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Festival of Contemporary Science WORKSHOPS 30th January 2010

Analytical Chemistry 1: Chromatography: GC-MS, HPLC and TLC.


Steve Croker MSc CChem FRSC

As an illustration of the above techniques we will focus on the analysis of a pharmaceutical


product available over the counter.

All of the above contain paracetamol and caffeine.

Using the chromatographic techniques of Thin Layer Chromatography (TLC) High Performance
Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS) we will
confirm the presence of both of these in the following product.

You will be provided with standards of caffeine and paracetamol for comparison.

Sample Preparation:

Crush a paracetamol plus pain relief tablet in a mortar and pestle until it is a fine powder, add 10
mL of ethanol and mix well. Transfer your sample to 25ml plastic syringe with a filter paper in the
bottom. Force your sample through the syringe and collect the liquid in a 25ml volumetric flask.
Make your flask up to the graduation mark with ethanol.

TLC

Obtain a 3.5 x 10 cm silica gel TLC plate. Do not touch the faces of the plate as fingerprints will
mark the plate, and interfere with the chromatography. Using a pencil draw a faint line 1.5 cm from
the bottom of the plate. Mark three faint x’s on this line equally spaced.

Below the line label pure caffeine (C), sample (S), and Paracetamol(P).
Apply a spot using a capillary tube from your sample in the 25ml volumetric flask. Apply spots for
each of the standards supplied (Caffeine and Paracetamol) Use a new capillary for each sample.

Place the plate in the developing jar to which you have already added your solvent to a depth of
1cm (ethyl acetate–hexane–acetic acid 80:20:1). Allow the solvent to come within ~1 cm of the top
of the plate then remove the plate from the jar and mark the solvent line. Allow the plate to dry and
then place the plate under the UV lamp. Carefully circle all the spots you observe. Determine the
Rf’s of the spots for each sample.

HPLC
Using a pipette half fill an HPLC vial with some of your sample from your 25ml volumetric flask.
The standard samples of caffeine and paracetamol are already prepared on the HPLC.
Place your sample in the HPLC rack and start the program on the HPLC.
You will be instructed on how to obtain your data and the instrument will be explained to you.

GC-MS
Place 40ul of your sample from the volumetric flask into a 2ml auto sampler vial using an
automatic pipette. Fill the vial with ethanol and screw on the blue top. Place your vial in the GC-
MS rack and start the program on the GC-MS. You will be instructed on how to obtain your data
and the instrument will be explained to you.
Results and information on techniques
TLC
Caffeine RF= 0.12
Paracetamol (Acetaminophen) RF= 0.44

HPLC
The caffeine and paracetamol separation is easily achieved using HPLC. Liquid chromatography can be
divided into several types, including normal-phase (e.g. a silica or alumina column), reversed-phase and
ion exchange. Reversed-phase partition chromatography uses a non-polar organic coating on a silica
structure for the stationary phase. The non-polar coating is commonly formed by reacting an
organochlorosilane with the OH groups on the silica surface. With normal-phase HPLC, the most commonly
used solvents are hexane, isopropanol or THF, whereas a more polar mobile phase such as
methanol/water or acetonitrile/water is commonly used for reversed-phase partition chromatography. In this
experiment, the separation will be done using a non-polar C18 reversed-phase column and a
methanol/ammonium acetate mobile phase.

The chemical structure of caffeine is shown here.


Spectrophotometry provides a sensitive method for
the detection and measurement of caffeine. The UV
absorption spectrum (see figure) of caffeine exhibits
a pair of absorption bands peaking at 205 nm and
273 nm with a characteristic absorption shoulder
between them. Typically, caffeine content is
determined by measuring the absorbance at 273 nm.
We can make use of the absorbance at 273nm by
using a diode array UV-VIS detector on the HPLC.

The chemical structure of paracetamol is shown


here. Spectrophotometry provides a sensitive
method for the detection and measurement of
paracetamol. The UV absorption spectrum (see
figure) of caffeine exhibits a pair of absorption
bands peaking at 205 nm and 247 nm. Typically,
paracetamol content is determined by measuring
the absorbance at 247 nm. We can make use of
the absorbance at 247nm by using a diode array
UV-VIS detector on the HPLC.
m AU
273nm ,4nm (1.00)
7.5

5.557
5.0

6.163
2.5

0.0

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 m in

m AU

5.557
15.0 247nm ,4nm (1.00)

12.5

10.0

7.5

5.0

2.5

0.0

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 m in

Paracetamol elutes at 5.557 min and Caffeine elutes at 6.163 min. In the the figure above you can see
how 247nm is selective for paracetamol.

GC-MS
Gas chromatography-mass spectroscopy (GC-MS) is one of the so-called hyphenated analytical techniques.
As the name implies, it is actually two techniques that are combined to form a single method of analyzing
mixtures of chemicals. Gas chromatography separates the components of a mixture and mass spectroscopy
characterizes each of the components individually. By combining the two techniques, an analytical chemist
can both qualitatively and quantitatively evaluate a solution containing a number of chemicals.

Gas Chromatography

In all chromatography, separation occurs when the sample mixture is introduced (injected) into a mobile
phase. In liquid chromatography (LC), the mobile phase is a solvent. In gas chromatography (GC), the
mobile phase is an inert gas such as helium.

The GC has a long, thin column containing a thin interior coating of a solid stationary phase (5% phenyl-,
95% dimethylsiloxane polymer). This 0.25 mm diameter column is referred to as a capillary column.

The capillary column is held in an oven that can be programmed to increase the temperature gradually (or in
GC terms, ramped). this helps the separation. As the temperature increases, those compounds that have low
boiling points elute from the column sooner than those that have higher boiling points. Therefore, there are
actually two distinct separating forces, temperature and stationary phase interactions.

As the compounds are separated, they elute from the column and enter a detector. The detector is capable of
creating an electronic signal whenever the presence of a compound is detected. The greater the concentration
in the sample, the bigger the signal. The signal is then processed by a computer. The time from when the
injection is made (time zero) to when elution occurs is referred to as the retention time (RT).

While the instrument runs, the computer generates a graph from the signal. (See figure below). This graph is
called a chromatogram. Each of the peaks in the chromatogram represents the signal created when a
compound elutes from the GC column into the detector. The x-axis shows the RT, and the y-axis shows the
intensity (abundance) of the signal.
Paracetamol has a retention time on GC of 2.8 min and Caffeine has a retention time on GC of 3.7min

Mass Spectroscopy

As the individual compounds elute from the GC column, they enter the electron ionization (mass spec)
detector. There, they are bombarded with a stream of electrons causing them to break apart into fragments.
These fragments can be large or small pieces of the original molecules.

The fragments are actually charged ions with a certain mass. The mass of the fragment divided by the charge
is called the mass to charge ratio (M/Z). Since most fragments have a charge of +1, the M/Z usually
represents the molecular weight of the fragment.

A group of 4 electromagnets (called a quadrapole, focuses each of the fragments through a slit and into the
detector. The quadropoles are programmed by the computer to direct only certain M/Z fragments through
the slit. The rest bounce away. The computer has the quadrapoles cycle through different M/Z's one at a time
until a range of M/Z's are covered. This occurs many times per second. Each cycle of ranges is referred to as
a scan.

The computer records a graph for each scan. The x-axis represents the M/Z ratios. The y-axis represents the
signal intensity (abundance) for each of the fragments detected during the scan. This graph is referred to as a
mass spectrum (see the spectra of caffeine and paracetamol below).

The mass spectrum produced by a given chemical compound is essentially the same every time. Therefore,
the mass spectrum is essentially a fingerprint for the molecule. This fingerprint can be used to identify the
compound. The computer on our GC-MS has a library of over 220,000 spectra that can be used to identify
an unknown chemical in the sample mixture. The library compares the mass spectrum from a sample
component and compares it to mass spectra in the library. It reports a list of likely identifications along with
the statistical probability of the match.

GC-MS

When GC is combined with MS, a powerful analytical tool is created. A researcher can take an organic
solution, inject it into the instrument, separate the individual components, and identify each of them.
Furthermore, the researcher can determine the quantities (concentrations) of each of the components.
%
100.0 194
109
75.0

50.0 55
67
82
25.0

110 137 165


79 94 122 151
0.0 207
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0

Mass spectrum obtained for caffeine

Mass spectral fragmentation of caffeine


%
100.0 109

75.0

50.0

151
25.0 80
53
63 68 9194 111 120 135 170 185
0.0
50.0 75.0 100.0 125.0 150.0 175.0 200.0

Mass spectrum obtained for paracetamol

Mass spectral fragmentation of paracetamol