CHM-757

Separation Techniques
Prepared by:

Dr. Syed Tariq Ali

Outline
Classical Techniques
Column Chromatography
Flash Chromatography
Planner Chromatography

Instrumentation Techniques

High Performance Liquid Chromatography

Gas Chromatography
Amino Acid Analyzer
Counter Current Chromatography
Gel Chromatography
Electrophoresis Chromatography
Densitometry

Outline
Hardware & Software

Types of Pumps
Types of Columns
Types of Detectors
Sample Preparation Techniques
Channels & Dimensions
Coupling of Detectors

Parameters

Acquisition Parameters
Processing Parameters
Statistical Parameters
Recording Parameters
Performance parameters

Classical Techniques
Prepared by:

Dr. Syed Tariq Ali

What is Separation?

Definition
The isolation of components of mixtures into their
purest form
Purpose
Enhance the Purity
Identify
Quantify

Classical Separation Techniques

Filtration
Mechanical Separation
Floatation
Centrifugation
Simple Distillation
Fractional Distillation
Crystallization
Chromatography
6

Filtration

Purpose
To separate
Insoluble Solid
from Liquid

Mechanical Separation

Purpose
Physical separation
methods that involve
the use of tools such
as forceps and sieves,
to separate the
components of a
mixture.

Floatation

Purpose
Separation method
in which some
solids of a
suspension
mixture are
allowed to settle
and the less dense
material is poured
off.

Centrifugation

Purpose
The separation of
suspended solid from
a liquid through the
centripetal force
developed during the
rotation of the
centrifuge.

10

Simple Distillation

Purpose
This is a technique
used to separate a
mixture of a
soluble substance
and a solvent.

11

Fractional Distillation

Purpose
This is the technique
used to separate a
mixture of two miscible
liquids with different
boiling points.

12

Crystallization
Purpose
Fractional
crystallization is
done by lowering
of temperature of a
mixture or solution
so that the more
insoluble
component
crystallizes out
first. (Uses
solubility to
separate
substances)

13

Principle
of
Chromatography
Prepared by:

Dr. Syed Tariq Ali

What is Chromatography?

Collective term for a family of laboratory techniques

for the separation of mixtures
Passing a mixture dissolved in a mobile phase
through a stationary phase
The main purpose of chromatography is to separate,
Identify and quantify the target components in the
matrix

15

What is Chromatography?

History

Greek
Chromato-Graphy
Chroma means color

Graphein to write

Color - Writing
16

What is Chromatography?

The Aim of Chemical Analysis

Qualitative Analysis:
Deals with chemical composition of the sample
Quantitative Analysis:
Deals with the quantity of the compounds that are present in
the sample

Compound A (Benzene)
Compound B (Toluene)
Compound C (Xylene)
Compound D (Mesitylene) Qualitative

Analysis

Quantitative
Analysis

Compound A (10ppm)
Compound B (10pbb)
Compound C (20ppm)
Compound D (25ppm)

17

Classification
of
Chromatography
Prepared by:

Dr. Syed Tariq Ali

Classification of Chromatography

Chromatography

Chromatographic
Bed Shape

Physical State
of
Mobile Phase

Separation
Mechanism

Special Techniques

19

Classification of Chromatography

Chromatographic Bed Shape

Chromatographic Bed Shape

Planer
Chromatography

Paper
Chromatography

Column
Chromatography

Thin layer
Chromatography
Classical Column
Chromatography

Flash Column
Chromatography

20

Chromatographic Bed Shape

Classification of Chromatography

Planer Chromatography
Paper
Chromatography

Ascending
Chromatography

Descending
Chromatography

21

Paper Chromatography

Chromatographic Bed Shape

Ascending Chromatography
Lid

Clamp

Paper

Solvent Front

Sample

Mobile Phase
Chromatography jar

22

Paper Chromatography

Chromatographic Bed Shape

Descending Chromatography
Lid
Clamp

Paper
Mobile Phase

Solvent Front
Chromatography jar

23

Chromatographic Bed Shape

Classification of Chromatography

Planer Chromatography

Distance Travel
by Compound
Solvent Front

Rf = 1.00
5
4

Distance Travel
by Solvent

3
2
1

Rf = 0.82
Rf = 0.65
Rf = 0.40
Rf = 0.38
Rf = 0.00

24

Chromatographic Bed Shape

Column Chromatography

Classical Column Chromatography

25

Classification of Chromatography

Physical State of Mobile Phase

Physical State
of
Mobile Phase

Gas
Chromatography

Liquid
Chromatography

Planer
Chromatography

Column
Chromatography
26

Classification of Chromatography

Separation Mechanism
Separation Mechanism

Ion Exchange
Chromatography

Cation
Chromatography

Electrophoresis
Chromatography

Size Exclusion
Chromatography

Anion
Chromatography

27

Classification of Chromatography

Special Techniques
Special Techniques

Moving-Bed
Chromatography

Supercritical Fluid
Chromatography

Two-dimensional
Chromatography

Pyrolysis Gas
Chromatography

Chiral
Chromatography

Countercurrent
Chromatography
28

Principle of HPLC

Prepared by:

Dr. Syed Tariq Ali

Outline

Concept and scope of HPLC

Separation Mechanism of reversed phase HPLC

30

Scope of HPLC
Simultaneous Analysis
High Resolution
High Sensitivity (ppm-ppb)
Good repeatability
Small sample size
Moderate analysis condition
- no need to vaporize the sample like GC
Easy to fractionate the sample and purify
Non-destructive

31

Scope of HPLC
Field

Typical mixtures

Pharmaceuticals

Antibiotics, sedatives, steroids, analgesics, crude drugs,

cosmetics

Biochemical

Amino acids, proteins, peptides, carbohydrates, lipids,

enzymes, medicines, hormone

Food products

Mycotoxins, additives, saccharides, amino acids, vitamins,

fatty acid, coloring agents, antibacterials

Industrial chemicals

Condensed aromatics, surfactants, propellants, dyes,

polymers, plasticizers

Forensic chemistry

Drugs, poisons, blood alcohol, narcotics

Environmental field

Inorganic ions, organic acids, agricultural chemicals,

pesticides, herbicides, phenols,

Clinical medicine

Bile acids, drug metabolites, urine extracts, estrogens

32

Scope of HPLC
106

Size
exclusion

Gel
filtration

105
104

Normal
phase

103

Reversed
phase

Ion
exchange

Nonionic polar

Nonpolar

Ionic

Water-insoluble

Water-soluble

Increasing polarity

102

Molecular weight

Gel
permeation

Concept of HPLC

Flow Diagram of HPLC

Column
Injector
Pump

Detector

Oven

Mobile Phase

Data Processing

34

Mobile Phase

Prepared by:

Dr. Syed Tariq Ali

Mobile Phase Modes

Isocratic system
Gradient system

Low-pressure gradient system

High-pressure gradient system

Isocratic

Gradient
B%

B%

Time

Time
36

Elution Modes
MeOH / H2O = 6 / 4

Long Time Analysis

Volts

Bad Separation

Isocratic

MeOH / H2O = 8 / 2

Isocratic

MeOH%

Gradient

Time

( Column : ODS )

37

Isocratic System

Column
Injector
Pump

Detector

Oven

Mobile Phase
Data
processor
Simple system with one pump and one solvent reservoir.
If more than one solvent is used, solvents should be premixed.

38

Low-pressure Gradient System

Column

Injector

Detector

Oven

Pump
Low pressure
gradient valve

Data
processor

One pump used to control 4 reservoirs;

Mixing is done before pump.

On-line degasser is necessary.
A

D
39

High-pressure Gradient System

pump

Mixer

pump

Injector

Column
Oven

Detector
Data
processor

pump

Excellent gradient accuracy.

2-3 pumps required - one pump per solvent used.
On-line degassing may not be critical.
40

Low vs. High pressure Gradient Systems

LOW PRESSURE SYSTEMS
Simpler, only one pump required
On-line degasser is necessary.
Compromise on gradient accuracy
HIGH PRESSURE SYSTEMS
Excellent gradient accuracy
Complicated system
- one pump per solvent used.
Depending on sensitivity required and detector
used, on-line degassing may not be as critical.

41

Injector

Prepared by:

Dr. Syed Tariq Ali

HPLC Injectors

Manual injector

Auto injector
43

HPLC Manual Injector

How to inject sample?
Insert a syringe at INJECT position.
Turn the knob to the LOAD position.
Load the sample.
Turn the knob to the INJECT position.
Remove the syringe.
Wash the injection port.

Rheodyne Manual injector

Cautions
Do not use pointed or beveled needle tip.
Must use square end type.
Do not use more than pH 10 solution.
Must change rotor seal.
44

Column

Prepared by:

Dr. Syed Tariq Ali

Separation Modes of HPLC

Reversed phase chromatography

Normal phase chromatography

Ion chromatography

Size exclusion chromatography

Affinity chromatography

46

Understand Columns
Column is the heart of the separation

Controlling a separation means understanding and controlling

the chemistry and physics going on inside the column
Different columns can vary in plate number, band symmetry,
retention, band spacing, and lifetime.

47

Separation Mechanism
Due to different interaction between stationary phase and
different sample, the molecules move at different rate,
therefore separation can be done.
Mobile Phase

1
Stronger
interaction

Weaker
interaction

Stationary Phase

48

Reversed Phase Mode

Stationary phase: Non-polar property

Mobile phase

: Polar property

This combination is defined as

Reversed Phase Mode

49

Reversed Phase Mode

C18 (ODS) type
C8 (octyl) type

CH3

C4 (butyl) type

Si O Si C
C
HH
Ph
CH
37
318
4H
9CN
3
8
366
17

Phenyl type

CH3

TMS type

Non-polar

Cyano type

Reversed phase HPLC

Stationary phase:

Non-polar property

Mobile phase:

Polar property
50

Reversed Phase Mode

Mobile Phase
Water / buffer + Organic solvent
Organic solvents:
Methanol
Acetonitrile
THF
Buffer:
Phosphate buffer
Acetate buffer
etc
Ratio of aqueous and organic solvents is important

51

Reversed Phase Mode

Interaction
Hydrophobic Interaction
A Less polar analyte
B More polar analyte
B
A

A
A
A

Support
particle

Nonpolar
bonded phase

Interstitial area
(mobile phase)

Less polar (more hydrophobic) analytes are more attracted and

spend more time associated with the hydrophobic bonded
phase, therefore, they are eluted last.
52

Reversed Phase Mode

Hydrophobicity
If the sample has more
CH3CH2CH2--

: Carbon chain
: Aromatic group

Hydrophobicity is stronger
If the sample has more
-COOH : Carboxyl group
-NH2
: Amino group
-OH
: Hydroxyl group

Hydrophobicity is weaker
53

Reversed Phase Mode

Retention Time and Hydrophobicity

OH
1

C18 (ODS)
Strong
OH

Weak
1

54

Reversed Phase Mode

Increase of Mobile Phase Polarity

20 % H2O

30 % H2O

40% H2O
1

Solvent : MeOH

1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate
55

Reversed Phase Mode

Effect of Stationary Phase

C8
Medium

C18 (ODS)

sample
Strong

C4

sample
Weak
sample

56

Reversed Phase Mode

Effect of Stationary Phase

C18(ODS )
1

2 3

C8

C4

Peaks
1. Methyl benzoate
2. Ethyl benzoate
3. n-Propyl benzoate
4. n- Butyl benzoate

57

Reversed Phase Mode

Residual Silanol

C18
OH
C18
silica gel
OH
C18
C18

Residual Silanol

58

Reversed Phase Mode

End Capping

silica gel

C18
OH
C18
OH
C18
C18

[Non-End capping type]

TMS treatment

silica gel

C18
O-TMS
C18
O-TMS
C18
C18

[End capping type]

59

Reversed Phase Mode

Comparison of End Capping Type and

Insufficient End Capping Type

Peaks
1. Propranolol
2. Acenaphthene
3. Ammitriptyline

Shim-pack VP-ODS

Other column
60

Reversed Phase Mode

Purity - Trace Metal

M+
C18
O-TMS
M+
C18
silica core
O-TMS
M+
C18
C18

Surface metal

O
O

OH

M+

Si

Internal metal
(activated silanol)

61

Detector

Prepared by:

Dr. Syed Tariq Ali

Detectors for HPLC

Ultraviolet / Visible detector

(UV/VIS)

Photodiode Array detector

(PDA)

Fluorescence detector

(RF)

Conductivity detector

(CDD)

Refractive Index detector

(RID)

Electrochemical detector

(ECD)

Evaporative light scattering detector

(ELSD)

Mass spectrometer detector

(MS)
63

Instrumentation
of
HPLC
Customer Support Centre

TECHNOLGY LINKS (Pvt.) Ltd

Instrumentations

HPLC
Solvent delivery pumps

Sample injectors
Columns
Column ovens
Detectors

65

Flow Diagram of HPLC

Column
Injector
Pump

Detector

Oven

Mobile Phase

Data Processing

66

Fully Loaded System

Low volume degasser
DGU-20A

Solvent delivery units

-Low pulsation LC-20AD
-General purpose LC-20AT
-Binary LC-20AB

World's highest sensitivity!

High Sensitive detector
SPD-20A/20A/ M20A

Rack Changer
Oven CTO-20A/C

Fast autosampler SIL-20A/C

World'
fastest!

Controller CBM-20A / CBM card

World first!
67

Pump

Customer Support Centre

TECHNOLGY LINKS (Pvt.) Ltd

Pump

Desirable Pump Performance

High Pressure Resistance
Precise Flow Rate
Low Pressure Fluctuation

Pump

Plunger Reciprocating
out
check valve

pump head
motor and cam

5 - 50L

plunger

check valve
plunger seal

in
Mobile phase

70

Pump

Plunger Reciprocating
Consists of a small chamber in which the solvent is pumped
by the back and forth motion of a motor-driven piston
Advantage

Low pressure fluctuation

Very easy to replace other solvent
Disadvantage

Change the plunger seal

71

Pump

Dual Plunger with Parallel Flow Line

LC-20AD/AB

check valve
plunger

plunger

check valve

Very low pressure fluctuation

Refractive index detector
Conductivity detector
Electrochemical detector
MS detector

The number of maintenance

parts is more.

72

Pump

Dual Plunger with Tandem Flow Line

LC-20AT

Main
plunger

check valve

Low pressure fluctuation

UV / PDA detector
Fluorescence detector
Sub
plunger

The number of maintenance

parts is less.
So this design is suitable for
routine analysis.

73

Injector

Customer Support Centre

TECHNOLGY LINKS (Pvt.) Ltd

Injector

HPLC Manual Injector

How to inject sample
Insert a syringe at INJECT position.
Turn the knob to the LOAD position.
Load the sample.
Turn the knob to the INJECT position.
Remove the syringe.
Wash the injection port.

Rheodyne Manual injector

Cautions
Do not use pointed or beveled needle tip.
Must use square end type.
Do not use more than pH 10 solution.
Must change rotor seal.
75

Injector

HPLC Manual Injector

from Pump

to Column

LOAD
from Pump

to Column

INJECT
76

Injector

Manual injector
- Sample measurement

Partial-filling
The volume of the sample loaded is limited to half
the sample loop volume.
Complete-filling
In order to replace all the mobile phase in the loop,
excess sample (two to five loop volumes) must be
used.

77

Injector

Injection Method
Partial Injection Method
better to inject less than half volume of sample loop
Loop Injection Method

Response of Detector

better to inject more than 3 times volume of sample loop

Loop injection
Partial
injection
Half volume
of sample loop

3 times volume
of sample loop

Volume of Injection

78

Injector

Washing for Injection Port

Use of Needle Port Cleaner

79

Injector

Caution

Do not use pointed or beveled needle tip.

Must use square end type.

Do not use more than pH 10 solution.

Must change rotor seal.

80

Injector

HPLC Auto Injectors

Inside of SIL-20AC

81

Injector

Principle of Auto Injectors

Sample Aspiration

82

Injector

Principle of Auto Injectors

Start of analysis

83

Column Oven

Customer Support Centre

TECHNOLGY LINKS (Pvt.) Ltd

Column Ovens
The temperature fluctuation of column will influence retention time
reproducibility.

Column temperature control devices are functioning to keep the

column temperature constant.
Column management with CMD

Optional column management device automatically

records column usage history.

CTO-20A/20AC
85

Column

Customer Support Centre

TECHNOLGY LINKS (Pvt.) Ltd

Normal Phase Mode

Customer Support Centre

TECHNOLGY LINKS (Pvt.) Ltd

Column

Normal Phase Mode

First developer used

CaCO3 as Separation Column
Petroleum ether as Developed Solvent
We defined this combination as

Normal Phase Mode

Column
Solvent

:
:

polar property
non polar property

88

Column

Normal Phase Mode

Silica gel type :

Cyano type :
Amino type :
Diol type
:

General use
General use
Sugar analysis
Protein analysis

-Si-CH2CH2CH2CN
-Si-CH2CH2CH2NH2
-Si-CH2CH2CH2OCH(OH)-CH2(OH)

Si
Si
Silica gel

Modified
89

Normal Phase Mode

Column

Interaction

OH

Non-polar

Si-OH

Si

O
Si-OH

Hexane
OH

Silica gel (polar)

Hydrogen bonding
90

Normal Phase Mode

Column

Interaction
If the sample has
-COOH :
Carboxyl group
-NH2
: Amino group
-OH
: Hydroxyl group
Hydrogen bonding becomes strong

If the sample has bulky group

Cause Steric Hindrance
Hydrogen bonding becomes weak

91

Normal Phase Mode

Column

Retention Time
Strong
HO

SiOH

SiOH

Si

Weak
OH

Very Weak
3

or

92

Normal Phase Mode

Column

Mobile Phase Solvents

Primary solvents (non-polar)

Hydrocarbons (Pentane, Hexane, Heptane, Octane)
Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
Methylene chloride
Chloroform
Carbon tetrachloride

Secondary solvents
Methyl-t-butyl ether (MTBE), Diethyl ether, Tetrahydrofuran (THF),
Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-Propaol,
Ethanol, Methanol

A primary solvent is used as mobile phase. Addition of secondary

solvents is to adjust retention time

Solvents which possess UV transparency are often preferred for easy

detection
93

Normal Phase Mode

Column

Increase of solvent polarity

0 %EtOAc
1

2 3

2 % EtOAc

5% EtOAc

1 : Dioctyl phthalate
2 : Dibutyl phthalate
3 : Diethyl phthalate
4 : Dimethyl phthalate

Solvent : Hexane
94

Normal Phase Mode

Column

Normal phase Vs Reversed phase

Parameter

Normal Phase

Reverse Phase

Polarity of Column

High

Low

Polarity of Solvent

Low

High

Elution Sequence

Low Polarity First

High Polarity First

Increase Solvent Polarity

Faster Elution

Slower Elution

95

Ion-Pair Mode

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Ion-Pair Chromatography

Column

Interaction
O

H2O/MeOH 1:1
C4
SO3-Na+

+ Cn

C4
O

Inject

C4
Mobile Phase
C4

Si-OSiC18

Si

C4 -OH

N+

Cn

SO3-

N+

C4

O
C4
Ion-Pair Reagent
O

97

C4

Ion-Pair Chromatography

Column

How it works?

98

Ion-Pair Chromatography

Column

Reagents
Anion Compounds
Tetra-n-butylammonium hydroxide (TBA)
Cation Compounds
Butanesulfonic acid sodium salt
Pentanesulfonic acid sodium salt
Hexanesulfonic acid sodium salt
Heptanesulfonic acid sodium salt
Octanesulfonic acid sodium salt
Decanesulfonic acid sodium salt

(C4)
(C5)
(C6)
(C7)
(C8)
(C10)

99

Ion-Pair Chromatography

Column

Important Considerations
Separation Example
Benzoic acid
Column:
C18 (ODS) Column
Mobile phase:

Toluic acid

H2O/MeOH 1:1
Tetrabutyl ammonium hydroxide

100

Ion-Pair Chromatography

Column

Important Considerations
Type of Ion-Pair reagents

Concentration of Ion-Pair reagents

pH of solvent

RCOO- + H+

R-COOH

(pKa=4.5)
R-NH2 + H+

R-NH3+

(pKa=6.0)

101

Ion-Pair Chromatography

Column

Ion-Pair Chromatography
Advantages

Share several features with reversed phase HPLC column and

mobile phase

By selecting ion pair reagent, concentration, or pH, selectivity can be

improved.

Ions and neutral compounds can usually be done in the same run.

Disadvantages

Equilibration time is long

Dedicated columns are often recommended.

Beware of ion pair reagent precipitation in organic solvents, eg.

methanol, acetonitrile, etc.

Wash the column carefully after using ion pair reagent. a

Ion-Pair Chromatography

Column

Washing Column After Using TBA

If amine modifier or TBA ion pair reagent is used as a mobile
phase, the column must be washed when analysis is finished.

In order to remove these amine modifier or TBA ion pair

reagent, sodium perchlorate is effectively working. Because
sodium perchlorate has strong affinity to amine compounds.
Following solution may be recommended for column washing :
0.1% phosphoric acid including 100 mM sodium perchlorate :
methanol = 50 : 50

103

Ion-Pair Chromatography

Column

Precautions
In case of amine modifier or TBA ion pair reagent used as a mobile
phase, the column must be washed when analysis is finished
In order to remove these amine modifier or TBA ion pair reagent,
sodium

perchlorate

is

effectively

working.

Because

sodium

perchlorate has strong affinity to amine compounds

Following solution may be recommended for column washing
0.1% phosphoric acid including 100 mM sodium perchlorate :
methanol = 1 : 1

104

Ion-Exchange Mode

Customer Support Centre

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Ion Chromatography

Column

What is Ion Chromatography ?

Ion Chromatography is a type of HPLC which seperates

ions based upon ion exchange interaction.

Anion Exchange or Cation Exchange columns are used.

Conductivity detector is the most common type of
detector used, but other types of detector can be used as
well (UV-Vis detector).

Applicable to inorganic ions, small organic ions, biological

compound (protein, peptide, amino acid).

106

Ion Chromatography

Column

TYPES
Biological Fields
(Protein, Peptide, Amino Acid Analysis)
Ion chromatography
Cation Exchangers

Strong Cation Exchange

(SCX)

(R-SO3 )

Weak Cation Exchange

(WCX)

(R-COO )

Anion Exchangers
Strong Anion Exchange
Weak Anion Exchange

(SAX)
(WAX)

(R4N )
(DEAE)

DEAE=Diethylaminoethyl cellulose
107

Ion Chromatography

Column

Ion Exchange Mode (anion)

Silica/resin

NR3

Sample

Anion
exchanger

xRN(CH3)3+OH- + Ax-

[RN(CH3)3+]xAx- + xOH-

108

Ion Chromatography

Column

Ion Exchange Mode (cation)

Silica/resin

SO3

Sample

Cation
exchanger

xRSO3-H+ + Mx+

(RSO3-)xMx+ + xH+

109

Ion Chromatography

Column

Ion Exchange Interaction

Ion Exchange Interaction takes place inside the column
SO3- H+

SO3-

K+

SO3- H+

SO3- H+

Resin or gel

Resin or gel

H+
SO3- H+

SO3- H+

Potassium Ion (K+) in the sample exchange with the H+ ion, before being
displaced again by the H+ ion and elute from the column.

110

Ion Chromatography

Column

Application Example (Anions)

Analytical Conditions
Column : Shim-pack IC-A3
Mobile phase :

8.0 mM p-hydroxybenzoic acid

3.2 mM Bis-Tris *
Flow rate : 1.5 mL/min
Temperature : 40C
Injection Volume : 100 L
Peaks
1. F(1.4 ppm)
2. Cl(10200 ppm)
3. NO2(10 ppm)
4. Br(43 ppm)
5. NO3(44 ppm)
6. SO42(431 ppm)

Bis-Tris : bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane

111

Ion Chromatography

Column

Application Example (Cations)

Analytical Conditions

Column : Shim-pack IC-C3

Mobile phase : 2.0 mM Oxalic Acid

Flow rate : 1.0 mL/min

Temperature : 40C

Injection volume : 100L

Peaks

1. Na+

(8.25 ppm)

2. NH4+

(0.01 ppm)

3. K+

(1.66 ppm)

4. Mg2+

(2.22 ppm)

5. Ca2+

(11.85 ppm)

112

Ion Chromatography

Column

Protein Analysis
Analytical Conditions
Column : Shim-pack WCX-1
Mobile phase :
[A] 20 mM phosphate buffer (pH=6.0)
[B] 0.25M sodium sulfate
[A] - [B] 30 min linear gradient
Flow rate : 1.0 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. albumin
2. myoglobin
3. a-chymotrypsinogen A
4. liponuclease A
5. lisozyme

113

Ion Chromatography

Column

Important Considerations

pH of Buffer solution
Concentration of Buffer solution
Elution Method
Isocratic elution
pH gradient elution
increasing Ionic strength gradient

114

Size Exclusion Mode

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Column

Size Exclusion Mode

Types

GPC (Gel Permeation chromatography)

Mainly Polymer Science Field
GFC (Gel Filtration Chromatography)
Mainly Biological Science Field

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Column

Size Exclusion Mode

Principle
No Interaction Force
Difference of traveling Time

117

Column

Size Exclusion Mode

Elution order

SEC Column
118

Column

Size Exclusion Mode

Molecular Weight (LogMW)

Relationship between MW and RT

Exclusion limit
Permeation limit

Time

119

Column

Size Exclusion Mode

Purpose

GPC
Molecular Weight, Measurement and Distribution of Polymer

GFC
Separation of Protein

120

Column

Size Exclusion Mode

Actual Sample Injection

To calculate the MW, GPC software is required.

121

Column

Size Exclusion Mode

Protein Separation

Analytical Conditions
Column : Asahipak GFA-50
Mobile phase :
0.1 M sodium phosphate
0.1 M NaCl (pH=7.0)
Flow rate : 0.5 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. glutamate dehydrogenase
2. lactate dehydrogenase
3. enolase
4. adenylate kinase
5. cytochrome C

122

Chiral Mode

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Column

Chiral Mode

Enantiomer
Chemical properties are all the same
Physical properties are all the same
except optical rotation
H

*Chiral Center

*C

C*
COOH

HOOC

NH2

NH2

(L) form

(D) form
124

Column

Chiral Mode

Absolute Configuration
Use Cahn, Ingold, Prelog priorities
Place the lowest priority group back
(focus down C - 4 bond)
draw arrow from 1-2-3
1
1
clockwise
counterclockwise
4
4
2
2
3
3
(R)
(S)

1
OH

1
OH

4
H
HO2C
2

Assign Priority to each Group on

Asymmetric Center

CH3
3

CH3
3

(S)
Lactic Acid

H
CO2H
2
(R)

(R)(+) Thalidomide
O O
H

(S)(-) Thalidomide
O O

H
N

N
O
a sedative and hypnotic

H
N

O
a teratogen

125

H
N
O

Column

Chiral Mode

Diastereomers
Stereo-isomers That Are Not Mirror Images

H OH
3

CO2H

H OH
3

CO2H

Br H
H Br
(2S,3S)
(2S,3R)
same stereochemistry at C2 (S)
opposite stereochemistry at C3
126

Column

Chiral Mode

Chiral Chromatography
A system that contains an appropriate chiral selector
Only chiral selectors or chiral irradiation (e.g. a polarised light beam
which consists of two chiral circular-polarised components) can
distinguish between two enantiomers
Chiral selectors can be an appropriate chiral molecule or a chiral
surface
Separation of enantiomers
Gas chromatography
Supercritical fluid chromatography
Capillary electrophoresis
Chiral HPLC
127

Column

Chiral Mode

Chiral HPLC

Chiral derivatization (indirect method)

Chiral mobile phase additive (direct method)
Chiral stationary phase (direct method)

128

Column

Chiral Mode

Chiral Derivatization
Involves reaction of an enantiomeric molecule with an enantiomerically
pure chiral derivatization agent (CDA) to form two diastereomeric
derivatives.
Diastereomeric derivatization

(S) + (R)

(S)-(R)

Enantiomer

Diastereomer

(R) + (R)

(R)-(R)

Diastereomer can be separated by reversed or normal phase column.

129

Column

Chiral Mode

Chiral Mobile Phase Additive (CMPA)

In this approach, an enantiomerically pure compound is added to the

HPLC mobile phase
Reversed phase or normal phase can be used to separate transient
diastereomeric complexes between the analyte and CMPA.

130

Column

Chiral Mode

Chiral Stationary Phase (CSP)

Use a chiral substance that is chemically bonded (or coated) to a

stationary phase support to form a CSP.
CSP interacts with analytes enantiomers to form short-lived,
transient diastereomeric complexes.
The binding strength of one of these complexes is stronger than the
other, resulting in differences in retention time.

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Column

Chiral Mode

Selecting a Chiral Column

Protein
Carbohydrate
Pirkle
Cyclodextrin
etc.

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Column

Chiral Mode

Protein-derived CSP
Bovine serum albumin
Human serum albumin

1-acid glycoprotein
Ovomucoid
Cellobiohydrolase
Pepsin

Some of most versatile stationary phase for separating enantiomers

are immobilized proteins.
These proteins are covalently bonded to silica or polymeric support
Compatible with buffered aqueous/organic mobile phase
Limited sample capacity
Relatively high column cost
133

Column

Chiral Mode

Mechanism of Chiral Separation

At pH values used with most protein stationary phase (3-7)
When hydrophobic interactions dominate
An increase in pH will tend to increase retention for bases, and
decrease retention for acids
When electrostatic interactions dominate
An increase in pH will tend to increase retention for acids, and
decrease retention for bases
So it is difficult to predict how pH will affect the retention of a given
sample (acid or base) on a protein stationary phase.
134

Column

Chiral Mode

Donor-Acceptor (Pirkle) CSP

Introduced by Regis Tech & Prof. William Pirkle in 1980

Pirkle columns are able to resolve enantiomers based on

preferential binding for one enantiomer to the CSP (forming a
diastereomeric complex) through a combination of - bonding,
hydrogen bonding, steric interaction, and dipole interaction.
Applied to a wide variety of compound groups, e.g. Aryl propionic,

Acid non-sterodial, Anti-inflammatory drugs

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Column

Chiral Mode

Derivatization
Derivatization with an achiral reagent often enhances enantiomeric
separation on Pirkle columns.
The most easily derivatizable groups are alcohols, amines, and
acids.
The drawbacks to derivatization are increased method development
time and the additional time needed for the derivatization reaction.
Common derivatizating reagents include N-imidazole-N-carbonic
acid-3,5-dinitroanilide, alpha-naphthylisocyanate, 3,5-dinitobenzoyl
chloride, 2-naphthoyl chloride, and 3,5-dinitroaniline, etc.
136

Column

Chiral Mode

Cavity Type CSP

A cavity type CSP allows inclusion of one or both enantiomers

into a chiral cavity, thereby providing enhanced chiral
discrimination between the two enantiomers.
Most cavity type CSP are cyclodextrin (CD).
synthetic chiral polymer can be used too.

137

Column

Selection
Mode

Solvent type used

Compound type

Reverse Phase

H2O/Buffer, ACN, MeOH

Neutral or Non-ionized
Compounds soluble in
water/organic mixtures

Ion-Pair RP

H2O/Buffer, ACN, MeOH

Ionic or Ionizable compounds

With addition of Ion-Pair

Normal Phase

Hexane, CH2Cl2

Mixtures of isomers and

compounds not soluble
in organic/water mixter

Ion Exchange

H2O/Buffer

Inorganic ions, Proteins

Nucleic Acid
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Column

Selection
Mode

Solvent type used

Compound type

GPC/GFC

H2O, THF, DMF, etc.

High Molecular Weight

Polymer, Sugars, Protein

Chiral

Organic/Water Mixtures

Optical Isomer

For reversed phase analysis

10-30 mM triethylamine may be added to suppress tailing of basic
compounds
1% acetic acid to suppress tailing of acidic compounds
Triethylamine is also used in normal phase analysis to partially deactivate
silica gel to give more consistent retention time
139

Column

Polarity of Common
Organic Functional Groups & Solvents
Functional groups

Organic solvents
Non-polar

Aliphatic hydrocarbons
Olfins
Aromatic hydrocarbons
Halids
Sulfides
Ethers
Nitro compounds
Esters, Aldehydes, Ketones
Alcohols, amine
Sulfones
Sulfoxides
Amides
Polar
Carboxylic acids

Hexane
Carbon tetrachloride
Ether
Benzene
Methylene chloride
THF
Iso-propanol
Chloroform
Ethyl acetate
Acetonitrile
Methanol
Water

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Column

Column Dimension
Type

Inner diameter
(cm)

Length
(cm)

Particle size
(m)

Analytical

0.3 - 0.46

3 - 25

3 - 10

Semimicro

0.1 0.21

3 25

38

Semipreparative

0.8 1.0

10 - 25

5 - 10

Preparative

2.0 5.0

10 25

10 - 20
141

Detector

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Detector

Detectors for HPLC

UV-VIS

Ultraviolet / Visible detector

PDA

Photodiode Array detector

RF

Fluorescence detector

CDD

Conductivity detector

RID

Refractive Index detector

ECD

Electrochemical detector

ELSD

Evaporative light scattering detector

MS

Mass spectrometer detector

143

Detector

Ultraviolet / Visible Detector

Grating

Sample Cell

M1
Ein

Eout

Ein

Eout

Photodiode

M2
Photodiode

Reference Cell
Light source
D2 / W lamp
144

Detector

Ultraviolet / Visible Detector

Lambert-Beers Law

Absorbance

A = C L = - log (Eout / Ein)

Ideal

2.5

Actual

A : absorbance
: molar absorptivity
C : analyte concentration
L : path length of the flow cell
E : energy

Backgroud absorbance
Concentration
145

Detector

Ultraviolet / Visible Detector

How to select wavelength
275 nm

208 nm

275 nm

Which one is
better???

Chromatogram obtained by
UV-Vis detector at 275nm.

208 nm

UV-Vis Spectrum
The compound has two absorbance
bands at 208nm & 275nm.

Chromatogram obtained by
UV-Vis detector at 208nm.
146

Detector

Additional Functions

Dual Wavelength mode

Ratio Plot mode

Wavelength Time Program mode

Wavelength Scan mode

147

Detector

Dual Wavelength Mode

148

Detector

Ratio Plot Mode

149

Detector

Wavelength Scan Mode

150

Detector

Ultraviolet / Visible Detector

Advantage:
Sensitivity is high
Relative robust to temperature and flow rate change
Compatible with gradient elution
Disadvantage:

Only compounds with UV or visible absorption could be

detected.
Additional Functions:

Dual Wavelength mode

Wavelength Time Program mode
Wavelength Scan mode
151

Detector

Photodiode Array Detector

Sample Cell

D2 / W lamp

Grating

One element detects

one absorbance at
one wavelength.

512 Elements Photodiode Array

152

Detector

Photodiode Array Detector

3-D Data
Spectrum

Absorbance

Chromatogram

Time

153

Detector

154

Detector

Example of PDA Application

- Checking Peak Purity
UV-Vis Spectrum of
Acetyl Salicylic Acid

Sample
Standard

155

Detector

PDA Detector
Advantages:
PDA Detector could analyze a sample simultaneously at
many different wavelengths.
UV Visible spectra are useful for compound identification,
checking peak purity, as well as finding the optimum
absorbance for the compounds.
UV Visible spectra of many compounds could be stored in the
spectrum libraries, which are useful for compound
identification.
Relatively robust to temperature and flow rate fluctuations
Compatible with gradient elution.
Disadvantages:
Slightly less sensitive than UV-Visible detector.
156

Detector

UV/Vis Absorbance Detectors

Light sources: Deuterium or tungsten filament sources

The Variable Wavelength UV

Detector uses a monochromator
(slits and a grating) to select one
wavelength of light to pass through
the sample cell.

The Photodiode Array Detector

passes all wavelengths of light
through the sample cell, then focuses
each wavelength on a single sensor
element.
157

Detector

Fluorescence detector
Excitation Wavelength

+ hn1

hn2+

A*

Emission Wavelength

hn2

hn1
A

Fluorescence
A
158

Detector

Cell Design

159

Detector

Derivatization Reagents
OPA reagent for primary amines

CHO
+ R-NH2
CHO

N-R

o-phthalaldhyde
(OPA)

ADAM reagent for fatty acids

+ R-COOH
CHN2

CH2OCOR

9-anthryldiazomethane
(ADAM)
160

Detector

Refractive Index detector

Optical System

161

Detector

Refractive Index detector

162

Detector

Conductivity detector
K (conductivity) = I [A] / E [V]
=A [cm2] / L [cm] * k
(k : specific conductivity)

V
I

k= (I/E)*(L/A)

L
electrode

163

Detector

Conductivity detector

Conductivity is very affected by temperature.

Should keep the cell within the temperature control device.

164

Detector

Electrochemical Detector

Working
electrode
Reference
electrode

AUX electrode

165

Detector

Principal of ECD Detection

O + H+

R
e-

Electrode
Glassy Carbon (GC)
Pt, Ag, Au

[ Applications ]
GC : Phenol compounds
general use
Pt : H2O2
Ag : Halogen ion
Au : Sugar analysis

166

Peak Resolution Parameters

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Peak Resolution Parameters

Chromatogram

Signal

tR
Peak

tR : Retention time
A : Area

h
A

h : Height

Time

168

Peak Resolution Parameters

Chromatogram

169

Peak Resolution Parameters

Peak Resolution Parameters

Resolution (R or Rs) value for complete separation of two
neighboring peaks should be >1.5.

170

Peak Resolution Parameters

Peak Resolution Parameters

Rs = 1.0

Rs = 1.50
Peak shape is not triangle
but Gaussian distribution.
171

Peak Resolution Parameters

Factors

Capacity factor (k)

Selectivity ()
Column efficiency (N)

1
R

k'
N

k '1

N : average of N1 and N2
k' : average of k'1 and k'2
172

Peak Resolution Parameters

Capacity Factor, k
k =

t1 - t0
t0

t1
t0
t1 = retention time of a solute peak
t0 = column void time solvent peak
(non retained peak)
173

Peak Resolution Parameters

Selectivity,

k2
k1

t2 - t0
t1 - t0

t1

t2

t0
Selectivity is an indication of the degree of
separation between two peaks
174

Peak Resolution Parameters

The Number of Theoretical Plate, N

Rt

Equation :
N = 16 x ( Rt / W )2
Area

H
W1/2
H1/2

Modified equation for

actual measurement :
N = 5.54 x ( Rt / W 1/2 )2
Modified equation for
integrator :
N = 6.28 x (Rt x H / Area)2

175

Peak Resolution Parameters

Contribution of Capacity
1
1

R 4

k'

N
k'1

k'
1
2
3
4
10
20
50
100

(k'/k'+1)
1/2
2/3
3/4
4/5
10/11
20/21
50/51
100/101

contribution
0.500
0.667
0.750
0.800
0.909
0.952
0.980
0.990
176

Peak Resolution Parameters

Contribution of Selectivity
1
1

R 4

k'

N
k'1

1
2
3
10
13
17
20

( -1/)
0/1
1/2
2/3
10/11
12/13
16/17
19/200

contribution
0.000
0.500
0.667
0.909
0.923
0.941
0.950
177

Peak Resolution Parameters

Contribution of Efficiency
1
1

R 4

k'

N
k'1

N
8000
10000
15000
20000
30000

1/2

(N)
89
100
122
141
173

contribution
--0.12
0.37
0.58
0.94

178

Peak Resolution Parameters

How to increase N ?

Van Deemter Equation

Optimum flow rate

H = Length of Column / N

Each column has a

optimum flow rate

Linear velocity

179

Peak Resolution Parameters

Optimum Flow Rate

To get a good column efficiency,

4.0 mmID
4.6 mmID
6.0 mmID

0.6 mL/min
0.8 mL/min
1.0 mL/min

These setting are preferable

180

Peak Resolution Parameters

Smaller Particle Size

3 mm
5 mm

Using smaller
particle size!
10 mm
Flow rate

4.0 mmID
4.6 mmID
6.0 mmID

0.6 mL/min
0.8 mL/min
1.0 mL/min
181

Peak Resolution Parameters

How to increase k ?
By decreasing % of organic solvent

k<2
Insufficient separation
k > 10
Long running time
Broad band, cause
(1) low sensitivity
(2) poor accuracy

Rs

0 2 4 6 8 10

k= 2 - 10 is preferable.

2- 10
1- 20

0 2 4 6 8 10 12 14 16 18 20

182

Peak Resolution Parameters

Solvent Optimization
100% MeOH
0.1<K<0.3

80% MeOH
0.6<K<1.7

MeOH / water mixtures for a mobile phase

sample component: nitorbenzene, benzene, 2,6-dinitrotoluene,
2-nitrotoluene, 4-nitrotoluene, 3-nitrotoluene, toluene, 2-nitro-1,3xylene
4-nitro-1,3-xylene, m-xylene
183

Peak Resolution Parameters

How to improve ?

By changing
mobile phase and composition
pH
concentration of buffer
column temperature
packing material (to C8, CN and Phenyl)

184

Peak Resolution Parameters

Change of Mobile Phase

a

[ MeOH/H2O ]
critical pair
: c,d

[ MeOH/THF/H2O ]
a

c
b

[ THF/H2O ]
critical pair
: a,b

c
b

Although one organic solvent cannot provide good

resolution, 3 combination of mobile phase has a possibility
to provide good resolution, if critical pair of resolution has
been changed.
185

Peak Resolution Parameters

Effect of mobile phase pH

1:benzoic acid
2:sorbic acid
3:methylparaben

[analytical conditions]
1mL/min
ODS column
10mM phosphate
buffer 75%
Acetonitrile 25%
40oC
UV-240 nm
186

Peak Resolution Parameters

Relationship Between k and pH

k of organic acid and amines varies with pH of mobile phase.

R-COOH
associated type

H+

RCOO- +

(pKa=4.5)
R-NH3+

R-NH2 + H+
(pKa=6.0)

pKa
dissociated type

pH (mobile phase)

In the range of pKa +/- 1.5, a linear

relationship between k and pH
exists.

187

Peak Resolution Parameters

Precaution of pH Adjustment
associated type

dissociated type
pH (mobile phase)
When pH of mobile phase is very close to pKa of target sample, pH
adjustment must be very careful, since pH of mobile phase will
influence k.
Normally, pH which does not affect k should be selected.
188