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28/05/2015

Kimia Analisis Modern

HPLC

Tujuan Perkuliahan
Memahami prinsip pemisahan menggunakan
HPLC
Mengetahui instrument-instrument dalam
HPLC
Memahami prinsip pemilihan metode dalam
HPLC
Mampu merancang suatu penelitian
menggunakan instrument HPLC

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Basic Principles of HPLC


HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution. HPLC instruments
consist of a reservoir of mobile phase, a pump, an injector, a
separation column, and a detector.

Compounds are separated by injecting a sample mixture onto


the column. The different component in the mixture pass
through the column at differentiates due to differences in their
partition behavior between the mobile phase and the stationary
phase. The mobile phase must be degassed to eliminate the
formation of air bubbles.

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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!

Flow Channel Diagram for High Performance


Liquid Chromatograph

Detector

Column

Pump Column oven


(thermostatic column
chamber)
Eluent Sample injection unit Drain
(mobile phase) (injector)
Data processor
Degasser

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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

Fields in Which High Performance


Liquid Chromatography Is Used
Food products
Biogenic substances Vitamins, food additives,
sugars, organic acids, amino
Sugars, lipids, nucleic acids, acids, etc.
amino acids, proteins,
peptides, steroids, amines, etc. Environmental samples
Inorganic ions
Medical products Hazardous organic substances,
Drugs, antibiotics, etc. etc.
Organic industrial
products
Synthetic polymers, additives,
surfactants, etc.

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HPLC Hardware: Part 1

Solvent Delivery System, Degasser,


Sample Injection Unit, Column Oven

Flow Channel Diagram for HPLC

Detector

Column

Pump Column Oven


(thermostatic column
chamber)
Eluent Sample injection unit Drain
(mobile phase) (injector)
Data processor
Degasser

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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.

11

Solvent Delivery Pump:


Representative Pumping Methods
Syringe pump
Plunger pump
Diaphragm pump

12

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Solvent Delivery Pump:


Schematic Diagram of Plunger Pump

Pump head
Motor and cam

Check
valves

Plunger
Plunger seal 10 -100L

13

Solvent Delivery Pump:


Single Plunger Type

Check valves

Plunger head

14

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Solvent Delivery Pump:


Dual Plunger Type
Check valves

Plunger heads

Type Type
15

Gradient System
Isocratic system
Constant eluent composition
Gradient system
Varying eluent composition
HPGE (High Pressure Gradient)
LPGE (Low Pressure Gradient)

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Aim of Gradient System (1)

In isocratic mode

CH3OH / H2O = 6 / 4
Long analysis time!!

Poor CH3OH / H2O = 8 / 2


separation!!

(Column: ODS type) 17

Aim of Gradient System (2)

If the eluent composition is changed gradually during analysis...

95%
Concentration of methanol in eluent

30%

18

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High- / Low-Pressure Gradient System

Low-pressure
gradient unit

Mixer
Mixer

High-pressure gradient Low-pressure gradient


19

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

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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.

21

Online Degasser

Regulator Vacuum chamber


Helium Polymeric film tube
cylinder

To pump
To pump
To draft

Drain valve

Eluent container Eluent container

Helium purge method Gas-liquid separation membrane method

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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

23

Manual Injector

From pump

To column
LOAD position
From pump

To column
INJECT position
24

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Manual Injector:
Operating Principle of Sample Injection

From pump From pump


Loop

Loop
To column
To column

LOAD INJECT

25

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.

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Autosampler
(Pressure Injection Method)

From pump To column From pump To column

Sample Loop

LOAD 27
INJECT

Autosampler
(Total-Volume Injection Method)
From pump To column From pump To column

Needle

Sample vial
LOAD INJECT
Measuring pump
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Column Oven
Air circulation heating type
Block heating type
Aluminum block heater
Insulated column jacket type
Water bath

29

Tubing and Preparation for


Solvent Delivery
Prior to Analysis

30

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Tubing
Material
Stainless steel (SUS) O.D. (outer diameter)
PEEK (polyether ether 1.6 mm
ketone) I.D. (inner diameter)
Fluororesin 0.1 mm
0.3 mm
0.5 mm
0.8 mm etc.

31

Connectors

Male nut (SUS) Ferrule


Ferrule (SUS)
Sealing possible up to 40
MPa Male nut

Male nut (PEEK)


Can be connected without
any tools Male nut (PEEK)
Resists pressures of up to
approx. 25 MPa

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Dead Volume
(Extra-column volume)

Dead volume can cause peaks broadening.

Male nut Dead volume

Tube

Excellent connection Poor connection


33

Mobile Phase
Water Organic Solvent
Ultrapure water can be HPLC-grade solvent can be
used with confidence. used with confidence.
Commercial distilled water Special-grade solvent is
for HPLC is also acceptable. acceptable depending on the
detection conditions.
Care is required regarding
solvents containing stabilizers
(e.g., tetrahydrofuran and
chloroform)

34

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Replacement of Eluent
Aqueous solutions containing salt
Mutually insoluble solvents must and organic solvents must not be
not be exchanged directly. exchanged directly.

Water Buffer solution

2-Propanol Water

Hexane Water-soluble
35 organic solvent

Mixing, Filtration, and Offline


Degassing of the Eluent

Decompression Membrane filter with pore


by aspirator size of approx. 0.45 m

Decompression
by aspirator

Ultrasonic
cleaning unit

36

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Reversed Phase Chromatography


Part 1
Basic Principles

37

Polarity of Substances

Polarity Miscibility of solvents


Property of a substance Solvents of similar polarities
whereby the positions of the can be easily dissolved
electrons give rise to positive together.
and negative poles Polar and nonpolar molecules
Water: Polar have a similar relationship to
Methane: Nonpolar that of water and oil.

H
H O
O
C H C C
H H O

H + H
H
H
Methane Water
38 Acetic acid

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Nonpolar (Hydrophobic) Functional Groups and Polar


(Hydrophilic) Functional Groups

Nonpolar Functional Polar Functional Groups


Groups -COOH
-(CH2)nCH3 Carboxyl groups
Alkyl groups -NH2
-C6H5 Amino groups
Phenyl groups -OH
Hydroxyl groups

39

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

40

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Normal Phase / Reversed Phase

Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity


phase (hydrophobic) (hydrophilic)

41

Reversed Phase Chromatography


Stationary phase: Low polarity
Octadecyl group-bonded silical gel (ODS)
Mobile phase: High polarity
Water, methanol, acetonitrile
Salt is sometimes added.

42

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Separation Column for Reversed Phase


Chromatography
C18 (ODS) type Phenyl type
C8 (octyl) type TMS type
C4 (butyl) type Cyano type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2


Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)

43

Effect of Chain Length of Stationary


Phase

C8

Medium
C18 (ODS)

Strong C4

Weak

44

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Hydrophobic Interaction

H2O H2O H2O


H2O
H2O
H2O Nonpolar solute H2O
H2O
H2O If a nonpolar H2O
H2O H2O substance is added... H2O H2O
Network of hydrogen bonds the network is broken and...

H2O H2O
H2O
H2O H2O the nonpolar substance
H2O H2O is pushed to a nonpolar
location.
Nonpolar solute

Nonpolar stationary
45 phase

Relationship Between Retention Time


and Polarity

C18 (ODS) OH

Weak
Strong
CH3

46

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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.

47

Difference in Solute Retention Strengths for Water


and Water-Soluble Organic Solvents

Tightly packed network Loose network

H2O H2O CH3OH CH3OH


H2O H2O
H2O CH3OH
H2O H2O CH3OH CH3OH

CH3OH
Nonpolar solute CH3OH
Nonpolar solute

Nonpolar stationary phase


48

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Relationship between Polarity of Eluent and


Retention Time in Reversed Phase Mode

Eluent: Methanol / Water

60/40

70/30

80/20
49

Chromatogram Parameters

Methods for Expressing Separation and


Column Performance

50

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Retention Factor, k

t R t0
Strength of detector signal

tR
k
t0
t0
tR: Retention time
t0: Non-retention time

Time

51

Theoretical Plate Number, N

2
t
N 16 R
W
2
t
5.54 R
H W1/ 2
W1/2
tR H
2
H1/2
2
W Area
52

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Evaluation of Column Efficiency Based on


Theoretical Plate Number

If the retention times are the If the peak width is the same,
same, the peak width is the retention time is longer
smaller for the one with the for the one with the larger
larger theoretical plate theoretical plate number.
number.
N: Large N: Small
N: Large

N: Small

53

Separation Factor, a
Separation factor: Ratio of ks of two peaks

k1 k2 k2

k1
(k 2 k1 )

54

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Resolution, RS

tR1 tR2

t R 2 t R1
RS
1
(W1 W2 )
2
t R 2 t R1
1.18
W1/ 2 h ,1 W1/ 2 h , 2
W1/2h,1 W1/2h,2 h1/2

W1 W2
55

Resolution Required for Complete


Separation
(tR2 - tR1) (tR2 - tR1)

W1 W2 W1 W2
tR2 - tR1 = W1 = W2 tR2 - tR1 = W1 = W2
RS = 1 RS = 1
If the peaks are isosceles triangles, If the peaks are Gaussian distributions,
they are completely separated. RS > 1.5 is necessary for complete separation.
56

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Relationship Between Resolution


and Other Parameters

The resolution is a
t t
function of the RS R 2 R1
separation factor, the 1
(W1 W2 )
theoretical plate number, 2
and the retention factor.
1 1 k2
The separation can be N
4 k2 1
improved by improving
these 3 parameters!

57

Contribution of Capacity Factor to


Resolution

Increasing the capacity 1.0


Contribution ratio for resolution

factor improves 0.8


separation!
0.6
A capacity factor of
around 3 to 10 is 0.4

appropriate. Exceeding 0.2


this just increases the 0.0
analysis time. 0 5 10 15 20
Capacity factor

58

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Contribution of Theoretical Plate


Number to Resolution

The resolution

Contribution factor for resolution


2,0
increases in
proportion to the
square root of the 1,0
theoretical plate
number.
0,0
0 10000 20000 30000
Theoretical plate number

59

To Improve Separation...

Before
adjustment

k increased Eluent replaced with one


of lower elution strength.

Column replaced with one of


N increased superior performance.
Column lengthened.

Column (packing material) replaced.


increased Eluent composition changed.
Column temperature changed.
60

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pH Buffer Solution Used for


Eluent
Selection and Preparation of Buffer
Solution

61

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 always shifts
the equilibrium shifts to in a way that offsets changes.
the right to offset the OH-
decrease in H+.
62

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Acid Dissociation Constant and pH-


Based Abundance Ratio
1,0
HA A - + H+
CH3COOH CH3COO-
0,8
The acid dissociation constant, Ka,

Abundance ratio
is defined as follows:
0,6
[A ][H ]
Ka 0,4
[HA]
[A ] 0,2
pH pK a log
[HA] 0,0
1 2 3 4 5 6 7 8 9
pH
pH log[ H ]
pKa

pK log K Relationship Between Abundance Ratio
a a and pH Value of Acetic Acid and Acetic Acid Ions
63

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.

64

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Buffer Solutions Used for HPLC Eluent

Requirements Commonly Used Acids


High buffering power at Phosphoric acid
prescribed pH. pKa 2.1, 7.2, 12.3

Does not adversely affect Acetic acid


pKa 4.8
detection.
Citric acid
Does not damage column pKa 3.1, 4.8, 6.4
or equipment.
Concentration
Inexpensive.
If only to adjust pH, 10
mmol/L is sufficient.

65

Characteristics of Phosphate Buffer


Solution
Advantages Disadvantages
Three dissociation states No volatility
(pKa 2.1, 7.2, 12.3) Difficult to use for LCMS
Possible to prepare buffer or evaporative light
solutions of various pH scattering detection.
values.
No UV absorption
Inexpensive

66

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Reversed Phase Chromatography


Part 2
Consideration of Analytical
Conditions

67

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

68

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pH of Eluent and Retention of Ionic


Solutes

COOH
Acidic
Increased
hydrophobicity
pH of eluent

COO
Alkaline
decreased
hydrophilicity
+
H
69

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!

70

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Analysis of Basic Substances (1)


Problems Encountered with Alkaline Eluents

With alkaline eluents, although the


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

OH Si
O

OH Si OH
silica gel dissolves in alkalis, so
the packing material deteriorates
OH rapidly.
OH
OH
71

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 N+
O H
Si

72

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Analysis of Basic Substances (3)


Addition of Sodium Perchlorate

ClO4 Ion pair


N+
H

Si
O

Si Basic substances form ion pairs with perchlorate ions,


thereby balancing the charge and increasing the
retention strength.

73

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!
74

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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 pair formation

Ion exchange-like effect Ion exchange-like effect

Basic Substance Acidic Substance 75

Representative Ion Pair Reagents


Anionic Compounds
Tetra-n-butylammonium hydroxide (TBA)
Cationic Compounds
Pentanesulfonic acid sodium salt (C5)
Hexanesulfonic acid sodium salt (C6)
Heptanesulfonic acid sodium salt (C7)
Octanesulfonic acid sodium salt (C8)

76

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Points to Note Concerning the Use of


Ion Pairs
Selection of Ion Pair Reagent
In general, the retention strength increases with the length of the
alkyl chain.
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 ion pair
concentration, but there is an upper limit.
Proportion of Organic Solvent in Eluent
Optimize the separation conditions by considering the type and
concentration of the ion pair reagent.

77

HPLC Separation Modes

Separation Modes Other Than


Reversed Phase Chromatography

78

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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

79

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.

80

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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

81

Normal Phase and Reversed Phase

Solid phase Mobile phase

Normal High polarity Low polarity


phase (hydrophilic) (hydrophobic)
Reversed Low polarity High polarity
phase (hydrophobic) (hydrophilic)

82

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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

83

Invention of Chromatography by M.
Tswett
Ether
Chromatography

Chlorophyll Colors

CaCO3

84

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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.

85

Relationship between Hydrogen Bonding and


Retention Time in Normal Phase Mode

SiOH HO
Strong
SiOH
Weak
Very weak
OH

Steric hindrance
86

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Relationship Between Eluent Polarity and Retention


Time in Normal Phase Mode

Eluent: Hexane/methanol

100/0

98/2

95/5
87

Comparison of Normal Phase and


Reversed Phase

Normal Phase Reversed Phase


Effective for separation of Wide range of applications
structural isomers Effective for separation of
Offers separation homologs
selectivity not available Stationary phase has long
with reversed phase service life
Stabilizes slowly and is Stabilizes quickly
prone to fluctuations in Eluents are inexpensive and
retention time easy to use
Eluents are expensive

88

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Ion Exchange Chromatography

R
Anion exchange N+ R
R

++++
Cation exchange SO3- + +
++++
Electrostatic interaction
(Coulomb force)
89

Stationary Phase Used in Ion Exchange


Mode
Base Material
Resin is often used.
Silica gel is also used.

Cation Exchange Column


Strong cation exchange (SCX) -SO3-
Week cation exchange (WCX) -COO-
Anion Exchange Column
Strong anion exchange (SAX) -NR3+
Week anion exchange (WAX) -NHR2+

90

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Dependence of Exchange Capacity of Ion Exchanger


on pH of Eluent

Strongly acidic Strongly basic


cation exchanger anion exchanger
Exchange capacity

Exchange capacity
Weakly acidic
cation exchanger Weakly basic anion
exchanger

0 7 14 0 7 14
pH pH
Cation exchange mode Anion exchange mode
91

Relationship between Retention Time and Salt


Concentration of Eluent in Ion Exchange Mode

Resin Resin Resin


The exchange groups are An eluent ion is The solute ion is driven
in equilibrium with driven away away by an eluent ion and
anions in the eluent. and a solute ion is is adsorbed by the next
adsorbed. exchange group.

Solute ions and eluent ions compete for ion exchange groups.

If the salt concentration of the eluent increases,


92
the solutes are eluted sooner.

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Ion Exclusion Chromatography

H+

H+

H+

Strong acid ions are repelled by charge Depending on the level of dissociation, some
and cannot enter the pore. weak acid ions can enter the pore.

93

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

94

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Principle of Size Exclusion Mode

The size of the solute molecules


determines whether or not they can
enter the pores.

Packing
material

95

Relationship Between Molecular Weight and


Retention Time in Size Exclusion Mode

Exclusion limit
Molecular weight
(logarithmic axis)

Permeability limit

Elution capacity

96

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Creating a Molecular Weight


Calibration Curve

For separation of large molecular weights

Molecular weight For wide-range separation


(logarithmic axis) (mix gel)

Elution capacity
For separation of small molecular weights
97

Calculating Molecular Weights

Chromatogram
Various Average Molecular
Weights
Mn: Number-average
molecular weight
Mw: Weight-average
Calibration curve molecular weight
Mz: Z-average molecular
weight, etc.
Molecular weights and
molecular weight distributions
are calculated using special
calculation software.
Retention time

98

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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.

99

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

100

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HPLC Hardware: Part 2

Detectors and Their Ranges of Application

101

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.

102

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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

103

UV-VIS Absorbance Detector

C: Concentration
Detection cell

Ein Eout
A

A = eCl = log (Eout / Ein) C


(A: absorbance, E: absorption coefficient)

104

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Optical System of UV-VIS Absorbance


Detector
Grating
Sample cell
l Ein Eout Photodiode

Ein Ein Photodiode

Reference cell

D2 / W lamp

105

Spectrum and Selection of Detection


Wavelength

The longer wavelength is


more selective.

200 250 300 350


Wavelength [nm]
106

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Optical System of Photodiode Array


Detector

Sample cell
Grating

A single photodiode
D2 / W lamp measures the absorbance for
the corresponding wavelength
at a resolution of approx. 1 nm.

Photodiode array

107

Data Obtained with a Photodiode


Array Detector
Spectrum

Chromatogram
Absorbance

Retention time 108

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Advantages of Photodiode Array


Detectors
Peak Identification Using Spectra
Complementation of identification based on
retention time
Library searches
Evaluation 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

109

Fluorescence Detector
Excitation wavelength

+ hv1 *

* hv2 +
Fluorescence wavelength
Excited state
Quasi-excited state
hv1
hv2
Fluorescence

110
Ground state

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Optical System of Fluorescence Detector

Xenon lamp
Fluorescence
grating
Photomultiplier tube

Fluorescence

Excitation
Excitation grating Sample cell
light

111

Fluorescence Derivatization Reagents

OPA Reagent (Reacts with Primary Amines)


S-R
CHO
+ R-NH2 N-R
CHO R-SH
o-phthalaldhyde
(OPA)

ADAM Reagent (Reacts with Fatty Acids)


+ R-COOH

CHN2 CH2OCOR
9-anthryldiazomethane
(ADAM)
112

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Differential Refractive Index Detector


(Deflection-Type)

Light-receiving unit
Reference cell

Light

Sample cell

113

Optical System of Differential Refractive Index


Detector (Deflection-Type)

Slit W lamp

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

Photodiode

114

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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.

115

Electrical Conductivity Detector

Pure water NaCl aqueous


solution

Cl- Na+

The bulb does not light with water. The bulb lights if there are ions.

116

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Principle of Electrical Conductivity Detector

I A
V K k
I E L
L
k K
A
A A K: Electrical conductivity [S]
I: Electric current [A]
E: Voltage [V]
A: Electrode surface area [cm2]
L Electrode L: Distance between electrodes [cm]
k: Specific electrical conductivity [Scm-1]

117

Limiting Equivalent Ion Conductance, l


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

Cation l Anion l
H+ 349.8 OH 198.3
Li+ 38.6 F 55.4
Na+ 50.1 Cl 76.3
K+ 73.5 Br 78.1
NH4+ 73.5 NO3 71.4
(CH3)3NH+ 47.2 CH3COO 40.9
Mg2+ 53.0 C6H5COO 32.3
Ca2+ 59.5 SO42 80.0

118

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Electrochemical Detector

Electrode
HO R

HO
2e-
O R
+ 2H+
O

119

Cell Structure of Electrochemical


Detector (Amperometric Type)

Reference electrode Working electrode


(Ag/AgCl) (glassy carbon)

Eluent

Electrode couple
120

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Mass Spectrometer (LCMS)

Atmospheric
pressure High vacuum

Quadrupole MS analyzer
API probe

Electron
multiplier tube

RP TMP1 TMP2
(high vacuum pumps)
121

Atmospheric Pressure Ionization

Electrospray Ionization (ESI)


+
-+ + +
++- + -- +
+ +- + - +-+-+- +
+-+-+-
- + + + +
+
3) n E

Liquid
LiquidSamples
Sample
1)

+
2) of S

Io
Co va
Ch

Neburaizing Gas High


HighVoltage
Ev o l

Nebulizing Gas Voltage


ul po
ar

ap ve

on ra
ge

ol nt

E x tio
d

at
D

cl n
io
ro

us
n
pl

io
et

Atmospheric Pressure Chemical Ionization (APCI)

Molecular ion reaction


Liquid Sample
Liquid Samples Heater
Heater
Nebulizing GasGas
Neburaizing Colona Discharge
Corona Discharge
122 Nnndle
Needle

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Advantages of LCMS (1)

Quantitative analysis with excellent selectivity


m/z=100
A
TIC A:100 B
B:100
C:150 D:150

m/z=150
C D

123

Advantages of LCMS (2)

Peaks can be identified with MS spectra.

M/Z

M/Z

M/Z 124

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Comparison of Detectors

Possibility of
Selectivity Sensitivity
Gradient System
Light-absorbing
Absorbance ng Possible
substances

Fluorescence Fluorescent substances pg Possible

Differential
None g Impossible
refractive index
Evaporative light
Nonvolatile substances g Possible
scattering
Electrical
Ionic substances ng Partially possible
conductivity
Oxidizing / reducing
Electrochemical pg Partially possible
substances
Note: The above table indicates general characteristics. There are exceptions. 125

Post-Column Derivatization

Reaction
chamber

Pump
Reaction
solution
126

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Application Examples of Post-Column


Methods

Amino Acids Bromate Ions


Orthophthalic acid, OPA Tribromide ionization
(fluorescence) (ultraviolet absorption)
o-Dianisidine
Ninhydrin (visible absorption)
(visible absorption)
Reducing Sugars Cyanide Ions
Arginine (fluorescence) Chlorination - pyrazolone
Carbamate Pesticides (visible absorption)
Alkaline hydrolysis - OPA Transition Metal Ions
(fluorescence) 4-(2-Pyridylazo) resorcinol,
PAR (visible absorption)

127

Quantitative Analysis

Absolute Calibration Curve Method and


Internal Standard Method

128

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Qualitative Analysis
Identification based on retention time
Acquisition of spectra with detector
UV spectra
MS spectra
Transfer to other analytical instruments after
preparative separation

129

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

130

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Calibration Curve for Absolute


Calibration Curve Method
Area
Concentration
A1 Calibration curve
C1
A4

A2 A3

Peak area
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
131 Concentration

Calibration Curve for Internal Standard


Method
Concentration Area
Area for target substance / Area for internal standard

Target Internal
substance standard A1 AIS Calibration curve
C1 CIS A4 /AIS

A2 AIS A3 /AIS
C2 CIS

A2 /AIS
A3 AIS
C3 CIS
A1/AIS

A4 AIS
C4 CIS C1/CIS C2 /CIS C3 /CIS C4 /CIS
Concentration of target substance /
132
Concentration of internal standard

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Advantages of Internal Standard


Method (1)

Not affected by inconsistencies in injection volume.


IS
X AX / AIS
10 L
injected

Same area
ratio
IS
X
9 L
injected
CX / CIS
133

Advantages of Internal Standard


Method (2)

Not affected by the pretreatment recovery rate.


IS
X
AX / AIS

100%
recovery
rate

Same area
ratio
IS
90% X
recovery
rate
CX / CIS
134

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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.

135

Sample Pretreatment

Tasks Performed Before Injection

136

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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

137

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

138

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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
Filter Syringe
are difficult to filter.

139

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

140

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Solid Phase Extraction

(1) (2) (3) (4)


Conditioning Sample addition Rinsing Elution
Solvent with
low elution
strength

Solvent with
high elution
strength

Target
component
Unwanted
components

141

Pre-Column Derivatization

OPA Reagent (Reacts with Primary Amines)


S-R
CHO
+ R-NH2 N-R
CHO R-SH
o-phthalaldhyde
(OPA)

2,4-DNPH (Reacts with Aldehydes and Ketones)


R
NHNH2 NHN=C
R
+ C=O R
R H+
O2 N NO2 O2 N NO2
2,4-dinitrophenylhydrazine
(2,4-DNPH)
142

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Evaluation of the Reliability of


Analysis
Validation of Analytical Methods

143

What Is Validation of Analytical


Methods?
Scientifically Validation characteristics
demonstrating that the Accuracy / trueness
analytical methods Precision
concur with the intended Specificity
purpose (i.e., that errors Detection limit
are within a permissible Quantitation limit
range) Linearity
Evaluating required items Range
from the validation (Robustness)
characteristics

144

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Accuracy / Trueness

Definition Evaluation Method


Degree of bias in measurements Comparison with theoretical
obtained with analytical values (or authenticated
procedures values)
Difference between true value
Comparison with results
and grand mean of
measurements
obtained using other
analytical procedures for
True value which the accuracy (trueness)
is known
Recovery test

Measurement

Average 95% confidence interval


145

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

146

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Specificity

Definition Evaluation Method


The ability to accurately analyze Confirmation that the target
the target substance in the substance can be
discriminated (separated)
presence of other expected
from co-existing components,
substances related substances,
The discrimination capability of decomposition products, etc.
the analytical methods If reference standards for
Multiple analytical procedures impurities cannot be obtained,
may be combined in order to the measurement results for
attain the required level of samples thought to contain
the impurities are compared.
discrimination

147

Detection Limit

Definition Evaluation Method


Calculated from the
The minimum quantity of standard deviation of
a target substance that can measurements and the
be detected. slope of the calibration
curve.
Quantitation is not
DL = 3.3 /slope
absolutely necessary. (: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)
Calculated from the signal-
to-noise ratio.
Concentration for which S/N
= 3 or 2

148

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Quantitation Limit

Definition Evaluation Method


The minimum quantity of a Calculated from the standard
target substance that can be deviation of measurements and
the slope of the calibration
quantified
curve.
Quantitation with an QL = 10 /slope
appropriate level of accuracy (: Standard deviation of
and precision must be measurements)
possible. (In general, the (Slope: Slope of calibration
curve)
relative standard deviation
must not exceed 10%.) Calculated from the signal-to-
noise ratio.
Concentration for which S/N =
10

149

Linearity
Definition Evaluation Method
The ability of the analytical Samples containing different
method to produce quantities of the target
measurements for the substance (usually 5
quantity of a target substance concentrations) are analyzed
that satisfy a linear repeatedly, and regression
relationship. equations and correlation
Values produced by coefficients are obtained.
converting quantities or Residuals obtained from the
measurements of the target regression equations of the
substance using a precisely measurements are plotted,
defined formula may be used. and it is confirmed that there
is no specific slope.

150

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Range

Definition Evaluation Method


The region between the The accuracy, precision,
lower and upper limits of and linearity are
the quantity of a target investigated for samples
substance that gives containing quantities of
appropriate levels of a target substance that
accuracy and precision correspond to the lower
limit, upper limit, and
approximate center of
the range.

151

Robustness

Definition Evaluation Method


The ability of an Some or all of the
analytical procedure to variable factors (i.e., the
remain unaffected by analytical conditions)
small changes in are changed and the
analytical conditions. effects are evaluated.

152

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Maintenance of Separation
Column
Extending the Columns Service Life

153

Silica-Based Packing Materials and Resin-


Based Packing Materials

Silica-Based Resin-Based

Generally a wide
pH range 2 - 7.5
range
Organic Significant
No restrictions
solvent restrictions
Pressure Low pressure
25 MPa max.
resistance resistance
Depends on
Temperature 60C max.
packing material
154

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General Handling of Columns

Observe restrictions Use as low a load pressure


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

155

Typical Problems (1)


Column Clogging

Preventive Measures Corrective Action


Filter samples. Check for clogging in parts
other than the column.
Check that samples
Rinse with an appropriate
dissolve in the eluent.
solvent.
Get in the habit of Connect the column in
observing pressure values. reverse and flush out the
insoluble substances at a
low flow rate.
Open the column end and
perform ultrasonic cleaning
of the filter.

156

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Typical Problems (2)


Peak Deformation
Cause Corrective Action
Sample overload Reduce the sample injection volume or
concentration.
Inappropriate sample Replace the sample solvent with one of a
solvent low elution capacity.

Dirt Rinse the column.


Gap in column inlet Repair the column by supplementing it
with packing material.

Influence of secondary Rinse the column.


retention effects Replace the column with one that is only
minimally influenced.
157

Typical Problems (3)


Decrease in Retention Time
If the column is
Check whether the identified as the cause...
cause of the problem is Rinsing
not the column. Replacement
Eluent composition
Eluent flow rate
Column temperature

158

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Typical Problems (4)


Baseline Drift
If the column is
Check whether the cause identified as the cause...
of the problem is not the Rinsing
column. Review of temperature
If the problem persists control
when the column is Replacement
removed, it is caused by
the eluent, the solvent
delivery system (pump or
degasser), or the detector.

159

Guard Column and Pre-column

Guard column

Pre-column

160

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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 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.

161

Checking Column Performance

2
t
N 16 R
W
2
t
5.54 R
H W1/ 2
W1/2
tR H
2
H1/2
2
W Area
162

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Column Storage

Storage Solution Storage Conditions


It is generally safe to use the Insert an airtight stopper in the
same storage solution as column end. Never allow the
used at shipment. packing material to dry.
In order to prevent Make a record of the storage
putrefaction, alcohol or solution and final usage
some other preservative conditions and store it together
substance may be added. with the column.
Store the column in a location
not subject to shocks or sudden
temperature changes.

163

82