High Performance Liquid Chromatography (HPLC)

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Science@CSCB
                       Chromatography and Instrumentation in Organic Chemistry
                                   Information pack for teachers


High Performance Liquid
Chromatography (HPLC)

What is chromatography?
                                                          Figure 2: Chemical composition of a C8 column
Chromatography is a way to
      separate mixtures of compounds                   The stationary phase is non polar or
      identify unknown compounds                       hydrophobic and the mobile phase is polar
      establish the purity or concentration            and usually consists of a mix of solvents,
      of compounds                                     such as water and acetonitrile. The mobile
      monitor product formation                        phase is pumped at high pressure through
                                                       the system.
All chromatography involves transporting a
sample along a mobile phase, which can be              The non polar part of the compound injected
a liquid (liquid chromatography) or a gas              will interact with the stationary phase which
(gas chromatography). High Performance                 slows it down as it moves through the
Liquid Chromatography (HPLC) was                       system. So, if a mixture of compounds is
developed along the principles of Thin Layer           injected, each one will interact differently
Chromatography (TLC) but is fully                      with the stationary phase and the mobile
automated and more quantitative.                       phase, thus facilitating separation.

How does HPLC work?                                    How are samples detected?
In HPLC, a sample is injected into a mobile            After the sample passes through the
liquid phase and it passes along a stationary          column, it is detected by ultraviolet
phase. Although manual injection of                    absorption. For many compounds this is
samples is still possible, most HPLCs are              usually at 254nm. The sample and the
fully automated and controlled by a PC,                mobile phase are collected as waste and the
allowing up to 200 or more samples to be               absorption spectrum is outputted as a
injected. This is particularly useful in both          chromatogram. This process is fully
research and pharmaceutical labs where                 automated and controlled by a PC. The time
samples often need to be analysed 24 hours             taken for a sample to pass through the
a day, 7 days a week.                                  system is recorded as its retention time and
                                                       is one of the characteristics used to identify
The stationary phase comprises a column                a compound. Retention times can vary from
which is usually stainless steel and packed            a few minutes to an hour. The area under a
with silica particles bonded with alkyl chains.        peak is used for calculating the
                                                       concentration of a sample.
The length of the chain will depend on the
type of molecule being analysed. For
example, for large protein molecules a C4
column could be used but for smaller
molecules C8 or even C18 may be more
appropriate.




             Figure 1: HPLC column
                                                                  Figure 3: HPLC chromatogram




                                       Science@CSCB Page 1 of 4
                                                      Science@CSCB
                         Chromatography and Instrumentation in Organic Chemistry
                                     Information pack for teachers


What are the main uses of HPLC?
In the pharmaceutical industry the purity of                Identifying and separating enantiomers is
drugs can be calculated by first running a                  key when developing drugs that have a
series of standards at known concentrations.                chiral centre. HPLC allows chemists to
A curve is then plotted of the concentration                confirm which enantiomer is present and
of the standards (x axis) versus their peak                 calculate the ratios where both are present.
area (y axis).

The drug samples to be tested are injected
and from the chromatogram peak areas, it is
possible to calculate the concentration of the
drug sample.




                                                                 Figure 6: Chromatogram of two enantiomers

                                                            HPLC is also used to monitor product
     Figure 4: Standard curve used to calculate             formation in chemical reactions. (See
                   concentrations                           Appendix 1)

                                                            Photographs of Shimadzu LC 2010A
HPLC can also be used to separate                           Liquid Chromatograph
enantiomers. Enantiomers are molecules
that contain the same number of atoms but
the different groups have different spatial
arrangements so that they are non
superimposable mirror images.




           Figure 5: Pair of enantiomers

Both forms often exhibit very different
characteristics in biological systems. The                             Figure 7: Shimadzu LC 2010A Liquid
most famous case being that of thalidomide,                                       Chromatograph
which was manufactured as a racemic
mixture (50:50) of two enantiomers. One
enantiomer was effective in treating morning
sickness but the other caused side effects.


                                            Science@CSCB Page 2 of 4
                                                     Science@CSCB
                        Chromatography and Instrumentation in Organic Chemistry
                                    Information pack for teachers


                                                           Mass Spectrometry
                                                           Many research chemists and chemical
                                                           biologists in the Centre for Synthesis and
                                                           Chemical Biology are involved in drug
                                                           discovery programmes where they
                                                           synthesise novel compounds which they
                                                           then evaluate for their biological activity.

                                                           Key to the success of this research is being
                                                           able to identify the structure of the
                                                           compounds they have synthesised in the
Figure 8: Autosampler with a capacity of up to 200         lab.
                    samples
                                                           What does a mass spectrometer do?
                                                           It measures mass to charge (m/z) ratio of a
                                                           molecular ion and hence determines the
                                                           mass of the molecule. It can also provide
                                                           information about the chemical structure of
                                                           the molecule.

                                                           Take an example of an isopropyl quinazoline
                                                           compound shown below. Its molecular
                                                           formula is C11H11ClN2.


       Figure 9: HPLC high pressure pump                   TSF107

                                                           LCT20060502DKR2351 47 (1.518)
                                                                                                                                                                02-May-200615:46:42

                                                                                                                                                           1: TOF MS ES+
                                                                                                               +
                                                            100
                                                                                        207.06    [M+H]                                                            2.06e4


                                                                                                                                  N

                                                                                                                                       N

                                                                                                                                  Cl
                                                                                              209.07
                                                                                                                            C11H11ClN2
                                                              %                                                           Mass [M] = 206.06




                                                                                                                          +
                                                                                              [M+CH3CN+H]
                                                                                                     248.09


                                                               0                                                                                                         m/z
                                                               100    125   150   175   200    225     250    275   300   325   350    375   400   425   450   475     500

                                                                    Figure 11: Mass spectrum of quinazoline derivative

     Figure 10: HPLC stainless steel column                So, the molecular mass is
                                                           C (12)          x      11                                                         = 132
                                                           H (1)           x      11                                                         = 11
                                                           N (14)          x      2                                                          = 28
                                                           Cl (35)         x      1                                                          = 35
                                                                                                                                                206

                                                           Thus, a peak at m/z 207.06 corresponding
                                                           to [M+H]+ is seen in the spectrum. The other
                                                           peak at m/z 248.09 corresponds to the
                                                           acetonitrile (CH3CN) adduct of the molecule




                                           Science@CSCB Page 3 of 4
                                                         Science@CSCB
                             Chromatography and Instrumentation in Organic Chemistry
                                         Information pack for teachers


since the sample was prepared in                             modern mass spectrometers provide
acetonitrile.                                                information on the intact molecule.

What types of mass spectrometers                             Each mass spectrometer has an ionisation
are in the CSCB?                                             source, an analyser (that sorts out m/z) and
                                                             a detector. In some cases HPLC is first used
The following two mass spectrometers
                                                             to separate compounds before they are
(Micromass/Waters Corp. USA) equipped
                                                             passed through the ionisation source.
with electrospray source are available:
                                                             The sample is ionised in the ionisation
                                                             source. There are different types of
                                                             ionisation techniques and at the CSCB we
                                                             use electrospray. This is suitable for the
                                                             detection of molecules from 100 Da to more
                                                             than 1,000,000 Da in molecular mass. It
                                                             uses an electric field to generate ions from a
                                                             sample in a small metal capillary.

                                                             These ions are then passed along to the
                                                             analyser, which can be time-of-flight, for
                        TM
Figure 12: Quattro micro LC-MS/MS (fully automated           example. The analyser separates out the
             for normal mass screening)                      charged species according to their m/z
                                                             ratios. Time-of-flight is so called because it
                                                             measures the length of time it takes the ions
                                                             to travel over a certain distance to the
                                                             detector.

                                                             In some instruments two mass analysers are
                                                             in tandem with a collision cell in between.
                                                             Tandem mass spectrometers allow the
                                                             fragmentation of the molecular ions of
                                                             interest for further structural
 Figure 13: Liquid chromatography time-of-flight (LCT)       characterisation.
                  mass spectrometer
                                                             Further information available on
                                                             http://www.ucd.ie/cscb/main_pages/facilities/
Where is mass spectrometry used?                             mass_spectrometry.htm
Mass spectrometry is used by research
scientists to identify unknown compounds
but it is also used to check biological
samples from athletes for banned
substances such as steroids and for
checking dioxins in contaminated fish.

How does a mass spectrometer
work?
Conventional mass spectrometry techniques
work on the principle of fragmentation,
where a molecule is broken down into its
component parts upon an impact from an                        Centre for Synthesis and Chemical Biology,
electron or a chemical compound. However,                    University College Dublin 2006


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