High Performance Liquid
Chromatography (HPLC )
Pharmaceutical Analytical Chemistry (PHC 121)
2nd year Pharm D Program
1440/1441 -2019/2020
Dr. Habibullah, PhD
Unaizah College of Pharmacy (UCP)
List the components of High Performance
Liquid Chromatography (HPLC)
Explain the function of major
components of High Performance Liquid
Chromatography (HPLC)
Explain the advantage of High
Performance Liquid Chromatography
(HPLC)
List the applications of High Performance
Liquid Chromatography (HPLC)
Sample
injector
Solvent port
mixing Pump
valve
Column
HPLC
Chart
Detector Recorder
Mobile phase Waste
Or solvent
reservoir
A schematic diagram of a typical HPLC unit
The HPLC consists of:
1.Mobile phase or solvent reservoir.
2.A high pressure pump.
3.A sample inlet port (hole).
4.Column
5.Detector
6.Recorder
1. Pump, capable of maintaining high pressures draws the solvent
(mobile liquid phase) from the reservoir and pushes it through the
column.
2. Sample is injected through a port into the high pressure liquid
carrier steam between pump and column.
3. The separation takes place on the columns, which vary, from 25-
100 cm length and 2-5 mm in internal diameter.
4. Typical flow rates are 1-2 ml/min with pressures up to several
thousands psi.
5. Column effluent passes through a non-destructive detector where
a property such as UV absorbance, RI or molecular fluorescence is
monitored amplified and recorded as a typical detector response
vs retention time to obtain a chart which is the chromatogram.
1. Mobile phase
a. isocratic elution - single solvent
separation technique
b. gradient elution - 2 or more solvents,
varied during separation
2. To carry sample into the column
1. To produce an appropriate pressure to push
solvent into the sample.
2. A pump capable of pumping solvent up to a
pressure of 4000 psi and at flows of up to 10
ml/min
Sample Injection System
Sample valve
Syringe/injector
1. Syringe :
Manual
Autoinjector
A fixed-volume loop of between 1 –
200 l (20 l is often used as standard)
Columns
straight, 15 to 150 cm in
length; 2 to 3 mm i.d.
packing - silica gel, alumina,
Celite
HPLC Detectors
UV/Vis
Refractive index
Fluorescence
Evaporative light scattering (ELSD)
MS
Diode Array Detector (DAD)
Using specific sowtare that is
connected to HPLC machine
Receive the information from HPLC
machine and present it as a graph
The graph describes about
qualitative data (Retention time)
and quantitative data (area under
curve)
1. Internal diameter of column
- the smaller in diameter, the higher in
sensitivity
2. Pump pressure
- the higher in pressure, the higher in
separation
3. Sample size
4. The polarity sample, solvent and column
5. Temperature
- the higher in temperature, the higher in
separation
a. High resolution and speedy analysis
b. High sensitivity
c. Reusable columns
d. No destruction of the components
e. Sample is recovered completely
f. Greater reproducibility due to close
control of the parameters affecting the
efficiency of separation
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Application of HPLC
1. Pharmaceuticals industry
a.To control the drug stability
b.Quantity of drug determination from
pharmaceutical dosage forms, ex. Paracetamol
determination in panadol tablet
c.Quantity of drug determination from biological
fluids, ex: blood glucose level
2. Analysis of natural contamination
- Phenol & Mercury from sea water
3. Forensic test
- Determination of steroid in blood, urine &
sweat.
- Detection of psychotropic drug in plasma
4. Clinical test
a. Monitoring of hepatic cirrhosis
patient through HPLC analysis
of the urine sample
5. Food and essence manufacture
a.Sweetener analysis in the fruit juice
b.Preservative analysis in sausage.
Separation in based upon differential
Injector
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Pumps Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Column
Detector
Waste
Solvents
High Performance Liquid Chromatograph
16
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
High Performance Liquid Chromatograph
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Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
18
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
19
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
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Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
21
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
22
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
23
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
24
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
25
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
26
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
27
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
28
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
29
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
30
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
31
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
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to - elution time of unretained peak
tR- retention time - determines sample identity
tR
tR
mAU Area or height is proportional
to the quantity of analyte.
to
Injection time
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Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant
