Professional Documents
Culture Documents
and HPLC
Various HPLC modes and applications
Normal
phase
Reversed phase
Reversed phase ion pairing
Ion exchange (IC)
SEC (GPC/GFC)
Chiral separation
HPLC
system modes
Isocratic elution
Gradient elution
What is chromatography?
Chromatography
technique.
The
river bed
Strong
Weak
Interaction is different
by gravity
column
packing material, 3-5um
sample A
packing
material
sample B
column
High
Advantage of Chromatography
Simultaneous Analysis
High
Resolution
High
Sensitivity (ppm-ppb)
Small
10
Advantage of HPLC
Moderate
analysis condition
11
pump
injector
column
oven
detector
12
13
M. Tswett :
first developer of chromatography
Ber. Deut. Botan. Ges., 24, 384 (1906)
Adsorptionsanalyse und chromatographische
Methode. Anwendung auf die Chemie des
Chlorophylls.
14
Chlorophyll's
CaCO3
Chromatograp
Chromato
h
Colors
15
developer used
CaCO3
as Separation Column
Petroleum
We defined
this combination as
:
:
polar property
non polar property
16
Si
Silica gel
-Si-CH2CH2CH2NH2
Si
-Si-CH2CH2CH2OCH(OH)-CH2(OH)
Modified Si
17
Non-polar
18
Hydrogen Bonding
Compounds contain
-COOH
-NH2
-OH
: Carboxyl group
: Amino group
: Hydroxyl group
HO
SiOH
Weak
OH
Very Weak
or
19
Secondary solvents
20
21
1 : Dioctyl phthalate
2 : Dibutyl phthalate
3 : Diethyl phthalate
4 : Dimethyl phthalate
2%
Solvent : Hexane
5% / MeOH
22
Column
Solvent
:
:
Non-polar property
Polar property
:
:
polar property
non polar property
23
(ODS) type
C8 (octyl) type
C4 (butyl) type
Phenyl type
TMS type
Cyano type
Non-polar property
-Si-C18H37
Si
24
Non-polar
polar solvent
25
Hydrophobicity
If
If
-COOH
-NH2
: Carboxyl group
: Amino group
-OH
: Hydroxyl group
Hydrophobicity
is stronger.
Hydrophobicity
is weaker.
C18 (ODS)
Strong
OH
Weak
26
+ Organic solvents
Optimization
27
28
30 %
1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate
40% / H2O
Solvent : MeOH
29
C18 (ODS)
sample
Strong
C4
sample
Weak
sample
30
C8 TMS
Analytical Conditions
31
Phase
good
Phase
Reversed Phase
Ion-Pair Chromatography
32
Ion-Pair Reagent
33
Ion-Pair Reagents
Anion
Compounds
Tetra-n-butylammonium
Cation
hydroxide (TBA)
Compounds
Butanesulfonic
Ion Pairing
Separation of Carboxylic Acids
34
Ion Pairing
Separation of Amino Compounds
Analytical Conditions
Peaks
1. Nicotinic acid
2. Nicotinamide
3. Pantothenate
4. Pyridoxine
5. Riboflavin
Phosphate
6. Thiamine
7. Caffeine
8. Folic acid
9. Biotin
10. Riboflavin
35
Ion Paring
Important Considerations
Type
of Ion-Pair reagents
Concentration of Ion-Pair reagents
pH
of solvent
RCOO- + H+
R-COOH
(pKa=4.5)
R-NH2 + H+
R-NH3+
(pKa=6.0)
36
37
Pentane Sulfonate
Concentration of
Ion-Pair Reagents
38
39
40
N+ R
R
SO3
Sample
+ + + +
+
+
Sample
+
+
+ + + + +
41
Fields
(protein, peptide, amino acid analysis)
Ion chromatography
Cation
Exchangers
Strong
(R-SO3-)
(R-COO-)
Exchangers
Strong Anion
Exchange
Weak Anion Exchange
(SAX)
(WAX)
(R4N+)
(DEAE)
Protein Analysis
using WCX column
Analytical
Conditions
Column
: Shim-pack WCX-1
Mobile phase :
[A] 20 mM phosphate buffer (pH=6.0)
[B] A+0.25M sodium sulfate
[A] - [B] 30 min linear gradient
Flow rate : 1.0 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. albumin
2. myoglobin
3. -chymotrypsinogen A
4. liponuclease A
5. lisozyme
42
of Buffer solution
Concentration of Buffer solution
Elution Method
Isocratic
elution
pH gradient elution
increasing Ionic strength gradient
43
44
What is SEC ?
Size Exclusion Chromatography (SEC)
GPC
mainly
GFC
mainly
45
Principle of SEC
No Interaction Force
Difference of Traveling Time
46
Elution order
SEC
Column
47
polymer
GFC
Separation of
protein
of
Relationship between
MW and RT
Exclusion limit
Permeation limit
Time
48
49
Calibration Curve
Inject
time(min)
22.0
22.6
23.4
25.0
27.4
31.0
33.8
38.4
39.8
42.8
46.8
mol. wet.
5500000
1800000
860000
400000
160000
50000
20000
4000
2000
600
80
50
the MW,
GPC software is required.
Protein Separation
using GFC column
Analytical
Conditions
Column
: Asahipak GFA-50
Mobile phase :
0.1 M sodium phosphate
0.1 M NaCl (pH=7.0)
Flow rate : 0.5 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. glutamate dehydrogenase
2. lactate dehydrogenase
3. enolase
4. adenylate kinase
5. cytochrome C
51
52
C*
NH2
M
ir
ro
r
*C
COOH
HOOC
*Chiral Center
NH2
(L) form
(D) form
Physical properties are all the same
except optical rotation.
53
(L)-(R)
Enantiomer
Diastereomer
(R) + (R)
(R)-(R)
Direct separation
using Chiral Column
Stationary
(R)
(R)
(L)
Strong interaction
Weak interaction
(R)
54
55
elution mode
One
Gradient
Multi
elution mode
56
pump
oven
injector
column
Single Solvent
detector
57
B
pump
oven
B concentration
pump
injector
column
Time
detector
58
Bad Separation
MeOH / H2O = 8 / 2
( column : ODS type )
59
95%
30%
MeOH concentration
60
Sample injector
Column oven
Detector
61
pump
injector
column
oven
detector
62
Pressure Resistance
Precise
Low
Flow Rate
Pressure Fluctuation
63
P=0.5
P=1.5
64
pump head
check
valve
plunger
plunger seal
10 -100uL
65
Advantage
Low
pressure fluctuation
Very easy to replace the another
solvent
Disadvantage
Change
Dual plunger
with parallel flow line
check valve
plunger head
66
Dual plunger
with tandem flow line
check valve
67
Sub plunger
Main plunger
68
Single plunger
check valve
High pressure-fluctuation
Low sensitivity analysis
using UV / PDA and
Fluorescence detector
The number of maintenance
parts is minimized.
69
HPLC Injectors
Manual injector
Auto injector
70
load position
pump
column
sample loop
pump
column
71
Manual Injector
72
Manual Injector
[LOAD]
Pump
Pump
Column
Column
[INJECT]
Response of Detector
3 times volume
of sample loop
Volume of Injection
73
74
Injection Method
Partial Injection Method
better
75
dr
ai
n
dr
m
u
p
loop
dr
ai
co
lu
m
ai n
m
u
p
co
lu
m
in
a
dr
Sample Zone
Dilution Zone
Dispersion Zone
loop
76
m
u
p
in
co
lu
m
in
a
dr
dr
a
loop
m
u
p
in
co
lu
m
in
a
dr
loop
Sample Zone
Dilution Zone
77
78
[INJECT ] position
[LOAD] position
79
Injection port
Must put the end of drain pipe
at higher than Injection port
80
81
Caution
Do
Must
Do
Must
Column Temperature
Control Devices
Column temperature control devices are functioning to keep the
column temperature constant. The temperature fluctuation of
column will influence retention time reproducibility.
Column
Aluminum block
Insulated
Column Jacket
Water bath
82
83
/ Visible detector
Photodiode Array detector
Fluorescence detector
Conductivity detector
Refractive Index detector
Electrochemical detector
Mass spectrometer detector
(UV/VIS)
(PDA)
(RF)
(CDD)
(RID)
(ECD)
(MS)
84
Cell
Ein
Eout
l
Lambert-Beer's law
85
Sample Cell
Ein
Eout
Photodiode
Ein
Ein
Photodiode
Reference Cell
D2 / W lamp
86
Lambert-Beer's law
Absorbance
linear range
Concentration
87
Spectrum
275 nm
208 nm
275 nm
208 nm
88
Additional Functions
Dual Wavelength
Ratio
mode
Plot mode
Wavelength Time Program mode
Wavelength Scan mode
89
90
Wavelength Time
Program Mode
91
92
93
D2 / W lamp
Grating
One element can
detect one absorbance
at one wavelength.
94
W
av
ele
n
gth
Absorbance
Spectrum
Chromatogram
Time
95
UV spectra
2
3
30%
1: Azelaic acid
2: Benzoic acid
3: Nitrobenzoic acid
Acetonitrile
concentration
15%
min
Identification by Spectrum
Elution sequence
30%
Peak 1
Peak 2
Peak 3
Acetonitrile
Azelaic acid
Benzoic acid
Nitrobenzoic acid
15%
98
99
Fluorescence detector
Excitation Wavelength
+ h 1
h 2+
A*
Emission Wavelength
h 2
h 1
A
Fluorescence
A
Cell design
of Fluorescence detector
100
101
Derivatization Reagents
OPA reagent for primary amines A
CHO
CHO
+ R-NH2
N-R
o-phthalaldhyde
(OPA)
9-anthryldiazomethane
(ADAM)
CH2OCOR
102
103
104
Chromatogram of sugars
Analytical Conditions
Peaks
1. Glycerol
2. Xylose
3. Fructose
4. Glucose
5. Sucrose
6. Manose
7. Lactose
105
Conductivity detector
V
I
A
L
k= (I/E)*(L/A)
electrode
106
Conductivity detector
Conductivity
107
108
Non-Suppresser Type
extremely low
ion exchanger
Low conductivity
mobile phase
109
Suppresser Type
110
Chromatogram of Anion
in the brine
Analytical Conditions
Peaks
1.
2.
3.
4.
5.
6.
F
Cl
NO2
Br
NO3
SO4
(1.4 ppm)
(10200 ppm)
(10 ppm)
(43 ppm)
(44 ppm)
(431 ppm)
111
Chromatogram of Cations
in Tap water
Analytical Conditions
112
Peaks
1. Na
2. NH4
3. K
4. Mg
5. Ca
(8.25 ppm)
(0.01 ppm)
(1.66 ppm)
(2.22 ppm)
(11.85 ppm)
113
Electrochemical detector
Working
electrode
Reference
electrode
AUX electrode
114
O + H+
e-
[ Applications ]
GC
Pt
Ag
Au
: phenol compounds
general use
: H2O2
: halogen ion
: sugar analysis
Electrode
Glassy Carbon (GC)
Pt, Ag, Au
115
Chromatogram of Catecolamines
Analytical Conditions
Peaks
1. Noradrenalin ( 5 ppb)
2. Adrenalin (5 ppb)
3. Dopamine (5 ppb)
LCMS
Atmospheric Pressure Ionization
116
High Voltage
+
+-+-++
+
+
+
+
+-+-+++
+
+
on
si
lu
xc n
E tio
n
lo ora
ou p
C va
3) n E
Io
n
io
at
or nt
ap ve
Ev o l
2) of S
+
++- - + -+ +- + - +
Heater
Corona Discharge
Needle
117
Design of LCMS
Atmosphere
High Vacuum
API Probe
MS
detector
RP TMP1 TMP2
(Vacuum pump system)
APCI
pesticides
steroids
drugs
118
119
quantitative capability
M/Z
M/Z
M/Z
120
A
TIC
B
A:100
B:100
D:150
C:150
m/z=150
C
121
Analysis of Pesticide
TIC
isoxation oxon
iprofenfos
EPN oxon
303
298
289
577
Analysis of Pesticide 1
(Mass Spectrum)
NO 2
O
P O
OC 2H5
EPN oxon
O
O
P
O
P OC 2H5
OC 2H5
OCH(CH 3)2
OCH(CH 3)2
isoxation oxon
iprofenfos
122
123
Mass Separation
TIC
HO
HO
HOH 2C
OH
O
HOH 2C
O
HO
Maltose
O
OH
OH
HOH 2C
HO
HO
OH
OH
Glucose
HO
HO
387
225
195
H 3C
Xylose
O
OH
OH
124
UV/ VIS
LOD 1 ppb
GC
Yes
RF
10 ppt
Yes
RID
100 ppb
No
CDD
1 ppb
No
ECD
10 ppt
No
MS
1 ppb
Yes