CHROMATOGRAPH

Y METHOD
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 PRESENTERS

NURSYAFIQAH BINTI
SITI BALQIS BINTI AZIZ
SALEH
PA20020 PA20021

MUHAMAD AZALI SALIHIN NUR SYAFEEZAH BINTI

BIN MUHAMAD JILI SHAFIE
PA20022 PA20023
 TABLE OF CONTENT

INTRODUCTION CONCEPT INSTRUMENTATION

WHAT IS HIGH HOW DOES HPLC WORKS? WHAT IS THE
PERFORMANCE LIQUID COMPONENTS OF HPLC?
CHROMATOGRAPHY?

APPLICATION CONCLUSION
WHAT ARE THE
WHAT HPLC USED FOR?
WHAT ARE THE TYPES PF ADVANTAGES AND
HPLC? DISADVANTAGES OF HPLC?
 01.
INTRODUCTION
What is HPLC?
 INTRODUCTION

High Performance Liquid Chromatography (HPLC) is the most widely used of all
analytical separation techniques.
It is a technique in analytical chemistry used to separate the components in a
mixture , to identify each component, and to quantify each component.
In the 1960s the column chromatography LC with its low-pressure suitable glass
columns was further developed to the HPLC with its high-pressure adapted metal
columns.
HPLC is thus basically a highly improved form of column liquid chromatography.
Instead of a solvent being allowed to drip through a column under gravity, it is
forced through under high pressures of up to 400 atmospheres.
It relies on pumps to pass a pressurized liquid solvent containing the sample
mixture through a column filled with a solid adsorbent material.
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.
The development of HPLC from classical column chromatography can be
attributed to the development of smaller particle is important.
They offer more surface area over the conventional larger particle sizes.
02.
CONCEPT OF
HPLC
How does the HPLC works?
HOW DOES HPLC WORK?
• In column chromatography a solvent drips through a column filled with an adsorbent under gravity. HPLC
is a highly improved form of column chromatography. A pump forces a solvent through a column under
high pressures of up to 400 atmospheres. The column packing material or adsorbent or stationary phase is
typically a granular material made of solid particles such as silica or polymers.

• The pressure makes the technique much faster compared to column chromatography. This allows using
much smaller particles for the column packing material. The smaller particles have a much greater surface
area for interactions between the stationary phase and the molecules flowing past it. This results in a much
better separation of the components of the mixture.

• The pressurized liquid is typically a mixture of solvents such as water, acetonitrile and/or methanol and is
referred to as the mobile phase.

• The components of a mixture are separated from each other due to their different degrees of interaction
with the absorbent particles. This causes different elution rates for the different components and leads to
the separation of the components as they flow out the column. Compared to column chromatography,
HPLC is highly automated and extremely sensitive.
 Step 1 Step 2 Step 3
The purification takes place in a The stationary phase is a The mobile phase, on the other
separation column between a granular material with very hand, is a solvent or solvent
stationary and a mobile phase. small porous particles in a mixture which is forced at high
separation column. pressure through the separation
column.

Step 4 Step 5 Step 6

Via a valve with a connected Subsequently, the individual
After leaving the column, the
sample loop, i.e., a small tube components of the sample
individual substances are
or a capillary made of stainless migrate through the column at
detected by a suitable detector
steel, the sample is injected into different rates because they are
and passed on as a signal to the
the mobile phase flow from the retained to a varying degree by
HPLC software on the
pump to the separation column interactions with the stationary
computer.
using a syringe phase.
 Step 7 Step 8

At the end of this operation/run, a The chromatogram allows the

chromatogram in the HPLC software identification and quantification of
on the computer is obtained. the different substances.
 03.
INSTRUMENTATI
ON
The Pump
• The development of HPLC led to the development of the pump system.
• The pump is positioned in the most upper stream of the liquid chromatography system and generates a
flow of eluent from the solvent reservoir into the system.
• High-pressure generation is a “standard” requirement of pumps besides which, it should also be able to
provide a consistent pressure at any condition and a controllable and reproducible flow rate.
• Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-driven
piston (reciprocating pumps). Because of this piston motion, it produces “pulses”.
Injector
• An injector is placed next to the pump.
• The simplest method is to use a syringe, and the sample is introduced to the flow of eluent.
• The most widely used injection method is based on sampling loops.
• The use of the autosampler (auto-injector) system is also widely used that allows repeated injections in a
set scheduled-timing.
Column
• The separation is performed inside the column.
• The recent columns are often prepared in a stainless-steel housing, instead of glass columns.
• The packing material generally used is silica or polymer gels compared to calcium carbonate.
• The eluent used for LC varies from acidic to basic solvents.
• Most column housing is made of stainless steel since stainless is tolerant towards a large variety of
solvents.
Detector
• Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained
separation.
• The composition of the eluent is consistent when no analyte is present. While the presence of analyte
changes the composition of the eluent. What detector does is to measure these differences.
• This difference is monitored as a form of an electronic signal. There are different types of detectors
available.
Recorder
• The change in eluent detected by a detector is in the form of an electronic signal, and thus it is still not visible
to our eyes.
• In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a computer-based data processor
(integrator) is more common.
• There are various types of data processors; from a simple system consisting of the in-built printer and word
processor while those with software that are specifically designed for an LC system which not only data
acquisition but features like peak-fitting, baseline correction, automatic concentration calculation, molecular
weight determination, etc.
Degasser
• The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our eyes.
• When gas is present in the eluent, this is detected as noise and causes an unstable baseline.
• Degasser uses special polymer membrane tubing to remove gases.
• The numerous very small pores on the surface of the polymer tube allow the air to go through while preventing
any liquid to go through the pore.
Column Heater
• The LC separation is often largely influenced by the column temperature.
• In order to obtain repeatable results, it is important to keep consistent temperature
conditions.
• Also, for some analysis, such as sugar and organic acid, better resolutions can be
obtained at elevated temperatures (50 to 80°C).
• Thus, columns are generally kept inside the column oven (column heater).
04.
APPLICATIONS
OF HPLC
The HPLC has developed into a universally applicable method so that it finds it use
in almost all areas of environmental, clinical, food and flavor, and pharmacy.
 APPLICATION OF HPLC

Identification of counterfeit
drug products

PHARMACEUTICAL

Complex molecules Shelf-life determination and

separation quality controls of
pharmaceutical products
 APPLICATION OF HPLC

Water monitoring – Phenol

content and toxic
componants checking

ENVIRONMENTAL CLINICAL

Biomonitoring of pollutents Analysis of antibiotics and

blood substances
 APPLICATION OF HPLC

Sugar analysis in fruit juices

FOOD AND FLAVOUR

Detection of endogenous Ensuring soft drink

neuropeptides in brain consistency and quality
extracellular fluids
 Types of High-Performance Liquid Chromatography

1. Normal phase:
• Column packing is polar (e.g., silica) and the mobile phase is non-polar. It is used for water-sensitive compounds,
geometric isomers, cis-trans isomers, and chiral compounds.
2. Reverse phase:
• The column packing is non-polar (e.g., C18), the mobile phase is water+ miscible solvent (e.g., methanol). It can be
used for polar, non-polar, ionizable and ionic samples.
3. Ion exchange:
• Column packing contains ionic groups, and the mobile phase is buffer. It is used to separate anions and cations.
4. Size exclusion:
• Molecules diffuse into pores of a porous medium and are separated according to their relative size to the pore size. Large
molecules elute first and smaller molecules elute later.
 05.
CONCLUSION
 It can be concluded from the entire review that HPLC is a versatile,
reproducible chromatographic technique for the
estimation of drug products.

ADVANTAGES OF HPLC DISADVANTAGES OF HPLC

• Speed • Cost: Despite its advantages, HPLC

• Efficiency can be costly, requiring large quantities
• Accuracy of expensive organics.
• Versatile and extremely precise • Complexity: HPLC does have low
when it comes to identifying and sensitivity for certain compounds, and
quantifying chemical some cannot be detected as they are
components irreversibly adsorbed. Volatile
substances are better separated by gas
chromatography.
Thanks!
Do you have any questions?
 QUESTION
1. How many types of HPLC? State the type.
2. What are the components of HPLC?