Transcript Chromatography Gas GSC GLC Liquid LSC LLC IEC GC: volatile solutes LC: any mobile phase soluble solutes GPC AC pre-column guard-column pump injector value analytical column inlet filter Eluent reservoir detector Recorder / integrator.

Chromatography
Gas
GSC
GLC
Liquid
LSC
LLC
IEC
GC: volatile solutes
LC: any mobile phase soluble solutes
GPC
AC
pre-column
guard-column
pump
injector
value
analytical
column
inlet filter
Eluent reservoir
detector
Recorder /
integrator
ionic
water soluble
HPLC ion pair
HPLC normal phase
non-ionic
M<2000
polar
organic soluble
non-polar
sample
IC
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
SEC gel fitration
water soluble
M>2000
organic soluble
HPLC reverse phase
IC
SEC gel fitration
Interaction between molecules
solute
S.P
M.P
N.P
R.P
packing material
polar (silica)
non-polar (C18)
sep. interaction
polarity
polarity & size
(weak to medium
polar)
n-hexane/polar
solvent
n-hexane
CH2Cl2
Ethyl acetate
MeCN
(strong to medium
polar)
low polarity (alkanes)
high polarity
(salts)
Eluent
weak
strong
most rapidly
eluented cpd.
H2O/organic
H2O
MeOH
MeCN
THF
CH2Cl2
Pump
• Motor driven syringe
• Piston
– Reciprocating
– Multiple reciprocating
• Advantage and disadvantage
– Solvent reservoir limited?
– Pressure pulse produced?
– Gradient can be produced?
沖提模式：isocratic elution (A% B%固定)
gradient elution
Gradient Elution
%B
100
%B
linear
100
50
%B
curve
100
50
10
20
tR
segmented
50
10
20
tR
Application
(1) sample with a wide k’ range
(2) sample composed of large molecule
(3) samples containing late-eluting interferences
(4) maximizing detection sensitivity (&前濃縮)
10
20
tR
使用HPLC須注意：
1. 氣泡→壓力、流速不穩(常為溶液混合所產生)
2. 顆粒→阻塞，壓力上升
Eluent
考慮(1) sample
(2) separation mode
Eluent之選擇
α
k’
phy. pro.
(1) phy. properties
b.p.、viscosity、UV cutoff、毒性、純度、價格
UV cutoff：
(2) 1 ≤ k’ ≤ 10 for simple components
0.5 ≤ k’ ≤ 25 for multiple components
solute/eluent interaction↑ k’↓
solute
S.P
M.P
4 major interaction
(a) dispersion interaction
(b) dipole interaction
(c) H-bonding interaction
(d) dielectric interaction
dispersion force：
x
(非極性分子)
s
Temporacy dipole moment from x will polarize the e in
adjacent molecule s (R.I.值大表示分散力大)
eluent strength
solvent polarity
N.P. solvent polarity ↑ k’↓
R.P. solvent polarity ↑ k’↓
N.P.
k2 '
 10
k1 '
P1 '  P2 '
2
P’：polarity
R.P.
k2 '
 10
k1 '
P2 '  P1 '
2
solvent mixture
P'  a Pa 'b Pb '
a , b ：volume ratio lf solvent a,b
30% MeOH 70% H2O
P'  0.3  5.1  0.7 10.2  8..67
40% MeOH 60% H2O
P   0.4  5.1  0.6  10.2  8.16
8.67 8.16
0.5
k2 '
 10 2
 10 2  1.8
k1 '
(3) solvent selectivity
proton donor
由proton acceptor 將solvent分類
dipole moment
column
由physical size分類
I.D.
L
1. Analytical column：
2.1~5.0 mm
10,15,25 cm
2. microbore column：
1.0~2.0 mm
‘’ ‘’
3. capillary column：
‘’ ‘’
同樣濃度，peak大小：
A>B>C
A：1 mm
B：2.1 mm
C：4.6 mm
50 μL/min
0.2 mL/min
1 mL/min
column A：1 mm i.d. and
50μL/min solvent flow
column B：2.1 mm i.d. and
200μL/min solvent flow
column C：4.6 mm i.d. and
1000μL/min solvent flow
structure of silica gel depicting the various types of
bonds and silanol groups present
column 材料
bonded-stationary phase
Si
OH +
Si
HOR
OR
MgBr
Si
SiOH + SOCl
Si
Cl
H2N(CH2)2NH2
Si
NH(CH2)2NH2
R
R
SiOH + Cl
Si
Si
R
O
Si
R
R
R
R¡G (H2C)17
CH3
C1 , C2 , C4 ,
(CH2)3CN ,
N+
R3 Cl-
SO3- H+
, C8 , C18 , C22
(CH2)3NH2
anion exchange
cation exchange
PS-DVB ( polystyrene – divinyl benzene)
C C
C C
C C C C C
+
C C
C C C
pH=1~13
Mechanically strong Tailing peak for base
Inorganic
High efficiency
limited pH range
( silica )
predictable retention chem. unstable
organic
wide pH range
no tailing
chemical stable
low efficiency
Mech. weak
unpredictable retention
silica：
Si
O
Si
residual silanol
R
Si OH CH
3
Si
O
Si
CH3
end-capped
CH3
2 < pH < 8
if pH > 8 Si-O-Si →SiOpH < 2 Si-R可能被破壞
pre-column的材料常為silica
hybrid (silicon-C)：
OEt
2 EtO
Si
OEt
OEt
+
1 EtO
OEt
OEt
pH=1~12
Si
O ( Si
Si
OH
Si
O ( Si
Si
polar
polar
CH3
Si
O
Si
R
Si
R
Si
O
Si
R
Si
R
Si
O
Si
Elution order most polar being eluted 1st
靜 相 ：C1，C8，C18
tR ：C1 < C8 < C18
polarity：C1 < C8 < C18
HPLC detectors
• Bulk property detectors
– respond to a mobile phase bulk property
– refractive index, dielectric constant, or density
• Solute property detectors
– respond to some property of solutes
– UV absorbance, fluorescence, or diffusion current
Light path in a micro flow cell of a spectrophotometric detector.
Cells that have a 0.5-cm pathlength and contain only 8μL of liquid
are common.
Refractive Index Detector
A refractive index detector responds to almost every solute, but its
detection limit is about 1000 times poorer than that of the ultraviolet
detector.
Refractive index detectors are useless in gradient elution because
it is impossible to match exactly the sample and the reference while
the solvent composition is changing.
Fluorescence detectors
Fluorescence detectors excite the eluant with a laser and measure
the fluorescence.
These detectors are very sensitive but respond only to the few
analytes that fluoresce.
Evaporative Light-Scattering Detector
An evaporative light-scattering
detector responds to any
analyte
that is significantly less volatile
than the mobile phase.
LC Detector
Commercially
Available
Mass LOD
(commercial
detectors)
Mass LOD
(state of the art)
Absorbance
Yes
100 pg – 1 ng
1 pg
Fluorescence
Yes
1 – 10 pg
10 fg
Electrochemical
Yes
10 pg – 1 ng
100 fg
Refractive index
Yes
100 ng – 1 ng
10 ng
Conductivity
Yes
500 pg – 1 ng
500 pg
Mass spectrometry
Yes
100 pg – 1 ng
1 pg
FT-IR
Yes
1μg
100 ng
Light scattering
Yes
10 μg
500 ng
Optical activity
No
─
1 ng
Element selective
No
─
10 ng
Photoionization
No
─
1pg – 1 ng
ionic
water soluble
M<2000
polar
organic soluble
non-polar
water soluble
M>2000
organic soluble
HPLC ion pair
HPLC normal phase
non-ionic
sample
IC
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
SEC gel fitration
HPLC reverse phase
IC
SEC gel fitration
Size-Exclusion Chromatography
(SEC)
‧固定相︰ silica or polymer particles
with pore size 102-106 Å
‧ Type︰
-Gel Filtration Chromatography (GFC)
stationary phase: hydrophilic
mobile phase: aqueous
適用: protein, amino acid, peptide.
-Gel Permeation Chromatography (GPC)
stationary phase: hydrophobic
mobile phase: nonpolar solvent
適用: oligomers
VR = V0 + KVi
Applications:
1. Molecular weight determination
2. Group fraction
ionic
water soluble
HPLC ion pair
HPLC normal phase
non-ionic
M<2000
polar
organic soluble
non-polar
sample
IC
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
SEC gel fitration
water soluble
M>2000
organic soluble
HPLC reverse phase
IC
SEC gel fitration
Ion-exchange chromatography
sample ion：X
mobile phase：Y
charged stationary phase：R
X- + R+Y-
Y- + R+X-
anion exchange
X+ + R-Y+
Y+ + R-X+
cation exchange
SCX：strong cation exchange –SO3-H+
WCX：weak cation exchange –COOH
SAX：strong anion exchange –NR3+OHWAX：weak anion exchange –NH2
SCX
exch.
capacity
WCX
COOH
COO-
pH
exch.
capacity
SAX
WAX
pH
selectivity
R-SO3-H+(R) + Na+ (S)+ Cl-
K
Na
H
R-SO3-Na+(R) + H+ (S)+ Cl-

Na  H 
 H  Na 


R

S

R
S
Na
selectivity coefficient： K H
counter ion (M)
H+
Li+
Na+
K+
Mg2+
Cu2+
Ba2+
Co2+
K HNa
1
0.76
1.2
1.76
2.23
3.14
5.66
2.46
Ba2+ < Pb2+ < Sr2+ < Ca2+ < Ni2+ < Cd2+ < Cu2+ < Zn2+ <
Mg2+ < Tl+ < Ag+ < Cs+ < K+ < NH4+ < Na+ < H+ < L+
HCl
KOH
pump
pump
HCl
NaCl
R-
NaOH
KCl

NaCl

R+ separator
OH- SAX
separator
H+
SCX
NaOH
KOH
R+
suppressor
OHSAX
HCl
HBr
R- suppressor
H+
SCX
conductivity
cell
R+OH- + HCl → R+Cl- + H2O
R+OH- + NaOH → R+Cl- + NaOH
R-H+ + NaOH → R-Na+ + H2O
R-H+ + NaCl → Na+ + HCl
ionic
water soluble
HPLC ion pair
HPLC normal phase
non-ionic
M<2000
polar
organic soluble
non-polar
sample
IC
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
HPLC normal phase
HPLC reversed phase
SEC gel fitration
water soluble
M>2000
organic soluble
HPLC reverse phase
IC
SEC gel fitration
Ion-pair chromatography (IPC)
IPC & RP-HPLC share several features：
column and mobile phase and similar
Compounds：weak acids, bases, ionic compound
Concept：solvent extraction
Aaq- + Baq-
(A+B- )aq
A+B-org
ion-pair
aq：aqueous phase org：organic phase
→Ionized compound Aaq+ in water add suitable counter ion Baqion-pair A+B- similar to non-ionic compound

A B 
E
A  B 

→In solvent extraction extraction constant

aq

org

aq
capacity (reaction) factor


A B  org
Vs
Vs
Vs

k'


K

E
B
VM
A aq
VM VM
 
 
K
E 
B aq
 

A B 
K 
A B 

A B 
k'
 K B 
A 
k '  B 


IP






aq

IP
aq
aq
sample
amine
acid
buffer
pH>7
3<pH<7
3>pH
retention
time
counter ion
OCl4- , C12OSO3N+R4 , acetic acid
CH3COOCH3COO- + CH3COOH
neutral CH3COOH
base
neutral compound
acid
pH
Electrochemical Detector
An electrochemical detector responds to analytes that can be
oxidized or reduced.
The detector is very sensitive to flow rate and temperature
changes.
Quantitation method
measurement of signal
peak height (trace analysis)
peak area
k’
N
μ
A
1/(1+k’)
N1/2
<μ -0.2
no change
no change
1/ μ
A
400
300
H
40
200
20
0
H
A
H
20
30
40
%B
H
A
H
400
300
40
A
200
20
0
μ(mL/min)
(1) External standard calibration
A
peak area
response factor 
conc.
P. A.
sam ple conc. 
RF
conc.
(2) Internal standard calibration
compensate for the change in sample size or conc.
due to instrumental variation
signal
ratio
RF 
signal ratio
conc .
conc.
(3) Method of standard addition
signal
RF  slope 
Δy
conc.
S0
y
x
original conc. 
S0
R.F .
Error
(1) constant systematic error
(2) proportional systematic error
S=a+bc
S=b’c
(1)
(2)
S=bc
Recovery (sample preparation)
(1)pure reference standard
y  a0  b0 x0
(2) pure reference standard + sample preparation
(3)
y  a0
xp 
b0
xp：mass or conc. of ref. analyte after sample preparation
x p  a p  bp x0
xp
a p  0 → constant systematic error
bp  1 → proportional systematic error
(4) recovery rate
x0
 ap


R.R    bp  100%
 x0
