Beruflich Dokumente
Kultur Dokumente
Applications
Strengths
Limitations
There
Modes of HPLC
Normal
Phase mode
Reverse Phase mode
Reverse Phase Ion Pairing mode
Ion Exchange mode
SEC mode ( GPC / GFC )
Chiral separation mode
5
(ODS) type
C8 (octyl) type
C4 (butyl) type
Phenyl type
TMS type
Cyano type
Non-polar property
-Si-C18H35
Si
polar solvent
Non-polar
7
Hydrophobicity
If
Hydrophobicity
becomes strong.
Hydrophobicity
becomes weak.
8
C18 (ODS)
Weak
Strong
OH
30 %
1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate
40% / H2O
Solvent : MeOH
10
C18 (ODS)
sample
Strong
C4
sample
Weak
sample
11
C8 TMS
Analytical Conditions
12
Ionizable compounds
Control of elution rate of ionisable compounds by adjustment of pH of mobile
phase. Untuk asam Kapp = K/(1+10pH-pKa)
untuk basa Kapp = K/(1+10pKa-pH)
The pH of the mobile phase can only be set within the range of ca 2-8.5 pH
units because of the tendency for extremes of pH to dissolve silica gel and
break the bonds between silane-coating agents and the silica gel support
13
Reverse Phase
Ion-Pair Chromatography
Ion-Pair Reagent
14
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)
15
Pentane Sulfonate
16
Concentration of
Ion-Pair Reagents
17
group
Residual heavy metal
18
Dead Volume
Dead
Sample
Injector Connection
Column Connection
Detector Connection
Good
Dead Volume
19
20 uL
20 uL
Caffeine
10 uL
Caffeine
Caffeine
Solubility effect
Low soluble solvent
21
Group
Even
Silanol group
negative charge
22
End capping
To
C18
C18
OH
O-TMS
TMS treatment
C18
C18
silica core
silica core
OH
O-TMS
TMS : trimethylsilyl group
C18
C18
C18
C18
[Non-End capping type]
[End capping type] 23
heavy metal
Normal
C18
O-TMS
+
C18
silicaMcore
O-TMS
C18
M+
C18
O
O
Caution
Do
Must
Do
Must
HPLC system
Isocratic
Single
Gradient
Multi
elution system
elution system
High
26
pump
oven
injector
detector
column
Single Solvent
27
B
pump
oven
detector
B concentration
pump
injector
column
Time
28
Bad Separation
MeOH / H2O = 8 / 2
( column : ODS type )
29
95%
30%
MeOH concentration
30
mixer
low pressure
gradient device
Low pressure
gradient system
Degasser
31
excellent
gradient accuracy
complicate system (more than two pumps)
Low
simple
system
degasser is required
32
33
Quantity of oxygen
for saturation (uL)
Solubility of oxygen in 1 mL of
water-ethanol solvent mixture
200
100
50
100
34
What's happen
in mixing two solvents?
H2O
Bubble will
form!!!
30
140
Mixing 50
140-50=90
EtOH 250
( 30 + 250 ) / 2 = 140
35
O2 concentration (mL)
0.6
0.4
0.2
10
20
Pressure (atm)
36
less than 1%
Column
k,
Performance check
N, HETP, , Rs
Calibration method
External
calibration method
Internal calibration method
Standard additive method
Correction Normalization method
38
Dilution
Dilution
Dilution
Dilution
39
Concentration
1000
2000
3000
4000
Peak Area
40
[Concentration]
Calculation of Results
Y = bX + a
125 ppm
2500
[Peak Area]
b : SLOPE
a : Y intercept
2500
41
Internal
Standard
Target Compounds
Dilution
Dilution
Dilution
Dilution
42
43
Analysis of Vanillin
4
3.5
3
2.5
2
1.5
1
0.5
0
0
10
15
Calculation of Results
Y = bX + a
b : SLOP
a : Y intercept
1.67
5.0
[Target Area / IS Area]
T 2500
500
IS
Target
Target
46
Disadvantage of external
standard calibration method
Injection
100 ppm
11 uL injection
110 ppm
47
10 uL injection
1000
IS
11 uL injection
2000
2000 / 1000 = 2
1100
IS
2200
T
2200 / 1100 = 2
48
to actual sample
T
IS
950
Disadvantage of internal
standard calibration method
Separation
IS
is slightly difficult.
IS
IS
50
Disadvantage of internal
standard calibration method
It
The
51
Calibration Method
External
standard calibration
Separation
is not difficult
Injection error will directly influence the
quantitative result
Internal
standard calibration
Injection
Standard additive
calibration method
Original
Sample
Target
T
53
Standard additive
calibration method
x104
100 ppm=10x104
??? ppm= 7x104
17
12
10x104
Peak Area
??=70 ppm
7x104
T
-70
50
100 ppm
Added amount
54
Accurate Quantitation
[ Detector Response ]
ty
i
r
a
e
n
Li
c
e
p
Ex
t
i
s
Sen
R
d
te
e
g
n
a
y
t
i
iv
[ Concentration of Solute ]
55
Derivatization - automatization
Precolumn Derivatization - good separation
Sample itself
Column
Reagent
Detector
Derivatized Sample
Column
Detector
56
o - phthaldialdehyde (OPA)
57
58
Preparation of Sample
Removal
of insoluble material
Filtration
Centrifuge
Control
(Precipitation)
of concentration
Concentration
Dilution
Extraction
Liquid
phase extraction
Solid phase extraction
Solvolysis
(Hydrolysis)
Derivatization for detection
59
Filtration
Membrane filter of 0.45 m mesh will be
used before injection in order to remove the
insoluble material.
60
Ultrafiltration
Separation of high
molecular weight
compounds such as
protein and
low molecular
weight compound
61
Centrifuge
62
Liquid-Liquid Extraction
Extracting
solvent
n-Hexane
Carbon
tetrachloride
Cyclohexane
Chloroform
Dichloromethane
1,1-Dichloroethane
Diethyl ether
Ethyl acetate
63
Liquid-Liquid Extraction
pH
control
Analyte
carboxyl
group (-COOH)
amine group (-NH2)
Mixing
Manual
or mechanical mixing
Minimize emulsions by less vigorous mixing
Centrifuge
64
Removal of Proteins
Serum 100 uL
Acetonitrile for denaturation of proteins
IS (20 ug/mL)
100 uL
3 min)
Organic phase
Filtration
0.1N HCl 1 mL
Organic phase
H2O 1 mL
Evaporation
Organic phase
HPLC
66
67
68
Benefit of SPE
Solid
Convenient
Inexpensive
Time
- saving
Reduction of organic solvent volume
alternative to liquid - liquid extraction, the method
traditionally used for cleaning and concentrating
analytical samples
69
Extraction Tubes
Normal Phase
71
Preventive Maintenance
Mobile Phase
Connection
Solvent grade
How to change mobile phase
Degassing
Selection of pipe
Dead volume
Injector
Column
Detector
72
Mobile phase
- solvent grade Pure
grade
water
Distilled water
Deionized water
Absorption
Water
pure
water
deionized
water
Wavelength (nm)
H2O / MeOH
gradient
a
r
G
t
n
e
di
e
v
r
cu
ODS Column
Ghost Peak
74
Difference of analytical
and HPLC grade
Methanol
Acetonitrile
Hexane
75
76
Chloroform
Cyclohexane
Ethanol
0.350
Ethyl Acetate
Ethyl Eter
0.750
Heptane
1.000
Hexane
0.250
1.000
Methanol
1.000
0.200
0.080
0.300
Methylene Chloride
Pentane
2-Propanol
THF
1.000
0.300
0.100
1.000
0.250
1.000 0.600
240
245
250
254
0.010
0.025
1.000 0.320 0.150
0.014
0.040
1.000
0.070
0.014
0.014
0.150
0.025
1.000 0.700 0.200
0.100
0.014
0.130
0.025
0.300
0.100
77
Tetrahydrofurn
78
Chloroform
79
How to change
the mobile phase
Buffer solution
Water
Aqueous Organic Solvent
( methanol, acetonitrile etc. )
2-propanol
80
Pipe
20 cm
How to change
the mobile phase
Do not
touch
directly
Suction Filter
81
Degassing
Off line degassing
Vacuum pump
or Aspirator
On line degassing
He purging
Vacuum Chamber
He
Pump
Pump
Ultrasonic bath
Teflon tube
Vacuum Chamber
82
Selection of pipe
Material
Stainless
Steal
Teflon
PEEK
Size
0.1
Pipe Material
Pipe Size
0.8 mmI.D.
connection for from pump to injector
drain pipe
0.5 mmI.D.
connection for from pump to injector
reaction coil
0.3 mmI.D.
connection for from injector to column
connection for from column to detector
back pressure coil
0.1 mmI.D.
resistor coil
85
Connector
Ferrule
Male Nut SUS
easy to handle
withstand up to 250 kgf/cm2
easy to make a dead volume
Plugged
87
LC column
Wash
000
xxxx
xxxx
xxxx
xxxx
000
xxxx
xxxx
xxxx
xxxx
xxxx
90
Voids
Voids
void
Hard to repair!!!
91
Chemical Attack of
Packing Material
Detector
- bubbles problems -
93
Detector
check