Gas Chromatography What Goes

on Inside the Box?

?
Agenda
Introductions
Objectives
Theory of Chromatography
Theory of Gas Chromatography (GC)
Activity #1 on Sample Flow through GC
Parts of the GC
Troubleshooting the GC Activity #2
Q&A / Discussion
Written Assessment
Introductions!
Objectives
Learners should be able to explain the theory behind
gas chromatography.
Learners should be able to recognize and
differentiate between the different parts of the GC.
Learners should be able to outline the process for
setting up the GC.
Learners should be able to investigate and resolve
common GC issues.
Chromatography
Chromatography is the analytical science of
separation.
The core of separation theory is that
molecules can be separated based upon
their properties, whether it be their polarity,
size, solubility, or vapor pressure.
Types of Chromatography
Thin Layer Chromatography (TLC)
High Performance Liquid Chromatography
(HPLC)
Liquid Chromatography (LC)
Gas Chromatography (GC)
Gas Chromatography
Gas chromatography is a technique used to separate
chemical compounds in the gas phase.
Gas chromatography is a technique employed in quality
control laboratories to quantify chemical compounds used
in the manufacture of pharmaceutical products.
Gas Chromatography
An injector system takes a liquid sample
and deposits the injection into the head of
the GC.
The liquid is subjected to high temperature
that vaporizes the sample.
The sample enters the column, also known
as the stationary phase.
An inert carrier gas, such as helium, forces
the sample through the column.
Gas Chromatography 2 Phases
1. Mobile Phase Carrier gas moving
through the system, carrying the
sample.
2. Stationary Phase A column with a
matrix or coating of particles that the
mobile phase passes through or across.
GC From Injection to Analysis
Sample enters column and separation
begins.
Gas phase molecules that have a stronger
affinity to the column than the mobile phase
will spend more time on the column.
Compounds that have a higher affinity to
the mobile phase will flow through the
column quickly and be detected first.
Separation
Separation occurs based upon each molecule's
physical properties and affinity for the mobile and
stationary phase.
What happens when similar compounds have similar
retention times? Compounds may coelute, resulting
in peaks on top of each other.
http://www.kromasil.com/www/img/notes/insulin_figure_3_impurity_coelution.png
How to achieve better separation?
Temperature ramping
The inlet temperature can be changed to
inhibit the entrance of compounds into the
column based upon their vapor pressure.
Ramping the temperature and forcing a
sample through a column can efficiently
separate most compounds.
Parts of the GC
Capillary columns are the most commonly
used type of columns in quality control
laboratories. The column is long and
narrow with the stationary phase coating
the inside of the column.
Parts of the GC
Packed column The stationary phase
coats large particles packed inside of the
column. The mobile phase forces the
sample through this particle matrix that
facilitates the separation.
Parts of the GC
Split injectors vaporize the sample and vent
the majority of the sample to waste. Only a
small portion of sample enters the column.
Venting fine tunes the injection and
prevents the capillary column from
overloading.
Parts of the GC
Splitless injectors can be used to introduce
a small amount of sample into the system
without being purged to waste during the
injection. Venting can make some
impurities impossible to detect if the sample
is small.
Parts of the GC
Headspace samplers remove volatile gases
from within a sample vial. The sample vial
is heated in a temperature controlled oven
within the sampler. A portion of the liquid
within the vial vaporizes and is injected into
the GC.
Parts of the GC
Flame Ionization Detectors (FIDs) are the most commonly
used GC detectors. This type of detector is a destructive
detector, or a detector that consumes the sample. The
gas exiting the column of the GC is passed through a
hydrogen flame that ignites and burns the sample. The
sample is ionized in the flame, and a resistor measures
the voltage difference generated by the ions passing in
the resistor.
Parts of the GC
Thermal conductivity detectors (TCDs) are detectors that
contain both a reference channel and a sample channel
which contain metal filaments. The reading on the
reference channel is compared to the sample channel. A
change in temperature between the two channels is
translated into a resistance signal which in turn
represents a peak and peak area.
Parts of the GC
Mass spectrometer detector (MS or Mass Spec)
bombards a sample with an electrical current or a
chemical reagent that fragments a molecule into ions.
Each molecule subjected to a similar bombardment will
fragment in a predictable manner. The fragmentation
pattern is unique for each compound.
Troubleshooting the GC
No peaks detected
Split peaks
Flat top peaks
Pregnant peaks
Ghost peaks
Peak response changes
Peak tailing
Peak fronting
Retention time shift
Loss of resolution
Noisy baseline
No Peaks Detected
Ensure that detector is operational. For
FID detectors, check to see if flame is
lit. Hold a piece of glassware over the
detector; if the glass fogs up, then the
detector is lit.
Ensure the column is installed properly,
both at the injector and detector end.
Check the carrier glas and ensure it is
flowing through the column. A flow
meter can be used to detect carrier flow.
Alternatively, remove the detector end of
the column and place in a small aliquot
of water. If not bubbles are produced,
then the carrier gas is not reaching the
detector.
Check the injector. Ensure the syringe
is installed and is not plugged or bent.
Split Peaks
Split peaks are a result
of poor sample
injection.
Check the injector to
ensure it is not leaking or
damaged.
Check the inlet
temperature. Not having
the inlet temperature high
enough can cause split
peaks.
Ensure appropriate split or
splitless liner is installed.
Flat Top Peaks
Flat top peaks are caused
by overloading the
detector.
Check that the injection volume
is correct.
Ensure the correct syringe size
is installed in the sampler and
software.
For a split injection, check the
split ratio.
For a splitless injection, shorten
the purge time on setting and
increase the purge flow setting.
Ghost Peaks
Ghost peaks are anomalous peaks that appear in the chromatography.
Contamination could be present in the column. Purge the column.
Inject a blank to determine if the solvent is contributing to the ghost
peaks.
The contamination could be from the syringe. Install a new syringe.
Peak Response Changes
Peak response changes can be an indication of a
malfunction.
Ensure the split ratio is set correctly in the software.
Ensure that the injector port temperature is set correctly.
If the analysis is splitless, ensure that the correct purge time is
used.
Peak Tailing
Tailing problems are typically
isolated to the column;
however, tailing is expected for
certain compounds (especially
alcohols).
Ensure that the appropriate liner is
installed.
Reinstall the column. A poor fitting
at the injector can cause tailing
problems.
Ensure that the injector end of the
column is not damaged or
discolored. Remove damaged
sections of column and reinstall.
Severe contamination may also
cause tailing.
Peak Fronting
Peak fronting is
uncommon, but is
caused primarily by
three problems:
Ensure the appropriate split
or splitless liner is installed.
Overloading a column can
cause fronting. Check
injection volume.
Reinstall the column. A poor
fitting at the injector can
cause fronting issues.
Retention Time Shift
Shifting retention times are
typically isolated to the column
or the gasses used in the
system.
Change the septum. A leaky septum
can cause retention time shifts.
Check the carrier gas flow of the GC.
Changes in the carrier gas velocity can
impact the retention time of the
samples.
Oven temperature can affect the
velocity of samples in the column.
Check the oven temperature program.
Column damage can cause retention
time shifts. Trim and reinstall column.
Loss of Resolution
Loss of resolution is
typically caused by
problems associated with
the flow path.
Column may no longer be
suitable for use. Ensure that
the column is not damaged or
discolored. Trim and reinstall
the column. If the issue
persists, discard the column
and use a newer column that
has acceptable resolution.
The injector liner is damaged.
Replace the column liner.
Noisy Baseline
Possible Sources:
Is detector operational? For FID detectors, check to see that the flame is lit.
Hold a piece of glassware over the detector. If the glass fogs up, then the
detector is lit.
Is carrier gas flowing through the column?
Is the injector syringe plugged or bent?