HPLC-COLUMNS

In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.

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 &
NBA (UG)
SIRO-DSIR
Raghavendra Institute of Pharmaceutical Education and Research -Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P-515721
2
S.NO CONTENT
1Introduction
2Types of Columns based on scale of preparation
3Typesof column based on mode of operation
4Chemistry and mechanism of RP-HPLC C18 column
5Chemistryinvolvedin RP-HPLC
6Chemistryinvolved inNP-HPLC
7Efficiency of Columns
8Van deemterequation
9Lx Table inUSP
10Reference
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
•TheHPLCcolumnsmostcommonlyusedaremadefromprecision-
borePolishedstainlesssteeltubing.
•Typicaldimensionsbeing10-30cmlongand4to5mminternal
diameter.
•Eachendwithstainlesssteelfritswithameshof2µmorless.
Introduction

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
Raghavendra Institute of Pharmaceutical Education and Research -Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P-515721
4
1.Precolumn(Guardcolumn).
•ItisshortcolumnpresentbetweenTheinjectorandanalytical
column.
•PackingcompositionofguardcolumnandAnalyticalcolumn
same.

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
Raghavendra Institute of Pharmaceutical Education and Research -Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P-515721
5
2.Analytical column.
•Thesuccessorfailureofanalysisdependsuponchoiceofcolumn.
•Actualseparationiscarriedoutbyanalyticalcolumn.
•Thecolumnisfilledwiththesmallparticles5-10micronandthesolid
supportcanbesilicagel.
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

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
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 pgto ng
Analytical nano0.075-0.1 Silica,
polymer
3 Sample size < 1 pg

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
Raghavendra Institute of Pharmaceutical Education and Research -Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P-515721
7
Analyticalcolumn:
Itisusedtoidentificationandquantificationofanalyte.
Flowrate0.5-2ml/min.
Particlesizeupto1000µg/ml.
Preparativecolumn:
Separationoflargequantityofsample.
Flowrete5-10ml/min.
Particlesize1-100mg/ml.
Types of Columns based on scale of
Preparation

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
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 phasePolar(silica gel) Non-polar (ODS)
Mobile phase Non-polar (organic
solvent)
Polar (aqueous/organic)
Sample movementNon polar fastest Polar fastest
Separation based
on
Different
polarities(functionality)
Different hydrocarbon
content
Use Long time analysis Drug analysis in QC

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
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.
Cyanotypecolumn.
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

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
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
Cyanotype: -Si-CH
2CH
2CH
2CN
Amino type: -Si-CH
2CH
2CH
2NH
2
Diol type: -Si-CH
2CH
2CH
2OCH(OH)-CH
2OH
Mobile phase: Low polarity
Hexane, cyclohexane, aromatic hydrocarbons.
Modified solvents: alcohol, ether, etc.
Normal-phase HPLC

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
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

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
Raghavendra Institute of Pharmaceutical Education and Research -Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P-515721
12
•In RP-HPLC, Strongly hydrophobicsubstances 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
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
Raghavendra Institute of Pharmaceutical Education and Research -Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P-515721
13
•In NP-HPLC, Hydrophilicsubstances 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

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
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
Equation1. Efficiencyequation
Equation2.Alternateequationforcalculatingefficiency

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO-DSIR
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 deemterequation

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
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
L1ODS-1.5 to 10µg
L2ODS-30-50µg
L3 Porous silica 1.5-10µg
L4
Silica gelcontrolled surface porosity
bonded 30-50µg
L5Alumina of controlled surface
porosity bonded 30-50µg
L7Octaylsilica bonded to porous silica
1.5-10µg
L9Totally porous silica gel bonded acidic
cation 3-10µg
L10Nitrile group bonded to porous silica
3-10µg
L11
Phenyl group bonded to porous silica
1.5-10µg
L13Trimethylsilanebonded to porous silica
3-10µg
L15 Hexylsilanebonded to porous silica 3-
10µg
L16
Dimethylsilianebonded to porous silica
5-10µg
L38Metacrylatebased size exclusive
packing.
L45 Betacyclodextrinebonded to porous
silica 5-10µg
L55 Cationic exchange resin 5µg
L59 Silica based 10µg (separation of
protein)

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
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”5
th
edition 1989 p.223.
2.Ed kim“A look at column choices” agilenttech slide, 2008.
3.Ravi sankar. “ Text book of pharmaceutical analysis”5
th
edition 2018
p.18.3-18.4
4.S. Malathi, Pallavimangeshpatil, Sunilkumar. “Thakur publication’s
instrumental methods of analysis”1
st
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