High Performance/Pressure

Liquid Chromatography
(HPLC)

Prof. Nelson A. Ochekpe

Learning Outcome
After studying this topic student should be
able to :
• Define HPLC
• Describe HPLC principle
• Explain major components of HPLC and their function
• Explain application of HPLC
• Explain factors affect function of HPLC
• Describe advantages & disadvantages of HPLC
HPLC MACHINE
HPLC…?
• Originally referred to as High-Pressure Liquid
Chromatography

• Now more commonly called High Performance Liquid

Chromatography

• HPLC is really the automation of traditional liquid

chromatography under conditions which provide for
enhanced separations during shorter periods of time,
utilizing very small particles, small column diameters,
and very high fluid pressures.
HPLC PRINCIPLE
Stationary Phases
• Polar (“Normal” Phase):
• Silica, alumina

• Non-Polar (“Reversed Phase”)

• ODS Silica gel
• C18, C8
The Mobile Phase
• Normal chromatography
Hexane ; dichloromethane; isopropanol; methanol

Increasing strength

• Reverse phase chromatography

water ; methanol; acetonitrile; tetrahydrofuran (THF)

Increasing strength
Components of HPLC

1. Solvent Reservoir
2. Pumps
3. Sample Injection System
4. Columns
5. Detectors
6. Data Processing
7. Waste
The pump:
• Capable of maintaining high pressures draws the solvent (mobile liquid phase) from the reservoir and
pushes it through the column.

The sample
• is injected through a port into the high pressure liquid, carrier between the pump and the column.

The separation
• takes place on the columns, which vary, from 25-100 cm length and 2-5 mm in internal diameter.
Typical flow rates are 1-2 ml/min with pressures up to several thousand psi.

The column effluent

• passes through a non-destructive detector where a property of sample such as :
UV absorbance,
Refractive index (RI) or
molecular fluorescence is detected

To increase the efficiency of separation, the mobile phase may be altered by changing its polarity, pH or
ionic strength. HPLC offers the advantages of speed, resolution and sensitivity.

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There are two types of HPLC procedures:

1.LLC:
• The column consists of an inert support usually silica gel on
which the stationary partitioning phase is adsorbed.
 In the normal phase mode, the stationary phase is polar
while the mobile phase is less polar (e.g. iso-octane,
chloroform or n-hexane). This mode is usually used for the
separation of non polar components.
 In the reverse phase LLC, the stationary phase is less
polar and the mobile phase is polar (e.g. methanol,
acetonitrile or water). It is usually used for the separation
of polar components.
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2. LSC: The packing may be silica (polar packing) or octadecylsilica, ODS (C18-
silica, non-polar packing). Adsorption mechanism is involved here.

• In the normal phase LSC, the packing is polar (silica) and the mobile
phase is less polar (e.g. n-hexane).

• In the reverse phase LSC, the packing is non-polar (e.g. ODS) and the
mobile phase is polar (e.g. acetonitrile-water or methanol-water).

• Again, as under LLC, normal phase LSC is used for non-polar solutes while
reverse phase LSC is used for separation of polar compounds.

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ELUTION APPROACHES

• Isocratic - constant mobile phase composition

• Gradient - variable mobile phase composition
• step - change accomplished sharply at a defined point in time
• continuous - change accomplished gradually over time

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GRADIENT ELUTION
• Profile

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 HPLC columns

Guard columns
Analytical columns •Shorter column 7.5
• Made of stainless steel mm
• Internal diameter 2.1 – 4.6 mm •Used to prevent the
• column length 30 – 300 mm adsorption of
• Particle size 3–10 micrometer substances on the
analytical column

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STATIONARY PHASES IN HPLC-properties

•Chemically inert

•Non-soluble in any imaginable mobile phase

•Thermal and chemical stability

•Appropriate physical sorption of analyte

•Shape: Uniform spherical particles

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STATIONARY PHASE TYPES IN HPLC
1. Unmodified silica stationary phases
Spherical and regular Particle size 3-10 uM;
Polar surface due to the silanol groups
Uses
For the separation and retention of non polar and moderately
polar compounds such as poly aromatics, fats, oil, isomers

2. Modified silica stationary phases

Spherical and regular particle size 3-10 uM:
Non-polar due to the modification of silanol groups by chlorooxysilane produces stable stationary
phases, e.g ODS Octadecylsilane the most used stationary phase in reversed phase chromatography
Uses
For the separation and retention of wider range of polar and
moderately polar substances such as drugs and amino acids
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18
 CH3 -HCl CH3
Si OH Cl Si R Si O Si R
+
CH3 CH3

Si OH Si O
-2HCl
+ Cl2SiR2 SiR2
Si OH Si O

amino
octadecyl
NH2

CN cyano Non-polar phase

octyl

O OH
OH phenyl
Polar phase diol
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A. Solvent delivery system-mobile phase
• The mobile phase in HPLC refers to the solvent being continuously
applied to the column or stationary phase

• The mobile phase acts as a carrier to the sample solution

• A sample solution is injected into the mobile phase of an assay

through the injector port

• As a sample solution flows through a column with the mobile phase,

the components of that solution migrate according to the non-
covalent interaction of the compound with the column
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• The chemical interaction of the mobile phase and sample, with
the column, determine the degree of migration and separation
of components contained in the sample

• The solvents or mobile phases used must be passed through the

column at high pressure at about 1000 to 3000 psi.

• This is because as the particle size of stationary phase is

around 5-10µm, the resistance to the flow of solvent is high.

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Pumps
• The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate,
expressed in milliliters per min (mL/min).

• Normal flow rates in HPLC are in the 1-to 2-mL/min range.

• Typical pumps can reach pressures in the range of 6000-9000 psi

(400-to 600-bar).

• During the chromatographic experiment, a pump can deliver a

constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).
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Types of pumps
• There are several types of pumps used for HPLC analysis- most commonly
used are reciprocating piston pump, syringe pump and constant pressure
pump

• 1. Reciprocating piston pumps:

• Consists of a small motor driven piston which moves rapidly back and
forth in a hydraulic chamber that may vary from 35-400µL in volume
• On the back stroke , the separation column valve is closed, and the piston
pulls in solvent from the mobile phase reservoir
• On the forward stroke, the pump pushes solvent out of the column from
the reservoir
• A wide range of flow rates can be attained by altering the piston stroke
volume during each cycle, or by altering the stroke frequency.
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• Dual and triple head pump consists of identical piston chamber
units which operate at 180 or 120 degrees out of phase (this
system is significantly smoother because one pump is filling while
the other is in the delivery cycle.

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2. Syringe type pump
• These are most suitable for small bore columns because this
pump delivers only a finite volume of mobile phase before it has
to be refilled. These pumps have a volume between 250 to 500mL
• The pump operates by a motorized lead screw that delivers
mobile phase to the column at a constant rate. The rate of
solvent delivery is controlled by changing the voltage on the
motor.

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• These are most suitable for small bore columns because this pump
delivers only a finite volume of mobile phase before it has to be
refilled. These pumps have a volume between 250 to 500mL

• The pump operates by a motorized lead screw that delivers

mobile phase to the column at a constant rate. The rate of solvent
delivery is controlled by changing the voltage on the motor.

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ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

• Column efficiency is inversely proportional to the square root of

the particle size of the stationary phase
• Main factor that held back use of very small particles was high
pressure required to pump solvent
• UPLC pumps can pump up to 600 bars through 1-7 µm particle size
• UPLC can be used to produce comparable separations to HPLC
• Has much shorter run time, narrower peaks meaning limits of
detection are lower than HPLC

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HPTLC

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HPTLC
• Sophisticated form of thin layer chromatography. It
involves the same theoretical principle of thin layer
chromatography.
• Traditional Thin Layer Chromatography & its modern
instrumental quantitative analysis version HPTLC are very
popular for many reasons such as :
• visual chromatogram,
• simplicity,
• multiple sample handling,
• low running and maintenance costs, disposable layer etc.

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Principle
• Separation may result due to adsorption or partition or by
both phenomenon
• depends upon the nature of adsorbents used on plates and
solvents system used for development.

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WHAT EXACTLY IS HPTLC?
• High Performance Thin Layer Chromatography (HPTLC) is not the
TLC that many people remember from the past where the extent
of discussion was filter paper being dipped into a beaker.
• It is also not the TLC that was used by many labs where 20 cm x
20 cm plates were hand spotted with a pipette and the resulting
blobs were crudely interpreted.
• HPTLC is a mature, sophisticated technique that involves using
modern instrumentation with sound science and standardized,
reproducible methodology.
• To get a visual impression of the major differences between
conventional TLC and modern HPTLC:

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32
Here are some of the reasons why these two techniques differ
in appearance:

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Methods for Identification
• Are usually part of quality monographs (pharmacopoeias).
• Should be fit for the purpose of satisfying the 100%
Identification rule under cGMP.
• Can identify the desired species with certainty.
• Distinguish adulterants (related or other species).
• Allow batch to batch comparison and determination of shelf-life.
• Could give semi-quantitative information about the potency of a
material.
• Include System Suitability Test (SST)

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Identification of incorrect species (and potency of extracts)

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Herbal extracts – detection of excipients

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Comparative HPTLC profile of Berberine in different
market samples

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Solvent Reservoir

• Mobile phase
• Isocratic elution - single solvent separation teachnique
• Gradient elution - 2 or more solvents, varied during
separation
• To carry sample into the column
HPLC Detectors

• UV/Vis
• Refractive index
• Fluorescence
• Evaporative light scattering (ELSD)
• MS
• Diode Array Detector (DAD)
Data Processing

• Using specific software 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)
Application of HPLC
1. Pharmaceuticals industry
• To control the drug stability
• Quantity of drug determination from pharmaceutical dosage forms, e.g.
Paracetamol determination in panadol tablet
• 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
Application of HPLC
4. Clinical test
- Monitoring of hepatic cirosis patient through aquaporin 2 in
the urine.

5. Food and essence manufacture

- Sweetener analysis in the fruit juice
- Preservative analysis in sausage.
The factors which influence the HPLC performance

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
Advantages

1. Needs a small sample with a high accuracy and precis

2. Non-destructed sample during operation compared to GC.
Disadvantages
• Need a skill to run the instruments
• Solvents consuming
Chromatographic Column

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HPLC CHROMATOGRAM
Predict the order of elution from first to last of the following morphinane
compounds from an ODS column in an acetonitrile/buffer mixture pH 8 (10:90).
Retention Time

• The retention time of a solute is taken as the elapsed time

between the time of injection of a solute and the time of
elution of the peak maximum of that solute.
GOODLUCK!