HPLC

1
4. Gradient elution method
2
4. Gradient elution method
For separation of a sample containing many
components
3
4. Gradient elution method
Advantage of gradient elution method
Gradient elution method
Isocratic elution method
A
A
Finepak SIL C18 MeOH/1 AcOH(40/60)
B
B


A
MeOH/1 AcOH 30/70?45/55 Linear
Gradient?16min
MeOH/1 AcOH(30/70)
A Chlorogenic acid B Rutin Impurity
B
4
4. Gradient elution method
Precautions in gradient elution method
- Can the gradient save time ? -
Reproducibility - Baseline - Ghost peak - Salt
5
4. Gradient elution method
Effect of temperature on retention time
Finepak SIL C18 CH3CN/H2O(90/10) Sample 1.
Benzene 2. Anthracene 3. Pyrene 4.
Benz(a)pyrene
6
Section 4
Review of
Gradient elution method Temperature effect
7
5. Detector
8
5. HPLC detectors
HPLC detectors UV-VIS(Absorption) PDA
(Absorption) Differential refractometer(Refractiv
e index) Fluorometric (Florescence) Electrochemi
cal (ECD) (Oxidation -reduction) Conductivity M
ass Chiral (OR) Circular dichroism (CD)
9
5. HPLC detectors
UV/Vis detector - Selective detection
minimizing effects from other components - High
sensitivity detection at maximum absorption
wavelength
10
5. HPLC detectors
Improved selectivity
Traditional medicine
berberine
berberine
impurity
impurity
7.385
7.395
Wavelength260nm
Wavelength340nm
11
5. HPLC detectors
0nm
Improved sensitivity
Saccharin (SAC) and sorbin acid (SOR)
265nm
230nm
SOR
SOR
SAC
SAC
10-
20-
10-
20-
3.575
3.608
12.467
12.250
Wavelength programming
Fixed wavelength at 265nm
12
5. HPLC detectors
UV spectrum measurement to find wavelength
effective for wavelength programming
1.0
Diazepam (DZP)
Nitrazepam (NZP)
Absorbance
0.5
Chronazepam (CZP)
0
210 250 300
350
Wavelength(nm)
13
5. HPLC detectors
Wavelength programming and fixed wavelength
Wavelength programming
Fixed wavelength
14
5. HPLC detectors
Optics of Multi-wavelength detector
Grating
I2 lamp
UV/Vis detector
D2 lamp
Photo diode
lamp
Grating
Photo diode
Cell
Cell
Photodiode array
15
5. HPLC detectors
Multi-wavelength detector 3D chromatogram
16
5. HPLC detectors
Multi-wavelength detector Cont. data
17
5. HPLC detectors
Features of Multi-wavelength detector
1. Spectrum collection at any time 2. Library
search 3. Purity check 4. Quantitative
analysis at 6 wavelengths
18
5. HPLC detectors
Principle of Fluorescence detector
(S1)
Excited state
(S2)
(S3)
H? (fluorescence)
excitation
emission
(S0)
Ground state
Mobile phase
19
5. HPLC detectors
Features of Fluorescence detector
1. Selective detection 2. Detection at any
excitation or emission wavelength 3. High
sensitivity
20
5. HPLC detectors
Wavelength programming by Fluorescence detector
Wavelength programming
Fixed wavelength Ex275nm Em400nm
Fixed wavelength Ex450nm Em525nm
VB2
VB2 Phosphate
VB2 Phosphate
VB6
VB2
VB6
VB1
0 10 20 min
0 10 20 min
0 10 20 min
Column Finepak SIL C18S Mobil phase
MeOH/Phosphate Buffer Gradient
21
5. HPLC detectors
Selectivity of UV detector and Fluorescence
detector
UV detector
Fluorescence detector
22
5. HPLC detectors
Principle of RI detection
light
light
i
i
r0
r
Solvent
Sample and solvent
r0gtr
23
5. HPLC detectors
UV detector and RI detector
RI detector
UV detector
24
5. HPLC detectors
Considerations for IR detection
1. Temperature change 2. Replacement of solvent
(reference cell and sample cell) 3. Unstable
when solvent mixed 4. Replacement of solvent
inside column
25
5. HPLC detectors
Detectors
UV Fluorescence RI Sensitivity ng pg µg Detec
tion selective highly selective universal selectiv
ity Temperature small small large Influence
Gradient elution possible possible impossible
26
5. HPLC detectors
Label method
Samples absorb less UV/Vis light . Samples do not
fluoresce. ? Improved sensitivity and selectivity
required ? Label method
27
5. HPLC detectors
Label method
Post-label method
Pre-label method
reagent
injector
sample
(reaction)
pump
injector
column
reagent
column
reactor
detector
detector
28
5. HPLC detectors
Label method
Post-label method
Aminoacid 0PA Fluorescence ninhydrine Absorption
in Visible range Sugar guanidine Fluorescence Or
ganic acid brom thymol blue Absorption in
Visible range Catecholamine ethylenediamine
Fluorescence THI Fluorescence Bile acid NAD
Fluorescence HSD Fluorescence
29
5. HPLC detectors
Pre and Post column derivatization method

Pre-column Post-column LC system
required Standard system Reaction system is
required Reproducibility less than
post-column good Operation for all samples only
reagents Reagents wide range limited Applicabil
ity spot routine
30
5. HPLC detectors
Pre-column derivatization method
Amino acid
Dabcyl-Cl
pH8.1?70??12min
H
Dabcyl - Amino acid
31
5. HPLC detectors
Separation of Dabcyl - Amino acid
40pmol each Wavelength 465nm
32
5. HPLC detectors
Post-column derivatization method
orthophthalaldehyde (OPA)
Derivative compound
33
5. HPLC detectors
Post column derivatization method
Ex 345nm Em 455nm
CysSO3H
Val
Thr
His
Ser
Asp
Met
Gly
Ile
Glu
Arg
Ala
Leu
Phe
Lys
Pro
Tyr
Cys
Trp
0 20
40 60 (min)
34
Section 5
Review of
Detectors Selectivity and sensitivity Pre-/Post-
column derivatization methods