INSTRUMENTAL

METHODS OF
ANALYSLIS
C H R O M A T O G R A P H I C
T E C H N I Q U E S
( P a r t 2 )

CHY 33 Lecture
Hannah Jean V. Esteban
Unit Objectives

• Demonstrate understanding of
the basic principles of
chromatographic separation
through :
• the illustration of the
instrumental block diagram,
• identifying applications from
published research, and
• stating the limitations of
each technique
High Pressure Liquid Chromatography (HPLC)

• a type of chromatography
that combines a liquid
mobile phase and a very
finely divided stationary
phase

• The sample, which is in

solution form, into a liquid
mobile phase

• The mobile phase carries the sample through a packed or capillary

column that separates the sample’s components based on their ability to
partition between the mobile phase and the stationary phase
High Pressure Liquid Chromatography (HPLC)
Typical Instrumentation
High Pressure Liquid Chromatography (HPLC)
Block diagram
High Pressure Liquid Chromatography (HPLC)
Block diagram

• Mobile phase reservoirs

• one or more glass reservoirs, each of which
contains 500 mL or more of a solvent.
• Provisions are often included to remove
dissolved gases and dust from the liquids
▪ Dissolved gases can lead to irreproducible
flow rates and band spreading
▪ both bubbles and dust interfere with the
performance of most detectors
➢ Sparging is a process in which dissolved
gases are swept out of a solvent by bubbles
of an inert, insoluble gas
High Pressure Liquid Chromatography (HPLC)
Block diagram

• Pumps/ Solvent delivery system

• plays dual role of passing mobile
phase through column bed under
high pressure and maintaining a
constant pulse free flow rate
throughout the separation
➢ An isocratic elution in HPLC is one in which the solvent
composition remains constant.
➢ A gradient elution in HPLC is one in which the composition of the
solvent is changed continuously or in a series of steps
High Pressure Liquid Chromatography (HPLC)
Block diagram

• Pumps/ Solvent delivery system

High Pressure Liquid Chromatography (HPLC)
Block diagram

• Injector/ Sample Management

• The operating pressure within an HPLC is
sufficiently high that it is not possible to inject the
sample into the mobile phase by inserting a
syringe through a septum like GC A sampling loop for
liquid chromatography. (Courtesy
of Beckman Coulter, Fullerton, CA.)
• Uses Loop injector
High Pressure Liquid Chromatography (HPLC)
Block diagram
• HPLC Column
• an essential part of HPLC based chromatographic
system and serve the purpose of holding
stationary phase
• separation of mixture into individual component
takes place interior of column onto the bed of
adsorbent by the phenomenon of
adsorption/partition
• Most common are made of steel
• Analytical column and precolumns
High Pressure Liquid Chromatography (HPLC)
Block diagram
• HPLC Column, Analytical
• range in length from 5 to 25 cm and have inside
diameters of 3 to 5 mm
• most common particle size of packings is 3 or 5 mm
• Commonly used columns are 10 or 15 cm long, 4.6 mm in
inside diameter, and packed with 5-mm particles
• HPLC Column, Precolumn
• scavenger column is between the mobile-phase
container and the injector is used to condition the
mobile phase.
• guard column is between the injector and the column
removes particulates and other solvent impurities.
High Pressure Liquid Chromatography (HPLC)
Block diagram
• HPLC Column, Analytical
• In liquid–liquid chromatography the stationary
phase is a liquid film coated on a packing material,
typically 3–10 μm porous silica particles
• Silica → most common packing in liquid
chromatography; often coated with thin organic
films which are chemically or physically bonded to
the surface (Bonded stationary phases)
 High Pressure Liquid Chromatography (HPLC)
Block diagram
• HPLC Column, Analytical

• R – polar functional group → stationary phase is polar, ex.

cyano (–C2H4CN), a diol (C3H6OCH2CHOHCH2OH), or an amino
(–C3H6NH2); SP is polar, MP is non-polar. The system is called
Normal phase chromatography
• Reversed-phase chromatography → non-polar stationary
phase (SP) and polar mobile phase (MP)
▪ More common form of HPLC
▪ R-group is an n-octyl (C8) or n-octyldecyl (C18)
hydrocarbon chain
 High Pressure Liquid Chromatography (HPLC)
Block diagram
• Detectors

• Bulk Property → measure

some of the physical
properties of analyte present
in mobile phase against
blank mobile phase
• Solute Property → measures
physical or chemical
properties of analyte
irrespective of mobile phase
and can be used effectively
with gradient elution
High Pressure Liquid Chromatography (HPLC)
Block diagram
• Detectors
High Pressure Liquid
Chromatography (HPLC)
Block diagram
HPLC separation of a mixture of flavonoids with UV/Vis
detection at 360 nm and, in the inset, at 260 nm. The
choice of wavelength affects each analyte’s signal. By
carefully choosing the wavelength, we can enhance the
signal for the analytes of greatest interest.
HPLC chromatogram for the determination
of riboflavin in urine using fluorescence detection with
excitation at a wavelength of 340 nm and detection at
450 nm. The peak corresponding to riboflavin is marked
with a red asterisk (*). The inset shows the same
chromatogram when using a less-selective UV/Vis
detector at a wavelength of 450 nm.
High Pressure Liquid Chromatography (HPLC)
Block diagram
Applications and Limitations of
HPLC

Comparison of GC and HPLC

Applications and Limitations of HPLC

• Wide array of applications

a. Detection of psychoactive drug (benzodiazepines, phenothiazine,
tricyclic antidepressant, and neuroleptic) in body fluid including
blood, urine, CSF can be done with an aid of C-18 reverse phase HPLC
column.
b. Analysis of cardiac glycosides, separation of bioactive alkaloids,
anthocynides, xanthines, isoflavones, tannin etc can be done with
highest grade of resolution with reverse phase HPLC technique using a
C-18 column.
c. HPLC can be used for study of various microbiological process of
industrial importance like HPLC controlled analysis of penicillin
production
Applications and Limitations of HPLC
a. Assay of human insulin by using a Vydac C-18 column.
b. A combination of HPLC with suitable detection technique allows an
accurate and precise identification of analyte like oestradiol,
catecholamines, opium alkaloids, aspirin, paracetamol tablets, verapamil
and its metabolites.
c. Analysis of vitamins both water and fat soluble via ion exchange HPLC.
• Compound identification may be limited unless HPLC is interfaced with other
techniques
• Resolution can be difficult to attain with complex samples
• Only one sample can be analyzed at a time
• Requires training in order to optimize operations
• Time analysis can be long
• Sample preparation required
UP NEXT…

• November 21
Supercritical Fluid Chromatography
Capillary Electrophoresis

• November 25
Unit 5. Spectroscopic methods

• November 28
Long exam, Unit 2-3

• December 2
Long exam, Unit 4-5