Reminder: These notes are meant to supplement, not replace, the lab manual.
Gas Chromatography notes
Application: Gas Chromatography (GC) provides a quick, accurate and relatively
inexpensive way to analyze very small samples for composition. Simple GC
machines may cost in the area of $5,000i. This is fairly inexpensive as far as
analytical instrumentation goes. They can be installed in a variety of laboratories
designs including mobile testing units. GC’s are easy to operate and provide
reliable results.
1.
Here is some terminology related to this experiment:
Chromatography is a technique in which compounds to be separated are
distributed between a mobile phase and a stationary phase. Different
distributions or partitioning between the two phases give rise to the
separation.
Volatile- Easily converted into a gas. Evaporating rapidlyii.
Gas Chromatography (GC) is useful for separating and analyzing volatile liquid or
gas mixtures. GC does not work on many solids.
The stationary phase in gas chromatography (GC) is almost always a relatively
nonvolatile liquid, like a wax. This liquid is coated on either solid particles
or the inside of a capillary tube. The stationary phase used in this
experiment is carbowax coated on small inert particles.
The mobile phase in gas chromatography is an inert (nonreactive) gas, usually
helium or nitrogen. Nitrogen will be used in this lab. This mobile phase is
often called the carrier gas.
The retention time is the time (in seconds or minutes) between the injection of a
sample and the middle of when the sample reaches the detector. The
retention time is measured on a chromatogram between the start mark
and the top of a peak. These times are used like Rf values in TLC, to
establish the identity of the compound giving the peak by comparison.
To elute a compound means to have it emerge from or come off of a column.
2.
The fact that the mobile phase is a gas usually makes it necessary to use a more
elaborate setup to confine the gas and direct its flow. Nevertheless, this is still
chromatography; there is a sample, a mobile phase, and a stationary phase. In
GC the stationary phase is a liquid and the mobile phase is a gas. Three different
types of Gas Chromatographs (GCs) may be used in this lab. The first two types
(Shimadzu and Gow-Mac) are the most common. A typical set up is drawn
below. The components are not drawn to scale. Each has an external
pressurized gas cylinder attached. In addition, new Vernier mini-GC’s may be
used. These mini-GC’s use air from the room and do not need a separate
pressurized gas tank.
The carrier gas is typically kept in a large metal cylinder under high pressure.
The gas flows through a valve into a regulator, which reduces the pressure to a
lower value the rest of the system can use. (Scuba divers use regulators for the
same purpose). The gas flows past the injection port, where samples are
injected using syringes. The injection port is heated, and the samples evaporate
there. The sample and carrier gas mixture are carried into an oven that contains
the chromatography column. (The heat prevents components of the sample from
condensing). The components partition and separate in the column. The
separated components of the mixture pass through the detector, which sends a
signal to the strip chart recorder or integrator.
3.
The separation occurs by different partitioning or distribution of compounds
between the stationary (non-volatile liquid) phase and the mobile (gas) phase.
What physical characteristic describes the energy needed to move a compound
from the liquid to the gas phase? A compound that spends more of its time in the
gas phase (mobile phase) would have a shorter retention time than a compound
that spends more of its time in the liquid phase (stationary phase). The boiling
point of compounds is the primary means of predicting the order of elution from a
GC. The lower the boiling point, the shorter the retention time (RT). Secondarily
to boiling point is polarity. If a mixture of similar compounds with known boiling
points is injected into a GC, the identity of the peaks can be made by correlating
the retention time to boiling point. The lowest boiling point material will spend a
higher portion of its time in the gas phase and will have the shortest retention
time.
4. To validate a peak identification, a pure authentic sample of the suspected compound
is injected into the same GC at the exact same conditions (temperature, flow
rate, column). The Rt of the authentic sample is then compared with the Rt of the
suspected peak. If the Rt’s are different, they are different compounds. If the Rt’s
are the same, a positive identification has been made. In this way, Rt values of
GC are very similar to Rf values of TLC.
5.
A photo of one of the Gow-Mac GC’s found in the organic lab is shown.
6.
The technique of gas chromatography is widespread in academic, industrial, and
governmental labs. It is used to separate the components of volatile liquid and
gas mixtures, identify the compounds (by comparison of retention times), and
determine the amounts of the compounds present.
7.
Samples for GC analysis must be able to be vaporized into the gas phase in
order to pass through the machine. Non-volatile liquids and non-volatile solids
cannot be analyzed using GC. The sample size necessary for GC is quite small.
A typical injection volume ranges between 1/10 and 5 microliters (l). One
microliter is equal to 1/1000 of a milliliter or 1/1,000,000 or 1x 10 -6 liters. If a drop
of liquid is equal to 1/20 of a milliliter, then one drop has enough volume for ten 5
l injections.
8.
The size of a peak (area) is proportional to the amount of compound present.
The relative size (area) of two peaks will indicate the relative composition of
those two materials in the sample. If one peak has an area of 2 cm 2 and another
peak has an area of 3 cm2, then the sample is composed of (2/(2+3) x 100%) or
40 % of the first compound and 60 % of the second compound.
9.
The volume of sample injected is not critical (within reasonable limits). It doesn’t
matter if 3, 4, 5, or 6 l of sample is used. It is not reasonable to inject 2 mL of a
sample into a GC. If double the volume is injected (3l vs 6 l) , the absolute
size of the peaks will double, but the ratio of the sizes of the two peaks will
remain the same, because the percent composition will remain the same. The Rt
will also not change.
10.
If a sample containing a mixture of three compounds is injected multiple times on
the same machine, with the same column, the same flow rate and the same
temperature of the column, the retention times of the three compounds will be the
same in each injection. If the relative composition of the samples are different,
the relative sizes of the peaks will change, but the retention time will remain the
same.
11.
Here are the structures of compounds which make up the mixtures used in the
2230L experiment. The exact % composition of each in the mixture will be
determined using the GC. Each unknown is made up of these two materials.
o
Ethyl acetate, B. P. 77 C
o
Butyl Acetate, B. P. 126 C
Both of these compounds are esters of acetic acid (acetate). They differ by two
CH2 groups. The addition of a single CH2 group raises the boiling point by over
20oC, so the addition of two methylene groups raise the boiling point by 49°C.
Most esters have a pleasant scent and a moderately low toxicity. These are no
exceptions to this rule. Which compound is expected to have the highest
retention time?
12.
Safety considerations for this experiment include:
Syringes are sharp and can easily prink skin. If a syringe is left too long in the
injection port without depressing plunger, the liquid will volatilize and expand,
shooting the plunger out at an unsuspecting student. The injection port is hot and
can easily burn skin. Be sure to wear goggles while operating GC.
Cylinders of gases under high pressure must be securely fastened to solid,
permanent furniture or walls. If such cylinders fall and break, the escaping gas
transforms them into potentially deadly missiles.
The organic materials analyzed in this experiment are highly flammable.
13.
Changes in the procedure can, as always, have a profound effect on the
outcome of the experiment. Here are some possible changes in procedure and
their effects.
Faster carrier gas flow rates and higher column temperatures give shorter
retention times, but peaks may not be resolved as well.
Slower flow rates and lower temperatures give longer retention times and better
resolution.
The primary characteristic which determines which compound will have shorter
retention times (elute quicker) is the boiling point. The more volatile
compounds have lower boiling points and will elute the column faster and
hence have shorter retention times.
Secondarily to boiling point, is polarity. Less polar compounds generally have
shorter retention times (there are exceptions to this).
Stationary phases should not evaporate at the temperatures of the experiment,
and should not react with the compounds under study.
Stationary phases have a significant impact on the retention time. To change a
stationary phase means to physically remove the column (with wrenches)
from the GC machine and replace it with another column packed with a
different stationary phase. This is not done frequently.
14.
Gas chromatography can be used for both qualitative (identification) and
quantitative (amounts) analyses. The RT is used for identification. The area of
the peak is used to determine the amount of each component present.
The area of a peak in a chromatograph is proportional to the amount of
compound present and various instrument factors. Integrators connected to gas
chromatographs provide RTs, peak area, and percent area. The percent area of
each peak is the same as the percent of each material present. This is read
directly off the integrator output. A sample output is below.
PK
1
2
3
4
RT
0.23
1.14
2.47
2.78
AREA
7054
642298
3205001
1816737
TYPE
BB
PB
BP
BP
AREA %
0.12439
11.32583
56.51473
32.03505
There are four peaks and hence four compounds in the above sample. The first
peak at 0.23 can be disregarded due to its minute size and insignificant presence
(0.12439%). The third peak with a retention time of 2.47 minutes is the major
component with 56.51 %. The fourth peak at 2.78 minutes is the second most
prevalent material and the peak at 1.14 minutes is present in 11.32%.
15. If an integrator is not available, the output of a GC detector is then recorded using a
plotter or strip chart recorder. The peak area will then have to be measured manually.
The method for manually determining peak area is described below.
A typical plotter or strip chart recorder output looks like the image below.
The area of each peak is defined by the height of the peak times the width of the peak
at half of the maximum height. (H x W 1/2H)
Area = Height (cm) x Width at Half maximum peak height (cm) = cm2
For the below chromatogram, the area of peak A is 4.68 cm times the width of the peak
at a height of 2.34 cm. This equals 4.68 cm x 0.18 cm = 0.84 cm2.
For the below chromatogram, the area of peak B is 6.50 cm times the width of the peak
at a height of 3.25 cm. This equals 6.50 cm x 0.17 cm = 1.1 cm 2.
For this chromatogram, the percent of each is calculated as follows.
The percent of each compound in the mixture is the area of that peak, divided by the
sum of all of the areas times 100%.
16. If an integrator is not available, the retention time can easily be calculated. This is
done by knowing the chart speed of the printer or strip chart recorder in cm per minute
(cm/min) and the distance from the injection to the center point of the peak. For peak B,
the distance from the injection point at 0.0 to the peak of peak B is 8.05 cm. If the chart
speed was 0.50 cm/min then the retention time for peak B is calculated as following:
(
i
)
Shimadzu Instrumentation Price http://www.shimadzu.com/an/gc/index.html (January 22, 2012)
ii
Grant, J. Hach’s Chemical Dictionary,McGraw-Hill, 1969, p 717
Revised October 13, 2014, S. L. Weaver