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Reading: Skoog, Holler and Crouch: Chapters 26 and 27.
A.
INTRODUCTION
Gas-liquid chromatography, or simply gas chromatography (GC), is a physical method of
separation based on the distribution of a solute (i.e., analyte) between a mobile gaseous
phase and a stationary liquid phase. When the sample is injected onto the column, the
individual molecules continuously transfer between the stationary and mobile phases as
the injected solute band moves down the column. Each solute moves at a rate determined
by its partition between the gas and liquid phase and the flow rate of carrier gas.
Selective partitioning between the mobile and stationary phases is highly dependent on
the analyte molecule’s solubility in each phase. Thus, solutes that are less soluble in the
stationary phase will elute from the column first while solutes that are more soluble will
elute later. Because solubility depends on the physical and chemical properties of a
compound, a series of compounds exhibiting a systematic variation in structure and thus,
chemical properties, would be expected to partition between the mobile and stationary
phases to different extents. It will be shown in this experiment that separations in
gas-liquid chromatography are related to boiling point differences in a solute mixture.
Column temperature is an important variable that must be controlled for precise work, so
the column is usually housed in a thermostated oven. The optimum temperature depends
on the boiling point of the sample and the degree of separation required. Roughly, a
temperature equal to or slightly above the average boiling point of a sample results in a
reasonable elution time (2 to 30 min). For samples with a broad boiling point range, it is
often desireable to employ temperature programming, whereby the column temperature
is increased either continuously or in steps as the separation proceeds. You will
investigate the effects of temperature programming on separations in this experiment.
B.
EXPERIMENT SUMMARY
In this experiment, you will:
•investigate the effect of analyte properties on retention in GC
•investigate the effect of carrier gas flow and column temperature on GC separations
•demonstrate how temperature programming may be used to speed GC analysis.
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C.
GC INSTRUMENTATION
The basic gas-chromatograph consists of the following components:
1. Column
The column is a metal (stainless steel or copper) or glass tube of various lengths.
Packed columns contain a granular solid of uniform particle size, evenly coated with
a thin layer of the liquid phase. Capillary columns are fabricated from very
narrow-bore tubing with the liquid phase distributed along the column walls.
2. Carrier Gas
An inert carrier gas passes through the column and transports the solute from the
injection port to the detector. H2, He, and N2 are the most common carrier gases.
3. Injection System
The sample is commonly injected from a microliter syringe by insertion through a
rubber septum. For routine sample injections a 1, 5 or 10 microliter syringe is
recommended. The entire sample should be vaporized immediately and swept
directly into the column.
Syringe loadings and sample injections vary with the chromatographer. The key is
that each GC operator must develop and perfect a technique that will produce the
highest degree of reproducibility from injection to injection. The following is a
recommended procedure:
a) Fill the syringe with sample and expel it into the waste several times .
b) Pump the syringe with the needle in the sample until several microliters of
bubble free sample are in the syringe. Try to get rid of the air bubbles by
repeatedly slowly retracting the plunger about half way then rapidly
depressing it.
c) Expel the sample from the syringe until the plunger is at the 1 µL mark.
d) Retract the syringe plunger, pulling about 2 µL of air into the syringe. This
gives an air peak, which can be used to indicate the dead time of the column,
and prevents premature loss of sample due to vaporization
e) Insert the needle to its full length into the injector port and inject the sample.
Quickly remove the syringe. Smoothness and quickness are necessary for
good results.
4. Detection System
The detector monitors the carrier gas stream as it passes out of the column. The
signal is generally proportional to the concentration or the total amount of solute
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present. The thermal conductivity detector (TCD) and the flame ionization detector
(FID) are the two most common types (see Skoog, Holler and Nieman, pp. 706-8).
D.
THEORY
(see also Skoog, Holler and Crouch, Chapter 26)
1. Height Equivalent to a Theoretical Plate (HETP)
The Height Equivalent to a Theoretical Plate (HETP or H) results from the
treatment of separations in terms of repeated equilibration between the mobile and
stationary phase. This is a very important parameter used for measuring separation
efficiency. The smaller H is, the more efficient the separation. The HETP for a
particular carrier gas flow rate is calculated from the total number of theoretical
plates (N) and column length (L), i.e.
H=
L
N
(1)
where:
⎛t ⎞
N = 16⎜⎜ r ⎟⎟
⎝ Wb ⎠
2
(2)
where t r is the retention time of the component and Wb is the peak width at the
baseline. Notice that t r and Wb can be directly obtained from the chromatogram.
2. van Deemter Theory
For a given GC column, the van Deemter theory is useful for determining the flow
rate which gives optimum efficiency at a given column temperature for a particular
compound. The van Deemter equation is
H = A+
B
+ C s u + Cmu
u
(3)
where A, B, C s and C m are constants and u is the carrier gas flow rate.
The first term in the van Deemter equation, A, accounts for the multiple pathways of
differing length that an analyte molecule may take through the column. B u accounts
for longitudinal diffusion. The third and fourth terms are called mass transfer terms and
account for the finite time required to reach equilibrium in the stationary phase ( C s ) and
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the mobile phase ( C m ). Detailed descriptions of these terms can be found in Skoog,
Holler and Crouch, section 26C-1. For a particular column at a constant temperature,
HETP varies with the flow rate of the carrier gas as shown in Figure 1. The curve in
Figure 1 is broken down into the contributions from each of the terms in the van Deemter
equation. The optimum velocity of carrier gas, uopt, is located at the point where the
HETP-velocity curve passes through a minimum as shown in Figure 1 (from reference 4,
p 772).
Figure 1:
Plot showing dependence of plate height, H, on carrier velocity u.
Symbols are from the van Deemter equation.
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3. Determination of optimum flow velocity
The velocity at the point where the HETP-velocity curve passes through a minimum.
i.e., uopt, can be determined by measuring the HETP at a sufficient number of flow
velocities to experimentally define the HETP-velocity curve. The optimum flow
velocity can then be read directly from the curve.
E.
EXPERIMENTAL
E.1. Samples
The following sample solutions are available:
Sample
Component
S1
1% heptane in pentane
S2
1% octane in pentane
S3
1% nonane in pentane
S4
1% decane in pentane
S5
1% octane and 2,2,5-trimethylhexane (TMH) in pentane
S6
1% heptane, octane, nonane, Decane, and TMH in pentane (5
components)
S7
1% heptane, octane, nonane, and decane in pentane (4 components)
E.2 Equipment and Settings
1. Gas Chromatograph
The Varian 3400 gas chromatograph will be used in this experiment. Since warm
up times are in the range of hours for certain parts of the instrument, it will already
be warmed up for you at the start of the laboratory period. Figure 2 shows the
3400 GC front panel with keyboard functional description. Operation of the GC is
described in a separate handout.
The GC is already configured to use the Thermal Conductivity Detector ( Front
injection port/A injects onto the column connected to the TC dector) with the
following settings:
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INJ A TEMP
200°C
COL TEMP
100°C
TCD TEMP
220°C
CARRIER GAS A
30 ml/min
You will build methods for running your samples. Building a method simply
means to construct a list of parameters to be used when that method is activated.
To start building a method BUILD/MODIFY and one Method keys are pressed.
The DISPLAY shows prompts for entry of experimental parameters such as the
type of run (isothermal or temperature programming), initial temperature, length of
run, rate of temperature increase etc.
3. Column
The column packing used is 3% (w/w) SP-2100. SP-2100 is a nonpolar polymeric
silicone stationary phase. It is a general purpose nonpolar stationary phase, useful
for hydrocarbons, steroids and PCBs.
The column length is 8 feet.
4. Thermal Conductivity Detector (TCD)
TCD will be used for this lab. You will adjust the ATTENUATION so that peaks
do not go off-scale (except for the solvent peak , i.e., the first major peak) or are
too small. Be sure to write down the value.
5. Chart Recorder
The recommended chart speed for the recorder is 1 cm /min. Write down the
speed you are using. This is important because you need it for your retention
time calculation. Your Chart Speed _________________.
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Figure 2:
Front panel of the Varian 3400 Gas Chromatograph.
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E.3. Experiment Procedures
1. Isothermal GC
Make sure the column temperature is 100°C and the carrier gas flow rate is
30 mL/min. Turn on the chart recorder and inject 1µL of sample S1 (with 2 µL
of air, see Sample Injection Techniques for explanation). Immediately mark
the injection point on the chart. Wash the syringe and prepare for the next
injection while waiting. When the last peak elutes, inject sample S2 in the
same fashion. Repeat for samples S3, S4, S5, and S6.
2. Effects of Carrier Gas Flow Rate
At column 100°C, change the carrier gas flow rate to 20 mL/min, inject
samples S2 and S5. Then change the flow rate to 25, 35, and 45 mL/min,
respectively. Inject sample S2 under each condition. Finally inject sample S5
at 45 mL/min.
3. Effects of Column Temperature
Change the flow rate to 30 mL/min and the column temperature to 120°C.
Inject sample S6. Repeat at column temperature 140°C and 160°C.
4. Programmed Temperature Gas Chromatography
In this section three programmed temperature chromatograms will be run of
the mixture of n-alkanes (S7). Set the starting temperature at 100°C and the
final temperature to 200°C. Make runs, at program rates of 5°Cmin, 10°C/min
and 15°C/min.
E.4. Instrument shut down (T.A. will do this)
1. Turn off the chart recorder. Take the pen out and cap it .
2. Turn off the TCD power
3. The column temperature should be reset to 60°C. (when you do this, the door
of the oven will be opened automatically. DO NOT force it, or you may
damage the instrument!).
4. Close the sample vials tightly and put them into the refrigerator.
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F.
DATA PROCESSING AND QUESTIONS
1. Plot log(tN) (net or corrected retention time) vs. carbon number for the homologous
series (S1 to S4) for the run at 100°C and flow rate of 30 mL/min. Also plot log(tN)
vs. boiling point. Which plot provides the best straight line relationship? Plot log tN
for 2,2,5 trimethylhexane (TMH) at 100°C on the first plot. Comment on the result in
terms of data interpretation in GC separations of homologous series.
2. Calculate N and HETP for each of the chromatograms of octane (S2) under each
carrier gas flow rate, 20, 25, 30, 35, 45 mL/min. Tabulate the results and show
sample calculations.
3. Calculate the resolution for S5 (octane and TMH mixture) at carrier gas flow rate 20,
30, and 45 mL/min. Discuss the effect of flow rate on resolution (if any).
4. Discuss how the resolution and total analysis time is affected by the column
temperature. Use the octane and TMH peaks in the chromatograms for sample S6 at
100, 120, 140, and 160°C.
5. For the five components in Sample S6, plot log(tN ) vs.1/T for the temperatures 100,
120, 140, and 160°C. Briefly discuss significance of this plot (see references 1 and 2).
6. For the programmed temperature GC, which heating program (i.e. 5°, 10°, or 15°C
per min) seems optimal? Factors to consider are the peak separation, peak shape,
time of analysis, etc. Plot log (tN) vs. carbon number for these programmed
temperature chromatograms. How does this plot differ from that for the isothermal
case.
G.
REFERENCES
1. S. Dal Nogare and R.S. Juvet, Gas-Liquid Chromatography, John Wiley, New York,
1962.
2. W.E. Harris and H.W. Habgood, Programmed Temperature Gas Chromatography,
John Wiley, New York, 1967.
3. J.A. Perry, Instroduction to Analytical Gas Chromatography, Marcel Dekker, INC.,
1981. (Get this from me).
4. Skoog, Holler and Crouch: Chapters 26 and 27.
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