INSTRUMENTAL ANALYSIS AND SEPARATIONS

GAS CHROMATOGRAPHY - LECTURE 20

Mobile and Stationary Phases

Columns for Gas Chromatography

Capillary columns are commonly made from high purity silica which contains no
significant metallic oxides that may react with the sample. The surface however does
contain many active silanol groups. Generally prior to the application of the stationary
phase coating, the inner surface is deactivated. One method of deactivation is to react the
free silanol groups with a reagent such as trimethylsilyl chloride. The stationary phase is
then applied, usually by a method known as static coating. The column is filled with the
stationary phase dissolved in a volatile solvent. Then one end of the column is sealed, a
vacuum is applied and the solvent is evaporated, leaving an even coat of the stationary
phase on the column wall. The stationary phase is held in place only by surface tension
forces and it can be disturbed by solvents in the sample or excessive temperature.
Increased stationary phase stability can be achieved either by polymerization of the
coating or by covalently linking it to the column surface. In addition to increasing
durability, immobilization of the stationary phase by these processes allows a much
thicker film to be applied which is important especially for separating highly volatile
compounds. It is also possible to regenerate immobilized stationary phases by washing
the column with solvents such as methylene chloride or tetrahydrofuran to remove
soluble contaminants that accumulate in the column(this can extent column life). The
outer surface of the column is coated with a polymer called polyimide which makes the
column strong and flexible. Any flaw in the coating, and the column can break very
easily.
There are three major types of gas chromatography columns, packed columns,
porous layer open tubular (PLOT) columns, and wall coated open tubular (WCOT)
columns.
The geometry of WCOT capillary columns is fairly simple, consisting of length,
internal diameter, and stationary phase thickness. Nevertheless, there are endless
possible combinations of these three factors that could be used for optimizing
chromatography. Doubling the column length effectively doubles the number of
theoretical plates but the resolution between any two compounds is proportional to the
square root of the plate number so doubling the column length only increases resolution
by about 40%. Doubling the column length also will result in longer analysis times and
small peak heights, so longer columns are not the answer to poor resolution. However,
longer columns do spread out the peaks and as a general rule, the more complex the
sample, the longer the column should be.

Column Diameter

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 The diameter of a column and the thickness of the stationary phase should always
be considered together because they interact with regards to column performance.
However, the general effect of decreasing column diameter is to increase the speed of
analysis. This is because the optimal carrier gas velocity increases as the diameter
decreases (assuming that the retention factor and plate number are held constant).
Smaller diameter columns usually have lower plate height (higher N) because of
improved mass transfer. Increasing carrier gas velocity results directly in faster analyses.
On the other hand, larger diameter columns have increased sample capacity which can
provide higher detectability with mass sensitive detectors such as an FID.
Another consideration of column diameter is how it affects the amount of sample
that can be injected. Larger samples require a larger amount of stationary phase to
interact with, otherwise the column will be overloaded, resulting in poor chromatography.
Larger diameter columns have higher capacity and less problems with overloading.
Overloading results when a band of a compound in the sample totally saturates the
stationary phase. Once the stationary phase is saturated, no more of the compound can
interact with the stationary phase so it will move through the column as if it were an
unretained compound. Since some of the compound is moving faster than it should, it
will elute from the column a little earlier which will show up on the chromatogram as a
“fronting peak.” If a compound overloads the mobile phase it will condense on the inner
surface of the column (without partitioning into the stationary phase). This effect is most
often seen as later eluting and tailing peaks. It is important to note that overloading is
related to specific compounds in the sample. Generally if complex samples are injected,
some compounds will be overloaded, while some compounds may not show up on the
detector because their concentration is below the detection limit. Therefore, the amount
of sample injected is based on a balance between these effects.

Thickness of Coating

The thickness of the stationary phase coating is very important. Thinner coatings
usually provide higher efficiency (higher N or lower HETP) because mass transfer is
quick between the mobile and stationary phase. However, the column capacity is low
and the column is easily overloaded.
Very volatile compounds should be analyzed using thick films because there is
not sufficient interaction with thin films and the result is poor separation. Very non-
volatile compounds should be analyzed using thin films, otherwise the retention in the
stationary phase is too long. Polar compounds have a tendency to have excess tailing of
peaks. This peak tailing can be reduced with thicker stationary phase coatings

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Stationary Phases

The different stationary phases available are described by the term “polarity.”
This term is used in a loose sense as a measure of the interactions between the sample
compounds and the stationary phase. These interactions are mostly based on dipole-
dipole attraction, induced dipole-dipole attraction and van der Waals forces. Because
separations are so strongly influenced by temperature, there is no great need for many
types of stationary phases. Common stationary phases are based either on polysiloxanes
or polyglycols. The methylsiloxane group [Si(CH3)2O] can be polymerized to a wide
range of thermally stable compounds ranging from low viscosity liquids gums to
silastomer rubbers. These polymers can also be easily modified by substituting polar
groups for methyl groups in the polysiloxane structure thereby producing a wide range of
polarities.

Table. A Few of the Available Stationary Phases For Gas Chromatography

Compound Polarity Max Temp oC Column ID Manufacturer
Poly(methyl siloxane) Low 300-350 HP-1 Agilent
AT-1 Alltech
DB-1, SE-30 J&W
OV-1 Ohio Valley
ZB-1 Phenomenex
RTx-1 Restek
BP-1 SGE
SPB-1 Supelco
CP-Sil 5 CB Varian
95% Dimethyl, 5%phenyl Low 300 HP-5 Agilent
Poly(methyl siloxane) AT-5, EC-5 Alltech
DB-5, SE-54 J&W
OV-5 Ohio Valley
ZB-5 Phenomenex
RTx-5 Restek
BP-5 SGE
SPB-5,MDN-5 Supelco
CP-Sil 8 CB Varian
Polyethylene glycol Medium 250 HP-20M Agilent
AT-Wax Alltech
DB-Wax J&W
Carbowax 20M Ohio Valley
ZB-Wax Phenomenex
Stabilwax Restek
BP20 SGE
Supelcowax 10 Supelco
CP-Wax 52 CB Varian
Polyethyleneglycol Medium 250 HP-FFAP Agilent
Nitroterephthalic acid ester AT-1000 Alltech
(free fatty acid phase) DB-FFAP J&W
OV-351 Ohio Valley
ZB-FFAP Phenomenex
Stabilwax-DA Restek
BP-21 SGE
Nukol, SPB-1000 Supelco
Varian

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Mobile Phase

Hydrogen, Helium and Nitrogen are the most common gases used as mobile
phases. The lighter the carrier gas, the higher the speed of analysis. Therefore, hydrogen
will give the most plates/sec. However, helium is often used because hydrogen is
potentially explosive. High-speed analysis also results in narrower peaks and better
detectibility. Longitudinal diffusion is lowest in the heavier gases such as nitrogen so
nitrogen can give the highest N (lowest H) at low mobile phase velocities where
longitudinal diffusion has the highest effect on efficiency.

Figure 1. Velocity vs Theoretical Plate Height for Common Carrier Gasses.

Gases are forced through the

column by an applied pressure at the
head of the column. The outlet end of
the column is usually at atmospheric
pressure, or even vacuum (when
attached to a mass spectrometer).
Therefore there is a drop in pressure as
the gas moves through the column.
This drop in pressure causes the gas to
expand which can result in peak
broadening. It also causes the gas
velocity to increase as it moves
through the column (Figure 2).
Therefore it becomes difficult to
optimize gas velocity. Figure 2 shows
the gas velocity profile along the
column. The value pi/po is the ratio of
the inlet velocity(pi) to the outlet
velocity (po).

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 As the column oven is heated, the viscosity of the mobile phase increases. (the
viscosity of gases generally increases with temperature, which is in contrast to liquids
where the viscosity decreases with increasing temperature). Therefore as the column is
heated during the temperature ramp, the flow rate goes down. In order to keep a constant
flow (and reduce peak spreading of later eluting peaks) a process of pressure
programming is used. The constant flow mode increases the pressure at the head end of
the column, and keeps the mobile phase velocity constant. Pressure programming can
also be used to increase mobile phase velocity as the temperature increases, further
decreasing analysis time and increasing peak height.

Kovats Retention Index

The retention time of an analyte provides at least some information on the

chemistry of the compound. However, retention time is dependant on many operational
factors such as temperature, column length, column diameter, coating thickness, etc.
Therefore it is more satisfactory to use a relative retention value whereby many of these
variations are compensated for. The Kovats retention index system is based on a scale
defined by the elution of a series of n-alkanes. It is widely used, and information on the
Kovats Index for many compounds can be found in the literature. In this system, normal
alkanes (pentane, hexane, heptane, etc.) are given an index value 100 times their carbon
number (ie. Pentane = 500). The elution time of an alkane gets longer with the addition
of each additional CH2 group such that the a plot of the log of retention time vs carbon
number is linear (there is some deviation, especially at lower carbon numbers). The

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calculation of the Kovats Index for a particular compound is obtained by interpretation of
its position between the two nearest alkanes in the reference series.

Kovats Retention Index = 100Cn + 100 [log tR(A) –log tR(n)]

[log tR(n+1) – log tR(n)

Where Cn is the carbon number of the alkane eluting just before the analyte
tR(n) is the retention time of the alkane eluting just before the analyte
tR(n+1) is the retention time of the alkane eluting just after the analyte
tR(A) is the retention time of the analyte

The classical Kováts retention index is measured under isothermal conditions.

However, in the case of temperature-programmed gas chromatography a similar value
can be calculated utilizing direct numbers instead of their logarithm. In other words, an
equation for the retention index can be developed using a polynomial regression of a
series of alkanes vs. their retention times.
This method of measuring reference retention is dependant only on the analyte,
the temperature and the stationary phase composition. A Kovats index for an analyte
should be the same on any column with the same stationary phase irregardless of column
length, mobile phase, flowrate, etc. However it has been shown that in open tubular
columns with thin films, adsorption effects can contribute to retention, affecting the
accuracy of the Kovats index especially at low temperatures and with polar stationary
phases.

References.

Grant, D. W. Capillary gas chromatography; John Wiley & Sons, Ltd: West Sussex,
England, 1996; pp 295.
Modern Practice of Gas Chromatography; 3rd. ed.; Grob, R. L., Ed.; John Wiley & Sons,
Inc.: New York, 1995; pp 888.

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