We have all seen forensic scientists in TV shows, but how do they really work? What is the science behind their work? The course aims to explain the scientific principles and techniques behind the work of forensic scientists and will be illustrated with numerous case studies from Singapore and around the world. Some questions which we will attempt to address include: How did forensics come about? What is the role of forensics in police work? Can these methods be used in non-criminal areas? Blood. What is it? How can traces of blood be found and used in evidence? Is DNA chemistry really so powerful? What happens (biologically and chemically) if someone tries to poison me? What happens if I try to poison myself? How can we tell how long someone has been dead? What if they have been dead for a really long time? Can a little piece of a carpet fluff, or a single hair, convict someone? Was Emperor Napoleon murdered by the perfidious British, or killed by his wallpaper? *For Nanyang Technological University (NTU) students, please be noted that this course will no longer be eligible for credit transfer.
Week 2B - 2 GC & HPLC
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來自 Nanyang Technological University, Singapore 的課程
Introduction to Forensic Science
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從本節課中
Chemical Analysis in Forensic Science
與講師見面
Roderick BatesAssociate Professor
Division of Chemistry & Biological Chemistry | School of Physical & Mathematical Sciences
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Gas Chromatography is superior to Thin Layer Chromatography in many ways.
Particularly, it's much more sensitive and it gives
much higher resolution than the very simple TLC plates.
There are many technical differences between the two methods,
even though they rely on the same physical principles.
For a start, in Gas Chromatography, as you might be able to figure out from the name,
the mobile phase is not a liquid, it's a gas often referred to as the carrier gas.
In addition, Gas Chromatography does not use a plate,
it uses something called a column
as the container for the stationary phase.
Now even though it's called a column, it doesn't actually look like a column.
The name is retained for historical reasons.
The stationary phase is packed inside a very long, fine metal tube,
and for reasons of space, that metal tube
is wrapped around in a coil inside the instrument.
So one end of the coil is where the material goes in, at the far
end of the coil the material comes out, and then it passes through a detector
and the signal from the detector is then recorded by a computer.
So here is another technical difference between TLC and GC.
In TLC, we were allowing the mobile phase to move a certain
distance, and then we measure the distance moved by the components.
That is, the distance moved by the components within a fixed time.
In GC it's different, because the distance moved by
the components is fixed, it's the length of the column.
So what we are measuring is the time it takes
the different components to get to the end of the column.
So we cannot use the Rf, the Retention Factor, that we did in TLC.
Instead we use the Retention Time, that is the
time for that component to travel through the column.
So the output, the data that we get from the GC looks a little bit like this.
We no longer have spots on a plate, we have peaks on a graph.
We're no longer measuring our Rf, we're measuring the
Retention Time, and this is typically measured in minutes.
But the principles are the same.
When we get peaks from our GC from our unknown sample,
we can then do a tentative identification by comparing them to known standards.
So if your
unknown material comes off at a particular time,
your standard comes off at that same time,
then tentatively we can say, 'that is the compound'.
Though just like with TLC, it's not a very firm identification.
There is another technical difference between GC and TLC, and that is your GC
chromatogram will always show a big peak after a very short time,
and this peak we always ignore because it is solvent.
To inject your sample on to the GC, you have to dissolve it into a solvent,
you have to make it into a solution.
So of course, the solvent will also be picked up by the detector.
But the solvent will come through the GC column very quickly and
it's this big peak you see at about one minute or so.
Now the sophistication of GC gives it yet another advantage over
TLC, and that is, GC is quantifiable.
With TLC when you see a spot, you cannot
say much about the quantity present from that spot.
But with GC, the amount of material present
is proportional to the area under the peak.
So, not only can we separate out the mixture into its different components, we
can also measure the relative amount of those components that are present.
So GC is a technique where we can quantify the components.
This is a Gas Chromatograph, or GC, and this one is fitted
with an auto-sampler so that it can run multiple samples one by one.
With the auto-sampler, which is effectively an injection robot,
the sample is injected into the instrument automatically by this syringe here.
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The column where the components of the mixture are separated
is inside this oven.
The oven temperature is programmable, it can be completely controlled.
And the column,
which is many meters long, is wrapped around here in this coil.
The output
from the column goes into the detector, which
is built into the top of the machine here.
The information from the detector then goes onto
the computer, where it can be displayed and recorded.
The other chromatographic technique that is
very important for forensic science is HPLC.
The initials HPLC stand for High Performance Liquid Chromatography.
They also stand for High Pressure Liquid Chromatography.
Now HPLC is similar to GC, but as the name
implies, the mobile phase is a liquid, not a gas.
And the mobile phase which is used are organic solvents, or
sometimes aqueous solutions, depending on what your trying to analyze.
HPLC does not use the very
long, fine metal tube to contain the stationary phase that GC used.
HPLC, we use steel columns, and we have to
use these steel columns because of the high pressures involved.
Why do we use high pressure?
Because the stationary phase is a very
fine material densely packed inside the column.
So to get the liquids to flow through the column at a useful rate,
we have to apply a high pressure, and that is done with a pump.
So we need the high pressure to get the good flow rates, and therefore,
we need the steel columns to have the strength to stand up to those pressures.
Like GC, with HPLC, we are measuring the time it takes for each individual
component to get from one end of the column to the other end of the column.
So once again, we are measuring Retention Times.
So the output, the data that you get from an HPLC, the
chromatogram does look rather like the chromatogram that you get from a GC,
with these sharp peaks appearing one for each components. And you see can from this
chromatogram that HPLC tends to give well
resolved sharp peaks, so you can easily separate
and resolve many different compounds.
Just like GC, HPLC can also be quantified, so the area under the peak
for a particular component, once again, is
proportional to the amount of that component
present in the mixture.
Okay,
this is an HPLC instrument.
On the top here, we have the different components that
mix together to make the mobile phase in these bottles.
Here we have the pump that pumps the mobile phase through the instrument
and provides the pressure.
The sample is injected into the system, once again, by an auto-sampler.
It then travels along this fairly fine tube here, to this part of the instrument.
And here we have the column.
And you can see, the HPLC column is quite different to the GC
column; this is made of steel, and the pressure inside is currently 157 bars.
The sample passes through the column and then passes to the detector,
and the information from the detector is fed to
the computer, and again it can be displayed and recorded.
So which is better, HPLC or GC?
Both methods are very efficient,
they're very selective.
They can resolve a great number of compounds,
and both of them are widely applicable.
In both cases, the detectors are very sensitive, so you only
need very small amounts of sample in order to complete the analysis.
They may be non-destructive of the sample, meaning that after the chromatography you
can get your sample back, though in practice this is very, very rarely done.
Both methods are quantitative.
Okay.
With both methods, you can measure the amount
of each component that is present in the mixture.
Both methods work with high resolution.
There are some differences.
Okay.
GC
has the advantage that the equipment is a little simpler
and it tends to be a little less expensive than HPLC,
and often times GC is also faster than HPLC.
HPLC, on the other hand, has the advantage that it can be used
for samples that are non-volatile and also for samples that are thermally unstable.
But both methods are very, very widely used for the analysis of compounds.
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