GAS CHROMATOGRAPHY

The objective of this experiment is to separate, by gas

chromatographic techniques, a mixture of the four isomeric butyl
alcohols and to determine the percentage of each an unknown
mixture.

Upon completing this experiment, you should:

 know the basic components of a chromatography instrument
 understand the importance of component separation to
chemical analysis
 understand the mechanism by which components are separated
on a GC column and the variables that affect separation
 understand the basic methods of calibration common in
chromatographic analysis

PRINCIPLES

Chromatography is a very important analytical tool because it
allows the chemist to separate components in a mixture for subsequent
In the fluoride and
use or quantification.  Most samples that chemists want to analyze are
manganese
mixtures.     If   the   method   of   quantification   is   selective   for   a   given experiments, for
example, the
component in the mixture, separation is not required. However, it is
“detector” is selective
often the case that the detector is not specific enough, and a separation for the component of
interest within the
must first be performed.   There are several types of chromatography
matrix of our
depending on the type of sample involved.   In this experiment, we’ll particular samples.
use gas chromatography.

Gas Chromatography
Page 1 of 25
 The   gas   chromatograph   makes   it   possible   to   separate   the
volatile   components   of   a   very   small   sample   and   to   determine   the
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
oven

Gas Chromatography
Page 3 of 25
 Air inlet H2 inlet N2 inlet
(detector) (detector) (make-up gas)

He inlet
(carrier gas)

heater fan

Figure 2.  Rear view of gas chromatograph

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 

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

Gas Chromatography
Page 5 of 25
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
In this experiment, you will use a gas chromatograph to
rationalized
separate  according to Raoult’s law and the relationship
and quantify mixtures containing various  between
vapor pressure and enthalpy of vaporization.  The rule of thumb is that
isomers of butyl alcohol (n-butyl alcohol, sec-butyl
retention times correlate with boiling points.  (Do not expect an exact
alcohol, iso-butyl alcohol, and t-butyl alcohol). Look up
Properties of liquids
the structures of these compounds, their normal boiling
quantitative correlation, i.e. one with an R­value close to one, for thisand solutions are
covered in chapter 6 of
points,
simple and   their
  model. You   enthalpies
will   be   usingof  avaporization. (There
  non­polar   column   and  are
the In particular,
Oxtoby.
reference books in the library that contain section 6-1 covers the
interaction between an alcohol molecule and the stationary phase will
thermodynamic data for organic compounds. Another relationship between
be dominated by weak van der Waals forces.) molecular structure
source of thermodynamic data is NIST's webpage,
and vapor pressure.
which can be accessed via the lab homepage. Quick Section 6-6 covers
information on structure and boiling points can beRaoult's law, which
obtained from a variety of sources including chemical relates the vapor
pressure of a solution
catalogs, the Merck Index, and Chemfinder.com) Based to the vapor pressure of
on the relationship between vapor pressure and the pure components.
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?

Structure Hvap
bp(K)
n-butyl alcohol
Gas Chromatography
Page 7 of 25
 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 (V)

peak retention
time

time (min)

Figure 3.  Sample chromatogram

Gas Chromatography
Page 9 of 25
 The detector voltage (y­axis) is plotted as a function of time (x­
axis).   Each peak corresponds to a separate component.   The time it
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.  

The voltage from the detector is proportional to the number of
molecules passing through the detector at any given time.   For well­
separated  peaks,  the  total  number   of  molecules  of  each  component
reaching the detector is then proportional to the area under the peak.
The   recorder   determines   the   area   of   each   peak   by   integration   and
reports this in the results table.   The proportionality factor between
area   and   amount   must   be   determined   by   a   calibration   experiment.
Note that the integrator will also determine areas for peaks that are not
well­separated   by  dropping  a  vertical  line  where  the   slope  changes
sign (Figure 4).   The results will be in error, however, because the
voltage   read  at  the  beginning   of peak  3  is  actually  the  sum  of the
response   due   to   the   third   component   plus   the   response   due   to   the
second component still exiting the column.
 Figure 4.  Chromatogram of non­separated peaks

There are several methods by which gas chromatographs are A similar
calibration method
typically   calibrated.     One   method   is   to   inject   standard   samples is used in the
containing varying concentrations of the compound to be analyzed and fluoride and
manganese
creating   a   calibration   curve   (area   vs.   concentration).     As   you’ll experiments.
discover, however, it is very difficult to reproducibly inject the same
volume onto the column each time.
A more advanced calibration method is to use something called
an internal standard.  In this method, a constant concentration of a non­
interfering compound is added to each sample before it is analyzed.
The ratio of the areas of the added compound and analyte are then
used to construct the calibration curve.  Using an area ratio instead of
an absolute area compensates for varying injection volumes.
The calibration method we will employ is similar to the use of
an internal standard in that it corrects for variability in the injection
volume.  It differs in that it determines relative response factors based
on the results of one standard sample of known composition.  (This is
Gas Chromatography
Page 11 of 25
possible because our standard sample contains only the four isomers of
butyl alcohol,  i.e. the sum of all the components adds up to 100%).
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.

The following symbols will be used in this derivation:

ai  =  area of peak for ith component (from chromatogram)
pi  =  percent   composition   of   ith  component   (known   for   standard
sample, unknown for unknown sample;  but in both cases the
sum of the pi’s is 100%) 
qi = quantity of ith component reaching the detector
ki = response factor of ith component 
fi = relative response factor of ith component 

The   quantity   of   each   component   passing   through   the   detector   is

proportional to the area of the chromatogram peak for that component.

qi = ki *   (1)

Since the percent composition for each component is the quantity of
that component divided by the sum of all the components,

 k i  a i   100%
pi  (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.

k
fi  i (4)
k4

Gas Chromatography
Page 13 of 25
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 re­arrange to solve for fi in terms of pi’s and ai’s.  

100%   ki  a i 
pi

  ki  a i    ki  a i 
p4 100%   k 4  a 4 
(5)
 k 4  a 4 

 i i
k  a 

k
fi  i  (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

The following paragraphs outline  the procedure to follow  in

analyzing liquid samples on the gas chromatograph.  Your first task is
to experimentally determine a set of column conditions that yield a
good   separation   of   the   four   isomers   in   your   standard   sample   in   a
reasonable   time   frame.     Once   you   have   determined   a   good   set   of
conditions,   repeat   this   analysis   three   times.     You   will   use   these
chromatograms to determine an average relative response factor for
each component.  Next, obtain three chromatograms of your unknown
mixture   (which   will   contain   3   of   the   4   isomers   in   varying   percent
compositions) from which you will determine the percent composition.
Finally, you will identify each peak by injecting pure samples of each
component and noting the retention time.   

1. Column.   In order to separate the four isomers of butyl alcohol

you will use a 5% diphenyl, 95% dimethyl polysiloxane capillary

column that is 15 feet long and has a 0.25 mm inner diameter.

The column is already installed in the gas chromatograph.

2. Chromatograph Conditions.   You will be given a set of initial

conditions for your standard sample.   Your task is to vary the

conditions  to achieve good separation of the peaks.   Although

many parameters affect the separation (e.g. column type, column

length, carrier gas flow rate, column temperature) to simplify the

experiment  you   will   only   make   changes   to   the   column

temperature.     

Gas Chromatography
Page 15 of 25
 The following conditions will be set prior to your arrival.

 Gas Pressures and Flows:

H2 (to FID detector) = 60 kPa
Air (to FID detector) = 50 kPa
N2 (make­up gas) = 60­70 kPa
He (carrier gas) = 0.5 mL/min
split ratio = 100

 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.  C­R8A 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 + 100­200 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.) 

4. Sample Injection.  The liquid sample is injected into the helium

gas stream by inserting the needle of a special expensive syringe

through a heavy­wall rubber septum.  

a. DO   NOT   PULL   THE   METAL   PLUNGER   OUT   OF

THE GLASS BODY OF THE SYRINGE.   IN FACT,
DON'T EVEN COME CLOSE TO DOING IT.  
b. With the plunger pushed all the way into the syringe, insert
Without careful
cleaning between the needle into the liquid and then withdraw 0.3 - 0.5 L of
runs, liquid; eject this into a Kimwipe. Repeat this rinsing
contamination operation several more times. Without careful cleaning
from one injection between runs, contamination from one injection to the next
to the next can be can be a problem. When changing to a new sample (e.g.
a problem. from standard to unknown), check carefully for
contamination peaks. Your standard contains four isomers
whereas your unknown contains only three isomers.
Therefore the presence of a “fourth” peak in your unknown
is clear indication of contamination and the run should be
repeated.
c. When you are ready to make an injection, withdraw  0.3 -
0.5 L of liquid into the syringe; then, holding the syringe
vertically   with   the   needle   pointing   upward,   push   the
plunger in until it reads 0.1 L (the volume to be injected).
With the syringe still held vertically, withdraw the plunger

Gas Chromatography
Page 17 of 25
 to about 0.5 liter; this will leave a protective air space at
the tip of the needle.  
d. Insert the needle through the rubber septum until stopped
by the protective tube around the needle and inject the
liquid by pushing the plunger all the way in. Immediately
push the start button on the instrument to start the run.
The recorder should start automatically. Then remove the
needle from the septum. The above sequence of operations
must be carried out as quickly as possible.
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.  

5.  Sequence of Measurements— Summary.  

a. Measure a series of chromatograms for the standard

mixture as a function of the column temperature.
Determine a column temperature that yields good
separation within a reasonable length of time. Obtain three
chromatograms of the standard under these optimal
conditions. The relative response factors will be
determined from these runs. The injection volume for all
runs should be 0.1 L.
b. Obtain three chromatograms of your unknown under the
same conditions. The percent composition of your
unknown will be determined from these runs.
 c. Run each of the pure alcohols under the same conditions (3
out of 4 will suffice). The retention time for each alcohol
will be determined from these runs.

CALCULATIONS

1. Label each peak on the chromatograms of your standard and 

unknown mixtures with the name of the alcohol associated with 

that peak.

2. Using the runs for your standard mixture, calculate the relative 

95% confidence interval of the mean for the area of the first 

peak.

3. For each run of your standard mixture, calculate the relative 

response factor (fi) for each of the butyl alcohols.   Calculate the

average value of each fi based on the three runs and determine 

the relative 95% confidence interval of the mean for each.

4. Use the average values for each fi obtained in 3 and the areas of 

the peaks in the chromatogram of your unknown to determine 

the percent composition of your unknown sample for each run.  

Use the values determined for each run to calculate  average 

percent compositions and the associated relative 95% 

confidence intervals of the mean.

Gas Chromatography
Page 19 of 25
 NAME LAB SECTION
Chromatograph Number Sample Number

Date Report Submitted

GAS CHROMATOGRAPHY

Results for Standard Mixture

Final Column Temperature: ____________________

Run 1 Run 2 Run 3

Component ret. time area ret. time area ret. time area
1
increasing ret. time

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.)

Run 1 Run 2 Run 3

Component ret. time area ret. time area ret. time area
1

Calculation of % Composition of Unknown

Run 1 Run 2 Run 3
Component fi*ai % fi*ai % fi*ai %
1

Total

Component Avg. % Comp. Rel. 95% CIm

1

4
Identification of Peaks

Name of Component Structure

1.

2.

3.

4.

Question
Discuss the difference in magnitude between the rel. 95% CI m for areas vs. relative
response factors. Why are they so different?
Show sample calculations on back side of this sheet.
Attach chromatograms.