8.

High-performance liquid chromatography (HPLC)

Introduction
• Type of chromatography that employe liquid mobile phase and a very finely
divided stationary phase packed in a column

• Since the particles of the stationary phase are very small (between 2 & 5
µm) , there is a need to pump the mobile phase through a column under a
high pressure

• High-performance liquid chromatography (HPLC) is the technique most

commonly used for the quantitation of drugs in formulations

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 Basic principle of HPLC
• Sample is injected into a HPLC system
• Interactions happen between the samples with the mobile phase and
stationary phase (column) which results in separation of samples which is
detected through the detector and converted into a chromatogram
• The main principle of separation is adsorption
• They travel according to their relative affinities towards the stationary
phase.
• The component which has more affinity towards the SP, travels slower. The
component which has less affinity towards the stationary phase travels faster.
• Since no two components have the same affinity towards the stationary
phase, the components are separated

2
 Introduction…………
• Based on the relative polarities of the mobile phase and stationary phases,
HPLC can be classified:

a. Normal phase chromatography

• Stationary phase is highly polar e.g. silica gel
• The mobile phase is relatively non polar solvent e.g. hexane
• The least polar component is eluted first
• Polar compounds travels slower & eluted slowly due to higher affinity to
stationary phase
• Non-polar compounds travels faster & eluted first due to lower affinity to
stationary phase

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 STATIONARY PHASES
(NORMAL POLARITY)

• Silica or alumina possess polar sites that interact with polar molecules

silica
O
Polar Group HO Si
O

Components elute in increasing

order of polarity.

4
 • Normal HPLC

Mobile 1
2
Phase

POLAR: NONPOLA
Stronger R
interaction Weaker
interaction
Stationary Phase (POLAR)
5
 Introduction
b. Reversed phase chromatography
• The stationary phase is non polar

• The stationary phase is usually Octyl- or Octyl Decyl Siloxane packing (ODS)
(C18, C8, etc)

• The mobile phase is relatively polar solvent

– Eg: methanol- water or acetonitrile- water

• Polar compounds travels faster & eluted 1st due to lesser affinity to
stationary phase

• Non-Polar compounds travels slower & eluted slowly due to higher affinity to
stationary phase

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 STATIONARY PHASES
(REVERSE POLARITY)

• If the polar sites on silica or alumina are capped with non-polar groups,
they interact strongly with non-polar molecules

silica
C18 phase Me O
Si O Si
Me O

Components elute in decreasing

order of polarity.

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 Normal vs. Reverse phase HPLC
Normal Phase Reversed Phase
Stationary phase Polar (silica gel) Non-polar
(C18)
Mobile phase
Non-polar Polar
(organic solvents) (aqueous/organ
ic)
Sample movement Non-polar fastest Polar fastest

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Structural factors which govern rate of elution of compounds from HPLC columns

• In a reverse-phase column, the more lipophilic a compound is the

more it will be retained

• For a polar column such as a silica gel column, the more polar a
compound is the more it will be retained

• Polarity can often be related to the number of the hydroxyl groups

present in the molecule

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 exercise
Predict the order of elution, from first to last, of the following steroids
from an ODS column with methanol/water (70:30) as the mobile phase

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 Exercise
Predict the expected order of elution from a reverse-phase column using
a mobile phase containing methanol/water (75:25),

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Instrumentation
The main components of modern LC (HPLC) are:
1. mobile phase reservoir
2. High pressure pump
3. An injection port
4. column
5. detectors
6. recording systems

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Schematic diagram of HPLC instrument

13
 Outline of LC-2010

Reservior Tray

System
Auto sampler Controller

Column Oven
UV detector
Degassing Unit
Pump Unit
Low
pressure
gradient
device 14
1. Mobile phase reservoirs and Solvents

• Solvent Reservoirs are used to store mobile phase

• A modern HPLC apparatus is equipped with one or more glass or
stainless steel reservoir, each of which contains 500 ml or more of a
solvent
• Multiple solvents are necessary for performing gradient elution's
• It can be transparent or can be amber colored.
• Solvent reservoirs are placed above HPLC system (at higher level) in a
tray

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Mobile phase reservoirs………………..
• Gases like O2 always present in solvents that form the mobile phase

• This affect separation by modifying the compressibility of the mobile phase and
eventual formation of bubbles

• Particularly, oxygen can interfere with electrochemical detection and shortens

the life time of a column

• Hence, de-gassing is very important and it can be done by various ways

(i) Vacuum filtration: applying a partial vacuum to the solvent container

(ii) Ultrasonication: ultra sonicator converts ultra high frequency to mechanical

vibrations

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There are two types of solvent delivery system:

1. Isocratic elution

• is one in which the composition of the solvent remains constant

• A separation that employs a single solvent or solvent mixture of constant

composition with single pump

2. Gradient elution

• composition of the mobile phase is continuously varied

• Incorporated to achieve a better or/and faster separation

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2. Pumping system

• High pressure pumps are needed to force solvents through packed

stationary phase beds

• HPLC Pump is very important component of the system

• The Pump delivers the constant flow of the Mobile Phase

• The pumps provide a steady high pressure with no pulsating, and can be
programmed to vary the composition of the solvent during the course of
the separation

• The role of the pump is to force the mobile phase through the LC system

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 Pumping system….cont’d
Requirements:
• Ability to generate pressures up to 6000 psi

• Pulse free out put (control flow rate)

• Typical flow rates ranging from 0.1 to 10 mL/minute

• Flow reproducibility (0.5% relative or better)

• Resistance to corrosion by solvent

• the pump must be inert

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Types of pump

Two basic pumping system:

i. Screw driven syringe type pump

• Produce Pulse free delivery whose flow rate is readily controlled

• Inconvenient when solvent must be changed

• Readily adaptable for isocratic elusion

ii. Reciprocating pump

• Most widely used

• Pulsed flow( due to back &forth motion of piston)

• High out put pressure ( up to 10,000 psi)

• Readily adaptablle for gradient elusion

• Not dependent on solvent viscosity &back pressure

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 High-pressure Gradient System

pump

B
Mixer
Column Detector
pump Injector
Oven
Data
processor
C

pump • Excellent gradient accuracy.

• 2-3 pumps required - one pump per solvent used.

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3. Sample injection system
• Introduces the liquid sample into the flow stream of the mobile phase
• Typical sample volumes: 20 μl
Common requirements:
• Must allow delivery of very constant smaller sample size
• Reproducibility of the sample into the column must be ensured
• There should not be adsorption and desorption of samples
• Must tolerate pressure up to 10,000 psi
• Sample volume can varies 5 to 500µl

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3. Sample Injector

a. Manual sample Injector:

• Manually load sample into the injector using a syringe

• and then injected sample → into the flowing mobile phase →which transports
the sample into the beginning (head) of the column, which is at high pressure

b. Auto sampler injector:

• User loads vials filled with sample solution into the auto sampler tray

• Auto sampler automatically

o Takes appropriate sample volume

o injects the sample

o then flushes the injector to be ready for the next sample

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4. HPLC column

• It is the back bone of the system where separation is takes place

• It may be steel, glass or plastic tube with 5-30 cm in length and 1-5 mm
internal diameter

• Packing materials can be silica, alumina

• Stationary phase is packed with 5 to 10 µm

• Columns are expensive and easily degraded by dust or particles in the

sample or solvent

• A pre column or guard column is introduced in front of the main column to

increase the life of the main column by removing particulate matter &
contaminants from the solvent

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Guard column
• A short guard column placed between the injector and analytical column
• Contain the same stationary phase as analytical column
• Used to remove particulate matter and contaminants from the solvent
- Increase the life of analytical column
5. Detectors
• The elution of a compound from the column is detected as a peak in the
chromatogram
Ideal HPLC Detector Properties:
1. Adequate sensitivity (typical range: 10-8 – 10-15 g solutes/s);
2. Good stability and reproducibility
3. A linear response to solutes
4. A short response time

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 HPLC Instrumentation
5. Detectors…cont’d
• Optical detectors are most frequently used.
• These detectors pass a beam of light through the flowing column
effluent as it passes through a low volume ( ~ 10 ml) flow cell.
• The most commonly used detector in LC is the ultraviolet
absorption detector

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 a. UV/visible absorbance detector
• Measure the analyte absorption at one or many wave lengths in the
UV or visible region
• Important for unsaturated and aromatic cpds
• Principle: beer’s law
• Sensitivity: 100 pg to 1 ng
UV-Visible detectors could be
• Fixed wave length
– Measures at one wave length
• Variable wave length
– Measures at one wave length at a time, but can detect over a wide
range of wave length
• Diode array detectors
– Simultaneously record the absorbance simultaneously
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b. Fluorescence detector

• Based on measurement of fluorescence

• Important for substances that exhibit fluorescence properties
• Compared to UV-Vis detectors , fluorescence detectors offer a
higher sensitivity and selectivity (trace level analysis).
• Detection limit: 1-10 pg
• Limitation: only fluorescent analytes measured.

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Other types of detectors
• Refractive index (RI) indicators
6. Recorder / Data System

• For higher control levels, a more intelligent device is necessary, such as

a data station or minicomputer
• Electronic signals generated by detectors are recorded in the form of
chromatographic peak at varied function of time
• Peak Area, height, retention time, base width of chromatographic
peak is measured to compute analyte concentration of each peak

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 Applications of HPLC in pharmaceutical analysis

1. Qualitative Analysis

– Identification of compound identity

– Require a known standard

– Identified by comparing retention time

2. Quantitative Analysis
– The majority of applications of HPLC in pharmaceutical analysis are to
the quantitative determinations of drugs in formulations
– Require a standard with known amount of concentrations
– Identified by interpolating the area of unknown into a set of standards
with known concentration
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 Exercise
• Assay of Paracetamol was done by HPLC and Calculate the percentage of stated
content in paracetamol tablets using the calibration curve given and the following
data:
Data
• Weight of 20 tablets=12.2243 g
• Weight of tablet powder taken=152.5 mg
• Stated content per tablet=500 mg
• Initial extraction volume=200 ml
Dilution steps
• 20 ml into 100 ml
• 10 ml into 100 ml
• Calibration curve for paracetamol: Y= 35656X + 80 where X is in mg/100 ml
• Area of peak obtained for paracetamol in diluted sample extract =44 519
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