useful for comprehensive
analysis of very complicated samples (e.g.
gasoline) in which one dimension does not
have enough resolving power
• Even greater resolution possible with GC x
GC x MS