INTRODUCTION

Chromatography is a technique to separate two or more mixtures of substances into its

components. This separation is based on their molecular structure and composition.
Chromatography has a stationary phase and mobile phase where the mobile phase flows
through the stationary phase and carries the components of the mixture with it. Sample used in
the experiment that has high affinity with the stationary phase will move more quickly through
the column. There are few types of chromatographic separations which are thin-layer
chromatography, gas chromatography, paper chromatography and liquid chromatography.

HPLC is basically an improved form of column liquid chromatography. The solvent is

being forced through under high pressures up to 400 atmospheres which makes it become faster.
Basically, it operates under the principle where the separation of a sample into its constituent
parts due to the difference in the relative affinities between molecules for the mobile phase and
the stationary phase.

There are few types of HPLC which are normal phase HPLC, reverse phase HPLC, size
exclusion HPLC, and ion exchange HPLC. Normal Phase HPLC separates analytes on the basis
of its polarity. It uses polar stationary phase and non-polar mobile phase. Next, reverse phase
HPLC’s stationary phase is different which is nonpolar or hydrophobic in nature, while the
mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. For the
size-exclusion HPLC’s column, it is filled with material that have controlled pore sizes, and
the particles are separated according to their molecular size. Larger molecules are rapidly
washed through the column while smaller molecules penetrate inside the porous of the packing
particles and elute later. Lastly, ion-exchange HPLC’s stationary phase has an ionically
charged surface of opposite charge to the ion of the sample. This technique is used either with
ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be
attracted to the ionic surface and the longer it will take time to elute. Its mobile phase is an
aqueous buffer, where both pH and ionic strength are used to control elution time.

Moreover, there are six instruments in HPLC which are solvent reservoir, pump, sample
injector, columns, detector and data collection devices.
 Mobile phase contents are contained in a solvent reservoir which is glass reservoir. The
mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid
components where its concentrations are varied depending on the composition of the sample.
Next, a pump aspirates the mobile phase from the solvent reservoir and forces it through the
system’s column and detector. While in sample injector, the injector can be a single injection
or an automated injection system. An injector for an HPLC system should provide injection of
the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and
under high pressure (up to 4000 psi).

While columns are usually made of polished stainless steel, are between 50 and 300
mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with
a stationary phase with a particle size of 3–10 µm. Columns with internal diameters of less than
2 mm is known as microbore columns. The HPLC detector that is located at the end of the
column detect the analytes as they elute from the chromatographic column. The common
detectors used are UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical
detectors. Lastly, for data collection devices, the signals from the detector may be collected on
chart recorders or electronic integrators that vary in complexity and in their ability to process,
store and reprocess chromatographic data. The computer integrates the response of the detector
to each component and places it into a chromatograph that is easy to read and interpret.

Nowadays, HPLC is used in pharmaceutical applications to control drug stability,

environmental applications in detection of phenolic compounds in drinking water, applications
in forensics for identifying the steroids in blood, food and flavour and applications in clinical
tests for urine analysis, antibiotics analysis in blood.

PROCEDURES

Pre-treatment of sample

i. 1 ml of sample was homogenised with 2 ml of methanol.

ii. The mixture was vortex and sonicate for 10 minutes in the ultrasonic water bath at 30 ̊C.
iii. 1 ml of the mixture was transferred into microcentrifuge and it was centrifuged at 3000
rpm for 10 minutes.
iv. 1 ml of the supernatant was mixed with 1 ml of methanol.
v. Then it was filtered using 0.45 µm nylon filter membrane.
vi. The content was transferred into HPLC vials.
vii. The vial was inserted in the machine.

Preparation of standard solutions

i. The standard was mixed with methanol and dissolved at a concentration of 1mg/ml as
a stock solution.
ii. The dilution is done by adding 9ml of methanol into 1 ml of ascorbic acid.
iii. The standard solution was inserted in the machine as a reference.