HIGH PERFORMANCE LIQUID CHROMATOGRAPHY -

HPLC

High performance liquid chromatography is a powerful tool in analysis. This page looks at
how it is carried out and shows how it uses the same principles as in thin layer
chromatography and column chromatography.

Note:  It is important to read the introductory page about thin layer chromatography before you continue with
this one - particularly the part about how thin layer chromatography works. High performance liquid
chromatography works on the same basic principle. HPLC is essentially an adaptation ofcolumn
chromatography - so it might be a good idea to have a (very quick) look at that as well.

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Carrying out HPLC

Introduction

High performance liquid chromatography is basically a highly improved form of column

chromatography. Instead of a solvent being allowed to drip through a column under gravity, it
is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

It also allows you to use a very much smaller particle size for the column packing material
which gives a much greater surface area for interactions between the stationary phase and
the molecules flowing past it. This allows a much better separation of the components of the
mixture.

The other major improvement over column chromatography concerns the detection methods
which can be used. These methods are highly automated and extremely sensitive.

The column and the solvent

Confusingly, there are two variants in use in HPLC depending on the relative polarity of the
solvent and the stationary phase.

Normal phase HPLC

This is essentially just the same as you will already have read about in thin layer
chromatography or column chromatography. Although it is described as "normal", it isn't the
most commonly used form of HPLC.

The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for
example. A typical column has an internal diameter of 4.6 mm (and may be less than that),
and a length of 150 to 250 mm.

Polar compounds in the mixture being passed through the column will stick longer to the
polar silica than non-polar compounds will. The non-polar ones will therefore pass more
quickly through the column.

Reversed phase HPLC

In this case, the column size is the same, but the silica is modified to make it non-polar by
attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms
in them. A polar solvent is used - for example, a mixture of water and an alcohol such as
methanol.

In this case, there will be a strong attraction between the polar solvent and polar molecules in
the mixture being passed through the column. There won't be as much attraction between
the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules
in the solution. Polar molecules in the mixture will therefore spend most of their time moving
with the solvent.

Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups
because of van der Waals dispersion forces. They will also be less soluble in the solvent
because of the need to break hydrogen bonds as they squeeze in between the water or
methanol molecules, for example. They therefore spend less time in solution in the solvent
and this will slow them down on their way through the column.

That means that now it is the polar molecules that will travel through the column more
quickly.

Reversed phase HPLC is the most commonly used form of HPLC.

Looking at the whole process

A flow scheme for HPLC

Injection of the sample

Injection of the sample is entirely automated, and you wouldn't be expected to know how this
is done at this introductory level. Because of the pressures involved, it is not the same as in
gas chromatography (if you have already studied that).

Retention time

The time taken for a particular compound to travel through the column to the detector is
known as its retention time. This time is measured from the time at which the sample is
injected to the point at which the display shows a maximum peak height for that compound.

Different compounds have different retention times. For a particular compound, the retention
time will vary depending on:

 the pressure used (because that affects the flow rate of the solvent)
 the nature of the stationary phase (not only what material it is made of, but also
particle size)
 the exact composition of the solvent
 the temperature of the column

That means that conditions have to be carefully controlled if you are using retention times as
a way of identifying compounds.

The detector
There are several ways of detecting when a substance has passed through the column. A
common method which is easy to explain uses ultra-violet absorption.

Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV
light shining through the stream of liquid coming out of the column, and a UV detector on the
opposite side of the stream, you can get a direct reading of how much of the light is
absorbed.

The amount of light absorbed will depend on the amount of a particular compound that is
passing through the beam at the time.

You might wonder why the solvents used don't absorb UV light. They do! But different
compounds absorb most strongly in different parts of the UV spectrum.

Methanol, for example, absorbs at wavelengths below 205 nm, and water below 190 nm. If
you were using a methanol-water mixture as the solvent, you would therefore have to use a
wavelength greater than 205 nm to avoid false readings from the solvent.

Interpreting the output from the detector

The output will be recorded as a series of peaks - each one representing a compound in the
mixture passing through the detector and absorbing UV light. As long as you were careful to
control the conditions on the column, you could use the retention times to help to identify the
compounds present - provided, of course, that you (or somebody else) had already
measured them for pure samples of the various compounds under those identical conditions.

But you can also use the peaks as a way of measuring the quantities of the compounds
present. Let's suppose that you are interested in a particular compound, X.

If you injected a solution containing a known amount of pure X into the machine, not only
could you record its retention time, but you could also relate the amount of X to the peak that
was formed.

The area under the peak is proportional to the amount of X which has passed the detector,
and this area can be calculated automatically by the computer linked to the display. The area
it would measure is shown in green in the (very simplified) diagram.

If the solution of X was less concentrated, the area under the peak would be less - although
the retention time will still be the same. For example:

This means that it is possible to calibrate the machine so that it can be used to find how
much of a substance is present - even in very small quantities.

Be careful, though! If you had two different substances in the mixture (X and Y) could you
say anything about their relative amounts? Not if you were using UV absorption as your
detection method.

In the diagram, the area under the peak for Y is less than that for X. That may be because
there is less Y than X, but it could equally well be because Y absorbs UV light at the
wavelength you are using less than X does. There might be large quantities of Y present, but
if it only absorbed weakly, it would only give a small peak.

Coupling HPLC to a mass spectrometer

This is where it gets really clever! When the detector is showing a peak, some of what is
passing through the detector at that time can be diverted to a mass spectrometer. There it
will give a fragmentation pattern which can be compared against a computer database of
known patterns. That means that the identity of a huge range of compounds can be found
without having to know their retention times.
Basic Principles of HPTLC

HPTLC- High Performance Thin Layer Chromatography is a sophisticated and automated

form of TLC.

Main Difference of HPTLC and TLC - Particle and Pore size of Sorbents.

The other differences are

HPTLC TLC
Layer of  100µm  250µm
Sorbent
 High due to smaller
 Less
Efficiency particle size generated

 3 - 5 cm  10 - 15 cm
Separations

 Shorter migration
distance and the
 Slower
Analysis Time analysis time is greatly
reduced

 Wide choice of
stationary phases like  Silica gel ,
silica gel for normal Alumina &
Solid support
phase and C8 , C18 for Kiesulguhr
reversed phase modes

 New type that require

Development less amount of mobile  More amount
chamber phase

 Manual
Sample  Auto sampler
spotting
spotting

 Use of UV/ Visible/

Fluorescence scanner
scans the entire
chromatogram
 Not possible
Scanning qualitatively and
quantitatively and the
scanner is an advanced
type of densitometer
Features of HPTLC

1. Simultaneous processing of sample and standard - better analytical precision and accuracy less
need for Internal Standard
2. Several analysts work simultaneously
3. Lower analysis time and less cost per analysis
4. Low maintenance cost
5. Simple sample preparation - handle samples of divergent nature
6. No prior treatment for solvents like filtration and degassing
7. Low mobile phase consumption per sample
8. No interference from previous analysis - fresh stationary and mobile phases for each analysis -
no contamination
9. Visual detection possible - open system
10. Non UV absorbing compounds detected by post-chromatographic derivatization

Steps involved in HPTLC

1. Selection of chromatographic layer

2. Sample and standard preparation
3. Layer pre-washing
4. Layer pre-conditioning
5. Application of sample and standard
6. Chromatographic development
7. Detection of spots
8. Scanning
9. Documentation of chromatic plate

Selection of chromatographic layer

· Precoated plates - different support materials - different Sorbents available 
· 80% of analysis - silica gel GF · Basic substances, alkaloids and steroids - Aluminum oxide
· Amino acids, dipeptides, sugars and alkaloids - cellulose 
· Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18
· Preservatives, barbiturates, analgesic and phenothiazines- Hybrid plates-RPWF254s
Sample and Standard Preparation
To avoid interference from impurities and water vapours 
Low signal to noise ratio - Straight base line- Improvement of LOD 
Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Chloroform:
Methanol: Ammonia (90:!0:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid 
Dry the plates and store in dust free atmosphere
Activation of pre-coated plates
Freshly open box of plates do not require activation 
Plates exposed to high humidity or kept o n hand for long time to be activated 
By placing in an oven at 110-120ºc for 30’ prior to spotting 
Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15
minutes.
Application of sample and standard
· Usual concentration range is 0.1-1µg / µl 
· Above this causes poor separation
· Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe o n TLC
plates as bands 
· Band wise application - better separation - high response to densitometer
Selection of mobile phase
- Trial and error 
- one’s own experience and Literature 
- Normal phase 
- Stationary phase is polar 
- Mobile phase is non polar
- Non-polar compounds eluted first because of lower affinity with stationary phase
- Polar compounds retained because of higher affinity with the stationary phase
· Reversed phase 
- Stationary phase is non polar 
- Mobile phase is polar 
- Polar compounds eluted first because of lower affinity with stationary phase 
- Non-Polar compounds retained because of higher affinity with the stationary phase
- 3 - 4 component mobile phase should be avoided 
- Multi component mobile phase o nce used not recommended for further use and solvent composition is
expressed by volumes (v/v) and sum of volumes is usually 100
- Twin trough chambers are used o nly 10 -15 ml of mobile phase is required 
-· Components of mobile phase should be mixed introduced into the twin - trough chamber
Pre- conditioning (Chamber saturation)
· Un- saturated chamber causes high Rf values 
· Saturated chamber by lining with filter paper for 30 minutes prior to development - uniform distribution of
solvent vapours - less solvent for the sample to travel - lower Rf values.
Chromatographic development and drying
· After development, remove the plate and mobile phase is removed from the plate - to avoid
contamination of lab atmosphere
· Dry in vacuum desiccator - avoid hair drier - essential oil components may evaporate
Detection and visualization
· Detection under UV light is first choice - non destructive 
· Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave
length) 
· Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF
· Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine
solution
· When individual component does not respond to UV - derivatisation required for detection
Quantification

· Sample and standard should be chromatographed o n same plate - after development chromatogram is
scanned 
· Camag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by absorbance
or by fluorescent mode - scanning speed is selectable up to 100 mm/s - spectra recording is fast - 36
tracks with up to 100 peak windows can be evaluated 
· Calibration of single and multiple levels with linear or non-linear regressions are possible · When target
values are to be verified such as stability testing anddissolution profile single level calibration is suitable
· Statistics such as RSD or CI report automatically 
· Concentration of analyte in the sample is calculated by considering the sample initially taken and dilution
factors