What Is HPLC?

Basic Principles

LAAQ-B-LC001B

Invention of Chromatography by
M. Tswett
Ether

Chlorophyll

Chromatography
Chromato

Colors

CaCO3

LAAQ-B-LC001B

Comparing Chromatography to the

Flow of a River...
Light leaf
Heavy stone

Water flow

Base

LAAQ-B-LC001B

Mobile Phase / Stationary Phase

A site in which a moving
phase (mobile phase) and
a non-moving phase
(stationary phase) make
contact via an interface
that is set up.
The affinity with the mobile
phase and stationary
phase varies with the
solute. Separation
occurs due to differences
in the speed of motion.

Mobile
phase
Strong
Stationary
phase

LAAQ-B-LC001B

Weak

Chromato-graphy / -graph / -gram /

-grapher
Chromatography:
Chromatograph:
Chromatogram:
Chromatographer:

LAAQ-B-LC001B

Analytical technique
Instrument
Obtained picture
Person

Three States of Matter and

Chromatography Types
Mobile phase
Gas

Liquid

Solid

Gas

Stationary
phase

Liquid
Gas
chromatography

Liquid
chromatography

Solid
LAAQ-B-LC001B

Liquid Chromatography
Chromatography

in which the mobile phase is

a liquid.
The

liquid used as the mobile phase is called

the eluent.

The

stationary phase is usually a solid or a

liquid.
In general, it is possible to analyze any
substance that can be stably dissolved in the
mobile phase.
LAAQ-B-LC001B

Interaction Between Solutes, Stationary

Phase, and Mobile Phase
Differences

in the interactions between the solutes and

stationary and mobile phases enable separation.

Solute
Degree of adsorption,
solubility, ionicity, etc.

Stationary
phase
LAAQ-B-LC001B

Mobile phase
8

Column Chromatography and

Planar Chromatography
Separation column

Paper or a
substrate coated
with particles

Packing material

Column Chromatography
LAAQ-B-LC001B

Paper Chromatography
Thin Layer Chromatography (TLC)
9

Separation Process and Chromatogram

Output
concentration

for Column Chromatography

LAAQ-B-LC001B

Chromatogram

Time

10

Intensity of detector signal

Chromatogram

tR
t0

Peak

tR : Retention time
t0 : Non-retention time

h
A

A : Peak area
h : Peak height

Time
LAAQ-B-LC001B

11

From Liquid Chromatography to High

Performance Liquid Chromatography
Higher

degree of separation!
Refinement of packing material (3 to 10 m)
Reduction of analysis time!
Delivery of eluent by pump
Demand for special equipment that can
withstand high pressures
The arrival of high performance liquid chromatography!
LAAQ-B-LC001B

12

Flow Channel Diagram for High

Performance Liquid Chromatograph

Detector
Column
Pump
Eluent
(mobile phase)

Sample injection unit

(injector)

Column oven
(thermostatic
column chamber)
Drain
Data processor

Degasser
LAAQ-B-LC001B

13

Advantages of High Performance

Liquid Chromatography
High

separation capacity, enabling the batch analysis

of multiple components
Superior quantitative capability and reproducibility
Moderate analytical conditions

Unlike GC, the sample does not need to be vaporized.

Generally

high sensitivity
Low sample consumption
Easy preparative separation and purification of
samples
LAAQ-B-LC001B

14

Fields in Which High Performance

Liquid Chromatography Is Used
Biogenic

Sugars, lipids, nucleic

acids, amino acids,
proteins, peptides, steroids,
amines, etc.

Medical

substances

Food products

Environmental samples

products

Drugs, antibiotics, etc.

Inorganic ions
Hazardous organic
substances, etc.

Organic industrial
products

LAAQ-B-LC001B

Vitamins, food additives,

sugars, organic acids,
amino acids, etc.

Synthetic polymers,
additives, surfactants, etc.
15

HPLC Hardware: Part 1

Solvent Delivery System,
Degasser, Sample Injection Unit,
Column Oven

LAAQ-B-LC001B

16

Flow Channel Diagram for HPLC

Detector
Column
Pump
Eluent
(mobile phase)

Sample injection unit

(injector)

Column Oven
(thermostatic
column chamber)
Drain
Data processor

Degasser
LAAQ-B-LC001B

17

Solvent Delivery Pump

Performance

Requirements

Capacity

to withstand high load pressures.

Pulsations that accompany pressure
fluctuations are small.
Flow rate does not fluctuate.
Solvent replacement is easy.
The flow rate setting range is wide and the
flow rate is accurate.
LAAQ-B-LC001B

18

Solvent Delivery Pump:

Representative Pumping Methods
Syringe

pump
Plunger pump
Diaphragm pump

LAAQ-B-LC001B

19

Solvent Delivery Pump:

Schematic Diagram of Plunger Pump

Motor and cam

Pump head

Check
valves

Plunger
Plunger seal
LAAQ-B-LC001B

10 -100L
20

Solvent Delivery Pump:

Single Plunger Type

Check valves

Plunger head

LAAQ-B-LC001B

21

Solvent Delivery Pump:

Dual Plunger Type
Check valves

Plunger heads

Type
LAAQ-B-LC001B

Type
22

Gradient System
Isocratic

system

Constant

Gradient
Varying

eluent composition

system
eluent composition

HPGE

(High Pressure Gradient)

LPGE (Low Pressure Gradient)

LAAQ-B-LC001B

23

Aim of Gradient System (1)

In

isocratic mode
CH3OH / H2O = 6 / 4

Long analysis time!!

Poor
separation!!

LAAQ-B-LC001B

(Column: ODS type)

CH3OH / H2O = 8 / 2

24

Aim of Gradient System (2)

If the eluent composition is changed gradually during
analysis...
Concentration of methanol in eluent

LAAQ-B-LC001B

95%

30%

25

High- / Low-Pressure Gradient System

Low-pressure
gradient unit

Mixer

High-pressure gradient
LAAQ-B-LC001B

Mixer

Low-pressure gradient
26

Advantages and Disadvantages of

High- / Low-Pressure Gradient Systems
High-pressure

gradient system

High

gradient accuracy
Complex system configuration (multiple
pumps required)
Low-pressure

gradient system

Simple

system configuration
Degasser required
LAAQ-B-LC001B

27

Degasser
Problems

caused by dissolved air in the eluent

Unstable delivery by pump

More noise and large baseline drift in detector cell

In order to avoid these problems, the eluent

must be degassed.

LAAQ-B-LC001B

28

Online Degasser
Regulator
Helium
cylinder

Polymeric film tube

Vacuum chamber

To pump

To pump
To draft
Drain valve

Eluent container

Helium purge method

LAAQ-B-LC001B

Eluent container

Gas-liquid separation membrane method

29

Sample Injection Unit (Injector)

Performance

Requirements

No

sample remaining in unit

Minimal broadening of sample band
Free adjustment of injection volume
Minimal loss
Superior durability and pressure resistance

LAAQ-B-LC001B

30

Manual Injector
From pump

To column

LOAD position
From pump

INJECT position
LAAQ-B-LC001B

To column
31

Manual Injector:
Operating Principle of Sample Injection

From pump

Loop

Loop

From pump

To column

To column

LOAD
LAAQ-B-LC001B

INJECT
32

Manual Injector:
Injection Method
Syringe

measurement method

It

is desirable that no more than half the loop

volume is injected.

Loop

measurement method

It

is desirable that at least 3 times the loop

volume is injected.

LAAQ-B-LC001B

33

Autosampler
(Pressure Injection Method)
From pump

To column

From pump

To column

Sample Loop

LAAQ-B-LC001B

LOAD

INJECT

34

Autosampler
(Total-Volume Injection Method)
From pump

To column

From pump

To column

Needle

Sample vial
Measuring pump
LAAQ-B-LC001B

LOAD

INJECT
35

Column Oven
Air

circulation heating type

Block heating type
Aluminum

Insulated
Water

LAAQ-B-LC001B

block heater

column jacket type

bath

36

Tubing and Preparation for

Solvent Delivery
Prior to Analysis

LAAQ-B-LC001B

37

Tubing
Material

Stainless steel (SUS)

PEEK (polyether
ether ketone)
Fluororesin

LAAQ-B-LC001B

O.D.

(outer diameter)

1.6 mm

I.D.

(inner diameter)

0.1 mm
0.3 mm
0.5 mm
0.8 mm etc.

38

Connectors
Male

nut (SUS)
Ferrule (SUS)

Sealing possible up to 40
MPa

Male

LAAQ-B-LC001B

Ferrule
Male nut

nut (PEEK)

Can be connected without

any tools
Resists pressures of up to
approx. 25 MPa

Male nut (PEEK)

39

Dead Volume
(Extra-column volume)
Dead

volume can cause peaks broadening.

Male nut

Dead volume

Tube

Excellent connection
LAAQ-B-LC001B

Poor connection
40

Mobile Phase
Water

Ultrapure water can be

used with confidence.
Commercial distilled
water for HPLC is also
acceptable.

Organic

LAAQ-B-LC001B

Solvent

HPLC-grade solvent can

be used with confidence.
Special-grade solvent is
acceptable depending on
the detection conditions.
Care is required regarding
solvents containing
stabilizers (e.g.,
tetrahydrofuran and
chloroform)

41

Replacement of Eluent

Mutually insoluble solvents

must not be exchanged directly.

Water
2-Propanol

Hexane
LAAQ-B-LC001B

Aqueous solutions containing

salt and organic solvents
must not be exchanged
directly.

Buffer solution

Water

Water-soluble
organic solvent

42

Mixing, Filtration, and Offline

Degassing of the Eluent

Decompression
by aspirator

Membrane filter with pore

size of approx. 0.45 m

Decompression
by aspirator
Ultrasonic
cleaning unit
LAAQ-B-LC001B

43

Reversed Phase Chromatography

Part 1
Basic Principles

LAAQ-B-LC001B

44

Polarity of Substances
Polarity

Miscibility

Property of a substance
whereby the positions of the
electrons give rise to
positive and negative poles
Water: Polar
Methane: Nonpolar

LAAQ-B-LC001B

C
H

Methane

Water

of solvents

Solvents of similar
polarities can be easily
dissolved together.
Polar and nonpolar
molecules have a similar
relationship to that of water
and oil.

H
O
H C C

O
H
Acetic acid

45

Nonpolar (Hydrophobic) Functional Groups

and Polar (Hydrophilic) Functional Groups
Nonpolar

Functional

Groups

-(CH2)nCH3
Alkyl

Polar

Functional
Groups

Carboxyl

groups

-C6H5
Phenyl

-COOH

-NH2
Amino

groups

groups

-OH
Hydroxyl

LAAQ-B-LC001B

groups

groups

46

Partition Chromatography
A liquid

(or a substance regarded as a

liquid) is used as the stationary phase,
and the solute is separated according to
whether it dissolves more readily in the
stationary or mobile phase.
Liquid-liquid chromatography

LAAQ-B-LC001B

47

Normal Phase / Reversed Phase

Stationary
phase

Mobile phase

Normal
phase

High polarity

Low polarity

(hydrophilic)

(hydrophobic)

Reversed
phase

Low polarity

High polarity

(hydrophobic)

(hydrophilic)

LAAQ-B-LC001B

48

Reversed Phase Chromatography

Stationary

phase: Low polarity

Octadecyl

Mobile

group-bonded silical gel (ODS)

phase: High polarity

Water,

methanol, acetonitrile
Salt is sometimes added.

LAAQ-B-LC001B

49

Separation Column for Reversed

Phase Chromatography
C18

(ODS) type
C8 (octyl) type
C4 (butyl) type

Si

-O-Si

Phenyl

type
TMS type
Cyano type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)
LAAQ-B-LC001B

50

Effect of Chain Length of

Stationary Phase
C8
Medium

C18 (ODS)
Strong

C4
Weak

LAAQ-B-LC001B

51

Hydrophobic Interaction
H2O

H2O

H2O
H2O

H2O

H2O

H2O

H2O
H2O

Nonpolar solute

H2O
If a nonpolar
substance is added...

H2O
H2O

H2O
H2O

H2O
H2O

Nonpolar solute
LAAQ-B-LC001B

H2O

H2O
H2O

the network is broken and...

Network of hydrogen bonds

H2O

H2O

Nonpolar stationary phase

the nonpolar substance

is pushed to a nonpolar
location.

52

Relationship Between Retention

Time and Polarity
OH

C18 (ODS)
Strong

Weak

CH3

LAAQ-B-LC001B

53

Basic Settings for Eluent Used in

Reversed Phase Mode
Water

(buffer solution) + water-soluble organic

solvent
Water-soluble organic solvent: Methanol
Acetonitrile
Tetrahydrofuran etc.
The mixing ratio of the water (buffer solution) and
organic solvent has the greatest influence on
separation.
If a buffer solution is used, its pH value is an
important separation parameter.

LAAQ-B-LC001B

54

Difference in Solute Retention Strengths

for Water and Water-Soluble Organic
Solvents
Tightly packed network

H2O

H2O

H2O
H2O

H2O

Loose network

CH3OH

H2O
H2O

CH3OH
CH3OH

CH3OH
CH3OH

Nonpolar solute

CH3OH
CH3OH

Nonpolar solute

Nonpolar stationary phase

LAAQ-B-LC001B

55

Relationship between Polarity of Eluent and

Retention Time in Reversed Phase Mode
Eluent: Methanol / Water

60/40

70/30

80/20
LAAQ-B-LC001B

56

Chromatogram Parameters
Methods for Expressing Separation
and Column Performance

LAAQ-B-LC001B

57

Strength of detector signal

Retention Factor, k

tR

t R t0
k
t0

t0

tR: Retention time

t0: Non-retention time
Time

LAAQ-B-LC001B

58

Theoretical Plate Number, N

tR
N 16
W
H
W1/2
W
LAAQ-B-LC001B

H1/2

tR
5.54
W1/ 2

tR H
Area

59

Evaluation of Column Efficiency Based on

Theoretical Plate Number

If the retention times are

the same, the peak width
is smaller for the one with
the larger theoretical plate
number.
N: Large

If the peak width is the

same, the retention time is
longer for the one with the
larger theoretical plate
number.
N: Small

N: Large

N: Small

LAAQ-B-LC001B

60

Separation Factor, a
Separation

k1

factor: Ratio of ks of two peaks

k2

k2

k1
(k 2 k1 )

LAAQ-B-LC001B

61

Resolution, RS
tR1

tR2

RS

W1/2h,1

W1/2h,2
W1

LAAQ-B-LC001B

h1/2

t R 2 t R1

1
(W1 W2 )
2
t R 2 t R1
1.18
W1/ 2 h ,1 W1/ 2 h , 2

W2
62

Resolution Required for Complete

Separation
(tR2 - tR1)

(tR2 - tR1)

W1 W2

W1 W2

tR2 - tR1 = W1 = W2

tR2 - tR1 = W1 = W2

RS = 1
LAAQ-B-LC001B

If the peaks are isosceles triangles,

they are completely separated.

RS = 1
If the peaks are Gaussian distributions,
RS > 1.5 is necessary for complete separation.

63

Relationship Between Resolution

and Other Parameters
The

resolution is a
function of the
separation factor, the
theoretical plate
number, and the
retention factor.
The separation can be
improved by improving
these 3 parameters!
LAAQ-B-LC001B

RS

t R 2 t R1

1
(W1 W2 )
2
1
1

k2
k2 1

64

 Increasing

the capacity
factor improves
separation!
A capacity factor of
around 3 to 10 is
appropriate. Exceeding
this just increases the
analysis time.

Contribution ratio for resolution

Contribution of Capacity Factor to

Resolution
1.0
0.8
0.6
0.4
0.2
0.0
0

10

15

20

Capacity factor

LAAQ-B-LC001B

65

Contribution of Theoretical Plate

Number to Resolution
The

resolution
increases in
proportion to the
square root of the
theoretical plate
number.

LAAQ-B-LC001B

66

To Improve Separation...

Before
adjustment
k increased

N increased

increased
LAAQ-B-LC001B

Eluent replaced with one

of lower elution strength.

Column replaced with one of

superior performance.
Column lengthened.
Column (packing material) replaced.
Eluent composition changed.
Column temperature changed.
67

pH Buffer Solution Used for Eluent

Selection and Preparation of
Buffer Solution

LAAQ-B-LC001B

68

Acid Dissociation Equilibrium

H+

If an acid is added...
...the equilibrium shifts to
the left to offset the
increase in H+.

HA

A- + H+

If an alkali is
added...
the equilibrium shifts
to the right to offset the
decrease in H+.
LAAQ-B-LC001B

OH-

The equilibrium always shifts

in a way that offsets changes.
69

Acid Dissociation Constant and

pH-Based Abundance Ratio
HA

A- + H+

CH3COOH

CH3COO-

The acid dissociation constant, Ka,

is defined as follows:

[A ][H ]
Ka
[HA]
[A ]
pH pK a log
[HA]

LAAQ-B-LC001B

pH log[H ]

pK log K
a
a

pKa
Relationship Between Abundance Ratio
and pH Value of Acetic Acid and Acetic Acid Ions
70

Preparing pH Buffer Solution

Use

a weak acid with a pKa value close to the

desired pH value.

Example: Preparing a buffer solution for a pH value of around

4.8.
Use acetic acid, which has a pKa value of 4.8.

Make

the concentrations of HA and A- roughly equal.

Mix an acid with its salt.

Example: Mix acetic acid and sodium acetate so that they

have the same molar concentration.

LAAQ-B-LC001B

71

Buffer Solutions Used for HPLC Eluent

Requirements

Commonly

Used Acids

Phosphoric acid
High buffering power at
pKa 2.1, 7.2, 12.3
prescribed pH.
Acetic acid
Does not adversely
pKa 4.8
affect detection.
Citric acid
Does not damage
pKa 3.1, 4.8, 6.4
column or equipment.
Concentration
Inexpensive.
If only to adjust pH, 10
mmol/L is sufficient.

LAAQ-B-LC001B

72

Characteristics of Phosphate
Buffer Solution
Advantages

Three dissociation
states
(pKa 2.1, 7.2, 12.3)
Possible

to prepare
buffer solutions of
various pH values.

Disadvantages

No volatility
Difficult

to use for
LCMS or evaporative
light scattering
detection.

No UV absorption
Inexpensive

LAAQ-B-LC001B

73

Reversed Phase Chromatography

Part 2
Consideration of Analytical
Conditions

LAAQ-B-LC001B

74

Guidelines for Setting Mobile Phase

Conditions (1)
Neutral (Nonionic) Substances
Eluent

Composition

Water

/ acetonitrile
Water / methanol
Separation

Adjustment

Changing

the mixing ratio of the water and

organic solvent
Changing the type of organic solvent
LAAQ-B-LC001B

75

pH of Eluent and Retention of Ionic

Solutes
Acidic

COOH
Increased
hydrophobicity

pH of eluent

COO
Alkaline

Increased
hydrophilicity

+
H
LAAQ-B-LC001B

76

Guidelines for Setting Mobile Phase

Conditions (2)
Acidic (Anionic) Substances
Eluent

Composition

Acidic buffer solution / acetonitrile

Acidic buffer solution / methanol

Increase retention strength by making

the eluent acidic and suppressing
ionization!

LAAQ-B-LC001B

77

Analysis of Basic Substances (1)

Problems Encountered with Alkaline Eluents

N+
H

OH

With alkaline eluents, although the

ionization of basic substances is
suppressed, and the retention
strength increases...

Si
O

OH
OH
LAAQ-B-LC001B

Si

OH
OH

silica gel dissolves in

alkalis, so the packing
material deteriorates rapidly.

OH
78

Analysis of Basic Substances (2)

Influence of Residual Silanol Groups

Basic substances interact with the

residual silanol groups, causing
delayed elution and tailing.

Si
O

Si

-O-Si-O
Residual silanol group

LAAQ-B-LC001B

Si

N+
H

79

Analysis of Basic Substances (3)

Addition of Sodium Perchlorate

ClO4

N+
H

Ion pair

Si
O

Si
LAAQ-B-LC001B

Basic substances form ion pairs with

perchlorate ions, thereby balancing the
charge and increasing the retention strength.
80

Guidelines for Setting Mobile Phase

Conditions (3)
Basic Substances (Cationic Substances)
Eluent

Composition

Acidic buffer solution containing anions with a low

charge density (e.g., perchlorate ions) / acetonitrile
As above / methanol

Making eluent acidic

Suppresses dissociation of residual silanol groups
Prevents tailing!
Adding perchlorate ions
Forms ion pairs Increases retention strength!
Suppresses tailing!
LAAQ-B-LC001B

81

Reversed Phase Ion Pair

Chromatography

Increase the retention strength by adding an ion pair

reagent with the opposite charge to the target
substance into the eluent.
Ion pair formation

Ion exchange-like effect

LAAQ-B-LC001B

Basic Substance

Ion pair formation

Ion exchange-like effect

Acidic Substance

82

Representative Ion Pair Reagents

Anionic

Compounds

Tetra-n-butylammonium

Cationic

hydroxide (TBA)

Compounds

Pentanesulfonic

acid sodium salt (C5)

Hexanesulfonic acid sodium salt (C6)
Heptanesulfonic acid sodium salt (C7)
Octanesulfonic acid sodium salt (C8)
LAAQ-B-LC001B

83

Points to Note Concerning the Use

of Ion Pairs

Selection of Ion Pair Reagent

pH of Eluent

The retention strength changes according to whether or not

ionization takes place.

Concentration of Ion Pair Reagent

In general, the retention strength increases with the length of the

alkyl chain.

In general, the retention strength increases with the ion pair

concentration, but there is an upper limit.

Proportion of Organic Solvent in Eluent

LAAQ-B-LC001B

Optimize the separation conditions by considering the type and

concentration of the ion pair reagent.
84

HPLC Separation Modes

Separation Modes Other Than
Reversed Phase Chromatography

LAAQ-B-LC001B

85

HPLC Separation Modes

Adsorption

(liquid-solid) chromatography
Partition (liquid-liquid) chromatography
Normal

phase partition chromatography

Reversed phase partition chromatography
Ion

exchange chromatography
Size exclusion chromatography

LAAQ-B-LC001B

86

Adsorption Chromatography
A solid

such as silica gel is used as the

stationary phase, and differences, mainly
in the degree of adsorption to its surface,
are used to separate the solutes.
Liquid-solid chromatography
The retention strength increases with the
hydrophilicity of the solute.
LAAQ-B-LC001B

87

Partition Chromatography
A liquid

(or a substance regarded as a

liquid) is used as the stationary phase,
and the solute is separated according to
whether it dissolves more readily in the
stationary or mobile phase.
Liquid-liquid chromatography

LAAQ-B-LC001B

88

Normal Phase and Reversed Phase

Solid phase

Mobile phase

Normal
phase

High polarity
(hydrophilic)

Low polarity
(hydrophobic)

Reversed
phase

Low polarity
(hydrophobic)

High polarity
(hydrophilic)

LAAQ-B-LC001B

89

Normal Phase (Partition)

Chromatography
Partition

chromatography in which the

stationary phase has a high polarity
(hydrophilic) and the mobile phase has a
low polarity (hydrophobic)
Essentially based on the same separation
mechanism as adsorption chromatography
in which the stationary phase has a
hydrophilic base, such as silica gel
LAAQ-B-LC001B

90

Invention of Chromatography by
M. Tswett
Ether

Chlorophyll

Chromatography
Chromato
Colors

CaCO3

LAAQ-B-LC001B

91

Stationary Phase and Mobile Phase Used

in Normal Phase Mode
Stationary

Phase

Silica gel: -Si-OH

Cyano type: -Si-CH2CH2CH2CN
Amino type: -Si-CH2CH2CH2NH2
Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH

Mobile

Phase

Basic solvents: Aliphatic hydrocarbons,

aromatic hydrocarbons, etc.
Additional solvents: Alcohols, ethers, etc.

LAAQ-B-LC001B

92

Relationship between Hydrogen Bonding

and Retention Time in Normal Phase Mode
SiOH

Strong

HO

SiOH
Weak
Very weak

OH

Steric hindrance
LAAQ-B-LC001B

93

Relationship Between Eluent Polarity and

Retention Time in Normal Phase Mode
Eluent: Hexane/methanol

100/0

98/2

95/5
LAAQ-B-LC001B

94

Comparison of Normal Phase and

Reversed Phase
Normal

LAAQ-B-LC001B

Phase

Effective for separation

of structural isomers
Offers separation
selectivity not available
with reversed phase
Stabilizes slowly and is
prone to fluctuations in
retention time
Eluents are expensive

Reversed

Phase

Wide range of applications

Effective for separation of
homologs
Stationary phase has long
service life
Stabilizes quickly
Eluents are inexpensive
and easy to use

95

Ion Exchange Chromatography

Anion exchange

Cation exchange

R
N+ R
R
SO3-

++++
+
+
++++

Electrostatic interaction
(Coulomb force)
LAAQ-B-LC001B

96

Stationary Phase Used in Ion

Exchange Mode
Base

Material

Resin is often used.

Silica gel is also used.

Cation

Exchange Column

Strong cation exchange

Week cation exchange

Anion

LAAQ-B-LC001B

-SO3-COO-

(SAX)
(WAX)

-NR3+
-NHR2+

Exchange Column

Strong anion exchange

Week anion exchange

(SCX)
(WCX)

97

Dependence of Exchange Capacity of Ion

Exchanger on pH of Eluent
Strongly basic
anion exchanger

Weakly acidic
cation exchanger
0

7
pH
Cation exchange mode

LAAQ-B-LC001B

14

Exchange capacity

Exchange capacity

Strongly acidic
cation exchanger

Weakly basic
anion exchanger
0

7
pH

14

Anion exchange mode

98

Relationship between Retention Time and

Salt Concentration of Eluent in Ion
Exchange Mode

Resin

Resin

Resin

The exchange groups

are in equilibrium with
anions in the eluent.

An eluent ion is
driven away
and a solute ion
is adsorbed.

The solute ion is driven

away by an eluent ion
and is adsorbed by the
next exchange group.

Solute ions and eluent ions compete for ion exchange groups.
If
the salt concentration of the eluent increases, the solutes are eluted sooner.
LAAQ-B-LC001B
99

Ion Exclusion Chromatography

H+

H+

H+

Strong acid ions are repelled by

charge and cannot enter the pore.
LAAQ-B-LC001B

Depending on the level of dissociation,

some weak acid ions can enter the pore.
100

Size Exclusion Chromatography

Separation

is based on the size (bulkiness)

of molecules.
The name varies with the application field!
Size

Exclusion Chromatography (SEC)

Gel Permeation Chromatography (GPC)
Chemical

industry fields, synthetic polymers,

nonaqueous systems

Gel

Filtration Chromatography (GFC)

Biochemical

fields, biological macromolecules,

aqueous systems

LAAQ-B-LC001B

101

Principle of Size Exclusion Mode

The size of the solute molecules
determines whether or not they can
enter the pores.

Packing
material

LAAQ-B-LC001B

102

Relationship Between Molecular Weight and

Retention Time in Size Exclusion Mode

Molecular weight
(logarithmic axis)

Exclusion limit
Permeability limit

Elution capacity

LAAQ-B-LC001B

103

Creating a Molecular Weight

Calibration Curve

Molecular weight
(logarithmic axis)

For separation of large molecular weights

For wide-range separation
(mix gel)

Elution capacity
For separation of small molecular weights
LAAQ-B-LC001B

104

Calculating Molecular Weights

Chromatogram

Various Average Molecular

Weights

Calibration curve

Retention time
LAAQ-B-LC001B

Mn: Number-average
molecular weight
Mw: Weight-average
molecular weight
Mz: Z-average molecular
weight, etc.

Molecular weights and

molecular weight distributions
are calculated using special
calculation software.
105

Guidelines for Selecting Separation Mode (1)

Required Information
Soluble

solvent
Molecular weight
Structural formula and chemical
properties
Do

the substances ionize?

Is there UV absorption or fluorescence?
Is derivatization possible? etc.
LAAQ-B-LC001B

106

Guidelines for Selecting Separation Mode (2)

Basic Policy
Reversed

phase mode using an ODS column is

the first choice!
Exceptions
Large molecular weight (> 2,000) Size exclusion
Optical isomers Chiral column
Stereoisomers, positional isomers Normal phase /
adsorption
Inorganic ions Ion chromatography
Sugars, amino acids, short-chain fatty acids
Special column

LAAQ-B-LC001B

107

HPLC Hardware: Part 2

Detectors and Their Ranges of Application

LAAQ-B-LC001B

108

Detection Condition Requirements

Sensitivity
The

detector must have the appropriate level of

sensitivity.

Selectivity
The

detector must be able to detect the target

substance without, if possible, detecting other
substances.

Adaptability

to separation conditions
Operability, etc.
LAAQ-B-LC001B

109

Representative HPLC Detectors

UV-VIS

absorbance detector
Photodiode array-type UV-VIS absorbance
detector
Fluorescence detector
Refractive index detector
Evaporative light scattering detector
Electrical conductivity detector
Electrochemical detector
Mass spectrometer
LAAQ-B-LC001B

110

UV-VIS Absorbance Detector

Ein

Eout

C: Concentration

Detection cell

l
A = Cl = log (Eout / Ein)

(A: absorbance, E: absorption coefficient)

LAAQ-B-LC001B

111

Optical System of UV-VIS

Absorbance Detector
Grating

Ein

Ein

Sample cell

Eout

Photodiode

Ein

Photodiode

Reference cell
D2 / W lamp

LAAQ-B-LC001B

112

Spectrum and Selection of

Detection Wavelength

The longer wavelength

is more selective.

200
LAAQ-B-LC001B

250

300
Wavelength [nm]

350
113

Optical System of Photodiode

Array Detector
Sample cell

Grating

A single photodiode
measures the absorbance for
the corresponding wavelength
at a resolution of approx. 1 nm.

D2 / W lamp

Photodiode array

LAAQ-B-LC001B

114

Data Obtained with a Photodiode

Array Detector
Spectrum

W
av
ele
ng
th

Absorbance

Chromatogram

LAAQ-B-LC001B

Retention time

115

Advantages of Photodiode Array

Detectors
Peak

Identification Using Spectra

Complementation

retention time
Library searches
Evaluation

of identification based on

of Peak Purity

Purity

evaluation performed by comparison

of the shape of spectra from the peak
detection start point to the peak detection
end point

LAAQ-B-LC001B

116

Fluorescence Detector
Excitation wavelength

+ hv1

hv2 +
Excited state

Fluorescence wavelength

Quasi-excited state

hv1

LAAQ-B-LC001B

hv2

Fluorescence
Ground state

117

Optical System of Fluorescence Detector

Xenon lamp
Fluorescence
grating
Photomultiplier tube

Fluorescence

Excitation grating

LAAQ-B-LC001B

Excitation
Sample cell
light
118

Fluorescence Derivatization Reagents

OPA Reagent

(Reacts with Primary Amines)

S-R

CHO
CHO

+ R-NH2

R-SH

N-R

o-phthalaldhyde
(OPA)

ADAM

Reagent (Reacts with Fatty Acids)

+ R-COOH
CHN2

LAAQ-B-LC001B

9-anthryldiazomethane
(ADAM)

CH2OCOR
119

Differential Refractive Index

Detector (Deflection-Type)
Light-receiving unit
Reference cell

Light
Sample cell

LAAQ-B-LC001B

120

Optical System of Differential Refractive

Index Detector (Deflection-Type)

Slit

Sample cell

W lamp

Reference cell
The slit image moves if the
refractive index inside the
flow cell changes.

Photodiode

LAAQ-B-LC001B

121

Evaporative Light Scattering Detector

Light-receiving unit
Drift tube
Nebulizer
Column eluate
Nebulizer gas
Drain

Assist gas
Light source

The column eluate is evaporated and the light scattered

by the particles of nonvolatile substances is detected.
LAAQ-B-LC001B

122

Electrical Conductivity Detector

Pure water

NaCl aqueous
solution

Cl-

The bulb does not light with water.

LAAQ-B-LC001B

Na+

The bulb lights if there are ions.

123

Principle of Electrical Conductivity Detector

V
I
A

A
L

LAAQ-B-LC001B

I A
K k
E L
L
k K
A

Electrode

K:
I:
E:
A:
L:
k:

Electrical conductivity [S]

Electric current [A]
Voltage [V]
Electrode surface area [cm2]
Distance between electrodes [cm]
Specific electrical conductivity [Scm-1]
124

Limiting Equivalent Ion Conductance,

[Scm2/mol], in Aqueous Solution (25C)

Cation
H+
Li+
Na+
K+
NH4+
(CH3)3NH+
Mg2+
Ca2+

LAAQ-B-LC001B

349.8
38.6
50.1
73.5
73.5
47.2
53.0
59.5

Anion
OH
F
Cl
Br
NO3
CH3COO
C6H5COO
SO42

198.3
55.4
76.3
78.1
71.4
40.9
32.3
80.0

125

Electrochemical Detector
Electrode

HO

HO
2eO

R
+ 2H+

O
LAAQ-B-LC001B

126

Cell Structure of Electrochemical

Detector (Amperometric Type)

Reference electrode
(Ag/AgCl)

Working electrode
(glassy carbon)

Eluent

Electrode couple
LAAQ-B-LC001B

127

Mass Spectrometer (LCMS)

Atmospheric
pressure
API probe

High vacuum
Quadrupole MS analyzer

Electron
multiplier tube

RP TMP1 TMP2
(high vacuum pumps)
LAAQ-B-LC001B

128

Atmospheric Pressure Ionization

Electrospray Ionization (ESI)
1)
C
ha
High
rg
High Voltage
Voltage
ed

+
+-+-++

E
So vap
lv or
en at
t ion

ro
pl
et

+
+

of

+
+
+-+-++
+

+
+

n
io
us
cl
Ex tion
b
a
m
or
lo
ap
ou
C
Ev
n
3)
Io

Liquid Samples
Sample
Liquid
Neburaizing
Nebulizing Gas
Gas

+
++- - + -+ +- + - +
2)

Atmospheric Pressure Chemical Ionization (APCI)

Molecular ion reaction
Liquid Sample
Liquid
Samples
Nebulizing Gas
Neburaizing
Gas
LAAQ-B-LC001B

Heater
Heater

Corona
Colona Discharge
Discharge
Needle
Nnndle

129

Advantages of LCMS (1)

Quantitative

analysis with excellent selectivity

m/z=100
A

TIC A:100

B:100
C:150 D:150

m/z=150
C

LAAQ-B-LC001B

130

Advantages of LCMS (2)

Peaks

can be identified with MS spectra.

M/Z

M/Z

LAAQ-B-LC001B

M/Z

131

Comparison of Detectors

Absorbance
Fluorescence
Differential
refractive index
Evaporative light
scattering
Electrical
conductivity
Electrochemical
LAAQ-B-LC001B

Selectivity

Sensitivity

Possibility of
Gradient System

Light-absorbing
substances

ng

Possible

Fluorescent substances

pg

Possible

None

Impossible

Nonvolatile substances

Possible

Ionic substances

ng

Partially possible

Oxidizing / reducing
substances

pg

Partially possible

Note: The above table indicates general characteristics. There are exceptions. 132

Post-Column Derivatization

Reaction
chamber
Pump
Reaction
solution
LAAQ-B-LC001B

133

Application Examples of PostColumn Methods

Amino

Acids

Orthophthalic acid, OPA

(fluorescence)
Ninhydrin (visible absorption)

Reducing

Sugars

Arginine (fluorescence)

Carbamate

LAAQ-B-LC001B

Bromate

Pesticides

Alkaline hydrolysis - OPA

(fluorescence)

Tribromide ionization
(ultraviolet absorption)
o-Dianisidine
(visible absorption)

Cyanide

Ions

Ions

Chlorination - pyrazolone
(visible absorption)

Transition

Metal Ions

4-(2-Pyridylazo)
resorcinol, PAR (visible
absorption)
134

Quantitative Analysis
Absolute Calibration Curve Method
and Internal Standard Method

LAAQ-B-LC001B

135

Qualitative Analysis
Identification

based on retention time

Acquisition of spectra with detector
UV

spectra
MS spectra
Transfer

to other analytical instruments

after preparative separation

LAAQ-B-LC001B

136

Quantitative Analysis
Quantitation

performed with peak area or

height.
Calibration curve created beforehand
using a standard.
Absolute

calibration curve method

Internal standard method
Standard addition method
LAAQ-B-LC001B

137

Calibration Curve for Absolute

Calibration Curve Method
Concentration

Area
A1

Calibration curve

C1

C3

C4
LAAQ-B-LC001B

A2

Peak area

C2

A4
A3
A2

A3
A1
A4

C1

C2
C3
Concentration

C4
138

Concentration
Target Internal
substance standard

C1

Area

A1

CIS
A2

C2

AIS

CIS
A4

C4

AIS

CIS
A3

C3

AIS

CIS

LAAQ-B-LC001B

AIS

Area for target substance / Area for internal standard

Calibration Curve for Internal

Standard Method
Calibration curve
A4 /AIS
A3 /AIS
A2 /AIS
A1/AIS

C1/CIS C2 /CIS C3 /CIS C4 /CIS

Concentration of target substance /
Concentration of internal standard

139

Advantages of Internal Standard

Method (1)
Not

affected by inconsistencies in injection volume.

X

IS

AX / AIS

10 L
injected
Same area
ratio
9 L
injected

IS

CX / CIS
LAAQ-B-LC001B

140

Advantages of Internal Standard

Method (2)

100%
recovery
rate

affected by the pretreatment recovery rate.

X

IS
AX / AIS

Not

Same area
ratio
90%
recovery
rate

IS

CX / CIS
LAAQ-B-LC001B

141

Selection Criteria for Internal Standard

It

must have similar chemical properties to the target

substance.
Its peak must appear relatively near that of the target
substance.
It must not already be contained in the actual
samples.
Its peak must be completely separated from those of
other sample components.
It must be chemically stable.
LAAQ-B-LC001B

142

Sample Pretreatment
Tasks Performed Before Injection

LAAQ-B-LC001B

143

Objectives of Pretreatment
To improve

the accuracy of quantitative

values
To improve sensitivity and selectivity
To protect and prevent the deterioration of
columns and analytical instruments
To simplify measurement operations and
procedures
To stabilize target substances
LAAQ-B-LC001B

144

Substances That Must Not Be

Injected into the Column
Insoluble

substances (e.g., microscopic

particles and precipitation)
Substances that are precipitated in the
eluent
Substances that irreversibly adsorb to the
packing material
Substances that dissolve, or chemically
react, with the packing material
LAAQ-B-LC001B

145

Filtration and Centrifugal Separation

In

general, filter every

sample before injection!
It is convenient to use a
disposable filter with a
pore diameter of approx.
0.45 m.
Centrifugal separation is
applicable for samples
that are difficult to filter.
LAAQ-B-LC001B

Filter

Syringe

146

Deproteinization
Precipitation
Addition

of organic solvent (e.g., acetonitrile)

Addition of acid (e.g., trichloroacetic acid,
perchloric acid)
Addition of heavy metal or neutral salt
Ultrafiltration

LAAQ-B-LC001B

147

Solid Phase Extraction

(1)

Conditioning

(2)
Sample addition

(3)
Rinsing

(4)
Elution
Solvent with
low elution
strength
Solvent with
high elution
strength
Target
component
Unwanted
components

LAAQ-B-LC001B

148

Pre-Column Derivatization
OPA Reagent

(Reacts with Primary Amines)

S-R

CHO
CHO

+ R-NH2

N-R

R-SH

o-phthalaldhyde
(OPA)

2,4-DNPH

(Reacts with Aldehydes and Ketones)

NHNH2
+
O2N

NO2

R
R

NHN=C
C=O

H+

O2N

NO2

2,4-dinitrophenylhydrazine
(2,4-DNPH)
LAAQ-B-LC001B

149

Evaluation of the Reliability of

Analysis
Validation of Analytical Methods

LAAQ-B-LC001B

150

What Is Validation of Analytical

Methods?
Scientifically
demonstrating that the
analytical methods
concur with the
intended purpose (i.e.,
that errors are within a
permissible range)
Evaluating required
items from the
validation
characteristics

LAAQ-B-LC001B

Validation

characteristics

Accuracy / trueness
Precision
Specificity
Detection limit
Quantitation limit
Linearity
Range
(Robustness)

151

Accuracy / Trueness
Definition

Evaluation

Degree of bias in
measurements obtained with
analytical procedures
Difference between true value
and grand mean of
measurements

True value

Method

Comparison with
theoretical values (or
authenticated values)
Comparison with results
obtained using other
analytical procedures for
which the accuracy
(trueness) is known
Recovery test

Measurement

LAAQ-B-LC001B

Average

95% confidence interval

152

Precision
Definition

LAAQ-B-LC001B

Repeatability / IntraAssay Precision

Degree of coincidence of
Precision of
series of measurements
measurements taken over
obtained by repeatedly
a short time period under
analyzing multiple samples
the same conditions
taken from a homogenous
test substance
Intermediate Precision
Variance, standard
Reproducibility
deviation, or relative
standard deviation of
measurements

153

Specificity
Definition

LAAQ-B-LC001B

The ability to accurately

analyze the target substance
in the presence of other
expected substances
The discrimination capability
of the analytical methods
Multiple analytical
procedures may be
combined in order to attain
the required level of
discrimination

Evaluation Method

Confirmation that the target

substance can be
discriminated (separated)
from co-existing
components, related
substances, decomposition
products, etc.
If reference standards for
impurities cannot be
obtained, the measurement
results for samples thought
to contain the impurities are
compared.
154

Detection Limit
Definition

The minimum quantity of

a target substance that
can be detected.
Quantitation is not
absolutely necessary.

Evaluation

Calculated from the standard

deviation of measurements
and the slope of the
calibration curve.

DL = 3.3 /slope
(: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)

Calculated from the signalto-noise ratio.

LAAQ-B-LC001B

Method

Concentration for which S/N =

3 or 2

155

Quantitation Limit
Definition

LAAQ-B-LC001B

The minimum quantity of a

target substance that can
be quantified
Quantitation with an
appropriate level of
accuracy and precision
must be possible. (In
general, the relative
standard deviation must
not exceed 10%.)

Evaluation Method

Calculated from the standard

deviation of measurements
and the slope of the
calibration curve.

QL = 10 /slope
(: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)

Calculated from the signal-tonoise ratio.

Concentration for which S/N

= 10
156

Linearity
Definition

LAAQ-B-LC001B

The ability of the analytical

method to produce
measurements for the
quantity of a target
substance that satisfy a
linear relationship.
Values produced by
converting quantities or
measurements of the
target substance using a
precisely defined formula
may be used.

Evaluation

Method

Samples containing
different quantities of the
target substance (usually 5
concentrations) are
analyzed repeatedly, and
regression equations and
correlation coefficients are
obtained.
Residuals obtained from the
regression equations of the
measurements are plotted,
and it is confirmed that
there is no specific slope.
157

Range
Definition

LAAQ-B-LC001B

The region between

the lower and upper
limits of the quantity of
a target substance that
gives appropriate
levels of accuracy and
precision

Evaluation

Method

The accuracy,
precision, and linearity
are investigated for
samples containing
quantities of a target
substance that
correspond to the lower
limit, upper limit, and
approximate center of
the range.
158

Robustness
Definition

LAAQ-B-LC001B

The ability of an
analytical procedure
to remain unaffected
by small changes in
analytical conditions.

Evaluation

Method

Some or all of the

variable factors (i.e.,
the analytical
conditions) are
changed and the
effects are evaluated.

159

Maintenance of Separation Column

Extending the Columns Service Life

LAAQ-B-LC001B

160

Silica-Based Packing Materials and

Resin-Based Packing Materials
Silica-Based

Resin-Based
Generally a wide
range

pH range

2 - 7.5

Organic
solvent

No restrictions

Significant
restrictions

Pressure
resistance

25 MPa max.

Low pressure
resistance

Temperature 60C max.

LAAQ-B-LC001B

Depends on packing
material
161

General Handling of Columns

Observe

Use as low a load

restrictions
related to solvents and
pressure as possible.
the pH range.
Do not exceed the upper
Never allow the
pressure limit.
Do not subject the column
packing material to dry.
to sudden pressure
Do not allow solids or
changes.
microscopic particles
Do not subject the
to enter the column.
Filter samples.
column to intense
shocks.
LAAQ-B-LC001B

162

Typical Problems (1)

Column Clogging
Preventive

Measures

Filter samples.
Check that samples
dissolve in the eluent.
Get in the habit of
observing pressure
values.

Corrective

LAAQ-B-LC001B

Action

Check for clogging in parts

other than the column.
Rinse with an appropriate
solvent.
Connect the column in
reverse and flush out the
insoluble substances at a
low flow rate.
Open the column end and
perform ultrasonic
cleaning of the filter.
163

Typical Problems (2)

Peak Deformation
Cause

Corrective Action

Sample overload

Reduce the sample injection volume or

concentration.

Inappropriate sample
solvent

Replace the sample solvent with one of a

low elution capacity.

Dirt

Rinse the column.

Gap in column inlet

Repair the column by supplementing it

with packing material.

Influence of secondary
retention effects

Rinse the column.

Replace the column with one that is only
minimally influenced.

LAAQ-B-LC001B

164

Typical Problems (3)

Decrease in Retention Time
Check

whether the
cause of the problem
is not the column.
Eluent composition
Eluent flow rate
Column temperature

LAAQ-B-LC001B

If

the column is
identified as the
cause...
Rinsing
Replacement

165

Typical Problems (4)

Baseline Drift
Check

whether the
cause of the problem
is not the column.

LAAQ-B-LC001B

If the problem persists

when the column is
removed, it is caused
by the eluent, the
solvent delivery system
(pump or degasser), or
the detector.

If

the column is
identified as the
cause...
Rinsing
Review of
temperature control
Replacement

166

Guard Column and Pre-column

Guard column

Pre-column

LAAQ-B-LC001B

167

Column Rinsing
Use

an eluent with a high elution capacity

Reversed phase mode: Solution with a high proportion of

organic solvent
Ion exchange mode: Solution with a high salt concentration

Consider

LAAQ-B-LC001B

secondary retention effects

To remove basic substances from a reversed phase column

Use an acidic solution and add an ion pair reagent.
To remove hydrophobic substances from an ion exchange
column Add an organic solvent.

168

Checking Column Performance

tR
N 16
W
H
W1/2
W
LAAQ-B-LC001B

H1/2

tR
5.54
W1/ 2

tR H
Area

169

Column Storage
Storage

Solution

It is generally safe to use

the same storage solution
as used at shipment.
In order to prevent
putrefaction, alcohol or
some other preservative
substance may be added.

Storage

LAAQ-B-LC001B

Conditions

Insert an airtight stopper in

the column end. Never allow
the packing material to dry.
Make a record of the storage
solution and final usage
conditions and store it
together with the column.
Store the column in a
location not subject to
shocks or sudden
temperature changes.
170