High-Performance

Liquid
Chromatography
Dr. Amany M. Elshamy

Lecturer of Medical Laboratory Science

PH.D of Biochemistry and Molecular biology

MLS, AUC, Cairo

Outlines
Introduction

GC an LC are two categories of

column chromatography.
Column
chromatography
• In column chromatography,
the stationary phase is
coated onto or is chemically
bonded to support particles
that are packed into a tube,
or the stationary phase is
coated onto the inner
surface of a tube.
 Advances in column
technology have improved the
(1) selectivity
Columns
chromatography (2) stability

(3) reproducibility.
Separation mode
• HPLC methods include
A. liquid/liquid (partition) chromatography
B. liquid/solid (adsorption) chromatography
C. ion exchange and size exclusion chromatography
 • Modern liquid chromatography
uses pressure for fast separations,
High- controlled temperature, in-line
Performance detectors, and gradient elution
Liquid techniques.
Chromatography • Separation for all substances
(organic, Volatile, nonvolatile)
• Molecular weight ( 80-6000).
 Advantages:
High- •High resolution
Performance
Liquid •High accuracy
Chromatography
•High sensitivity
•Speed with min
•Automatic sys
 Mobile phase is liquid.

The components of the mobphasephas based

High- on the mode of chromatographic separation
Performance and the type of detector use .
Liquid
Chromatography the mobile phase commonly used is
acetonitrile, methanol, water, or any
combination of solvents

the stationary-phase is particles of small

diameter
Basic
component
 • 1. A solvent reservoir to hold the mobile
phase.
• 2. Degasser (remove dissolved gases).
• 2. One or more pumps to force the liquid
mobile phase through the system.
Basic • 3. An injector to introduce a sample into the
column.
component
• 4. A chromatographic column to separate
the solutes.
• 5. detector(s) to detect analytes as they elute
from the column.
• 6. A computer to control the system an to
process data.
Basic
components
 • In their simplest forms, the reservoirs are
glass bottles or flasks.
• Before putting solvent reservoir should
remove particles from solvents.
Solvent • Dissolved gases (O2 , N2) are removed by
Reservoir vacuum degassing (result in the formation
of bubbles in detectors or unstable signal ).
• Solvent should be high purity with low
viscosity.
• Inert chemically towards analytes and S.P.
• Compatible with the detector.
 • A pump function: it forces the mobile
phase through the column at a much
greater velocity than that
Pump accomplished by gravity flow columns.
• Pumps used with HPLC must deliver a
constant flow with low pulsation.
 Pump includes:

•Pneumatic.
•Syringe.
Pump
•Reciprocating
pumps (most widely
used ).
• The limitation of Syringe
type pump is that it has a
limited solvent capacity
and is inconvenient when
solvents are to be changed.
 • A small syringe can be used to introduce the
sample into the path of the mobile phase
that carries it into the column .
• The best and most widely used method,
however, is the loop injector.
Sample • The sample is introduced into a fixed-volume
Injectors loop.
• When the loop is switched, the sample is
placed in the path of the flowing mobile
phase and flushed onto the column.
• Loop injectors have high reproducibility and
are used at high pressures.
 Sample Injectors

Important characteristics (1) reproducibility (2) amount of carryover (3) range of injected
of injection systems volumes.
include their
 • Both packed and capillary columns are use
in LC, and there is great diversity in sizes of
columns and types of column packing.
Columns • Length (5-25 cm)
• Diameter (4-10mm)
• Particle size (3-10micro meter)
 • The stationary phase is packed into long
stainless steel columns.
• The packing can also be pellicular (an inert
Column core with a porous layer), inert and small
Stationary particles, or macroporous particles.
• The most common material used for column
Phase packing is silica gel. It is very stable and can
be used in different ways.
porous particles.
Column Stationary Phase

• The stronger affinity between the component and

the mobile phase, the faster the component moves
through the column along with the mobile phase.
• The stronger the affinity with the stationary phase,
the slower it moves through the column.
 • Modern HPLC detectors monitor the eluate
as it leaves the column and, ideally, produce
an electronic signal proportional to the
concentration of each separated
component.
Detectors • Spectrophotometers that detect visible or
UV light absorbances are most commonly
used. A photodiode array (PDA) is used for
spectral comparisons and compound
identification and purity. These detectors
have been used for drug analyses in urine.
 • The recorder is used to record the detector
signal versus the time the mobile phase
passes through the instrument, starting
from the time of sample injection.
• The graph is called a chromatogram.
Recorders • The retention time is used to identify
compounds when compared with standard
retention times run under identical
conditions.
• The peak area is proportional to the
concentration of the compounds that
produced the peaks.
chromatogram
Chromatogram

• The word "chromatogram" means a plot obtained via

chromatography.
• The chromatogram is a two-dimensional plot with the
vertical axis showing concentration in terms of the
detector signal intensity and the horizontal axis
representing the analysis time.
Chromatogram

• When no compounds are eluted from the column, a line

parallel to the horizontal axis is plotted. This is called the
baseline.
• The detector responds based on the concentration of
the target compound in the elution band.
• The obtained plot is more like the shape of a bell rather
than a triangle. This shape is called a “peak”.
 • Retention time (tR) is the time interval
between sample injection point and the
apex of the peak.

Chromatogram
 • The required time for non-retained
compounds (compounds with no
interaction for the stationary phase) to go
from the injector to the detector is called
the dead time (t0=t zero).

Chromatogram
Results
• Results will be
used for the
qualitative and
quantitative
analysis of a
sample's
components.
Limitations
Summary
• Which of the following chromatography systems may be described as having a stationary phase that is liquid
absorbed on particles packed in a column and a liquid moving phase that is pumped through a column?
• A. Thin-layer
• B. High-performance liquid
• C. Ion-exchange
• D. Gas-liquid
• Which of the following techniques is more commonly used to measure vitamins?
• A. High-performance liquid chromatography
• B. Spectrophotometry
• C. Nephelometry
• D. Microbiological

• High pressure liquid chromatography can be performed only

in columns.
a) True
b) False
• Which of the following is not true about High pressure liquid
chromatography (HPLC)?
a) It requires high pressure for the separation of the
specious
b) There is no need to vaporise the samples
c) It is performed in columns
d) It has high sensitivity
• Which of the following is not an advantage of Syringe type
pumps used in High pressure liquid chromatography?
a) Independent of viscosity
b) Pulse-less flow
c) High pressure capability
d) Unlimited solvent capacity
• HPLC stands for
A.High Pressure Liquid Chromatography
B. High Performance Liquid Chromatography
C. both (a) and (b)
D. Highly Placed Liquid Chromatography
• HPLC methods include
A. liquid/liquid (partition) chromatography
B. liquid/solid (adsorption) chromatography
C. ion exchange and size exclusion chromatography
D. all of the above
Which can be used as a mobile phase in HPLC applications?
A. Any compound with solubility in liquid
B. Any compound with limited solubility in liquid
C. Any compound with non-solubility in liquid
D. Any of the above