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Liquid
Chromatography
ALAKESH PRADHAN
COCHIN UNIVERSITY OF
SCIENCE AND TECHNOLOGY
School of
Industrial Fisheries
M. Sc IInd
Overview:
● Liquid chromatography
● The chromatogram.
Latest instrument
Backgroun
d
● Chromatography and its
Principle
● Chromatography is a separation technique which is used to separate a
mixture of compounds into its individual components based on
certain physical and chemical properties.
Some important terms:
● Mobile phase: The solvent system which carries the mixture to be
separated.
● Stationary phase: Immobile surface which is particulate in nature. This is the region
over which the compound gets separated.
Principl
e:The process involves the interaction of the compounds in the analyte (which travels
●
along with a mobile phase) across an immobile surface (stationary phase).
In principle, LC and HPLC work the same way except the speed ,
efficiency, sensitivity and ease of operation of HPLC is vastly
superior.
HPLC
system
Ready
inject
Rheodyne
Injector
Varian 9010 Solvent Delivery to injector
System
through pump
Column
through C
pulse
dampener
A B
from solvent to
Ternary Pump reservoir detector
Picture of HPLC
instrument
COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
Solvent
Solvent Delivery System (Pump)
Injector
Sample Column
Detectors Waste
Collector
Recorder (Data
Collection)
Instrumentation of HPLC
( Describing the 5 major components and
their functions….)
Solvent
reservoirs
and degassing
1
Not shown 2
here 5
3
1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
1.
Pump:
• The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed in
milliliters per min (mL /min).
• The injector serves to introduce the liquid sample into the flow stream of the
mobile phase.
• The injector must also be able to withstand the high pressures of the
liquid system.
• An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .
Sample
…… how is a sample actually put into an LC
system Injection
Manual Injector:
1.User manually loads sample into the injector using a syringe 2.and then
turns the handle to inject sample into the flowing mobile
phase… which transports the sample into the beginning (head) of the
column, which is at high pressure
Auto sampler:
1.User loads vials filled with sample solution into the auto sampler tray (100
samples)
2. and the auto sampler automatically
a. measures the appropriate sample volume,
b. injects the sample,
c. then flushes the injector to be ready for the next sample, etc.,
until all sample vials are processed ……
…….for unattended automatic operation
Manual Injectors Sample Loop
Load - Inject
Inject
23
Automatic Injectors
Step 1 Step 2
Step 3
24
3. Column:
•Considered the “heart of the chromatograph” the column’s stationary phase
separates the sample components of interest using various physical and chemical
parameters.
• The small particles inside the column are what cause the high back
pressure at normal flow rates.
• The pump must push hard to move the mobile phase through the
column and this resistance causes a high pressure within the
chromatograph.
Several Column Types
( can be classified
as)
● Normal phase
● Reverse phase
● Size exclusion
● Ion exchange
Normal
●phase
In this column type, the retention is governed by the
interaction of the polar parts of the stationary phase
and solute.
● For retention to occur in normal phase, the
packing must be more polar than the mobile
phase with respect to the sample
STATIONARY
PHASES
(NORMAL POLARITY)
Silica or alumina possess polar sites that
interact with polar molecules.
silica
O
Polar Group HO Si
O
28
Reverse
with respect phase
● In this column the packing material is relatively nonpolar and the solvent is polar
to the sample. Retention is the result of the interaction of the
nonpolar components of the solutes and the nonpolar stationary phase.
● Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous- organic
mixtures such as methanol-water or acetonitrile-water.
Methanol CH3OH
Acetonitrile CH3CN
Tetrahydrofuran
Water H2O
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
30
Size
●
exclusion
In size exclusion the HPLC column is consisted of
substances which have controlled pore sizes and is
able to be filtered in an ordinarily phase according to
its molecular size.
● Small molecules penetrate into the pores within the
packing while larger molecules only partially
penetrate the pores. The large molecules elute
before the smaller molecules.
STATIONARY
PHASES
(SIZE EXCLUSION)
Size exclusion gels separate on the basis of molecular size.
Individual gel beads have pores of set size, that restrict
entry to molecules of a minium size.
32
Ion
●
exchange
In this column type the sample components are
separated based upon attractive ionic forces
between molecules carrying charged groups of
opposite charge to those charges on the
stationary phase.
● Separations are made between a polar mobile
liquid, usually water containing salts or small
amounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.
STATIONARY
PHASES
(CATION EXCHANGE)
Silica is substituted with anionic residues that interact
strongly with cationic species (+ve charged)
Cations exchange Na+ silica
O
Na O S
O
+ve
+ charged species adhere to the support and
are later eluted
a with acid (H+)
34
STATIONARY
PHASES
(ANION EXCHANGE)
Silica is substituted with cationic residues that interact
strongly with anionic species (-ve charged)
Anions exchange Cl- silica
Me
Cl Me N
CH2
M
e
-ve
- charged species adhere to the support and t
are later eluted
a with acid (H+)
35
HPLC s
Column
Within the Column is where separation occurs.
Key Point –Proper choice of column is critical for success in HPLC
Packing material:
The packing material is prepared from SILICA particle, ALUMINA particle
and ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columns to
retain the packing material.
According to the mode of HPLC , they are available in different size , diameters,
pore size or they can have special materials attached ( such as antigen or
antibody ) for immuno affinity chromatography.
Modes of High Performance Liquid
Chromatography
37
Types of columns in HPLC:
● Guard Column
● Fast Column
● Preparative(i.d.
● > 4.6 mm;
● lengths 50 –250
mm)
●
Capillary(i.d.
0.1 -1.0 mm;
various lengths)
Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm) Analytical[internal
diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]
Guard
●
Column
These are placed anterior to the separating column. This serves as
protective factor.
● They are dependable columns designed to filter or remove :
Particles that clog the separation column
● Here internal diameter is same but length is short and packed with smaller
particles , that are 3 μm diameter.
● Advantages-
Increased sensitivity Decreased
analysis time Decreased mobile
phase usage Increase
reproducibility
Capillary
●
Column
It is also known as micro columns
● It has a diameter much less than a millimeter and there 3 types: Open
tubular
Partially packed Tightly packed
They allow the user to work with nanoliter sample volume , decreased flow
rate and decreased solvent usage volume , led to cost effectiveness
Preparatory
Column
● Used when objective is to prepare bulk ( milligrams) of sample for
laboratory preparatory application.
• Light Scattering
• Electrochemical
• Radioactivity
• Conductivity
45
UV-Vis Detectors
Ready
5. Computer:
• Frequently called the data system,
Chromatogram
ABS.
Reset
Wavelength
Rt Rt
ABS.
Time
Chromatogram
Varian 9060 {shows peaks, Rt}
Polychrom
Detector
What is HPLC used
for ?
Separation and analysis of non-volatile
or thermally
HPLC unstable
is optimum for compounds
the separation of chemical and biological
compounds that are non-volatile .
54
Separations
Injector
stationary
mobile phases
Mixer Stationary Phase - the phase
which remains fixed in the
column, e.g. C18, Silica
Pumps
Mobile Phase - carries the sample
through the stationary phase as
it moves through the column.
Column
Detector
Solvents
55
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
56
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
57
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
58
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
59
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
60
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
61
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
62
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
63
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
64
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
65
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
66
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
67
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
68
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
69
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
70
rations
Se p In j
Chromatogram
Mixer
a
ecto r
Pumps
Start Injection
Column
Detector
Solvents
71
The
Chromatogram
R
72
HPLC used for
Qualitative
Analysis
HPLC used for
Quantitative
Analysis
HPLC
uses
chemistry
This technique and biochemistry
is used for - research analyzing
complex mixtures
Bioscience
Chemical
Pharmaceuticals
Consumer Products
Environmental
Clinical
Referenc
●
●
es
HPLC instrumentation – Agilent Technologies
Introduction to HPLC – Agilent Technologies
● Principles and Technique of Biochemistry and Moleculer Biology –
Wilson.Keith and Walker. John
● HPLC THEORY INTRODUCTION AND INSTRUMENTATION
HARDWARE
6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical
Chemistry (Separation Science) at KCL
WEB REFERENCES
● http://192.215.107.101/ebn/942/tech/techfocus/1071main.html
● Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed.
Orlando: Harcourt Brace & Co., 1998.
● http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm http://www.che
● mistry.nmsu.edu/Instrumentation/Lqd_Chroma.html
● http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLC
HP1090/HPLCINJ.HTM
● http://test- equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/
Analyti cal_Instruments/Chromatographs/HPLC_Columns
• THANK YOU…