From the Desk of Abhijit Bhar
Lession – 1
Objectives: Method development by GC
Introduction to temperature programming
Observe the effect of carrier flow rate
Observe the effect of the split ratio
Observe the precision of multiple injections
Obtain a final chromatographic separation
Calculate k’, for each pair of peaks
Use of internal standard for quantitative work
Perform a quantitative determination of an unknown
Turn in:
Notebook pages (With sample calculations for each analyte quantified)
Chromatograms
Method development
Gas chromatography works for volatile compounds only; The more volatile the
compound, the more rapidly it elutes from a GC column at a given temperature. While
rough estimates of the elution order can be made for non-polar stationary phases (such
as the DN-5 column in lab) based on boiling point, the only definitive answer of elution
order is obtained by doing method development on the separation in question.
Because GC is a technique that works largely on volatility, it only makes sense to use
the most extreme conditions (highest temperature) first. A preliminary injection is made
at a oven temperature close to the maximum temperature of the column stationary
phase. It is important to not exceed the manufacturer’s specification for maximum
temperature. An series of isothermal separations are performed at progressively lower
temperatures to determine the approximate temperature range for elution of the
components in your sample. A balance is struck (often based on operator experience)
between the elution time of the last peak and the separation needed for the analysis; As
the total separationtime increases, the separation usually improves. However, if you
“over-separate” you simply waste time.
To do:
Sample preparation: Quantitatively (three decimal places) weigh out ~0.10 g each of
naphthalene and the three phenols assigned to you; Mix them together in a beaker and
dissolve in MeOH. Make the solution up to volume in a 10 mL volumetric flask. Store
the diluted solution in the septum vial provided by your instructor.
Quantitatively weigh out ~0.10 g each of naphthalene and the three phenols assigned to
you; Place them separately in beakers and dissolve in MeOH. Make each solution up
to volume in a 10 mL volumetric flask. Store the diluted solution in the septum vial
provided by your instructor.
Injection: Your instructor will show you proper injection technique the first time. You
will be using a 1-uL syringe. Both the needle and the plunger are susceptible to
bending,
which will render the syringe useless. The sample to be injected should always be
“rinsed” out of the syringe by following it with clean solvent (see diagram below).
Column specifications: With the GC oven cool, open the oven and record the column
specifications in your notebook. Specifically note the column length, inner diameter,
stationary phase thickness and the type and maximum temperature limit of the
stationary phase.
Method development: Isothermal Separations
a. Set the oven temperature at 2000C. When the temperature has stabilized (as
indicated
by a “Ready” signal from the GC) make an injection of your mixed components.
Observe the resulting chromatogram. Wait about three minutes and then stop the run.
b. Repeat the above paragraph at 1600C and 1200C, stopping the run when the last
component has finished eluting.
c. Based on the above results, adjust the oven temperature to give you a separation of
the four components (excluding MeOH) in about five minutes. Verify with an injection.
d. When the components are well-separated, make a series of five injections of your
mixed sample. Record the individual peak areas or heights and calculate the precision
of your repeated injections.
e. Using the isothermal conditions established in c. and d., inject your individual
standards to identify the individual peaks.
Clean solvent Sample
Read sample volume from syringe barrel
Method development: Separations using temperature programming
The whole point to temperature programming is to improve the separation of solution
components by controlling the column temperature in a prescribed manner during the
course of the elution process. Compared to isothermal separations, temperature
programming can provide the operator the ability to retard the elution of low-boiling (or
early-eluting) components, and speed up the elution of high-boiling (or late-eluting)
components.
a. Set up a temperature program as follows:
Initial temperature 900C
Hold time 1 min
Temperature ramp 100C/min
Final temperaure 1200C
Hold time 30 min
b. When the temperature has stabilized (as indicated by a “Ready” signal from the GC)
make an injection of your mixed components. Observe the resulting chromatogram.
Stop the run when the last component has finished eluting.
c. Based on the above results, adjust the temperature program to give you a separation
of the four components (excluding MeOH) in about five minutes. Verify with an injection.
d. Using the temperature program established in c., inject your individual standards to
identify the individual peaks.
Temperature
Time
Isothermal
Temperature
programmed
Method development: The effect of carrier flow rate
Although often difficult to observe, there is an optimum carrier flow rate at which the
column efficiency is at a maximum. Determination of this optimum flow rate requires
careful measurement of peak width and the calculation of the number of theoretical
plates
in the column. With the kind of printouts available to the lab, this determination is nearly
impossible. However, you can still observe subtle changes in peak shape and
symmetry.
To do:
Set the oven temperature to 1200C and the split ratio to 50. Set the flow rate to 7
mL/min and make an injection of your mixed phenols and naphthalene solution. Repeat
at flow rates of 5, 3, and 2 mL/min. Note any changes in peak area and shape.
Method development: The effect of the split ratio
It is impossible to inject a sufficiently small volume of sample onto a high-efficiency
capillary GC column without overloading the column. Therefore, the injected sample is
typically “split” pneumatically in the injection port, with only a small fraction actually
getting into the column. Increases in the split ratio often improve the column efficiency
(seen as sharper, more narrow peaks) at the cost of peak area.
To do:
Set the oven temperature to 1200C and the flow rate to 3 mL/min. Set the split ratio to
100 and make an injection of your mixed phenols and naphthalene solution. Repeat at
split ratios of 50, 25, and 15. Note any changes in peak area and shape.
Quantitative work in gas chromatography
As you have already found out, the precision of repeated injections in GC is not
particularly good – certainly worse than the loop injectors used in HPLC. Because of
this limitation, it is problematical to generate an external calibration curve in the same
manner as HPLC. Therefore, it is common when using GC to use the internal standard
method ofcalibration.
Single Point Internal Standard
Internal standard methods account for instrumental variables, particularly varying
injection volumes!!
each sample contains an internal standard of known amount with a predictable
response
this method requires at least two analyses per sample
First, a known amount of internal standard is added to a sample containing known
amounts of the analytes of interest
the internal response factor (IRF) is calculated
Next, a known amount of the internal standard is added to the unknown sample
(containing the analytes of interest, but with unknown concentrations)
The amount of each analyte is calculated as follows:
Example Calculation: Single Point Standard Method
A sample is prepared which contains 2000 ug/mL of toluene (the internal standard) and
1000 ug/mL benzene (the analyte). The sample is injected. The resulting peaks have
the
following areas:
toluene 120,000
benzene 67,000
Now, a sample containing 2000 ug/mL of toluene and an unknown amount of benzene
is injected using the same chromatographic conditions. The resulting peak areas are:
toluene 122,000
benzene 43,000
To do:
Testing the internal standard concept
Using the chromatograms already generated in part d. of the isothermal separations
above, choose the naphthalene peak as the internal standard. Knowing the composition
of the mixed sample, calculate the IRF for each of the phenols in the first
chromatogram.
Then apply these IRFs to the subsequent chromatograms and tabulate how close the
internal standard calibration results are to the actual concentrations in your mixed
sample.
