The slide covers types of HPLC Columns and plate theories.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
M.MALARVANNAN. (20L81S0704).
M.PHARM
Department of Pharmaceutical Analysis.
Under the guidance/Mentorship of
Dr. P. Ramalingam., Ph.D.
Director- R&D Division,
Professor of pharmaceutical analysis
and medicinal chemistry
HPLC-COLUMNS

2. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
S.NO CONTENT
1 Introduction
2 Types of Columns based on scale of preparation
3 Types of column based on mode of operation
4 Chemistry and mechanism of RP-HPLC C18 column
5 Chemistry involved in RP-HPLC
6 Chemistry involved in NP-HPLC
7 Efficiency of Columns
8 Van deemter equation
9 Lx Table in USP
10 Reference
Content Table

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• The HPLC columns most commonly used are made from precision-
bore Polished stainless steel tubing.
• Typical dimensions being 10-30cm long and 4 to 5mm internal
diameter.
• Each end with stainless steel frits with a mesh of 2µm or less.
Introduction

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NAAC &
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
1.Pre column (Guard column).
• It is short column present between The injector and analytical
column.
• Packing composition of guard column and Analytical column
same.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
2.Analytical column.
• The success or failure of analysis depends upon choice of column.
• Actual separation is carried out by analytical column.
• The column is filled with the small particles 5-10micron and the solid
support can be silica gel.
PROPERTY GUARD COLUMN ANALYTICAL COLUMN
Particle size Large Small (5-10microns)
Length 2-10cm 25-100cm
Use Remove the impurities from the
solvent
It can protect the analytical
column
Effective separation of compounds

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AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
Different columns and uses
Column
construction
Column type Column internal
diameter (mm)
Particle
type
Particle size (µm) Use
HPLC
Stainless steel
Analytical 4.0-4.6 Silica,
polymer
1.8-10 Traditional
quantitative analysis
Analytical solvent
saver
3 Silica 1.8-5 Reduced solvent
consumption
Analytical narrow
bore
2.0-2.1 Silica 1.8-5 Reduced solvent
consumption
Analytical
microbore
1 Silica,
polymer
3-5 Increased sensitivity,
sample size ng to µg
Analytical
capillary
0.3-0.5 Silica,
polymer
3-5 Sample size pg to ng
Analytical nano 0.075-0.1 Silica,
polymer
3 Sample size < 1 pg

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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
Analytical column:
It is used to identification and quantification of analyte.
Flow rate 0.5-2ml/min.
Particle size up to 1000µg/ml.
Preparative column:
Separation of large quantity of sample.
Flow rete 5-10ml/min.
Particle size 1-100mg/ml.
Types of Columns based on scale of
Preparation

8. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Types of columns based on mode of
operation
NORMAL PHASE REVERSED PHASE
Stationary phase Polar(silica gel) Non-polar (ODS)
Mobile phase Non- polar (organic
solvent)
Polar (aqueous/organic)
Sample movement Non polar fastest Polar fastest
Separation based
on
Different
polarities(functionality)
Different hydrocarbon
content
Use Long time analysis Drug analysis in QC

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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
Stationary phase: Low polarity
Octadecyl (C18) group-bonded silica gel (ODS).
Phenyl type column.
Cyano type column.
Amino type column.
Mobile phase: High polarity
Water, methanol, acetone, acetonitrile, tetrahydrofuran etc.
Salt (or buffer) is sometime added to adjust the pH.
Reversed phase HPLC

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
Stationary phase: High polarity
Silica gel: -Si-OH
Cyano type: -Si-CH2CH2CH2CN
Amino type: -Si-CH2CH2CH2NH2
Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
Mobile phase: Low polarity
Hexane, cyclohexane, aromatic hydrocarbons.
Modified solvents: alcohol, ether, etc.
Normal-phase HPLC

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
• In partition HPLC uses liquid
bonded phase column,
where the liquid stationary
phase is chemically bonded
to the packing material.
• The packing material usually
hydrolysed silica.
• PH more then 7, The bond
will broken so it is called pH
sensitive column (3-7)
Chemistry and mechanism of
RP-HPLC C18 column

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
• In RP-HPLC, Strongly hydrophobic substances are strongly retained
by the stationary phase.
• Thus have relatively long retention times.
• The chromatogram containing multiple peaks, The substances are
generally eluted in decreasing order of polarity.
Chemistry involved in RP-HPLC

13. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
• In NP-HPLC, Hydrophilic substances are strongly retained by the
stationary phase.
• Thus have relatively long retention times.
• The chromatogram containing multiple peaks, The substances are
generally eluted in increasing order of polarity.
Chemistry involved in NP-HPLC

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NBA (UG)
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• Column efficiency is used to compare the performance of different
columns. It is probably the most frequently cited parameter of
column performance and is expressed as the theoretical plates
number. N
• Factors affecting column efficiency:
• Column length
• Particle size
• Packing quantity
• Linear viscosity(flow)
• Instrument quality
Efficiency of column
Equation 1. Efficiency equation
Equation 2. Alternate equation for calculating efficiency

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• It is evaluates the efficiency as a function of liner viscosity or
flowrate.
• HETP- Height equivalent to theoretical plate.
• L-column length, N-number of theoretical plate.
H∝L/N
Van deemter equation

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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
• It is nothing but the USP standard code for HPLC columns based on
the particle size and chemical compounds.
Lx Table in USP
L1 ODS- 1.5 to 10µg
L2 ODS- 30-50µg
L3 Porous silica 1.5-10µg
L4
Silica gel controlled surface porosity
bonded 30-50µg
L5 Alumina of controlled surface
porosity bonded 30-50µg
L7 Octayl silica bonded to porous silica
1.5-10µg
L9 Totally porous silica gel bonded acidic
cation 3-10µg
L10 Nitrile group bonded to porous silica
3-10µg
L11
Phenyl group bonded to porous silica
1.5-10µg
L13 Trimethylsilane bonded to porous silica
3-10µg
L15 Hexylsilane bonded to porous silica 3-
10µg
L16
Dimethylsiliane bonded to porous silica
5-10µg
L38 Metacrylate based size exclusive
packing.
L45 Beta cyclodextrine bonded to porous
silica 5-10µg
L55 Cationic exchange resin 5µg
L59 Silica based 10µg (separation of
protein)

17. RIPER
AUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
1. G.H.Jeffery, J.Bassett, J.Menham, R.C.Denny. “Vogel’s Textbook of
quantitative chemical analysis” 5th edition 1989 p.223.
2. Ed kim “A look at column choices” agilent tech slide, 2008.
3. Ravi sankar. “ Text book of pharmaceutical analysis” 5th edition 2018
p.18.3-18.4
4. S. Malathi, Pallavi mangesh patil, Sunilkumar. “Thakur publication’s
instrumental methods of analysis” 1st edition 2020 p.241.
5. Agilent's technologies' “The LC handbook for column and method
development” p.7,30.
6. Usama alshan. NEPHER 201 “Analytical chemistry-Ⅱ” chapter 7.
Reference