Professional Documents
Culture Documents
HPLC
HPLC
Basic Principles
LAAQ-B-LC001B
Invention of Chromatography by
M. Tswett
Ether
Chlorophyll
Chromatography
Chromato
Colors
CaCO3
LAAQ-B-LC001B
Water flow
Base
LAAQ-B-LC001B
Mobile
phase
Strong
Stationary
phase
LAAQ-B-LC001B
Weak
LAAQ-B-LC001B
Analytical technique
Instrument
Obtained picture
Person
Liquid
Solid
Gas
Stationary
phase
Liquid
Gas
chromatography
Liquid
chromatography
Solid
LAAQ-B-LC001B
Liquid Chromatography
Chromatography
a liquid.
The
The
Solute
Degree of adsorption,
solubility, ionicity, etc.
Stationary
phase
LAAQ-B-LC001B
Mobile phase
8
Paper or a
substrate coated
with particles
Packing material
Column Chromatography
LAAQ-B-LC001B
Paper Chromatography
Thin Layer Chromatography (TLC)
9
Output
concentration
LAAQ-B-LC001B
Chromatogram
Time
10
Chromatogram
tR
t0
Peak
tR : Retention time
t0 : Non-retention time
h
A
A : Peak area
h : Peak height
Time
LAAQ-B-LC001B
11
degree of separation!
Refinement of packing material (3 to 10 m)
Reduction of analysis time!
Delivery of eluent by pump
Demand for special equipment that can
withstand high pressures
The arrival of high performance liquid chromatography!
LAAQ-B-LC001B
12
Detector
Column
Pump
Eluent
(mobile phase)
Column oven
(thermostatic
column chamber)
Drain
Data processor
Degasser
LAAQ-B-LC001B
13
Generally
high sensitivity
Low sample consumption
Easy preparative separation and purification of
samples
LAAQ-B-LC001B
14
Medical
substances
Food products
Environmental samples
products
Inorganic ions
Hazardous organic
substances, etc.
Organic industrial
products
LAAQ-B-LC001B
Synthetic polymers,
additives, surfactants, etc.
15
LAAQ-B-LC001B
16
Detector
Column
Pump
Eluent
(mobile phase)
Column Oven
(thermostatic
column chamber)
Drain
Data processor
Degasser
LAAQ-B-LC001B
17
Requirements
Capacity
18
pump
Plunger pump
Diaphragm pump
LAAQ-B-LC001B
19
Pump head
Check
valves
Plunger
Plunger seal
LAAQ-B-LC001B
10 -100L
20
Check valves
Plunger head
LAAQ-B-LC001B
21
Plunger heads
Type
LAAQ-B-LC001B
Type
22
Gradient System
Isocratic
system
Constant
Gradient
Varying
eluent composition
system
eluent composition
HPGE
LAAQ-B-LC001B
23
isocratic mode
CH3OH / H2O = 6 / 4
Poor
separation!!
LAAQ-B-LC001B
CH3OH / H2O = 8 / 2
24
LAAQ-B-LC001B
95%
30%
25
Mixer
High-pressure gradient
LAAQ-B-LC001B
Mixer
Low-pressure gradient
26
gradient system
High
gradient accuracy
Complex system configuration (multiple
pumps required)
Low-pressure
gradient system
Simple
system configuration
Degasser required
LAAQ-B-LC001B
27
Degasser
Problems
LAAQ-B-LC001B
28
Online Degasser
Regulator
Helium
cylinder
Vacuum chamber
To pump
To pump
To draft
Drain valve
Eluent container
Eluent container
Requirements
No
LAAQ-B-LC001B
30
Manual Injector
From pump
To column
LOAD position
From pump
INJECT position
LAAQ-B-LC001B
To column
31
Manual Injector:
Operating Principle of Sample Injection
From pump
Loop
Loop
From pump
To column
To column
LOAD
LAAQ-B-LC001B
INJECT
32
Manual Injector:
Injection Method
Syringe
measurement method
It
Loop
measurement method
It
LAAQ-B-LC001B
33
Autosampler
(Pressure Injection Method)
From pump
To column
From pump
To column
Sample Loop
LAAQ-B-LC001B
LOAD
INJECT
34
Autosampler
(Total-Volume Injection Method)
From pump
To column
From pump
To column
Needle
Sample vial
Measuring pump
LAAQ-B-LC001B
LOAD
INJECT
35
Column Oven
Air
Insulated
Water
LAAQ-B-LC001B
block heater
bath
36
LAAQ-B-LC001B
37
Tubing
Material
LAAQ-B-LC001B
O.D.
(outer diameter)
1.6 mm
I.D.
(inner diameter)
0.1 mm
0.3 mm
0.5 mm
0.8 mm etc.
38
Connectors
Male
nut (SUS)
Ferrule (SUS)
Sealing possible up to 40
MPa
Male
LAAQ-B-LC001B
Ferrule
Male nut
nut (PEEK)
39
Dead Volume
(Extra-column volume)
Dead
Dead volume
Tube
Excellent connection
LAAQ-B-LC001B
Poor connection
40
Mobile Phase
Water
Organic
LAAQ-B-LC001B
Solvent
41
Replacement of Eluent
Water
2-Propanol
Hexane
LAAQ-B-LC001B
Buffer solution
Water
Water-soluble
organic solvent
42
Decompression
by aspirator
Decompression
by aspirator
Ultrasonic
cleaning unit
LAAQ-B-LC001B
43
LAAQ-B-LC001B
44
Polarity of Substances
Polarity
Miscibility
Property of a substance
whereby the positions of the
electrons give rise to
positive and negative poles
Water: Polar
Methane: Nonpolar
LAAQ-B-LC001B
C
H
Methane
Water
of solvents
Solvents of similar
polarities can be easily
dissolved together.
Polar and nonpolar
molecules have a similar
relationship to that of water
and oil.
H
O
H C C
O
H
Acetic acid
45
Functional
Groups
-(CH2)nCH3
Alkyl
Polar
Functional
Groups
Carboxyl
groups
-C6H5
Phenyl
-COOH
-NH2
Amino
groups
groups
-OH
Hydroxyl
LAAQ-B-LC001B
groups
groups
46
Partition Chromatography
A liquid
LAAQ-B-LC001B
47
Stationary
phase
Mobile phase
Normal
phase
High polarity
Low polarity
(hydrophilic)
(hydrophobic)
Reversed
phase
Low polarity
High polarity
(hydrophobic)
(hydrophilic)
LAAQ-B-LC001B
48
Octadecyl
Mobile
Water,
methanol, acetonitrile
Salt is sometimes added.
LAAQ-B-LC001B
49
(ODS) type
C8 (octyl) type
C4 (butyl) type
Si
-O-Si
Phenyl
type
TMS type
Cyano type
C18 (ODS)
LAAQ-B-LC001B
50
C18 (ODS)
Strong
C4
Weak
LAAQ-B-LC001B
51
Hydrophobic Interaction
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
Nonpolar solute
H2O
If a nonpolar
substance is added...
H2O
H2O
H2O
H2O
H2O
H2O
Nonpolar solute
LAAQ-B-LC001B
H2O
H2O
H2O
H2O
H2O
52
C18 (ODS)
Strong
Weak
CH3
LAAQ-B-LC001B
53
LAAQ-B-LC001B
54
H2O
H2O
H2O
H2O
H2O
Loose network
CH3OH
H2O
H2O
CH3OH
CH3OH
CH3OH
CH3OH
Nonpolar solute
CH3OH
CH3OH
Nonpolar solute
55
60/40
70/30
80/20
LAAQ-B-LC001B
56
Chromatogram Parameters
Methods for Expressing Separation
and Column Performance
LAAQ-B-LC001B
57
Retention Factor, k
tR
t R t0
k
t0
t0
LAAQ-B-LC001B
58
tR
N 16
W
H
W1/2
W
LAAQ-B-LC001B
H1/2
tR
5.54
W1/ 2
tR H
Area
59
N: Large
N: Small
LAAQ-B-LC001B
60
Separation Factor, a
Separation
k1
k2
k1
(k 2 k1 )
LAAQ-B-LC001B
61
Resolution, RS
tR1
tR2
RS
W1/2h,1
W1/2h,2
W1
LAAQ-B-LC001B
h1/2
t R 2 t R1
1
(W1 W2 )
2
t R 2 t R1
1.18
W1/ 2 h ,1 W1/ 2 h , 2
W2
62
(tR2 - tR1)
W1 W2
W1 W2
tR2 - tR1 = W1 = W2
tR2 - tR1 = W1 = W2
RS = 1
LAAQ-B-LC001B
RS = 1
If the peaks are Gaussian distributions,
RS > 1.5 is necessary for complete separation.
63
resolution is a
function of the
separation factor, the
theoretical plate
number, and the
retention factor.
The separation can be
improved by improving
these 3 parameters!
LAAQ-B-LC001B
RS
t R 2 t R1
1
(W1 W2 )
2
1
1
k2
k2 1
64
Increasing
the capacity
factor improves
separation!
A capacity factor of
around 3 to 10 is
appropriate. Exceeding
this just increases the
analysis time.
10
15
20
Capacity factor
LAAQ-B-LC001B
65
resolution
increases in
proportion to the
square root of the
theoretical plate
number.
LAAQ-B-LC001B
66
To Improve Separation...
Before
adjustment
k increased
N increased
increased
LAAQ-B-LC001B
LAAQ-B-LC001B
68
If an acid is added...
...the equilibrium shifts to
the left to offset the
increase in H+.
HA
A- + H+
If an alkali is
added...
the equilibrium shifts
to the right to offset the
decrease in H+.
LAAQ-B-LC001B
OH-
A- + H+
CH3COOH
CH3COO-
[A ][H ]
Ka
[HA]
[A ]
pH pK a log
[HA]
LAAQ-B-LC001B
pH log[H ]
pK log K
a
a
pKa
Relationship Between Abundance Ratio
and pH Value of Acetic Acid and Acetic Acid Ions
70
Make
LAAQ-B-LC001B
71
Commonly
Used Acids
Phosphoric acid
High buffering power at
pKa 2.1, 7.2, 12.3
prescribed pH.
Acetic acid
Does not adversely
pKa 4.8
affect detection.
Citric acid
Does not damage
pKa 3.1, 4.8, 6.4
column or equipment.
Concentration
Inexpensive.
If only to adjust pH, 10
mmol/L is sufficient.
LAAQ-B-LC001B
72
Characteristics of Phosphate
Buffer Solution
Advantages
Three dissociation
states
(pKa 2.1, 7.2, 12.3)
Possible
to prepare
buffer solutions of
various pH values.
Disadvantages
No volatility
Difficult
to use for
LCMS or evaporative
light scattering
detection.
No UV absorption
Inexpensive
LAAQ-B-LC001B
73
LAAQ-B-LC001B
74
Composition
Water
/ acetonitrile
Water / methanol
Separation
Adjustment
Changing
75
COOH
Increased
hydrophobicity
pH of eluent
COO
Alkaline
Increased
hydrophilicity
+
H
LAAQ-B-LC001B
76
Composition
LAAQ-B-LC001B
77
N+
H
OH
Si
O
OH
OH
LAAQ-B-LC001B
Si
OH
OH
OH
78
Si
O
Si
-O-Si-O
Residual silanol group
LAAQ-B-LC001B
Si
N+
H
79
ClO4
N+
H
Ion pair
Si
O
Si
LAAQ-B-LC001B
Composition
81
Basic Substance
82
Compounds
Tetra-n-butylammonium
Cationic
hydroxide (TBA)
Compounds
Pentanesulfonic
83
pH of Eluent
LAAQ-B-LC001B
LAAQ-B-LC001B
85
(liquid-solid) chromatography
Partition (liquid-liquid) chromatography
Normal
exchange chromatography
Size exclusion chromatography
LAAQ-B-LC001B
86
Adsorption Chromatography
A solid
87
Partition Chromatography
A liquid
LAAQ-B-LC001B
88
Solid phase
Mobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)
LAAQ-B-LC001B
89
90
Invention of Chromatography by
M. Tswett
Ether
Chlorophyll
Chromatography
Chromato
Colors
CaCO3
LAAQ-B-LC001B
91
Phase
Mobile
Phase
LAAQ-B-LC001B
92
Strong
HO
SiOH
Weak
Very weak
OH
Steric hindrance
LAAQ-B-LC001B
93
100/0
98/2
95/5
LAAQ-B-LC001B
94
LAAQ-B-LC001B
Phase
Reversed
Phase
95
Anion exchange
Cation exchange
R
N+ R
R
SO3-
++++
+
+
++++
Electrostatic interaction
(Coulomb force)
LAAQ-B-LC001B
96
Material
Cation
Exchange Column
Anion
LAAQ-B-LC001B
-SO3-COO-
(SAX)
(WAX)
-NR3+
-NHR2+
Exchange Column
(SCX)
(WCX)
97
Weakly acidic
cation exchanger
0
7
pH
Cation exchange mode
LAAQ-B-LC001B
14
Exchange capacity
Exchange capacity
Strongly acidic
cation exchanger
Weakly basic
anion exchanger
0
7
pH
14
Resin
Resin
Resin
An eluent ion is
driven away
and a solute ion
is adsorbed.
Solute ions and eluent ions compete for ion exchange groups.
If
the salt concentration of the eluent increases, the solutes are eluted sooner.
LAAQ-B-LC001B
99
H+
H+
Gel
Biochemical
LAAQ-B-LC001B
101
Packing
material
LAAQ-B-LC001B
102
Molecular weight
(logarithmic axis)
Exclusion limit
Permeability limit
Elution capacity
LAAQ-B-LC001B
103
Molecular weight
(logarithmic axis)
Elution capacity
For separation of small molecular weights
LAAQ-B-LC001B
104
Calibration curve
Retention time
LAAQ-B-LC001B
Mn: Number-average
molecular weight
Mw: Weight-average
molecular weight
Mz: Z-average molecular
weight, etc.
solvent
Molecular weight
Structural formula and chemical
properties
Do
106
LAAQ-B-LC001B
107
LAAQ-B-LC001B
108
Selectivity
The
Adaptability
to separation conditions
Operability, etc.
LAAQ-B-LC001B
109
absorbance detector
Photodiode array-type UV-VIS absorbance
detector
Fluorescence detector
Refractive index detector
Evaporative light scattering detector
Electrical conductivity detector
Electrochemical detector
Mass spectrometer
LAAQ-B-LC001B
110
Ein
Eout
C: Concentration
Detection cell
l
A = Cl = log (Eout / Ein)
111
Ein
Ein
Sample cell
Eout
Photodiode
Ein
Photodiode
Reference cell
D2 / W lamp
LAAQ-B-LC001B
112
200
LAAQ-B-LC001B
250
300
Wavelength [nm]
350
113
Grating
A single photodiode
measures the absorbance for
the corresponding wavelength
at a resolution of approx. 1 nm.
D2 / W lamp
Photodiode array
LAAQ-B-LC001B
114
W
av
ele
ng
th
Absorbance
Chromatogram
LAAQ-B-LC001B
Retention time
115
Complementation
retention time
Library searches
Evaluation
of identification based on
of Peak Purity
Purity
LAAQ-B-LC001B
116
Fluorescence Detector
Excitation wavelength
+ hv1
hv2 +
Excited state
Fluorescence wavelength
Quasi-excited state
hv1
LAAQ-B-LC001B
hv2
Fluorescence
Ground state
117
Xenon lamp
Fluorescence
grating
Photomultiplier tube
Fluorescence
Excitation grating
LAAQ-B-LC001B
Excitation
Sample cell
light
118
CHO
CHO
+ R-NH2
R-SH
N-R
o-phthalaldhyde
(OPA)
ADAM
LAAQ-B-LC001B
9-anthryldiazomethane
(ADAM)
CH2OCOR
119
Light
Sample cell
LAAQ-B-LC001B
120
Slit
Sample cell
W lamp
Reference cell
The slit image moves if the
refractive index inside the
flow cell changes.
Photodiode
LAAQ-B-LC001B
121
Light-receiving unit
Drift tube
Nebulizer
Column eluate
Nebulizer gas
Drain
Assist gas
Light source
122
Pure water
NaCl aqueous
solution
Cl-
LAAQ-B-LC001B
Na+
123
V
I
A
A
L
LAAQ-B-LC001B
I A
K k
E L
L
k K
A
Electrode
K:
I:
E:
A:
L:
k:
Cation
H+
Li+
Na+
K+
NH4+
(CH3)3NH+
Mg2+
Ca2+
LAAQ-B-LC001B
349.8
38.6
50.1
73.5
73.5
47.2
53.0
59.5
Anion
OH
F
Cl
Br
NO3
CH3COO
C6H5COO
SO42
198.3
55.4
76.3
78.1
71.4
40.9
32.3
80.0
125
Electrochemical Detector
Electrode
HO
HO
2eO
R
+ 2H+
O
LAAQ-B-LC001B
126
Reference electrode
(Ag/AgCl)
Working electrode
(glassy carbon)
Eluent
Electrode couple
LAAQ-B-LC001B
127
High vacuum
Quadrupole MS analyzer
Electron
multiplier tube
RP TMP1 TMP2
(high vacuum pumps)
LAAQ-B-LC001B
128
+
+-+-++
E
So vap
lv or
en at
t ion
ro
pl
et
+
+
of
+
+
+-+-++
+
+
+
n
io
us
cl
Ex tion
b
a
m
or
lo
ap
ou
C
Ev
n
3)
Io
Liquid Samples
Sample
Liquid
Neburaizing
Nebulizing Gas
Gas
+
++- - + -+ +- + - +
2)
Heater
Heater
Corona
Colona Discharge
Discharge
Needle
Nnndle
129
TIC A:100
B:100
C:150 D:150
m/z=150
C
LAAQ-B-LC001B
130
M/Z
M/Z
LAAQ-B-LC001B
M/Z
131
Comparison of Detectors
Absorbance
Fluorescence
Differential
refractive index
Evaporative light
scattering
Electrical
conductivity
Electrochemical
LAAQ-B-LC001B
Selectivity
Sensitivity
Possibility of
Gradient System
Light-absorbing
substances
ng
Possible
Fluorescent substances
pg
Possible
None
Impossible
Nonvolatile substances
Possible
Ionic substances
ng
Partially possible
Oxidizing / reducing
substances
pg
Partially possible
Note: The above table indicates general characteristics. There are exceptions. 132
Post-Column Derivatization
Reaction
chamber
Pump
Reaction
solution
LAAQ-B-LC001B
133
Acids
Reducing
Sugars
Arginine (fluorescence)
Carbamate
LAAQ-B-LC001B
Bromate
Pesticides
Tribromide ionization
(ultraviolet absorption)
o-Dianisidine
(visible absorption)
Cyanide
Ions
Ions
Chlorination - pyrazolone
(visible absorption)
Transition
Metal Ions
4-(2-Pyridylazo)
resorcinol, PAR (visible
absorption)
134
Quantitative Analysis
Absolute Calibration Curve Method
and Internal Standard Method
LAAQ-B-LC001B
135
Qualitative Analysis
Identification
spectra
MS spectra
Transfer
LAAQ-B-LC001B
136
Quantitative Analysis
Quantitation
height.
Calibration curve created beforehand
using a standard.
Absolute
137
Area
A1
Calibration curve
C1
C3
C4
LAAQ-B-LC001B
A2
Peak area
C2
A4
A3
A2
A3
A1
A4
C1
C2
C3
Concentration
C4
138
Concentration
Target Internal
substance standard
C1
Area
A1
CIS
A2
C2
AIS
CIS
A4
C4
AIS
CIS
A3
C3
AIS
CIS
LAAQ-B-LC001B
AIS
139
IS
AX / AIS
10 L
injected
Same area
ratio
9 L
injected
IS
CX / CIS
LAAQ-B-LC001B
140
100%
recovery
rate
IS
AX / AIS
Not
Same area
ratio
90%
recovery
rate
IS
CX / CIS
LAAQ-B-LC001B
141
142
Sample Pretreatment
Tasks Performed Before Injection
LAAQ-B-LC001B
143
Objectives of Pretreatment
To improve
values
To improve sensitivity and selectivity
To protect and prevent the deterioration of
columns and analytical instruments
To simplify measurement operations and
procedures
To stabilize target substances
LAAQ-B-LC001B
144
145
Filter
Syringe
146
Deproteinization
Precipitation
Addition
LAAQ-B-LC001B
147
Conditioning
(2)
Sample addition
(3)
Rinsing
(4)
Elution
Solvent with
low elution
strength
Solvent with
high elution
strength
Target
component
Unwanted
components
LAAQ-B-LC001B
148
Pre-Column Derivatization
OPA Reagent
CHO
CHO
+ R-NH2
N-R
R-SH
o-phthalaldhyde
(OPA)
2,4-DNPH
NHNH2
+
O2N
NO2
R
R
NHN=C
C=O
H+
O2N
NO2
2,4-dinitrophenylhydrazine
(2,4-DNPH)
LAAQ-B-LC001B
149
LAAQ-B-LC001B
150
LAAQ-B-LC001B
Validation
characteristics
Accuracy / trueness
Precision
Specificity
Detection limit
Quantitation limit
Linearity
Range
(Robustness)
151
Accuracy / Trueness
Definition
Evaluation
Degree of bias in
measurements obtained with
analytical procedures
Difference between true value
and grand mean of
measurements
True value
Method
Comparison with
theoretical values (or
authenticated values)
Comparison with results
obtained using other
analytical procedures for
which the accuracy
(trueness) is known
Recovery test
Measurement
LAAQ-B-LC001B
Average
Precision
Definition
LAAQ-B-LC001B
Degree of coincidence of
Precision of
series of measurements
measurements taken over
obtained by repeatedly
a short time period under
analyzing multiple samples
the same conditions
taken from a homogenous
test substance
Intermediate Precision
Variance, standard
Reproducibility
deviation, or relative
standard deviation of
measurements
153
Specificity
Definition
LAAQ-B-LC001B
Evaluation Method
Detection Limit
Definition
Evaluation
DL = 3.3 /slope
(: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)
LAAQ-B-LC001B
Method
155
Quantitation Limit
Definition
LAAQ-B-LC001B
Evaluation Method
QL = 10 /slope
(: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)
Linearity
Definition
LAAQ-B-LC001B
Evaluation
Method
Samples containing
different quantities of the
target substance (usually 5
concentrations) are
analyzed repeatedly, and
regression equations and
correlation coefficients are
obtained.
Residuals obtained from the
regression equations of the
measurements are plotted,
and it is confirmed that
there is no specific slope.
157
Range
Definition
LAAQ-B-LC001B
Evaluation
Method
The accuracy,
precision, and linearity
are investigated for
samples containing
quantities of a target
substance that
correspond to the lower
limit, upper limit, and
approximate center of
the range.
158
Robustness
Definition
LAAQ-B-LC001B
The ability of an
analytical procedure
to remain unaffected
by small changes in
analytical conditions.
Evaluation
Method
159
LAAQ-B-LC001B
160
Resin-Based
Generally a wide
range
pH range
2 - 7.5
Organic
solvent
No restrictions
Significant
restrictions
Pressure
resistance
25 MPa max.
Low pressure
resistance
Depends on packing
material
161
162
Measures
Filter samples.
Check that samples
dissolve in the eluent.
Get in the habit of
observing pressure
values.
Corrective
LAAQ-B-LC001B
Action
Corrective Action
Sample overload
Inappropriate sample
solvent
Dirt
Influence of secondary
retention effects
LAAQ-B-LC001B
164
whether the
cause of the problem
is not the column.
Eluent composition
Eluent flow rate
Column temperature
LAAQ-B-LC001B
If
the column is
identified as the
cause...
Rinsing
Replacement
165
whether the
cause of the problem
is not the column.
LAAQ-B-LC001B
If
the column is
identified as the
cause...
Rinsing
Review of
temperature control
Replacement
166
Pre-column
LAAQ-B-LC001B
167
Column Rinsing
Use
Consider
LAAQ-B-LC001B
168
tR
N 16
W
H
W1/2
W
LAAQ-B-LC001B
H1/2
tR
5.54
W1/ 2
tR H
Area
169
Column Storage
Storage
Solution
Storage
LAAQ-B-LC001B
Conditions