CHAPTER 3 High Performance Liquid

Chromatography (HPLC)

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INTENDED LEARNING OUTCOME
Explain fundamental principles
01 of HPLC

Draw and label schematic

02 diagram of a HPLC
Draw and label schematic
03 diagram of a HPLC

Explain types of HPLC

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Explain stationary phase and
05 normal and reversed phase
chromatography.
06 Explain types of detector used
in HPLC 2
 PRINCIPLES
▪ HPLC is a form of liquid chromatograph used to separate compounds that are
dissolved in solution.

▪ To understand the principle of HPLC, we must know the principle behind

liquid chromatography.

▪ Liquid chromatography is a separation techniques that involved:

-The placement (injection) of a small volume of liquid sample into a tube
packed with porous particles (stationary phase).
-The individual component of the sample are transported along the packed
tube (column) by a liquid moved by gravity.

▪ The main principle is adsorption.

▪ When a mixture of components are introduces into the column. Various

chemical ad/or physical interaction take place between the sample molecules
and the particle of the column packing.
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LIQUID CHROMATOGRAPHY

They travel according to their relatives affinities towards the stationary phase.

The component which has more affinity towards the absorbent, travels slower.

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PRINCIPLES OF HPLC

▪ HPLC is a separation technique that involves:

▪ The injection of a small volume of liquid sample into a tube

packed with tiny particles called the stationary phase.

▪ The individual components of the sample moved down the

packed tube (column) with a liquid (mobile phase) forced
through the column by high pressure delivered by a
pump.

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PRINCIPLES OF HPLC
▪ These components are separated from one another by the
column packing that involves various chemical and /or
physical interactions between their molecules and the column
materials.
▪ The separated component are detected at the exit of the
column by a detector that measures the amount.

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 PRINCIPLES OF HPLC
▪ HPLC, is a technique used to separate the components in a
mixture, to identify each component, and to quantify each
component.

▪ It is important to determination of volatile and non volatile

compounds.

▪ It is important for determination of qualitative and qualitative

analysis.

▪ It is also important for determination of retention time (the time

required, after sample injection maximum angle peak reaches
to detector)
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PRINCIPLES OF HPLC
▪ It relies on pumps to pass a pressurized
liquid solvent containing the sample mixture
through a column.

▪ Each component in the sample interacts

slightly differently with the adsorbent
material, causing different flow rates for the
different components and leading to the
separation of the components as they flow
out the column.
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SCHEMATIC DIAGRAM OF HPLC

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HPLC SYSTEM

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MAIN COMPONENT IN HPLC

▪ Reservoir (Solvent)
▪ Solvent Delivery System (Pump)
▪ Injector
▪ Sample
▪ Column
▪ Detectors
▪ Waste Collector
▪ Recorder (Data Collection)

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MAIN COMPONENT IN HPLC
Solvent reservoir (mobile phase)

▪ An HPLC system begins with the solvent

reservoir, which contains the solvent used to
carry the sample through the system.

▪ The solvent should be filtered with an

inlet solvent filter to remove any particles that
could potentially damage the system's
sensitive components.

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MAIN COMPONENT IN HPLC

▪ The choice of mobile phase is very important in HPLC and the

eluting power of the mobile phase is determined by its overall
polarity, the polarity of the stationary phase and the nature of
the sample components.

▪ Mixing unit is used to mix the solvents in different proportions

and pass through the column. There are 2 types of mixing
units.

▪ They are low pressure mixing chamber and high pressure

mixing chamber. Mixing of the solvents is done either with a
static mixer or dynamic mixer.
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MAIN COMPONENT IN HPLC

▪ In an isocratic separation, mobile phase is prepared

by using solvent of same eluting power of polarity.

▪ But in gradient elution technique the polarity of the

solvent is gradually increase and hence the solvent
composition has to be changed. Hence a gradient
controller is used when 2 or more solvent pumps are
used for such separation.

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COMMON MOBILE PHASES

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COMPONENT IN HPLC
❑ Several gases are soluble in organic solvents.

❑ When solvents are pumped under high pressure, gas bubbles are
formed which will interfere with the separation process, steady base
line and the shape of the peak.

❑ Hence degassing of solvent is important. Dissolved air (O2,N2) in

mobile phase will caused unstable delivery :

- Unstable delivery in pump

- Bigger noise and large baseline drift in detector cell.

❑ To overcome the problem, mobile phase should be degassed.

-vacuum pumping systems
-distillation system
-sparging system-bubbles an inert gas of low solubility through the
solvent.

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PUMP

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MAIN
COMPONENT IN
HPLC

Pump System

Mobile phase pressures up to 6000 psi are necessary to achieve

reasonable column elution times (~ minutes). Typical flow rates are
0.1 to 10 mL/minute.
The solvents or mobile phases 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 few µ (5-

10 µ), the resistance to the flow of solvent is high. Hence such high
pressure is recommended. 22
INJECTOR

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MAIN COMPONENT IN HPLC
Injector
Sample Injection System
-sample valve
-syringe
The function of the injector is to place the sample (0.1 to 500 µL) into the
high-pressure flow in as narrow volume as possible so that the sample enters
the column as a homogeneous, low-volume plug.

Several devices are available either for manual or auto injection of the
sample. Different devices are:

1.Septum injectors
2.Stop flow injectors
3.Rheodyne injectors (loop valve type)

Rheodyne injectors is the most popular Injector. This has a fixed volume loop
like 20µl or 50 µl or more. Injector has two modes. Load position and inject
mode.
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MAIN COMPONENT IN HPLC
Injector

When an injection is started, an air actuator rotates the valve:

solvent goes directly to the column; and the injector needle is

connected to the syringe.

The air pressure lifts the needle and the vial is moved into position

beneath the needle. Then, the needle is lowered to the vial.

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COLUMN

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 MAIN COMPONENT IN HPLC
Chromatographic Column.

• The column is one of the most important components of the HPLC

chromatograph because the separation of the sample components is
achieved when those components pass through the column.

• The High performance liquid chromatography apparatus is made out of

stainless steel tubes with a diameter of 3 to 5mm and a length ranging
from 10 to 30cm.

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MAIN COMPONENT IN HPLC
Chromatographic Column.

• Normally, columns are filled with silica gel because its particle
shape, surface properties, and pore structure help to get a good
separation.

• Silica is wetted by nearly every potential mobile phase, is inert to

most compounds and has a high surface activity which can be
modified easily with water and other agents.

• Silica can be used to separate a wide variety of chemical

compounds, and its chromatographic behavior is generally 30
MAIN COMPONENT IN HPLC

Detector:
▪ HPLC detectors monitor the elute as it leaves the column.
▪ Its produce an electronic signal proportional to the concentration
of each separated component.
▪ No universal or versatile detector
▪ Types
▪ General – respond to mobile phase bulk properties which vary
in the presence of solutes (e.g. refractive index)
▪ Specific – respond to some property of the solute (not possessed
by the mobile phase (e.g. UV adsorption)
▪ “Hyphenated” detector – LC-MS

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REFRACTIVE INDEX
DETECTOR
▪ The RI detector is one of the few universal
detector
▪ Principle:
▪ The RI detectors measure a bulk property of the
mobile phase leaving the column: its ability to
refract to bend light (i.e., its refractive index).
▪ This property changes as the composition of the
mobile phase changes, such as when solutes from
the column.
▪ By detecting this change, the presence of solutes
can be detected.

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THE UV, VIS, AND PDA
DETECTORS
▪ The UV, VIS, and PDA detectors are
categorized as absorbance detectors.

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THE UV-VIS DETECTORS
▪ During the analysis, sample goes through a clear
color-less glass cell, called flow cell.
▪ When UV light is irradiated on the flow cell, sample
absorbs a part of UV light.
▪ Thus, the intensity of UV light observed for the
mobile phase (without sample) and the eluent
containing sample will differ.
▪ By measuring this difference, the amount of sample
can be determined.
▪ Since the UV absorbance also differs depend on what
wavelength is used, it is important to choose an
appropriate wavelength based on the type of analyte.
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THE UV, VIS, AND PDA DETECTORS

▪ A standard UV detector allows user to choose

wavelength between 195 to 370 nm. Most commonly
used is 254 nm.
▪ Compared to a UV detector, a VIS detector uses longer
wavelength (400~700 nm).
▪ There are detectors that provide wider wavelength
selection, covering both UV and VIS ranges (195~700
nm) called UV/VIS detector.

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PHOTO DIODE ARRAY
DETECTOR (PDA)
• PDA detects an entire spectrum simultaneously.

• UV and VIS detectors visualize the obtained result in

two dimensions (light intensity and time), but PDA adds
the third dimension (wavelength).

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PHOTO DIODE ARRAY
DETECTOR (PDA)
• Photo diode array detectors
operate by simultaneously
monitoring absorbance of solutes
at different wavelength.
• The result is that an entire
spectrum of a solute can be taken
in a minimum amount of time.

• Allow for the recording of the entire

spectrum of each solute as it
passed through the diode array
detector.

• The resulting spectra is a 3-D or

three dimensional plot of
Response Vs time Vs Wave length
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FLUORESCENCE DETECTOR
A fluorescence detector is an example of a selective detector,
with limits of detection smaller than those by either RI or
absorbance monitors.

It can be used to detect any compound absorbing and emitting

light at the given excitation and emission wavelength.

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CONDUCTIVITY DETECTOR
A conductivity detector is an example of a ‘universal’ detector for ionic
compound.

This detector measures the ability of a solution to conduct a current when

placed in an electrical field.

This ability depends on the number of ions or ionic compounds present in

the solution.

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 ELECTROCHEMICAL
DETECTOR
• It can be used to detect an compound which
can undergo an electrochemical reaction.
• This detector measure the ability of a solute
to undergo either oxidation (i.e., loss of
electrons) or reduction (i.e. gain of electrons)
• One way in which such a reaction can be
monitored is by measuring the change in
current under a constant electric field.
• Another way is to measure the change in the
electric field produced when a constant
current is present.
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HPLC DETECTORS

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RECORDER AND INTEGRATORS

● Recorders are used to record the responses obtained from detectors after
amplication.

● They record the base line and all the peaks obtained, with respect to the
time.

● Retention time for all the peaks can be found out from such recordings, but
the area of individual peaks cannot be known.

● Integrator are improved version of recorders with some data processing

capabilities.

● They can record the individual peaks with retention time, height and width
of the peaks than recorders. Now a days computers and printers are used
for recording and processing the obtained data and for controlling several
operations.

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TYPES OF HPLC
A) Based on mode of separation
1. Normal phase chromatography
2. Reverse phase chromatography

B) Based on elution technique

1. Isocratic elution
A separation in which the mobile phase composition remains constant
throughout the procedure is termed isocratic elution.
2. Gradient elution
A separation in which the mobile phase composition is changed during
the separation process is describe as gradient elution.

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TYPES OF HPLC
C) Based on scale of operation

Analytical HPLC -no recovery of individual component of substance

Preparative HPLC - individual components of substance can be

recovered.

D) Based on type of analysis

1. Qualitative analysis
Analysis of substance in order to ascertain of its chemical constituents.
We can separate individual component but cannot access the quantity
in this analysis.

2. Quantitative analysis
Determining the amount and proportions of its chemical constituents.
Quantify of the impurity and individual components can be assessed.
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E) Based on principle of separation

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Affinity Chromatography (AC)
Separates based on the use of immobilized biological molecules (and related compounds)
as the stationary phase

Based on the selective, reversible interactions that characterize most biological systems
- binding of an enzyme with its substrate or a hormone with its receptor
- immobilize one of a pair of interacting molecules onto a solid support
- immobilized molecule on column is referred to as the affinity ligand
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NORMAL AND REVERSED
PHASED (PARTITION)
CHROMATOGRAPHY
Characteristics of Normal Phase Chromatography
▪ Highly polar stationary phase
▪ Silica or alumina oxides

▪ Relatively non-polar solvent

▪ e.g. hexane or i-propylether

▪ Least polar solutes elute first

▪ Increasing mobile phase polarity decreases
elution times (i.e. polar compounds remain in
the mobile phase longer)
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NORMAL AND REVERSED
PHASED (PARTITION)
CHROMATOGRAPHY
Characteristics of Reversed Phase
Chromatography
▪ Non-polar stationary phase
▪ e.g. a hydrocarbon

▪ Relatively polar mobile phase

▪ e.g. water, methanol or acetonitrile

▪ More polar solutes elute first

▪ Increasing the mobile phase polarity increases
elution time

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 Mobile phase chromatographic polarity spectrum

Stationary phase chromatographic polarity spectrum

Compound/Analyte Chromatographic Polarity Spectrum

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NORMAL AND REVERSE PHASE
CHROMATOGRAPHY
Reversed
order
of elution

Increasing Mobil
phase Polarity,
Decreases
Elution Time

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ROUTINE OPERATION
PROCEDURE:
EG: DETERMINATION OF CAFFEINE IN SOFT DRINK

▪ Use the four caffeine standards to prepare a

calibration curve (graph) that plots peak area vs.
concentration.
▪ Draw a “best fit” straight line on your graph.
▪ Use your calibration curve to determine the
concentration of caffeine in the sample you prepared.
▪ Use the concentration of caffeine in your sample,
along with the dilution equation, to determine the
concentration of caffeine in the soft drink you used.

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Chromatogram I: HPLC
HPLC Chromatogram of Standard 1

0.500 x 10-4 M caffeine

Chromatogram II: HPLC
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