Transcript HPLC – High Performance Liquid Chromatography

HPLC – High Performance Liquid Chromatography
1. Solvent reservoir
2. Degasser
7. Detector
8. Computer
6. Column
5. Injector
9. Waste
4. Pump
3. Mixing chamber
What is HPLC and what is it used for?
High Performance Liquid Chromatography is a widely used analytical separation
technique. This form of liquid chromatography or liquid separation is highly
sensitive and automated which leads to accurate quantitative determinations.
It is used in order to separate and identify the different analytes/compounds
within a sample and to find the concentration of these various analytes.
This separation technique can be used to analyse –
Proteins
Drugs
Amino acids
Pesticides
Fats
Antibiotics
Carbohydrates
Steroids
Nucleic acids
Foods e.g. amount of caffeine in fizzy drinks
HPLC instrument
• This technique involves injecting a small amount of a liquid sample into a
column which contains the stationary phase.
• It is this solid stationary phase that interacts with the sample and causes the
different compounds within the sample to separate.
• There are various chemical and physical interactions between the sample
molecules and the packing particles within the stationary phase that cause the
analytes within the sample to separate.
• The sample ‘moves’ down through the column with a liquid mobile phase.
• The sample and mobile phase are both interacting with the stationary phase
and are forced through the column at high pressures by the pump.
• The individual separated compounds from the sample are then eluted (exit)
from the column and are identified by the detector.
Components of the HPLC instrument
• There are 9 main components within the HPLC instrument
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1.
2.
3.
4.
5.
6.
7.
8.
9.
Solvent reservoir
Degasser
Mixing chamber
Pump
Injector
Column
Detector
Computer
Waste
7
2
5
6
9
3
4
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A simply schematic diagram of the HPLC components
1. Solvent reservoir
2. Degasser
3. Mixing
chamber
4. Pump
5. Injector
6. Column
7. Detector
8. Computer
9. Waste
Mixing Chamber
1. Solvent Reservoir
• This is the area at the top of the HPLC system
where the mobile phase is placed.
• The mobile phase is kept in large glass
reagent bottles.
• Tubes are placed into the reagent bottles
to ‘draw out’ the mobile phase into the system.
2. Degasser
• The degasser is like an ultra-sonic bath, it removes any gas
(de-fizzes) and air bubbles from the mobile phase.
• It is important for the mobile phase to be degassed prior to
entering the column as air bubbles in the mobile phase can
affect the flow of the mobile phase and can even block the column.
The presence of air bubbles means a non-continuous flow of mobile
phase through the system.
3. Mixing chamber
• Ensures that the mobile phase is thoroughly mixed to provide a homogenous mixture
to enter into the system.
• Mobile phases are generally made up of more than 1 component i.e. a mixture, e.g.
70:30 mix of methanol and water.
• An unmixed mobile phase can affect the interactions between the mobile phase and
the stationary phase (column) and therefore can affect the results.
4. Pump
• The role of the pump is to force the liquid mobile phase and the liquid sample through
the system at a specific flow rate.
• The flow rate is a changeable programme feature and allows for a continuous flow of
the mobile phase though the system e.g. 2ml/min, 5ml/min.
5. Injector
• The role of the injector is to introduce (inject) the
liquid sample into the flow stream of the mobile
phase which then travels into the column.
Injector
• Samples are contained within small glass vials .
• The injection volumes of the sample are a
changeable programme feature, e.g. 5 - 20µl.
• Most modern HPLC systems use automated
injection systems. This is called an autosampler
and is used when many samples require analysis.
This automatic system saves time and is more accurate than manual injection.
Vials
6. Column
• The role of the column is to separate the different
components/analytes in samples.
• The column is packed with a solid stationary phase.
This solid stationary phase is usually silica particles which
are coated to the inside of the column. The type of coating used
depends on the polarity of the sample and the mobile phase.
It is this stationary phase that interacts with the sample and
causes the different analytes within the sample to separate.
• The pump is required to ‘push hard’ in order for the mobile
phase to move through the column. As there is pressure within
the column created by the small particles of the stationary phase.
• Most columns are 5 – 20 cm in length, have an inside diameter
of 3 – 5mm and the particle size of the packing is 3 – 5 µm.
7. Detector
• The detector is used to detect or ‘see’ the individual components of the sample as they
elute from the column.
• The separated components will all elute (leave) from the column at different times. This
allows the detector to sense each different one.
• The detector measures the amount of each component/analyte so that there is
quantitative analysis of the sample components.
• The results from the detector are transferred onto the computer programme and are
presented in the form of a ‘chromatogram’.
• A UV-Vis detector is the type of detector used for the HPLC analysis.
Waste
8. Computer
• The computer programme is used to control the entire
HPLC system. It can ‘stop’ and ‘start’ runs, programme
the injection volumes, the number of injections, the
flow rate etc.
• The results from the detector appear on the computer
programme as a ‘chromatogram’. A chromatogram is a
graph that represents and illustrates the separated
components of the sample and the times at which they
separated. Each component/analyte of the sample that
has separated is represented as a peak on the
chromatogram.
9. Waste
• When the analysis is complete and the ‘run’ is over,
the used mobile phase and sample is directed into the
‘waste’ reagent bottle.
HPLC is used for the
separation and
identification of the
different components
within a sample.
UV-Vis detectors
are used for the
analysis of the
individual analytes
The column can be said to
be the ‘heart’ of the HPLC,
as it is the column that
separates the sample.
What have I learned?
The column is coated inside
with tiny particles (µm)e.g.
silica, (SP) which interacts
with the sample and MP
and causes separation.
Stationary phase – solid
particles inside the column
Mobile phase – liquid
phase which flows through
the system
The size of the stationary
phase particles (µm)
determines if the sample
particles will separate and
be eluted from the
column.