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HPLC - 2

The document provides an overview of High Performance Liquid Chromatography (HPLC), detailing the steps involved in analytical method development, sample injection systems, and the various types of columns used in HPLC. It discusses the significance of bonded phase stationary phases and outlines the applications of HPLC in analytical separation. The document emphasizes the importance of columns in the separation process and describes different column types and their characteristics.

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8 views24 pages

HPLC - 2

The document provides an overview of High Performance Liquid Chromatography (HPLC), detailing the steps involved in analytical method development, sample injection systems, and the various types of columns used in HPLC. It discusses the significance of bonded phase stationary phases and outlines the applications of HPLC in analytical separation. The document emphasizes the importance of columns in the separation process and describes different column types and their characteristics.

Uploaded by

rs7488498372
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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HIGH PERFOMANCE LIQUID

CHROMATOGRAPHY
Dr. Judy Jays

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HPLC - High Performance Liquid
Chromatography

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Lecture Objectives

By the end of this lecture, student will be able to,


• Summarize the various steps involved in analytical method
development
• Describe in detail the sample injection system
• Differentiate between the columns used in HPLC
• Discuss the significance of Bonded phase stationary phase
• Outline the applications

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Sample injection system

Septum injection port


• Syringe is used to inject the sample through a self sealing
inert septum directly into the mobile phase
• Drawback: leaching effect of the mobile phase with the septum
resulting in the formation of ghost peaks
Stop flow septumless injection
• Flow of mobile phase through the column is stopped for
a while
• Syringe is used to inject the sample
• Drawback: formation of ghost peak

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Sample injection system

Rheodyne injector /
loop valve type
• Sophisticated -
good precision
• Sample introduced
without causing
interruption to
mobile phase flow
• Volume of sample
ranges between 2
Micro volume sampling valve operation
µl to over 100 µl of a Sampling loop
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Sample injection system

• Operation of sample loop


• Sampling mode
• Injection mode
• Sample is loaded into an external loop in the micro
volume sampling valve - subsequently injected into the
mobile phase by rotation of the valve
• Auto sampler: inject continuously variable volume of
1μL – 1 mL

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Column

 Columns are the “Heart of the HPLC”

 Resolves a mixture into its different components

 Available in different material packings and sizes according to the


requirements

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Column

• Made of stainless steel or


heavy glass to withstand the
pressure
• Long (10 –30 cm),narrow
tubes- Contains stationary
phase at particle diameters of
25 µm or less
• Interior of column should be
smooth and uniform
• Column end fittings are
designed to have a zero void
volume
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Column

Column

Main column Guard column

Analytical column Preparative column

Micro preparative
Standard column
column
Narrow bore
Macro preparative
Short fast column
column 9
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Column

Classifaction -based on components


• Bonded phase column

• Column where liquid is impregnated on solid inert


support

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Column

Standard column
• Internal diameter 4 – 5 mm & length 10 – 30 cm
•Size of stationary phase is 3 – 5 µm in diameter
•Used for the estimation of drugs, metabolites,
pharmaceutical preparation and body fluids like plasma
Narrow bore column
• Internal diameter is 2 – 4 mm
• Require high pressure to propel mobile phase
• For high resolution analytical work of compounds with
very high Retention time

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Column

Short fast column


• Length 3 – 6 cm
• Used for substances which have good affinity towards
the stationery phase
• Analysis time less (1- 4 min for gradient elusion & 15 –
120 sec for isocratic elusion)
Preparative column
• Used for analytical separation i.e. to isolate or purify
sample in the range of 10-100 mg from complex mixture
• Length: 25 - 100 cm
• Internal diameter: 6 mm or more 12
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Column

• Preparative columns are of three type


• Micro preparative or semi preparative column
• Modified version of analytical column
• Uses same packaging and meant for purifying sample less then 100 mg
• Preparative column
• Inner diameter: 25 mm
• Stationary phase diameter: 15 - 100 µm
• Macro Preparative Column
• Column length: 20 - 30 cm
• Inner diameter: 600 mm

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Guard column

• They are placed anterior to the separating column


• Contains a packing chemically identical to that in analytical column
• Serve as a protective factor that prolongs the life and usefulness of
the column
• They are dependable columns designed to filter or remove
• Particles that clog the separation column
• Compounds and ions that could ultimately cause baseline drift, decrease
resolution, decrease sensitivity and create false peaks

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Bonded phase column

• Molecules, comprising the stationary phase i.e. the surface of the


silica particles, are covalently bonded to a silica based support
particles
• The most popular bonded phase, siloxanes, are formed by heating
the silica particles in dilute acid for a day so as to generate the
reactive Silanol group

OH OH OH
‫ו‬ ‫ו‬ ‫ו‬
- Si – O – Si - O - Si –
I I I

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Bonded phase column

• Silanol group is treated with


organochlorosilane
• Stable between pH range 2 – 9
and upto temperature of 80°C
• Bonded phase is made with a
linear C-18 hydrocarbon-ODS
(octadecyl silane) bonded phase
• Used in pharmaceutical analysis
or separation of less polar
components

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Bonded phase column

• Alkyl nitrile column


• Moderately polar column
• Amino alkyl column
• Polar column
• aldehydes, peptides, amino acids
Advantages
• Withstand high pressure
• Life of column is more
• No bleeding effect
Disadvantages
• Very expensive
• Cannot be fabricated manually

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Column-stationary phase on inert support

• Not use widely now days


• Stationary phase dose not have the strength to stay in the column on
account of the physical forces exerted by the mobile phase at very
high pressure
• Amount of loading on inner support is minimum
• Stationary phase starts bleeding out of the column and can cause
resistance to mass transfer

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Packing

Depends on the mechanical strength of stationary phase


• Particle size of the stationary phase
• Particles of greater then 20 µm – dry packing
• Particles of lesser then 20 µm – slurry packing / wet packing
• Dry packing
• Particle size greater then 20 µm filled into vertical clamped column in small quantity
• Deposition is done by tapping or vibrating the column
• Column is unclamped ,tapped on the firm surface to obtain dense and reproducible
packing
• Wet/Slurry Packing
• Particle size with diameter less then 20 µm can only be placed wet as a suspension
• Suspension should be stable, should not sediment, and agglomeration should be
avoided

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Summary

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Summary

• The heart of the system - column where separation


occurs- Since the stationary phase is micrometer size
particles, a high pressure pump is required to move the
mobile phase through the column
• Process begins by injecting the solute onto the top of the
column - Rheodyne valve injector
• Separation of components occurs as the analytes and mobile phase are
pumped through the column - each component elutes from the column
as a narrow band (or peak) on the recorder

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Thank you

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