Introduction:
In chromatography the corrected retention times (retention times) and for each analyte under a defined set of (physical) experimental conditions is unique. The identification of analytes in a mixture, therefore, can be made based on the corrected retention times. In this experiment the corrected retention times of some pure substances will be established first. Then from the chromatogram of a mixture of unknowns the identity of its constituents of the mixture is made. This is an isothermal run.
Chromatograms of Known Substances
Procedure:
1. Set up the Gas Chromatograph for an isothermal
run; follow the instructions relevant to the instrument.
The injector and detector temperatures must be higher than
the boiling point of the analyte with the highest boiling point.
The column temperature must be lower than the boiling point
of the analyte with the lowest boiling point.
(For this part of the experiment a temperature program of 45oC for 12min; ramp of 25oC/min to 145oC and holding at 145oC for 2min. works. You may have to change the chromatographic parameters to obtain a reasonable elution time and peak size)
2. Mix in a 10mL volumetric flask partially filled with pure water, 100uL each of the the known substances (methanol, ethanol, 1-propanol, 1-butanol).
Draw 1uL of the solution made above, carefully draw 0.1uL (or less) of hexane (unretained solute, dead-time marker) into the same syringe; do not push the plunger into hexane at this point. Inject the syringe contents into the column and measure (and tabulate) the corrected retention time for each substance. Clean the syringe and make sure that the plunger operates smoothly.
Allow the substance(s) to completely leave from the column perform two more injections (of the same sample) to find the mean corrected retention time and calculate its standard error. If time alloted for the experiment is limited you may skip the triplication.
Indicate the following on the chromatogram and in your notebook. The parameters determined from this run must be used throughout the experiment.
Name of the operator
Diameter, length, stationary phase and loading of the column (obtain from the tag)
Temperature of the injection port
Temperature of the column
Temperature of the detector
Carrier gas
Flow rate/pressure of carrier gas
Flow rate/pressure of hydrogen gas
Flow rate/pressure of oxygen gas
Sample size
Recorder speed
Tabulate all data appropriately.
Chromatogram of Unknown Mixture
Procedure:
(For this part of the experiment a temperature program: 45oC for 22min; ramp of 25oC/min to 145oC and holding at 145oC for 2min)
1. Mix in a 10mL volumetric flask partially filled with pure water, 200uL of the the unknown substances as before.
2. Inject 1.0uL of the mixture dissolved in the solvent (water) and 0.1uL of hexane (as above) without changing the chromatographic parameters determined above.
3. Measure the retention times of all the peaks on your chromatogram. Calculate the corrected retention times, t'.
Treatment of Results:
1. Plot a graph of log t' vs retention index of the (appropriate) known substances (retention index for homologous series = 100n, where n = number of carbon atoms on the molecule).
2. Using plot generated above identify as many components in the 'unknown' mixture as possible.
3. Assume the compounds not identified in the above step are positional isomers (any isomer that differs from another isomer only in the position of a substituent, or of a double or triple bond) of the compound identified, what would they be (speculate)? Justify your speculation.
4. Calculate the mobile phase velocity of the carrier gas for your experimental conditions.
Reference: Qualitative Analysis of Gas Chromatography, Harry G. Hajian and Robert L. Pecsok, 1990, New Jersey: Prentice Hall
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