HPLC course 1

HPLC course

1. LAAQ-B-LC001B 1
What Is HPLC?What Is HPLC?
Basic Principles

2. LAAQ-B-LC001B 2
Invention of Chromatography by Invention of Chromatography by
M. TswettM. Tswett
Ether
CaCO
3
Chlorophyll
ChromatoChromatography
ColorsColors

3. LAAQ-B-LC001B 3
Comparing Chromatography to the Comparing Chromatography to the
Flow of a River... Flow of a River...
Base
Water flow
Light leaf
Heavy stone

4. LAAQ-B-LC001B 4
Mobile Phase / Stationary PhaseMobile 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.
Strong
Weak
Mobile Mobile
phasephase
Stationary Stationary
phasephase

5. LAAQ-B-LC001B 5
Chromato-graphy / -graph / -gram / Chromato-graphy / -graph / -gram /
-grapher-grapher
Chromatography: Analytical technique
Chromatograph: Instrument
Chromatogram: Obtained “picture”
Chromatographer: Person

6. LAAQ-B-LC001B 6
Three States of Matter and Three States of Matter and
Chromatography TypesChromatography Types
Mobile phase
Gas Liquid Solid
Stationary
phase
Gas
Liquid
Solid
GasGas
chromatographychromatography
LiquidLiquid
chromatographychromatography

7. LAAQ-B-LC001B 7
Liquid ChromatographyLiquid 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.

8. LAAQ-B-LC001B 8
Interaction Between Solutes, Stationary Interaction Between Solutes, Stationary
Phase, and Mobile PhasePhase, and Mobile Phase
Differences in the interactions between the solutes and
stationary and mobile phases enable separation.
Solute
Stationary
phase
Mobile phase
Degree of adsorption,
solubility, ionicity, etc.

9. LAAQ-B-LC001B 9
Column Chromatography and Column Chromatography and
Planar ChromatographyPlanar Chromatography
Separation column
Packing material
Column Chromatography
Paper or a
substrate coated
with particles
Paper Chromatography
Thin Layer Chromatography (TLC)

10. LAAQ-B-LC001B 10
Separation Process and ChromatogramSeparation Process and Chromatogram
for Column Chromatographyfor Column Chromatography
O
u
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p
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c
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Time
ChromatogramChromatogram

11. LAAQ-B-LC001B 11
ChromatogramChromatogram
t
R
t
0
I
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s
it
y

o
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d
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Time
Peak
t
R: Retention time
h
A
t
0: Non-retention time
A: Peak area
h: Peak height

12. LAAQ-B-LC001B 12
From Liquid Chromatography to High From Liquid Chromatography to High
Performance Liquid ChromatographyPerformance Liquid Chromatography
Higher degree of separation!
®Refinement of packing material (3 to 10 µm)
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!

13. LAAQ-B-LC001B 13
Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic
column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Degasser
Flow Channel Diagram for High Flow Channel Diagram for High
Performance Liquid ChromatographPerformance Liquid Chromatograph

14. LAAQ-B-LC001B 14
Advantages of High Performance Advantages of High Performance
Liquid ChromatographyLiquid 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

15. LAAQ-B-LC001B 15
Fields in Which High Performance Fields in Which High Performance
Liquid Chromatography Is UsedLiquid Chromatography Is Used
Biogenic substances
Sugars, lipids, nucleic
acids, amino acids,
proteins, peptides, steroids,
amines, etc.
Medical products
Drugs, antibiotics, etc.
Food products
Vitamins, food additives,
sugars, organic acids,
amino acids, etc.
Environmental
samples
Inorganic ions
Hazardous organic
substances, etc.
Organic industrial
products
Synthetic polymers,
additives, surfactants, etc.

16. LAAQ-B-LC001B 16
HPLC Hardware: Part 1HPLC Hardware: Part 1
Solvent Delivery System,
Degasser, Sample Injection Unit,
Column Oven

17. LAAQ-B-LC001B 17
Pump
Sample injection unit
(injector)
Column
Column Oven
(thermostatic
column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Flow Channel Diagram for HPLCFlow Channel Diagram for HPLC
Degasser

18. LAAQ-B-LC001B 18
Solvent Delivery PumpSolvent 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.

19. LAAQ-B-LC001B 19
Solvent Delivery Pump:Solvent Delivery Pump:
Representative Pumping MethodsRepresentative Pumping Methods
Syringe pump
Plunger pump
Diaphragm pump

20. LAAQ-B-LC001B 20
Solvent Delivery Pump:Solvent Delivery Pump:
Schematic Diagram of Plunger PumpSchematic Diagram of Plunger Pump
Motor and cam
Plunger
Plunger seal
Check
valves
Pump head
10 -100µL

21. LAAQ-B-LC001B 21
Solvent Delivery Pump:Solvent Delivery Pump:
Single Plunger Type Single Plunger Type
Check valves
Plunger head

22. LAAQ-B-LC001B 22
Solvent Delivery Pump:Solvent Delivery Pump:
Dual Plunger TypeDual Plunger Type
Check valves
Plunger heads
Type Type

23. LAAQ-B-LC001B 23
Gradient SystemGradient System
Isocratic system
Constant eluent composition
Gradient system
Varying eluent composition
HPGE (High Pressure Gradient)
LPGE (Low Pressure Gradient)

24. LAAQ-B-LC001B 24
Aim of Gradient System (1)Aim of Gradient System (1)
In isocratic mode
Long analysis time!!Long analysis time!!
Poor Poor
separation!! separation!!
CH
3OH / H
2O = 6 / 4
CH
3OH / H
2O = 8 / 2
(Column: ODS type)

25. LAAQ-B-LC001B 25
Aim of Gradient System (2)Aim of Gradient System (2)
If the eluent composition is changed gradually during
analysis...
95%
30%
C
o
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m
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in

e
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t

26. LAAQ-B-LC001B 26
High- / Low-Pressure Gradient SystemHigh- / Low-Pressure Gradient System
High-pressure gradient
Mixer
Low-pressure
gradient unit
Low-pressure gradient
Mixer

27. LAAQ-B-LC001B 27
Advantages and Disadvantages of Advantages and Disadvantages of
High- / Low-Pressure Gradient Systems 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

28. LAAQ-B-LC001B 28
DegasserDegasser
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.

29. LAAQ-B-LC001B 29
Online DegasserOnline Degasser
Gas-liquid separation membrane methodHelium purge method
Helium
cylinder
To draft
To pump
Eluent container
Regulator
Drain valve
To pump
Eluent container
Polymeric film tube
Vacuum chamber

30. LAAQ-B-LC001B 30
Sample Injection Unit (Injector)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

31. LAAQ-B-LC001B 31
Manual InjectorManual Injector
INJECT positionINJECT position
LOAD positionLOAD position
From pump
To column
From pump
To column

32. LAAQ-B-LC001B 32
Manual Injector:Manual Injector:
Operating Principle of Sample InjectionOperating Principle of Sample Injection
LOADLOAD INJECTINJECT
To column
From pump
To column
From pump
L
o
o
p
L
o
o
p

33. LAAQ-B-LC001B 33
Manual Injector:Manual Injector:
Injection MethodInjection 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.

34. LAAQ-B-LC001B 34
AutosamplerAutosampler
(Pressure Injection Method)(Pressure Injection Method)
To columnFrom pump From pump To column
Sample Loop
LOADLOAD INJECTINJECT

35. LAAQ-B-LC001B 35
AutosamplerAutosampler
(Total-Volume Injection Method)(Total-Volume Injection Method)
From pump From pump To column
Sample vial
Needle
Measuring pump
To column
LOADLOAD INJECTINJECT

36. LAAQ-B-LC001B 36
Column OvenColumn Oven
Air circulation heating type
Block heating type
Aluminum block heater
Insulated column jacket type
Water bath

37. LAAQ-B-LC001B 37
Tubing and Preparation for Tubing and Preparation for
Solvent DeliverySolvent Delivery
Prior to Analysis

38. LAAQ-B-LC001B 38
TubingTubing
Material
Stainless steel (SUS)
PEEK (polyether
ether ketone)
Fluororesin
O.D. (outer diameter)
1.6 mm
I.D. (inner diameter)
0.1 mm
0.3 mm
0.5 mm
0.8 mm etc.

39. LAAQ-B-LC001B 39
ConnectorsConnectors
Male nut (SUS)
Ferrule (SUS)
Sealing possible up to 40
MPa
Male nut (PEEK)
Can be connected without
any tools
Resists pressures of up to
approx. 25 MPa
Male nut
Ferrule
Male nut (PEEK)

40. LAAQ-B-LC001B 40
Dead Volume Dead Volume
(Extra-column volume)(Extra-column volume)
Dead volume can cause peaks broadening.
Tube
Male nut Dead volumeDead volume
Excellent connection Poor connection

41. LAAQ-B-LC001B 41
Mobile PhaseMobile Phase
Water
“Ultrapure water” can be
used with confidence.
Commercial “distilled
water for HPLC” is also
acceptable.
Organic 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)

42. LAAQ-B-LC001B 42
Replacement of EluentReplacement of Eluent
Mutually insoluble solvents
must not be exchanged directly.
Aqueous solutions containing
salt and organic solvents
must not be exchanged
directly.
Water
Hexane
2-Propanol
Buffer solution
Water-soluble
organic solvent
Water

43. LAAQ-B-LC001B 43
Mixing, Filtration, and Offline Mixing, Filtration, and Offline
Degassing of the EluentDegassing of the Eluent
Decompression
by aspirator
Ultrasonic
cleaning unit
Decompression
by aspirator
Membrane filter with pore
size of approx. 0.45 µm

44. LAAQ-B-LC001B 44
Reversed Phase Chromatography Reversed Phase Chromatography
Part 1Part 1
Basic Principles

45. LAAQ-B-LC001B 45
Polarity of SubstancesPolarity of Substances
Polarity
Property of a substance
whereby the positions of the
electrons give rise to
positive and negative poles
Water: Polar
Methane: Nonpolar
Miscibility 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.
O
H H
–
+
C
H H
H
H
WaterMethane Acetic acid
CCH
H
O
O
–
H

46. LAAQ-B-LC001B 46
Nonpolar (Hydrophobic) Functional Groups Nonpolar (Hydrophobic) Functional Groups
and Polar (Hydrophilic) Functional Groupsand Polar (Hydrophilic) Functional Groups
Nonpolar Functional
Groups
-(CH
2)
nCH
3
Alkyl groups
-C
6H
5
Phenyl groups
Polar Functional
Groups
-COOH
Carboxyl groups
-NH
2
Amino groups
-OH
Hydroxyl groups

47. LAAQ-B-LC001B 47
Partition ChromatographyPartition 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

48. LAAQ-B-LC001B 48
Normal Phase / Reversed PhaseNormal Phase / Reversed Phase
Stationary
phase
Mobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)

49. LAAQ-B-LC001B 49
Reversed Phase ChromatographyReversed Phase Chromatography
Stationary phase: Low polarity
Octadecyl group-bonded silical gel (ODS)
Mobile phase: High polarity
Water, methanol, acetonitrile
Salt is sometimes added.

50. LAAQ-B-LC001B 50
Separation Column for Reversed Separation Column for Reversed
Phase ChromatographyPhase Chromatography
C
18 (ODS) type
C
8 (octyl) type
C
4 (butyl) type
Phenyl type
TMS type
Cyano type
Si
-O-Si
C
18 (ODS)
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

51. LAAQ-B-LC001B 51
Effect of Chain Length of Effect of Chain Length of
Stationary PhaseStationary Phase
C
18 (ODS)
Strong
C
8
C
4
Medium
Weak

52. LAAQ-B-LC001B 52
Hydrophobic InteractionHydrophobic Interaction
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
Network of hydrogen bonds
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
Nonpolar solute
If a nonpolar
substance is added...
…the network is broken and...
H
2O
H
2O H
2O
H
2O
H
2O H
2O
H
2O
Nonpolar solute
Nonpolar stationary phase
…the nonpolar substance
is pushed to a nonpolar
location.

53. LAAQ-B-LC001B 53
Relationship Between Retention Relationship Between Retention
Time and PolarityTime and Polarity
C
18 (ODS)
CH
3
StrongStrong
WeakWeak
OH

54. LAAQ-B-LC001B 54
Basic Settings for Eluent Used in Basic Settings for Eluent Used in
Reversed Phase ModeReversed 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.

55. LAAQ-B-LC001B 55
Difference in Solute Retention Strengths Difference in Solute Retention Strengths
for Water and Water-Soluble Organic for Water and Water-Soluble Organic
SolventsSolvents
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
Tightly packed network
CH
3OH
Nonpolar solute
Nonpolar solute
Nonpolar stationary phase
Loose network
CH
3OH
CH
3OH
CH
3OH
CH
3OH
CH
3OH
CH
3OH

56. LAAQ-B-LC001B 56
Relationship between Polarity of Eluent and Relationship between Polarity of Eluent and
Retention Time in Reversed Phase ModeRetention Time in Reversed Phase Mode
60/40
Eluent: Methanol / Water
80/20
70/30

57. LAAQ-B-LC001B 57
Chromatogram ParametersChromatogram Parameters
Methods for Expressing Separation
and Column Performance

58. LAAQ-B-LC001B 58
Retention Factor, Retention Factor, kk
t
R
t
0
S
t
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g
t
h

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d
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Time
t
R: Retention time
t
0: Non-retention time
0
0R
t
tt
k
-
=

59. LAAQ-B-LC001B 59
Theoretical Plate Number, Theoretical Plate Number, NN
W
W
1/2
H
1/2
H
2
.
21
R
R
/
R
W
t
W
2
2
2
p
545
16
·
=
=
=
Area
Ht
t
N

60. LAAQ-B-LC001B 60
Evaluation of Column Efficiency Based on Evaluation of Column Efficiency Based on
Theoretical Plate NumberTheoretical Plate Number
If the retention times are
the same, the peak width
is smaller for the one with
the larger theoretical plate
number.
If the peak width is the
same, the retention time is
longer for the one with the
larger theoretical plate
number.
N: Large
N: Small
N: Small
N: Large

61. LAAQ-B-LC001B 61
Separation Factor, Separation Factor, aa
Separation factor: Ratio of k’s of two peaks
)(
12
1
2
kk
k
k
>
=a
k
1 k
2

62. LAAQ-B-LC001B 62
Resolution, Resolution, RR
SS
2,2/11,2/1
RR
21
RR
S
12
12
18.1
)(
2
1
hhWW
tt
WW
tt
R
+
-
´=
+
-
=
t
R1 t
R2
W
1 W
2
W
1/2h,1 W
1/2h,2 h
1/2

63. LAAQ-B-LC001B 63
Resolution Required for Complete Resolution Required for Complete
SeparationSeparation
If the peaks are isosceles triangles,
they are completely separated.
t
R2 - t
R1 = W
1 = W
2
R
S = 1
(t
R2 - t
R1)
W
1W
2 W
1W
2
If the peaks are Gaussian distributions,
R
S > 1.5 is necessary for complete separation.
t
R2 - t
R1 = W
1 = W
2
R
S = 1
(t
R2 - t
R1)

64. LAAQ-B-LC001B 64
Relationship Between Resolution Relationship Between Resolution
and Other Parametersand 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!
+
-
=
+
-
=
1
1
4
1
)(
2
1
2
2
21
1R2R
S
k’
k’
N
WW
tt
R
a
a

65. LAAQ-B-LC001B 65
Contribution of Capacity Factor to Contribution of Capacity Factor to
ResolutionResolution
Increasing the capacity
factor improves
separation!
A capacity factor of
around 3 to 10 is
appropriate. Exceeding
this just increases the
analysis time.
0.0
0.2
0.4
0.6
0.8
1.0
0 5101520
Capacity factor
C
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n

66. LAAQ-B-LC001B 66
Contribution of Theoretical Plate Contribution of Theoretical Plate
Number to ResolutionNumber to Resolution
The resolution
increases in
proportion to the
square root of the
theoretical plate
number.
0.0
1.0
2.0
0100002000030000
Theoretical plate number
C
o
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f
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f
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s
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67. LAAQ-B-LC001B 67
To Improve Separation...To Improve Separation...
k’ increased
N increased
a increased
Before
adjustment
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.

68. LAAQ-B-LC001B 68
pH Buffer Solution Used for EluentpH Buffer Solution Used for Eluent
Selection and Preparation of
Buffer Solution

69. LAAQ-B-LC001B 69
Acid Dissociation EquilibriumAcid Dissociation Equilibrium
HA A
-
H
+
+
H
+
OH
-
If an acid is added...
...the equilibrium shifts to
the left to offset the
increase in H
+
.
…the equilibrium shifts
to the right to offset the
decrease in H
+
.
If an alkali is
added...
The equilibrium always shiftsThe equilibrium always shifts
in a way that offsets changes.in a way that offsets changes.

70. LAAQ-B-LC001B 70
Acid Dissociation Constant and Acid Dissociation Constant and
pH-Based Abundance RatiopH-Based Abundance Ratio
]HA[
]A[
logppH
]HA[
]H][A[
a
a
-
+-
+=
=
K
K
The acid dissociation constant, K
a,
is defined as follows:
÷
÷
ø
ö
ç
ç
è
æ
-=
-=
+
aa
logp
]Hlog[pH
KK
HA A
-
+ H
+
0.0
0.2
0.4
0.6
0.8
1.0
1 2 3 4 5 6 7 8 9
pH
A
b
u
n
d
a
n
c
e

r
a
t
io
pK
a
CH
3COOH CH
3COO
-
Relationship Between Abundance Ratio
and pH Value of Acetic Acid and Acetic Acid Ions

71. LAAQ-B-LC001B 71
Preparing pH Buffer SolutionPreparing pH Buffer Solution
Use a weak acid with a pK
a 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 pK
a 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.

72. LAAQ-B-LC001B 72
Buffer Solutions Used for HPLC EluentBuffer Solutions Used for HPLC Eluent
Requirements
High buffering power at
prescribed pH.
Does not adversely
affect detection.
Does not damage
column or equipment.
Inexpensive.
Commonly Used Acids
Phosphoric acid
pK
a 2.1, 7.2, 12.3
Acetic acid
pK
a 4.8
Citric acid
pK
a 3.1, 4.8, 6.4
Concentration
If only to adjust pH, 10
mmol/L is sufficient.

73. LAAQ-B-LC001B 73
Characteristics of Phosphate Characteristics of Phosphate
Buffer SolutionBuffer Solution
Advantages
Three dissociation
states
(pK
a 2.1, 7.2, 12.3)
Possible to prepare
buffer solutions of
various pH values.
No UV absorption
Inexpensive
Disadvantages
No volatility
Difficult to use for
LCMS or evaporative
light scattering
detection.

74. LAAQ-B-LC001B 74
Reversed Phase Chromatography Reversed Phase Chromatography
Part 2Part 2
Consideration of Analytical
Conditions

75. LAAQ-B-LC001B 75
Guidelines for Setting Mobile Phase Guidelines for Setting Mobile Phase
Conditions (1)Conditions (1)
Neutral (Nonionic) SubstancesNeutral (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

76. LAAQ-B-LC001B 76
pH of Eluent and Retention of Ionic pH of Eluent and Retention of Ionic
SolutesSolutes
COOH
COO
H
pH of eluent
Acidic
Alkaline
+
Increased
hydrophobicity
Increased
hydrophilicity

77. LAAQ-B-LC001B 77
Guidelines for Setting Mobile Phase Guidelines for Setting Mobile Phase
Conditions (2)Conditions (2)
Acidic (Anionic) SubstancesAcidic (Anionic) Substances
Eluent Composition
Acidic buffer solution / acetonitrile
Acidic buffer solution / methanol
Increase retention strength by making Increase retention strength by making
the eluent acidic and suppressing the eluent acidic and suppressing
ionization!ionization!

78. LAAQ-B-LC001B 78
Analysis of Basic Substances (1)Analysis of Basic Substances (1)
Problems Encountered with Alkaline EluentsProblems Encountered with Alkaline Eluents
O
Si
Si
With alkaline eluents, although the
ionization of basic substances is
suppressed, and the retention
strength increases...
OH
N
H
+
N
OH
OH
OH
OH
OH
…silica gel dissolves in
alkalis, so the packing
material deteriorates rapidly.

79. LAAQ-B-LC001B 79
Analysis of Basic Substances (2)Analysis of Basic Substances (2)
Influence of Residual Silanol GroupsInfluence of Residual Silanol Groups
O
-O-Si-O
Si
Si
O
Si
H
+
Residual silanol group
Basic substances interact with the
residual silanol groups, causing
delayed elution and tailing.
N

80. LAAQ-B-LC001B 80
Analysis of Basic Substances (3)Analysis of Basic Substances (3)
Addition of Sodium PerchlorateAddition of Sodium Perchlorate
O
Si
Si
Basic substances form ion pairs with
perchlorate ions, thereby balancing the
charge and increasing the retention strength.
H
+
N
ClO
4 Ion pairIon pair

81. LAAQ-B-LC001B 81
Guidelines for Setting Mobile Phase Guidelines for Setting Mobile Phase
Conditions (3) Conditions (3)
Basic Substances (Cationic Substances)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 acidicMaking eluent acidic
®® Suppresses dissociation of residual silanol groupsSuppresses dissociation of residual silanol groups
®® Prevents tailing!Prevents tailing!
Adding perchlorate ionsAdding perchlorate ions
®® Forms ion pairs Forms ion pairs ®® Increases retention strength! Increases retention strength!
®® Suppresses tailing!Suppresses tailing!

82. LAAQ-B-LC001B 82
Reversed Phase Ion Pair Reversed Phase Ion Pair
ChromatographyChromatography
Increase the retention strength by adding an ion pair
reagent with the opposite charge to the target
substance into the eluent.
Ion pair formationIon pair formation Ion pair formationIon pair formation
Ion exchange-like effectIon exchange-like effect Ion exchange-like effectIon exchange-like effect
Basic Substance Acidic Substance

83. LAAQ-B-LC001B 83
Representative Ion Pair Reagents 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)

84. LAAQ-B-LC001B 84
Points to Note Concerning the Use Points to Note Concerning the Use
of Ion Pairsof 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.

85. LAAQ-B-LC001B 85
HPLC Separation ModesHPLC Separation Modes
Separation Modes Other Than
Reversed Phase Chromatography

86. LAAQ-B-LC001B 86
HPLC Separation ModesHPLC Separation Modes
Adsorption (liquid-solid) chromatography
Partition (liquid-liquid) chromatography
Normal phase partition chromatography
Reversed phase partition chromatography
Ion exchange chromatography
Size exclusion chromatography

87. LAAQ-B-LC001B 87
Adsorption ChromatographyAdsorption 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.

88. LAAQ-B-LC001B 88
Partition ChromatographyPartition 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

89. LAAQ-B-LC001B 89
Normal Phase and Reversed PhaseNormal Phase and Reversed Phase
Solid phaseMobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)

90. LAAQ-B-LC001B 90
Normal Phase (Partition) Normal Phase (Partition)
ChromatographyChromatography
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

91. LAAQ-B-LC001B 91
Invention of Chromatography by Invention of Chromatography by
M. TswettM. Tswett
Ether
CaCO
3
Chlorophyll
ChromatoChromatography

ColorsColors

92. LAAQ-B-LC001B 92
Stationary Phase and Mobile Phase Used Stationary Phase and Mobile Phase Used
in Normal Phase Modein Normal Phase Mode
Stationary Phase
Silica gel: -Si-OH
Cyano type: -Si-CH
2CH
2CH
2CN
Amino type: -Si-CH
2CH
2CH
2NH
2
Diol type: -Si-CH
2CH
2CH
2OCH(OH)-CH
2OH
Mobile Phase
Basic solvents: Aliphatic hydrocarbons,
aromatic hydrocarbons, etc.
Additional solvents: Alcohols, ethers, etc.

93. LAAQ-B-LC001B 93
Relationship between Hydrogen Bonding Relationship between Hydrogen Bonding
and Retention Time in Normal Phase Modeand Retention Time in Normal Phase Mode
OH
HO
SiOH
SiOH
StrongStrong
WeakWeak
Steric hindrance
Very weakVery weak

94. LAAQ-B-LC001B 94
Relationship Between Eluent Polarity and Relationship Between Eluent Polarity and
Retention Time in Normal Phase ModeRetention Time in Normal Phase Mode
100/0
Eluent: Hexane/methanol
95/5
98/2

95. LAAQ-B-LC001B 95
Comparison of Normal Phase and Comparison of Normal Phase and
Reversed PhaseReversed Phase
Normal 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

96. LAAQ-B-LC001B 96
Ion Exchange ChromatographyIon Exchange Chromatography
N
+
R
R
R
SO
3
-
+
+
+
++
++
+
+
+
Electrostatic interaction
(Coulomb force)
Anion exchange
Cation exchange

97. LAAQ-B-LC001B 97
Stationary Phase Used in Ion Stationary Phase Used in Ion
Exchange ModeExchange Mode
Base Material
Resin is often used.
Silica gel is also used.
Cation Exchange Column
Strong cation exchange(SCX) -SO
3
-
Week cation exchange(WCX)-COO
-
Anion Exchange Column
Strong anion exchange (SAX)-NR
3
+
Week anion exchange (WAX)-NHR
2
+

98. LAAQ-B-LC001B 98
Dependence of Exchange Capacity of Ion Dependence of Exchange Capacity of Ion
Exchanger on pH of EluentExchanger on pH of Eluent
E
x
c
h
a
n
g
e

c
a
p
a
c
it
y
E
x
c
h
a
n
g
e

c
a
p
a
c
it
y
pH
0 7 14
pH
0 7 14
Weakly acidic
cation exchanger
Strongly acidic
cation exchanger
Weakly basic
anion exchanger
Strongly basic
anion exchanger
Cation exchange mode Anion exchange mode

99. LAAQ-B-LC001B 99
Relationship between Retention Time and Relationship between Retention Time and
Salt Concentration of Eluent in Ion Salt Concentration of Eluent in Ion
Exchange ModeExchange Mode
Resin ResinResin
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.
If the salt concentration of the eluent increases, the solutes are eluted sooner.
Solute ions and eluent ions compete for ion exchange groups.

100. LAAQ-B-LC001B 100
Ion Exclusion ChromatographyIon Exclusion Chromatography
H
+
H
+
H
+
Strong acid ions are repelled by
charge and cannot enter the pore.
Depending on the level of dissociation,
some weak acid ions can enter the pore.

101. LAAQ-B-LC001B 101
Size Exclusion ChromatographySize 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

102. LAAQ-B-LC001B 102
Principle of Size Exclusion ModePrinciple of Size Exclusion Mode
Packing
material
The size of the solute molecules
determines whether or not they can
enter the pores.

103. LAAQ-B-LC001B 103
Relationship Between Molecular Weight and Relationship Between Molecular Weight and
Retention Time in Size Exclusion ModeRetention Time in Size Exclusion Mode
Exclusion limitExclusion limit
Permeability limitPermeability limit
Elution capacity
M
o
le
c
u
la
r

w
e
ig
h
t

(
lo
g
a
r
it
h
m
ic

a
x
is
)

104. LAAQ-B-LC001B 104
Creating a Molecular Weight Creating a Molecular Weight
Calibration CurveCalibration Curve
M
o
le
c
u
la
r

w
e
ig
h
t

(
lo
g
a
r
it
h
m
ic

a
x
is
)
Elution capacity
For separation of large molecular weights
For separation of small molecular weights
For wide-range separation
(mix gel)

105. LAAQ-B-LC001B 105
Calculating Molecular WeightsCalculating Molecular Weights
Various Average Molecular
Weights
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.
Retention time
Chromatogram
Calibration curve

106. LAAQ-B-LC001B 106
Guidelines for Selecting Separation Mode (1)Guidelines for Selecting Separation Mode (1)
Required InformationRequired Information
Soluble solvent
Molecular weight
Structural formula and chemical
properties
Do the substances ionize?
Is there UV absorption or fluorescence?
Is derivatization possible? etc.

107. LAAQ-B-LC001B 107
Guidelines for Selecting Separation Mode (2)Guidelines for Selecting Separation Mode (2)
Basic PolicyBasic 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

108. LAAQ-B-LC001B 108
HPLC Hardware: Part 2HPLC Hardware: Part 2
Detectors and Their Ranges of Application

109. LAAQ-B-LC001B 109
Detection Condition RequirementsDetection 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.

110. LAAQ-B-LC001B 110
Representative HPLC DetectorsRepresentative 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

111. LAAQ-B-LC001B 111
UV-VIS Absorbance DetectorUV-VIS Absorbance Detector
A = e·C·l = –log (E
out / E
in)
l
C: Concentration
(A: absorbance, E: absorption coefficient)
Detection cell
E
in E
out
A
C

112. LAAQ-B-LC001B 112
Optical System of UV-VIS Optical System of UV-VIS
Absorbance DetectorAbsorbance Detector
Sample cell
Reference cell
Photodiode
Photodiode
E
in
E
inE
in
Grating
D
2
/ W lamp
E
outl

113. LAAQ-B-LC001B 113
Spectrum and Selection of Spectrum and Selection of
Detection WavelengthDetection Wavelength
200 250 300 350
Wavelength [nm]
The longer wavelength
is more selective.

114. LAAQ-B-LC001B 114
Optical System of Photodiode Optical System of Photodiode
Array DetectorArray Detector
Sample cell
Photodiode arrayPhotodiode array
Grating
D
2
/ W lamp
A single photodiode
measures the absorbance for
the corresponding wavelength
at a resolution of approx. 1 nm.

115. LAAQ-B-LC001B 115
Data Obtained with a Photodiode Data Obtained with a Photodiode
Array DetectorArray Detector
Retention time
W
avelength
A
b
s
o
r
b
a
n
c
e
Chromatogram
Spectrum

116. LAAQ-B-LC001B 116
Advantages of Photodiode Array Advantages of Photodiode Array
DetectorsDetectors
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

117. LAAQ-B-LC001B 117
Fluorescence DetectorFluorescence Detector
+ hv
1
*
Excitation wavelength
Fluorescence wavelength
FluorescenceFluorescence
*
hv
2 +
hv
1
hv
2
Excited state
Quasi-excited state
Ground state

118. LAAQ-B-LC001B 118
Optical System of Fluorescence DetectorOptical System of Fluorescence Detector
Xenon lamp
Excitation grating
Excitation
light
Fluorescence
Fluorescence
grating
Sample cell
Photomultiplier tube

119. LAAQ-B-LC001B 119
Fluorescence Derivatization ReagentsFluorescence Derivatization Reagents
OPA Reagent (Reacts with Primary Amines)
o-phthalaldhyde
(OPA)
+ R-NH
2 N-R
S-R’
R’-SH
CHN
2
9-anthryldiazomethane
(ADAM)
+ R-COOH
CH
2OCOR
CHO
CHO
ADAM Reagent (Reacts with Fatty Acids)

120. LAAQ-B-LC001B 120
Differential Refractive Index Differential Refractive Index
Detector (Deflection-Type)Detector (Deflection-Type)
Light
Sample cell
Reference cell
Light-receiving unit

121. LAAQ-B-LC001B 121
Optical System of Differential Refractive Optical System of Differential Refractive
Index Detector (Deflection-Type)Index Detector (Deflection-Type)
W lamp
Slit
Sample cell
Reference cell
Photodiode
The slit image moves if the The slit image moves if the
refractive index inside the refractive index inside the
flow cell changes.flow cell changes.

122. LAAQ-B-LC001B 122
Evaporative Light Scattering DetectorEvaporative Light Scattering Detector
The column eluate is evaporated and the light scattered
by the particles of nonvolatile substances is detected.
Drift tube
Nebulizer
Column eluate
Nebulizer gas
Drain
Assist gas
Light-receiving unit
Light source

123. LAAQ-B-LC001B 123
Electrical Conductivity DetectorElectrical Conductivity Detector
Pure water
NaCl aqueous
solution
Na
+
Cl
-
The bulb does not light with water.The bulb lights if there are ions.

124. LAAQ-B-LC001B 124
Principle of Electrical Conductivity DetectorPrinciple of Electrical Conductivity Detector
L
Electrode
V
I
A A
k
L
A
E
I
K ·==
K
A
L
k·=
K:Electrical conductivity [S]
I:Electric current [A]
E:Voltage [V]
A:Electrode surface area [cm
2
]
L:Distance between electrodes [cm]
k:Specific electrical conductivity [S•cm
-1
]

125. LAAQ-B-LC001B 125
Limiting Equivalent Ion Conductance, Limiting Equivalent Ion Conductance, ll
[S•cm[S•cm
22
/mol], in Aqueous Solution (25ºC)/mol], in Aqueous Solution (25ºC)
Cationl Anionl
H
+
349.8 OH
–
198.3
Li
+
38.6 F
–
55.4
Na
+
50.1 Cl
–
76.3
K
+
73.5 Br
–
78.1
NH
4
+
73.5 NO
3
–
71.4
(CH
3)
3NH
+
47.2 CH
3COO
–
40.9
Mg
2+
53.0 C
6H
5COO
–
32.3
Ca
2+
59.5 SO
4
2–
80.0

126. LAAQ-B-LC001B 126
Electrochemical DetectorElectrochemical Detector
RHO
HO
RO
O
+ 2H
+
2e2e
--
Electrode

127. LAAQ-B-LC001B 127
Cell Structure of Electrochemical Cell Structure of Electrochemical
Detector (Amperometric Type)Detector (Amperometric Type)
Working electrode
(glassy carbon)
Electrode couple
Reference electrode
(Ag/AgCl)
Eluent

128. LAAQ-B-LC001B 128
Mass Spectrometer (LCMS)Mass Spectrometer (LCMS)
API probe
Atmospheric
pressure
High vacuum
RP TMP1 TMP2
(high vacuum pumps)
Electron
multiplier tube
Quadrupole MS analyzer

129. LAAQ-B-LC001B 129
Atmospheric Pressure IonizationAtmospheric Pressure Ionization
Electrospray Ionization (ESI)
Atmospheric Pressure Chemical Ionization (APCI)
High Voltage
3
)

C
o
u
l
o
n

E
x
c
l
u
s
i
o
n

I
o
n

E
v
a
p
o
r
a
t
i
o
n

2
)

E
v
a
p
o
l
a
t
i
o
n




o
f

S
o
l
v
e
n
t

1
)

C
h
a
r
g
e
d

D
r
o
p
l
e
t
Liquid Samples
Neburaizing Gas
+
-+
-
+
-
+
-
+
-+
+
+
-
-
+
-
+
+
+
-
+-
+
-+
+
+
-
+
-
+
+
+
+
+
+
Liquid Sample
Nebulizing
Gas
2
)

E
v
a
p
o
r
a
t
i
o
n

o
f

S
o
l
v
e
n
t
3
)

C
o
u
l
o
m
b

E
x
c
l
u
s
i
o
n

I
o
n

E
v
a
p
o
r
a
t
i
o
n
1
)

C
h
a
r
g
e
d

D
r
o
p
l
e
t
High Voltage
Liquid Samples
Neburaizing Gas
Heater
Colona Discharge
Nnndle
Molecular ion reaction
Liquid Sample
Nebulizing Gas Corona Discharge
Needle
Heater

130. LAAQ-B-LC001B 130
Advantages of LCMS (1)Advantages of LCMS (1)
Quantitative analysis with excellent selectivity
A:100
D:150
B:100
C:150
m/z=150m/z=150
TIC TIC
m/z=100m/z=100
A
B
C D

131. LAAQ-B-LC001B 131
Advantages of LCMS (2)Advantages of LCMS (2)
Peaks can be identified with MS spectra.
M/Z
M/Z
M/Z

132. LAAQ-B-LC001B 132
Comparison of DetectorsComparison of Detectors
Note: The above table indicates general characteristics. There are exceptions.
Selectivity Sensitivity
Possibility of
Gradient System
Absorbance
Light-absorbing
substances
ng Possible
Fluorescence Fluorescent substances pg Possible
Differential
refractive index
None µg Impossible
Evaporative light
scattering
Nonvolatile substances µg Possible
Electrical
conductivity
Ionic substances ng Partially possible
Electrochemical
Oxidizing / reducing
substances
pg Partially possible

133. LAAQ-B-LC001B 133
Post-Column DerivatizationPost-Column Derivatization
Reaction
chamber
Pump
Reaction
solution

134. LAAQ-B-LC001B 134
Application Examples of Post-Application Examples of Post-
Column MethodsColumn Methods
Amino Acids
Orthophthalic acid, OPA
(fluorescence)
Ninhydrin (visible absorption)
Reducing Sugars
Arginine (fluorescence)
Carbamate Pesticides
Alkaline hydrolysis - OPA
(fluorescence)
Bromate Ions
Tribromide ionization
(ultraviolet absorption)
o-Dianisidine
(visible absorption)
Cyanide Ions
Chlorination - pyrazolone
(visible absorption)
Transition Metal Ions
4-(2-Pyridylazo)
resorcinol, PAR (visible
absorption)

135. LAAQ-B-LC001B 135
Quantitative AnalysisQuantitative Analysis
Absolute Calibration Curve Method
and Internal Standard Method

136. LAAQ-B-LC001B 136
Qualitative AnalysisQualitative Analysis
Identification based on retention time
Acquisition of spectra with detector
UV spectra
MS spectra
Transfer to other analytical instruments
after preparative separation

137. LAAQ-B-LC001B 137
Quantitative AnalysisQuantitative 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

138. LAAQ-B-LC001B 138
Calibration Curve for Absolute Calibration Curve for Absolute
Calibration Curve MethodCalibration Curve Method
C
1
C
4
C
3
C
2
Concentration
Area
A
1
A
2
A
3
A
4
C
1 C
2 C
3 C
4
A
1
A
2
A
3
A
4
Concentration
P
e
a
k

a
r
e
a
Calibration curveCalibration curve

139. LAAQ-B-LC001B 139
Calibration Curve for Internal Calibration Curve for Internal
Standard MethodStandard Method
C
1
C
4
C
3
C
2
Concentration Area
A
1
A
2
A
3
A
4
C
1/C
ISC
2 /C
ISC
3 /C
ISC
4 /C
IS
A
1/A
IS
A
2 /A
IS
A
3 /A
IS
A
4 /A
IS
Concentration of target substance /
Concentration of internal standard
A
r
e
a

f
o
r

t
a
r
g
e
t

s
u
b
s
t
a
n
c
e

/

A
r
e
a

f
o
r

in
t
e
r
n
a
l
s
t
a
n
d
a
r
d
Calibration curveCalibration curve
Target
substance
Internal
standard
C
IS
C
IS
C
IS
C
IS
A
IS
A
IS
A
IS
A
IS

140. LAAQ-B-LC001B 140
Advantages of Internal Standard Advantages of Internal Standard
Method (1)Method (1)
Not affected by inconsistencies in injection volume.
10 µL
injected
9 µL
injected
C
X / C
IS
A
X / A
IS
X
IS
X
IS
Same area Same area
ratioratio

141. LAAQ-B-LC001B 141
Advantages of Internal Standard Advantages of Internal Standard
Method (2)Method (2)
Not affected by the pretreatment recovery rate.
100%
recovery
rate
90%
recovery
rate
C
X / C
IS
A
X

/

A
I
S
X
IS
X
IS
Same area Same area
ratioratio

142. LAAQ-B-LC001B 142
Selection Criteria for Internal StandardSelection 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.

143. LAAQ-B-LC001B 143
Sample PretreatmentSample Pretreatment
Tasks Performed Before Injection

144. LAAQ-B-LC001B 144
Objectives of PretreatmentObjectives 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

145. LAAQ-B-LC001B 145
Substances That Must Not Be Substances That Must Not Be
Injected into the ColumnInjected 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

146. LAAQ-B-LC001B 146
Filtration and Centrifugal SeparationFiltration 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 µm.
Centrifugal separation is
applicable for samples
that are difficult to filter.
Filter Syringe

147. LAAQ-B-LC001B 147
DeproteinizationDeproteinization
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

148. LAAQ-B-LC001B 148
Solid Phase ExtractionSolid 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

149. LAAQ-B-LC001B 149
Pre-Column DerivatizationPre-Column Derivatization
OPA Reagent (Reacts with Primary Amines)
o-phthalaldhyde
(OPA)
+ R-NH
2 N-R
S-R’
R’-SH
NO
2
2,4-dinitrophenylhydrazine
(2,4-DNPH)
+
CHO
CHO
2,4-DNPH (Reacts with Aldehydes and Ketones)
O
2N
NHNH
2
C=O
R
R’
NO
2
O
2N
NHN=C
H
+
R
R’

150. LAAQ-B-LC001B 150
Evaluation of the Reliability of Evaluation of the Reliability of
AnalysisAnalysis
Validation of Analytical Methods

151. LAAQ-B-LC001B 151
What Is “Validation of Analytical What Is “Validation of Analytical
Methods”?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
Validation
characteristics
Accuracy / trueness
Precision
Specificity
Detection limit
Quantitation limit
Linearity
Range
(Robustness)

152. LAAQ-B-LC001B 152
Accuracy / TruenessAccuracy / Trueness
Definition
Degree of bias in
measurements obtained with
analytical procedures
Difference between true value
and grand mean of
measurements
Evaluation 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
True valueTrue value
Average
95% confidence interval

153. LAAQ-B-LC001B 153
PrecisionPrecision
Definition
Degree of coincidence of
series of measurements
obtained by repeatedly
analyzing multiple
samples taken from a
homogenous test
substance
Variance, standard
deviation, or relative
standard deviation of
measurements
Repeatability / Intra-
Assay Precision
Precision of
measurements taken over
a short time period under
the same conditions
Intermediate Precision
Reproducibility

154. LAAQ-B-LC001B 154
SpecificitySpecificity
Definition
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.

155. LAAQ-B-LC001B 155
Detection LimitDetection Limit
Definition
The minimum quantity of
a target substance that
can be detected.
Quantitation is not
absolutely necessary.
Evaluation Method
Calculated from the
standard deviation of
measurements and the
slope of the calibration
curve.
DL = 3.3 s/slope
(s: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)
Calculated from the
signal-to-noise ratio.
Concentration for which
S/N = 3 or 2

156. LAAQ-B-LC001B 156
Quantitation LimitQuantitation Limit
Definition
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 s/slope
(s: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)
Calculated from the signal-to-
noise ratio.
Concentration for which S/N
= 10

157. LAAQ-B-LC001B 157
LinearityLinearity
Definition
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.

158. LAAQ-B-LC001B 158
RangeRange
Definition
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.

159. LAAQ-B-LC001B 159
RobustnessRobustness
Definition
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.

160. LAAQ-B-LC001B 160
Maintenance of Separation ColumnMaintenance of Separation Column
Extending the Column’s Service Life

161. LAAQ-B-LC001B 161
Silica-Based Packing Materials and Silica-Based Packing Materials and
Resin-Based Packing MaterialsResin-Based Packing Materials
Silica-Based Resin-Based
pH range 2 - 7.5
Generally a wide
range
Organic
solvent
No restrictions
Significant
restrictions
Pressure
resistance
25 MPa max.
Low pressure
resistance
Temperature60ºC max.
Depends on packing
material

162. LAAQ-B-LC001B 162
General Handling of ColumnsGeneral Handling of Columns
Observe restrictions
related to solvents and
the pH range.
Never allow the
packing material to dry.
Do not allow solids or
microscopic particles
to enter the column.
Filter samples.
Use as low a load
pressure as possible.
Do not exceed the upper
pressure limit.
Do not subject the column
to sudden pressure
changes.
Do not subject the
column to intense
shocks.

163. LAAQ-B-LC001B 163
Typical Problems (1)Typical Problems (1)
Column CloggingColumn Clogging
Preventive Measures
Filter samples.
Check that samples
dissolve in the eluent.
Get in the habit of
observing pressure
values.
Corrective 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.

164. LAAQ-B-LC001B 164
Typical Problems (2) Typical Problems (2)
Peak DeformationPeak 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.

165. LAAQ-B-LC001B 165
Typical Problems (3)Typical Problems (3)
Decrease in Retention TimeDecrease in Retention Time
Check whether the
cause of the problem
is not the column.
Eluent composition
Eluent flow rate
Column temperature
If the column is
identified as the
cause...
Rinsing
Replacement

166. LAAQ-B-LC001B 166
Typical Problems (4)Typical Problems (4)
Baseline DriftBaseline Drift
Check whether the
cause of the problem
is not the column.
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

167. LAAQ-B-LC001B 167
Guard Column and Pre-columnGuard Column and Pre-column
Guard columnGuard column
Pre-columnPre-column

168. LAAQ-B-LC001B 168
Column RinsingColumn 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.

169. LAAQ-B-LC001B 169
Checking Column PerformanceChecking Column Performance
W
W
1/2
H
1/2
H
2
.
21
R
R
/
R
W
t
W
2
2
2
p
545
16
·
=
=
=
Area
Ht
t
N

170. LAAQ-B-LC001B 170
Column StorageColumn 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 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.