HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY
(HPLC)
High performance liquid chromatography is a column chromatography
performed by eluting under pressure.
This is done by pumping the eluting solvent(mobile phase) under a pressure of
up to 40MPa and a flow rate of 3 mL/min through a column packed with smaller
particle size(stationary phase).
Important terms to note:
Mobile phase: Carries the sample through the stationary phase as it moves
through the column
Stationary phase: The phase which remains fixed in the HPLC column,eg;Silica
Retention time(tR): Is a measure of the time taken for a solute to pass through
the HPLC column.
- it serves for qualitative analysis by comparing it with a reference pure substance
SETTING AND PRINCIPLE OF HPLC
• The setting consist of Mobile solvent,Solvent delivery system(pump),
Injector,sample,HPLC Column,detector(diode array),Waste collector
and Recorder
• The principle behind HPLC is Adsorption
• To achieve good separation ,the analyte should interact with the
stationary phase,but not too strong or the retention time will become
very long
1.Normal-phase HPLC
It is performed by eluting a silica-packed
column which is hydrophilic with a non
polar mobile phase which is usually
hexane, in addition of Chloroform( CHCl3),
Tetrahydrofuran(THF) or
Methanol(MeOH) so as to increase
polarity of the mobile phase.
Separation is achieved during the passage
of mobile phase through the column
whereby the polar solutes are retained
and more lipophilic molecules are not .
2.Reverse-phase HPLC
It is performed when when the solute is eluted by a polar mobile phase over a
hydrophobic stationary phase. Hydrophobicity of the stationary phase is
achieved by binding a coating on to the silica support.
-In general polar solutes have short retention times on reverse phase whereas non-
polar compounds are retained.
APPLICATION OF HPLC IN PHARMACY
Ensures consistency of active pharmaceutical ingredients
Evaluating stability of pharmaceutical products.
Detection and Identification of impurity that can pose a serious safety risk to
patients.
Useful in determining shelf life.Example some biotherapeutics are sensitive to
aggregation over time or if not stored properly, and HPLC can be used to monitor
this aggregation
TYPES OF MIXTURES TO BE SEPARATED BY HPLC
METHOD
Mixtures of Drugs
- Neutral or weakly acidic drugs can be chromatographed on a reverse phase HPLC
system
- Acidic drugs such as paracetamol, cannabis are separated either by ion-
suppression or ion-pair chromatography on a reversed-phase packing material
Plasma concentration of cortisol after oral administration
ADVANTAGES OF HPLC METHOD
Ability to determine the quantities of impurities in pharmaceutical formulations.
Suitable for compounds that are not easly volatilized, thermally unstable, and
have high molecular weight.
It can quantify a drug in its pure and dosage form
Compared to Thin Layer Chromatography, HPLC is extremely quick and efficient
It is accurate and reproducible
Basic HPLC runs, can be performed with minimal training
DISADVANTAGES OF HPLC METHOD
Cost and complexity
It requires large quantities of expensive organics.
Low sensitive to certain compounds and some can not be detected as they are
irreversibly adsorbed example Volatile substances are better separated by Gas
chromatography
REFERRENCES
https://pubmed.ncbi.nlm.nih.gov
Aulton, Michael E. 2002. Aulton’s pharmaceutics: the science of dosage form
design. High performance liquid chromatograpy(pg:128-129). Edinburgh:New
York: Churchill livingstone
https://www.pharmaguideline.com/2013/07/principle-of-hplc-liquid-
chromatography.html?m=1
GROUP MEMBERS
1. JONATHAN Z MBWILO
2. NYAMBIKIWE BENJAMIN
3. INNOCENT SUMARI
4. DIDAS MUSHI
5. EUNICE MBISE
6. PATRICK GEORGE
7. IRENE ANTIPAS
