2. CONTENT
INTRODUCTION
TYPES OF HPLC TECHNIQUES
PRINCIPLE
INSTRUMENTATON
PARAMETERS USED IN HPLC
ADVANTAGES OF HPLC
DERIVATISATION IN HPLC
APPLICATIONS
3. INTRODUCTION
• Chromatography" is a technique for separation,
“Chromatogram" is the result of chromatography, and
“Chromatograph" is the instrument used to conduct chromatography.
• Devices dedicated for molecular separation called columns and high-performance pumps
for delivering solvent at a stable flow rate are some of the key components of
chromatographs.
• HPLC separates compounds dissolved in a liquid sample and allows qualitative and
quantitative analysis of what components and how much of each component are contained
in the sample.
4. PRINCIPLE
• The separation principle of HPLC is based on the distribution of the analyte (sample)
between a mobile phase (eluent) and a stationary phase (packing material of the column).
• The specific intermolecular interactions between the molecules of a sample and the
packing material define their time "on-column". Hence, different constituents of a sample
are eluted at different times. Thereby, the separation of the sample ingredients is achieved.
• A detection unit (e.g. UV detector) recognizes the analytes after leaving the column.
• The signals are converted and recorded by a data management system (computer
software) and then shown in a chromatogram.
• After passing the detector unit, the mobile phase can be subjected to additional detector
units, a fraction collection unit or to the waste.
5.
6.
7. TYPES OF HPLC
NORMAL PHASE HPLC
• Mobile phase in Normal Phase HPLC are based on non polar solvents with addition of
small quantity of polar solvent.
• The stronger the mobile phase interactions with the stationary phase, the lower the
difference between the stationary phase interaction and the analyte interactions, thus
lower analyte retention.
• Packing materials used in normal phase HPLC are generally Silica or Alumina.
8. Reverse Phase HPLC
• Mobile phase in Reverse Phase HPLC is Polar and the stationary phase is
• Hydrophobic
• Employs mainly dispersive forces (hydrophobic or van der wall interactions.
• 90% of analysis are done using this technique.
• It has the advantage of low energy generation due to the presence of weak forces of
interaction.
9. INSTRUMENTATION
Solvent Reservoirs
• Clean and inert container.
• Storage of sufficient amount of solvent.
• Cap is placed with a tube that feeds mobile phase to the solvent delivery system.
• In-line degassers are also present, which are primarily used to remove small gas bubbles
and reduce dissolved air.
• An additional filter is often placed at the end of the mobile phase inlet line to remove any
particulates or precipitates that may form in the mobile phase during its use.
10. Pump:
• Continuous flow of mobile phase through the system.
• Pressure from 500 to 20,000 psi.
• The purpose of the pump is to deliver a precise, accurate, reproducible, constant, and
pulse-free flow of mobile phase to the column.
• The majority of commercial high pressure pumps available today are designed around a
simple reciprocating piston pump.
Injector:
• Introduce analyte mixture into the stream of mobile phase without depressurizing the
HPLC system.
11. TYPES OF PUMPS
PUMP PRINCIPLE APPLICATIONS PUMP CAPACITY
Constant Pressure
Pump
Mobile phase is driven
through the column with
the use of pressure from
the gas cylinder.
Suitable for isocratic
elution.
70 mL
Reciprocating
Piston Pump
It has a small motor-driven
piston which pushed the
mobile phase due to the
forward and backward
action of the piston
present.
The reciprocating
pump can work as an
isocratic and gradient
mode.
35-400 µL per
cycle
Syringe Type Pump This type of pump consist
of big syringe that is
motorized by an electronic
motor used to drive a
steady flow rate.
Suitable for isocratic
elution but
inconvenient for the
gradient elution.
250-500 mL
12. Column:
• Produces separation of the analytes in the mixture.
• Currently columns are made of heavy-walled glass, titanium, and plastic (e.g., PEEK) to
offer the analyst better performance for particular analytes.
• Common dimensions for analytical scale columns are in the range of 10-30 cm long and
4-10mm in diameter.
• The common particle sizes of packing are 3, 5, and 10 µm.
• Columns are easily degraded by the irreversible adsorption of impurities from samples
and solvents. Hence, a guard column is often used to protect the integrity of the analytical
column, which is much more expensive.
• Also, for analytes that may contain particulates, an in-line filter can be placed between the
injector and guard column.
13. TYPES OF COLUMNS
COLUMN PRINCILPE MOBILE PHASE STATIONARY
PHASE
Normal Phase Columns More polar stationary phase
than mobile phase; packing
material should be more polar
than the mobile phase.
Methyl chloride, hexane,
chloroform.
Amino bonded with
siloxane.
Reverse Phase Column It has non-polar or less polar
stationary phase than the
more polar mobile phase.
Aqueous organic solution
like water-methanol is
used.
Bonded hydrocarbons
like C8, C18, C4.
Ion Exchange Column Stationary phase is acidic or
basic while mobile phase is
polar liquid.
Aqueous buffer. Charged surface like
Sulfonic acid or
primary or tertiary
amine.
14. Detector:
• Continuous registration of specific physical properties of the column effluent.
• HPLC detectors are classified as either bulk property detectors, which respond to a bulk
property of the eluent (e.g., refractive index (RI) or conductivity), or solute property
detectors, which respond to some property of the analyte (e.g., UV absorbance).
• In either case, the response of the detector is modulated by the presence and amount of
the analyte.
15. PERFORMANCE CHARACTERISTICS OF COMMON HPLC
DETECTORS
Parameter UV/VIS
Absorptio
n
Fluorescence Electrochemi
cal
Conductivity Refractive
Index
Mass
Spectromet
ry
Classificatio
n
Solute
property
Solute
property
Solute
property
Bulk property Bulk
property
Solute
property
Response Selective Selective Selective Selective Universal Selective
Sensitivity Nanogram Picogram Picogram Nanogram Microgram Picometer
Gradient
Compatibilit
y
Yes Yes Limited Yes(suppress
ed); No(non-
suppressed)
No Yes
Flow
Sensitivity
No No Yes Yes Yes Yes
Temperatur
e Sensitivity
No No Yes Yes Yes Yes
16. Data acquisition and control system:
• Computer based system that controls all parameters of HPLC instrument.
• The data collection device takes the electronic signal produced by the detector and
outputs a plot of response versus time.
• This resulting chromatogram can then be evaluated for both qualitative and quantitative
information.
17.
18. APPLICATIONS
• HPLC is an integral analytical tool applied in all stages of drug discovery, development,
and production.
• It can help (bio-) pharmaceutical researchers and manufacturing facilities fully
characterize potential drug or treatment candidates, and ensure the medicines are
manufactured in a safe and consistent way.
• HPLC can be useful in ensuring critical quality attributes such as strength/concentration,
content uniformity, the detection and quantification of impurities, and the quality and
identity of raw material.
• It can be used in evaluating the pharmaceutical stability of a product.
19. • Impurities can pose a serious safety risk to patients, and their detection and identification
is often facilitated by the use of HPLC.
• HPLC can also be useful in determining shelf life; “for example, some biotherapeutics are
sensitive to aggregation over time, or if not stored properly, and HPLC can be used to
monitor this aggregation.”
• Widely used in forensic science for the separation and identification of morphine from
blood plasma.
• Used for detection of different poisons such as carbon mono oxides, alcohol, heavy metals
etc.
20. ADVANTAGES:
• Ensure lot to lot product purity and consistency.
• Efficiency for large-scale production.
• Sensitivity for monitoring product degradation and shelf-life.
• Allow versatility for contract manufacturing organizations.
• Meets regulatory compliance for on-market products.
• Lower your operating costs.
21. DISADVANTAGES:
• HPLC has high cost.
• High quality components are needed.
• The solvents and columns used in HPLC are expensive.
• Regular maintenance and calibration are needed which add extra cost.
• Sophisticated software is required for data analysis.
• Research and development cost.
