Gas Chromatography
Introduction:
A Gas Chromatograph is used to
detect the components based on the selective affinity of components towards the
adsorbent materials. The sample is introduced in the liquid/gas form with the
help of GC syringe into the injection port, it gets vaporized at injection port
then passes through column with the help of continuously flowing carrier stream
(mobile phase), mainly H2 (for TCD), and gets separated/detected at the
detection port with suitable temperature programming. We visualize this on
computer in the form of peaks. Carrier medium can be liquid (e.g. HPLC) or gas
(e.g. GC) for the ease of separation/detection, if it is gas then called gas
chromatography otherwise called liquid chromatography. Different chemical
constituents of the sample travel through the column at different rates .
It depends upon
1. Physical properties
2. Chemical properties
3. Interaction with a specific column filling
(stationary phase).
As the chemicals exit the end of the
column, they are detected and identified electronically. The function of the
stationary phase in the column is to separate different components, causing
each one to exit the column at a different time (retention time). Other
parameters that can be used to alter the order or time of retention are the carrier
gas flow rate, and the temperature. Physical Components involve inlet port,
Adsorption column, detector port, flow controller (to control the flow of
carrier gas), etc.
Types of columns:
Packed Column: Packed columns are
1.5 - 10 m in length and have an internal diameter of 2 – 4 mm. The tubing is
usually made of stainless steel or glass and contains a packing of finely
divided, inert, solid support material (eg. diatomaceous earth) that is coated
with a liquid or solid stationary phase. The nature of the coating material
determines what type of materials will be most strongly adsorbed.
Capillary Column: Capillary columns
have a very small internal diameter, on the order of a few tenths of
millimeters, and lengths between 25-60 meters are common. The inner column
walls are coated with the active materials.
Some columns are quasi solid filled
with many parallel micro pores.Most capillary columns are made of fused silica
with a polyimide outer coating. These columns are flexible, so a very long
column can be wound into a small coil.
Effect of temperature:
Temperature
dependence of molecular adsorption and of the rate of progression
along the column necessitates a careful control of the column temperature to
within a few tenths of a degree for precise work. Reducing the temperature
produces the greatest level of separation, but can result in very long elution
times.
Carrier gas:
The choice of carrier gas or mobile phase is important, with hydrogen
being the most efficient and providing the best separation. However, helium has
a larger range of flow rates that are comparable to hydrogen in efficiency,
with the added advantage that helium is non-flammable, and works with a greater
number of detectors. Therefore, helium is the most common carrier gas used.
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)
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 retention times
correlate with boiling points. (Do not
expect an exact quantitative correlation, i.e. one with an R-value close to
one, for this simple model. You will be
using a non-polar column and the interaction between an alcohol molecule and
the stationary phase will be subject by weak van der Waals forces.)
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. Not only do you waste valuable resources (your
time and chart paper) but broadening of the peaks and loss of resolution will
become marked when the elution times are too long. This broadening is an expected 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.
Detectors:
A number of detectors are used in
gas chromatography. The most common are the Flame ionization detector (FID) and
the thermal conductivity detector (TCD). While TCDs are essentially universal
and can be used to detect any component other than the carrier gas (as long as
their thermal conductivities are different than that of the carrier gas, at
detector temperature), FIDs are sensitive primarily to hydrocarbons, and are
more sensitive to them than TCD.
Both detectors are also quite
robust. Since TCD is nondestructive, it can be operated in-series before an FID
(destructive), thus providing complementary detection of the same eluents.
Applications:
Gas
Chromatography is widely used in pharmaceutical industries for analytical
research and development, quality control, quality assurance, production, pilot
plants departments for active pharmaceutical ingredients (API), bulk drugs and
formulations. It is used for process and method development, identification of
impurities in API. It is an integral part of research associated with medicinal
chemistry (synthesis and characterization of compounds), pharmaceutical
analysis (stability testing, impurity profiling), pharmacognosy, pharmaceutical
process control (Figure 7), pharmaceutical biotechnology etc
Alok Sharma
