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PRINCIPLES
Gas Chromatography
Page 1 of 23
amount of each component present. The essentials required for the
method are an injection port through which samples are loaded, a
"column" on which the components are separated, a regulated flow of
a carrier gas (often helium) which carries the sample through the
instrument, a detector, and a data processor. In gas chromatography,
the temperature of the injection port, column, and detector are
controlled by thermostatted heaters. Figures 1 and 2 are pictures of the
instrument from the front and rear respectively, with important
components labeled. The following sections describe in detail the
function of each component.
FID detector
control panel
injection port
on/off switch
capillary column
wound around holder
fan
Detail of column in oven
Figure 1. Front view of gas chromatograph
detail of column in
Air inlet H2 inlet N2 inlet
(detector) (detector) (makeup gas)
He inlet
(carrier gas)
heater fan
INJECTION PORT
The sample to be analyzed is loaded at the injection port via a
hypodermic syringe. The injection port is heated in order to volatilize
the sample. Once in the gas phase, the sample is carried onto the
column by the carrier gas, typically helium. The carrier gas is also
called the mobile phase. Gas chromatographs are very sensitive
instruments. Typically samples of one microliter or less are injected
Gas Chromatography
Page 3 of 23
on the column. These volumes can be further reduced by using what is
called a split injection system in which a controlled fraction of the
injected sample is carried away by a gas stream before entering the
column.
COLUMN
The column is where the components of the sample are
separated. The column contains the stationary phase. Gas
chromatography columns are of two types—packed and capillary.
Capillary columns are those in which the stationary phase is coated on
the interior walls of a tubular column with a small inner diameter. We
will use a capillary column in this experiment.
The stationary phase in our column is a polysiloxane material.
The basic structure of the polymeric molecules is shown below, where
n indicates a variable number of repeating units and R indicates an
organic functional group. In our columns, 5% of the “R’s” are methyl
groups (-CH3) and 95% of the “R’s” are phenyl groups (-C6H5)
CH3 R CH3
H3C Si O Si O Si CH3
CH3 R CH3
This polymeric liquid has a high boiling point that prevents it from
evaporating off the column during the experiment.
The components in the sample get separated on the column
because they take different amounts of time to travel through the
column depending on how strongly they interact with the stationary
phase. As the components move into the column from the injection
port they dissolve in the stationary phase and are retained. Upon re-
vaporization into the mobile phase they are carried further down the
column. This process is repeated many times as the components
migrate through the column. Components that interact more strongly
with the stationary phase spend proportionally less time in the mobile
phase and therefore move through the column more slowly. Normally
the column is chosen such that it’s polarity matches that of the sample.
When this is the case, the interaction and elution times can be
rationalized according to Raoult’s law and the relationship between
vapor pressure and enthalpy of vaporization. The rule of thumb is that
Properties of liquids
retention times correlate with boiling points. (Do not expect an exact and solutions are
quantitative correlation, i.e. one with an R-value close to one, for this covered in chapter 6 of
simple model. You will be using a non-polar column and the Oxtoby. In particular,
section 6-1 covers the
interaction between an alcohol molecule and the stationary phase will relationship between
be dominated by weak van der Waals forces.) molecular structure
and vapor pressure.
Section 6-6 covers
In this experiment, you will use a gas chromatograph to separate and
Raoult's law, which
quantify mixtures containing various isomers of butyl alcohol (n-butyl relates the vapor
alcohol, sec-butyl alcohol, iso-butyl alcohol, and t-butyl alcohol). Look pressure of a solution
up the structures of these compounds, their normal boiling points, and to the vapor pressure of
the pure components.
their enthalpies of vaporization. (There are reference books in the
library that contain thermodynamic data for organic compounds.
Another source of thermodynamic data is NIST's webpage, which can
be accessed via the lab homepage. Quick information on structure
and boiling points can be obtained from a variety of sources including
chemical catalogs, the Merck Index, and Chemfinder.com) Based on
the relationship between vapor pressure and enthalpy of vaporization,
which component do you expect to travel fastest through the column?
Boiling points are another indicator of intermolecular forces. Do your
predictions based on the trends in ∆ Hvap agree with the trends in
boiling points?
isobutyl alchol
sec-butyl alchol
Gas Chromatography
t-butyl alchol Page 5 of 23
As described above, the rate at which compounds move
through the column depends on the nature of the interaction between
the compound and the stationary phase. Other variables that affect this
rate are column temperature and carrier gas flow rate. In this
experiment, you will be provided a set of initial column conditions to
analyze your samples. Based on the results of your first run, you will
then vary the column temperature in order to achieve good separation
of the peaks in the shortest possible time. One should avoid
experimental conditions that lead to excessively long elution times.
Not only do you waste valuable resources (your time and chart paper)
but broadening of the peaks and loss of resolution will become evident
when the elution times are too long. This broadening is an inevitable
consequence of diffusion. The theory of diffusion shows that the
width of a peak is roughly proportional to the square root of elution
time. Thus the optimum conditions are those that result in complete
separation of the peaks in the shortest possible time.
If your peaks are very far apart such that the analysis
takes a long time, should you increase or decrease the
column temperature?
DETECTOR
If the column conditions are chosen correctly, the components
in the sample will exit the column and flow past the detector one at a
time. There are several different types of detectors common to gas
chromatography instruments. The choice of detector is determined by
the general class of compounds being analyzed and the sensitivity
required. Our gas chromatographs are equipped with flame ionization
detectors (FIDs)—the most widely used detectors for organic samples.
FIDs use an air/hydrogen flame to pyrolyze the effluent sample. The
pyrolysis of the compounds in the flame creates ions. A voltage is
applied across the flame and the resulting flow of ions is detected as a
current. The number of ions produced, and therefore the resulting
current, depends on the flame conditions and the identity of the
molecule in question. (As a rough approximation, the current is
proportional to the number of reduced carbons in the molecule.) In
other words, the detector shows a different response to each
compound. For this reason, separate calibrations must be performed
for each compound analyzed.
INTEGRATING RECORDER
The output of the detector (converted from current to voltage)
is sent to an integrating recorder that plots, stores, and analyzes the
data. A typical chromatogram is shown in Figure 3.
response (µ V)
peak retention
time
time (min)
Gas Chromatography
Page 7 of 23
takes for a given peak to appear after injection is called the retention
time. If the column conditions are kept constant, the retention time for
each component is quite reproducible from one sample and injection to
the next. The identity of each peak can be determined by injecting
pure samples of the individual components of the mixture and noting
their retention times.
Gas Chromatography
Page 9 of 23
The next section guides you through the calculation of relative
response factors from a single standard mixture.
RELATIVE RESPONSE FACTORS
As described above, if the detector were equally sensitive to
each component in a mixture, the peak areas could be used directly to
give the percentage composition of the mixture by dividing the area of
each peak by the total area under all of the peaks. Since the detector is
not equally sensitive to the different components, each peak area must
be multiplied by a suitable factor (called the response factor, k) to
correct for this difference. The corrected areas are then used for the
calculation of the percentage composition of the mixture. We will use
something called a relative response factor, f, which ratios each
response factor to that of a chosen component. The relative response
factors are determined by measuring the peak areas for a mixture of
known composition. These relative response factors can then be used
to determine the percent composition of an unknown mixture of the
same components.
The following paragraphs walk you through the derivation of
relative response factors in terms of chromatogram peak areas and
percent compositions. You will need to fill in the boxes to complete
the derivation.
qi = ki * (1)
pi =
( k i )( a i ) • 100%
(2)
∑ [ ( k i )( a i ) ]
or
p1 =
( k1 )( a1 ) • 100% etc. (3)
( k1 )( a1 ) + ( k 2 )( a 2 ) + ( k3 )( a 3 ) + ( k 4 )( a 4 )
Using the four known values of p and the four measured areas yields
four equations and four unknowns that can be solved simultaneously.
However, to simplify the analysis we can define a relative response
factor fi, which compares each response factor to a common reference
—we’ll choose component four, the component with the longest
elution time.
ki
fi = (4)
k4
The beauty of this approach can be seen when we express the
percentage of each component relative to the percentage of component
four and then re-arrange to solve for fi in terms of pi’s and ai’s.
1 0 0% • ( k i )( a i )
pi
= ∑ ( ki )( a i ) = ( ki )( ai )
p4 1 0 0% • ( k 4 )( a 4 )
(5)
( k 4 )( a 4 )
∑ ( ki )( ai )
Gas Chromatography
Page 11 of 23
ki
fi = = (6)
k4
Now each relative response factor can be calculated directly from the
known percent composition of the standard mixture and the
experimentally measured peak areas.
As mentioned above, the relative response factors can then be
used to calculate the percent composition of an unknown mixture of
these components. To determine the percent composition of a given
component in the unknown mixture, pi, divide both the numerator and
denominator of the appropriate equation 3 by k4 and substitute in the
appropriate fi’s for each ratio of k’s to obtain an expression for each pi
as a function of appropriate fi’s and ai’s.
f a
p1 = etc. (7)
f a + f a + f a + f a
EXPERIMENTAL PROCEDURE
Gas Chromatography
Page 13 of 23
The following conditions will be set prior to your arrival.
• Temperatures:
column = 100 ºC
injector = 200 ºC
detector = 200 ºC
After making your first run, you will adjust the column
temperature to improve the separation of your peaks. To do
this, press col on the instrument, enter the desired temperature
using the numerical keypad, and press enter . The LCD panel
displays both the setpoint and the actual temperatures. When
the column reaches the setpoint and has stabilized, the green
ready light will appear indicating that the instrument is ready
for an injection.
3. C-R8A Recorder.
a. Turn the recorder switch ON at the back left.
b. Use the monit button to monitor the voltage from the
detector when no sample is injected. Use the zero
button on the gas chromatograph to set the output voltage
to approximately + 100-200 µV. This sets the baseline for
the chromatogram. (The range of the output is –5000 µV to
+1V)
c. Set the recorder attenuation using the Atten button on
the recorder. Try a value of 7 to start. You may have to
adjust this if your peaks are too small or too large.
(Choosing a larger number makes the peaks appear
smaller.)
Gas Chromatography
Page 15 of 23
e. When all components of the liquid have emerged from the
column (including the tail from the last peak), press the
start1/ stop 1 button on the recorder and the stop button
on the instrument to stop the run. Label the chromatogram
and record the retention times and peak areas in your
notebook. When you are ready to run the next sample,
repeat steps (a) - (e) above.
f. When analyses have been completed, carefully tear the
paper from the recorder. Cut out each chromatogram with
their associated peak analysis charts and tape to a sheet of
paper for inclusion in your report.
CALCULATIONS
Gas Chromatography
Page 17 of 23
NAME
LAB SECTION
Chromatograph Number
Sample Number
Date Report Submitted
GAS CHROMATOGRAPHY
Results for Standard Mixture
Final Column Temperature: ____________________
Avg. retention time of 1st peak _______________ Rel. 95% CIm __________________
Avg. area of 1st peak __________________ Rel. 95% CIm __________________
Calculation of Relative Response Factors for Standard Mixture
Rel.
Response Run 1 Run 2 Run 3 Avg. 95% CIm
Factor
f1
f2
f3
f4
Results for Unknown Mixture
(Use same component designation as above; note that one line will be blank as there are
only 3 components in your unknown.)
Calculation of % Composition of Unknown
Run 1 Run 2 Run 3
Component
fi*ai % fi*ai % fi*ai %
1
Total
4
Identification of Peaks
Name of Component Structure
1.
2.
3.
4.
Question
Discuss the difference in magnitude between the rel. 95% CIm for areas vs. relative
response factors. Why are they so different?
Show sample calculations on back side of this sheet.
Attach chromatograms.