Transcript - Catalyst - University of Washington

CHEM 429 / 529
Chemical Separation Techniques
Robert E. Synovec, Professor
Department of Chemistry
University of Washington
Lecture 15
Supercritical Fluid Chromatography (SFC):
SF behavior as a mobile phase, SFC
Instrumentation; High temperature water extraction.
Phase Diagram for a Hypothetical Mobile Phase
2D projection of 3D
data: P, T, and r
Issue: Selectivity depends upon mobile phase density, r.
= discontinuity in r.
SF region = continuous changes in r between liquid-like
and gas-like.
Supercritical Fluid Chromatography (SFC)
Popular as an extraction tool (SFE)
Pressure, P (bar)
Phase Diagram of CO2 (Most common mobile phase):
For CO2:
Solid
Liquid
Supercritical
Fluid
Critical
Point
Pc
Critical point,
Tc = critical temperature
31 ºC
Pc = critical pressure
Gas
73 bar = 1060 psi
Temperature, T (ºC)
Tc
Pressure Gradient is applied isothermally in SFC to control
mobile phase strength.
High P (high density) liquid-like stronger mobile phase at end of run.
P gradient, compress the supercritical fluid
Thermodynamics:
k’ = KD(P,T,%M)(VS/VM)
%M = Addition of modifier
k’
as P
Analyte solubility in SF
Low P (low density, r) gas-like weaker mobile phase initially.
M.P. density  P
Mobile Phase Selection and Modification
Gas SF TC(°C) PC(bar) d*
CO2
31
73
7.5
Xe
17
58
6.1
CClF3
29
39
5.4
C3H8
97
42
5.3
NH3
132
111
9.3
H2O
374
218
13.5
*d for SF at 1.02 x TC and 2 x PC
CO2 is best mobile phase choice, but d is still low
and doesn’t change enough with P.
Therefore, a modifier is often needed to raise d !
1 bar = 14.5 psi
Modification of CO2 as a Supercritical Fluid
Modifier
TC (°C) PC(bar)
Methanol 239
Acetonitrile 275
80
48
Chloroform 263
54
1-Hexanol
40
337
dliq = 14.5
(polar)
Modifiers change net TC and PC!
TC = X(CO2)TC(CO2) + X(M)TC(M)
PC = X(CO2)PC(CO2) + X(M)PC(M)
X = mole fraction
M = modifier
Be Careful not to add too much M as it raises TC significantly!
Syringe
Pump
Capillary SFC Schematic
(Flow Rate Control, Pressure Measurement)
Po
inlet
outlet
Pi
Sample Injector
Oven
Capillary Column
10 to 50 mm I.D.
Oven at constant T  70 to 100C for CO2
Pressure Program Conditions:
DP = Pi - Pambient = F(t)
Need: Pi = Po so Pi - Po = 0
GC Detectors and
Abs, FI, MS
Detector
Decompression
after detection!
Pressure
KEY!
Restrictor
CO2 to atmosphere
Pambient
To maintain mobile phase strength throughout column L !
How is DP changed with time?
Use Flow Rate Program
How is Pi - Po = 0 along analytical column?
Use Restrictor
8LhF
….laminar flow
4
pr
• r (density of SF) increases with DP, thus F at constant T
• Pressure restrictor keeps SF compressed to the detector,
maintaining its density (solvating power)
Density, r
Capillary:
3F
DP =
Increasing DP
2F
F
Weaker m.p.
0, inlet
Column Length
L, outlet/detector
Additional Capillary
(restrictor)
Packed Column Supercritical Fluid
Chromatography of PAHs with N = 220,000
130 bar
80
bar
P and %
methanol
gradients
combined!
Composition program: initially 2% methanol in carbon dioxide for 5 min., then 3% to 5%, hold
5 min., then 1%/min to 20%, hold 10 min. And then 20%/min. to 2%. Pressure program:
initial 80 bar for 5 min, then 5 bar/min to 130 bar, hold 21 min, and then 20 bar/min to 80 bar.
Berger, T.A.; Wilson, W.H. Anal. Chem. 1993, 65, 1451-1455.
“Many rings with PAHs”
Packed Column SFC of
PAHs with N = 220,000
Chromatogram of concentrated
extract of scrapings from a
wood-burning stove chimney
Composition program: initially 2% methanol in
carbon dioxide for 5 min., then 3% to 5%, hold 5
min., then 1%/min to 20%, hold 10 min. And
then 20%/min. to 2%. Pressure program: initial
80 bar for 5 min, then 5 bar/min to 130 bar, hold
21 min, and then 20 bar/min to 80 bar.
Berger, T.A.; Wilson, W.H. Anal. Chem. 1993, 65, 1451-1455.
Phase Diagram of Water
d = 13
d = 23
K
Solubility Parameter, d
d vs. e
20 Solvents:
Room T and P
Polar organics, eg.,
methanol, acetone
Dielectric Constant, e
Dielectric Constant (e)
H2O at room T,P
e vs. T
for Water
Good
Region
liquid to steam
Temperature (ºC)
1450 psi
2900 psi
4350 psi
5800 psi
Effect of T and P on the dielectric constant of water
compared to the dielectric constants of some
representative organic compounds.
Can Water Behave Like an Organic Solvent?
e
d
YES, under suitable T and P ! !
PAH Recoveries from Soil Extraction: EPA vs. H20 vs. CO2
HT-HP-H2O
@ 250 oC in 15 min
% recovery (% RSD)
Watera
CO2b
Naphthalene
2-methylnaphthalene
Acenaphthene
Dibenzofuran
Fluorene
Phenanthrene
Anthracene
Fluroanthene
Pyrene
Benz(a)anthracene
Chrysene
Benzo(b+k)fluroanthene
Benzo(a)pyrene
aPercent
recovery versus certified value based on Soxhlet and sonication
performed according to EPA SW-846, methods 3540 and 3550. Relative std.
Dev. Are based on triplicate water extractions at each condition.
bRecoveries resulting from triplicate SFE extractions with CO .
2
Analytical Chemistry 1994, 66, 2912.
Recoveries of PAHs from Urban Air Particulates (NIST 1649)
% recovery (% RSD)
Analytical Chemistry 1994, 66, 2912.
Lecture 16
Size Exclusion Chromatography: stationary
phase design, separation mechanism, theory,
data analysis for molar mass determination.
Size - Exclusion Chromatography (SEC)
1,060
5,030
40
19,880
1,373,000
Signal
60
63,350
• Polystyrene analytes at molar mass (g/mol) indicated
• MP = methylene chloride
• SP = PS-DVB (PS-DVB is polystyrene – divinyl benzene
20
0
16
20
24
28
32
36
Time (min)
dSP = dMP = danalytes = 9
A closer look at stationary phase particle morphology…
Porous
Pore = Cave
Used
for
SEC
Monoliths
behave
this way
What Does “Porous” Mean?
Consider Spherical Substrates:
Non-porous
r
5 mm
Surface area = 80 mm2
(particle)
Surface area = 100 m2/g
Mass
 SA pores  100 SA sphere
Porous
Model
Pore
Lpore
Surface area = 1 m2/g
Mass
Let dpore = 60 Å, Lpore = 1 mm
N pores (particle) = 400,000 !!
Pores cover 14% of sphere surface.
dpore
Stationary Phase Particle - Porous
dpore
5 to 10 mm
(diameter)
SEC Retention Mechanism and Calibration
Differential permeation into pores of stationary phase leads to
differences in migration rates through the column.
Effective analyte
diameter, deff
correlates to
to molar mass, M
a tR, min
High Speed SEC of Polystyrenes (PS) and Toluene
V’M = 80 mL
k’SEC = 0
VP + V’M = 190 mL
k’SEC = 1
A = 1,100,000 PS
B = 9000 PS
VP = 110 mL
C = Toluene
Microbore SEC
•
•
•
•
•
1 mm i.d. x 25 cm
5 mm particles
60 Å ave pore diameter
25 C
100 mL/min
Analytical Chemistry, 64 (1992) 479-484.
Application of SEC:
deff = p Mq
Correlation between VR (and tR) and Analyte M
p, q dependent upon molecular shape
and solvent selection
Example, for linear polystyrenes in typical SEC solvent:
Deff = 0.3 M0.6, in Å. At M = 106 g/mol, deff = 1200 Å
Since log deff = (slope)VR + y-int
and
log deff = qlog M + log p
and since tR is proportional to VR ....
 log M = -atR + b
Calibrate with standards with same p,q as “unknown” samples.
Measure tR for two standards of known M to determine a and b.
Relating tR to Molar Mass, M
M range defined by dpore packing
Log Munk
tR,unk
t
Lecture 17
Monolithic stationary phases for HPLC, fluid
dynamics and band broadening; Pellicular
stationary phases for HPLC; Chiral stationary
phases & separations for HPLC.
Monolithic Stationary Phase Column
• Whole
column
stationary
phase
• Porous rod
• Synthesized
in-situ
in column
tube
Silica Monolithic Rod
(a)
1 mm
(b)
(a) Macropores (large) ~ 2 mm
through pores, not “dead end” pores
(b) Mesopores (small) ~ 13 nm
10 nm
Electron micrograph of the
(a) macroporous and (b) mesoporous
structures in a silica monolith rod
H vs. u (with u a F)
Monolith has similar BB to 3.5 mm particles but much
higher flowrate is possible….why?
particles
particles
H
Monolith
F
Permeability, KF
Monoliths and particles at same effective dP = 3.5 mm
Monolithic SP
KF(monolith) ~ 7x KF(particle)
7-fold lower pressure, f is 7-fold less
Particle SP
Flowrate, F, mL/min
Monoliths and particles at same effective dP = 3.5 mm
Particles
4200 psi
DP
(bar)
Monoliths
No Column,
just tubing,etc
1 bar =
14.5 psi
F, mL/min
Higher flow rates with lower H provide
for rapid, efficient separations
k’ = 0
k’ = 9.2
Wb ~ 0.1 to 0.2 min, very high N !
H vs. u plots for the test mixture at 10 flow rates
H, mm
u, mm/s
The H vs. u curves are independent of k’ !! Why?
Pellicular Stationary Phase Particles
• Performance like a sub-2 μm column
but much lower back pressure
• Poroshell 120 is made with a NEW
single-step porous shell process,
for superior reproducibility
• Works on any HPLC or UHPLC
dp = 2.7 mm
df = 0.5 mm
DP a 1/dp2 so pressure
determined by 2.7 mm
H kept small with thin df
since Csu term is minimized
Agilent Poroshell 120 Stable Bond C18 – Flowrate Study
0
1
3
2
Time, minutes
4
1 bar ~ 1 atm ~ 14.5 psi, so 200 bar ~ 3000 psi
Poroshell comparison to another product
0
10
0
10
Time, minutes
Superficially porous silica microspheres for fast high-performance
liquid chromatography of macromolecules
Journal of Chromatography A, Volume 890, Issue 1,
18 August 2000, Pages 3-13
J.J. Kirkland, F.A. Truszkowski, C.H. Dilks Jr., G.S. Engel
Evaluation of columns packed with shell particles with compounds
of pharmaceutical interest
Journal of Chromatography A, Volume 1228, 9 March 2012, Pages 221-231
Joséphine Ruta, Daria Zurlino, Candice Grivel, Sabine Heinisch,
Jean-Luc Veuthey, Davy Guillarme
Analytical Chemistry
1997, 69, 61-65.
Analytes
Studied:
Amino Acid
+/- derivatives
GMA – EDMA
Substrate Beads
6 mm diameter
33 nm pores
A fair comparison:
Both phases have the
same number of bonded
chiral selector groups
per column.
Differential pore size distribution
curves for the silica beads (dotted
line) and the monodisperse polymer
beads (solid line) as determined by
mercury intrusion porosimetry.
Yuelong, L., et. al. Anal. Chem., 1997, 69, 61-65.
Synthetic Preparation Strategies
Polymer-based phase
Silica-based phase
Yuelong, L., et. al. Anal. Chem., 1997, 69, 61-65.
Compare Selectivity: a
Silica-based
Polymer-based
Absorbance (AU)
a = 4.09
a = 7.11
Separation of 3,5dinitrobenzamidoleucineN,N-diallylamide (la)
enantiomers on chiral
stationary phases CSP1
and CSP2. Conditions:
column size, 150 mm x
4.6 mm i.d.; mobile
phase, 20 % hexane in
dichloromethane; flow
rate, 1 mL/min; injection,
7 mg; peaks, 1,3,5-tri-tertbutylbenzene (1), Renantiomer (2), Senantiomer (2’).
Anal. Chem., 1997, 69, 61-65.
Retention Time (min)
Retention Factors k’ and Separation Factors a Obtained for Enantioselective
Separations of Racemic Compounds on Columns Packed with Chiral
Stationary Phases CSP1 (silica-based) and CSP2 (polymer-based)
Yuelong, L., et. al. Anal. Chem., 1997, 69, 61-65.
Lecture 18
Ion Chromatography: stationary phase design,
ion-exchange theory, selectivity and separation
of anions.
Ion Exchange Chromatography (IEC): Stationary Phase Chemistry
Substrate = Co-polymer resins for wide pH range
stability and high pressure applications
Polystyrene - Divinyl benzene (PS-DVB)
H
H
C
CH2 C
H
CH2
C
CH2
C
H
H
C
H
CH2
C
H
CH2
C
Cross-link with
• reduce porosity
• make more rigid
Ion Exchangers
Cation Exchange (CATEX)
Sulfonic acid cation exchanger
PS-DVB + H2SO4
H
C
CH2
Mobile Cation
Reaction at surface
benzene groups
SO3- H+
Fixed Anion
Anion Exchange (ANEX)
Quaternary ammonium chloride (salt) anion exchanger
H
PS-DVB
ClCH2OCH3
AlCl3
C
N(CH3)3
CH2
Fixed Cation
Mobile Anion
CH2N+(CH3)3Cl-
“Nanotechnology”
Ion-Exchange Chromatography - High Performance Stationary Phase:
High Efficiency yet Low Pressure and High Surface Area
Substrate diameter
20 mm…. Now, 5 mm
Colloidal particles
(microbeads)
3000 Å = 300 nm
Now, 50 nm to 100 nm
The microbeads form
a monolayer on each
substate particle
Scanning electron micrograph of surface agglomerated anion exchange resin
particles. The diameter of the substrate particles is about 20 mm and of the
colloidal particles about 3000 Å. (Micrograph was prepared by E. Bradford of the Physical
Research Laboratory of the Dow Chemical Company.)
Dionex
H. Small, “Ion Chromatography,” Plenum Press, New York, 1989.
Dionex Stationary Phase: Consider binding of
one colloidal latex microbead particle to substrate
ANEX shown
Substrate ~ 5 mm
Latex particle ~ 0.050 mm = 50 nm
PS-DVB
Non-porous
Small particles on surface are
ion exchanger (fixed cation
for ANEX separations)
Opposite ion exchanger charge is
on substrate surface (fixed anion)
Electrostatic Linkage - multiple sites, VERY strong!
For ANEX:
+NR -OH
3
One electrostatic linkage
Each microbead has multiple
linkages, stable to ~4 M NaOH!
SO3- +NR3
Anex
etc…
+NR
Catex substrate
-
3 OH
Mobile
anions
Hydrodynamic Mass Transfer Issues
Substrate particle (5 - 25 mm dp) is highly cross-linked nonporous PS/DVB with +/- surface charge (depending upon
cation or anion exchange). Colloidal particle (50 to 100 nm df)
is a latex particle with -/+ charge…..dp >> df
Cation exchange shown (anion exchange similar)
s
Stationary phase cross-section (not proportional scale)
Csu term
This “pellicular” design keeps DP and H low, so high N!
General Ion-Exchange Theory
Shown for ANIONS, with OH - mobile phase. Competition between OHand X- for ANEX Sites…..
S NR3+OH- + X-
Keq
S
NR3+X- + OH-
[S NR3+X-] [OH-] ~ k’XKeq= k’OH[X ] [S NR3+OH-]
Keq, relative to Cl- on common ANEX:
• For proper retention,
want analyte to have
MORE affinity for ion
exchange site(s) than
mobile phase.
Keq,rel = Keq,rel,X- / Keq,rel,Cl-
X- = analyte
S = stationary
phase
ANION Keq,rel
Cl-
1.00
OH-
0.65
HCO3-
0.53
CO32-
4.0
Br -
2.3
NO3-
3.3
Control of Retention
Rearranging Keq equation: k’ =
x-
Keq • {CAP} • rst
[OH-]
log k’X- = log Keq + log {CAP} + log rSP - log [OH-]
meq ANEX sites
CAP = grams stationary phase

[S-NR3+OH-]
Typical CAP: 0.1 to 0.001 meq/g …meq = milliequivalent
• k’ increases with Keq and CAP
• k’ decreases with increasing [OH-] (for anions)
General Solution
For charged analyte -n in charged mobile phase -m
log k’ = constant - (n/m)log[E]
The constant
contains Keq,
analyte selectivity
Where [E] has units of mol/L
Example:
Let E = OH- = eluent (mobile phase)
-2
Log k’
Slope = n/m
-1
Analyte charge, also
provides selectivity
Gradient Elution
Start at low [E] and
increase [E] with time.
Log [E]
Conductance
IEC Gradient Elution of Anions
Gradient elution of inorganic and organic anions on a
pellicular anion exchange resin. Eluent: gradient of 0.75 mM
to 100 mM NaOH. Detection: suppressed conductometric;
Anion MicroMembrane Suppressor.
Lecture 19
Ion Chromatography: Detection and instrumentation,
conductivity detection, micromembrane suppression,
post-column derivatization chemistry with
absorbance detection.
IEC: Detection and Instrument Development
Conductivity Detection is common, especially for
inorganic anions.
Conductivity Signal
S(t) = k[SaiCi,+(t)li,+ + SaiCi,-(t)li,-]
all cations
all anions
i = all species present, + = cation, - = anion
C = concentration
a = degree of dissociation, a = 1 strong electrolyte, a ~ 10-7 H20
k = detector “constant”
l = equivalent ionic conductance (ability to transport charge)
l = Fm = F(v/E) where F = Faraday constant, m = ion mobility,
v = steady-state velocity, and E = electric field strength
Conductance
IEC Gradient Elution of Anions
Gradient elution of inorganic and organic anions on a
pellicular anion exchange resin. Eluent: gradient of 0.75 mM
to 100 mM NaOH. Detection: suppressed conductometric;
Anion MicroMembrane Suppressor.
lo, Equivalent Ionic Conductance (at º25 C)
Ion
lo
H+
350
OH
-
Big!
198
Na+
50
K+
74
HCO23-
45
CO32-
69
F-
55
Cl -
76
Br -
78
NO3 -
71
SO4 2-
80
Mobile Phase
Components for
ANEX (H+ for
CATEX)
Typical Analytes
lo much less than
OH-, typical MP !
Baseline Signal, SBL
Subscript m indicates mobile phase only signal:
SBL = kam [Cm,+lm,+ + Cm,-lm,-]
Consider NaOH as mobile phase for anion separations:
am = 1
lNa+ = 50
lOH- = 198
High Background at 1 to 100 mM NaOH
AND
Limiting Noise  0.1% SBL due to variations in
flowrate and temperature, therefore High Noise too.
Challenge: How to minimize SBL and
thus the BL noise, while optimizing
DS(t) = Net Analyte Signal ?
Net Analyte Signal
DS(t), Analyte Signal Relative to Baseline
DS(t) = S(t) - SBL
For an anionic analyte, X, it is associated with the same
cation as the mobile phase m, so the cation contribution
to DS(t) cancels, and X displaces m in a given volume
element to maintain electrical neutrality.
Solving, CX,-(t) = analyte concentration
DS(t) = k[aXCX,-(t)lX,- - amCX,-(t)lm,-]
For X and m strong electrolytes, a = 1,
DS(t) = kCX,-(t) [ lX,- - lm,-]
Resulting Data from Strong Electrolytes
S(t), absolute conductance
DS(tR)
NaCl sample elution, “Cl peak”
(negative since lOH-  lCl-)
NaOH baseline, high background
POOR S/N
0
0
Time
Solution to Conductivity Detection Problem
After the separation and before detection, convert
the mobile phase into a low/non-conducting species
while not sacrificing analyte conductance and not
introducing significant peak broadening or addition
“retention.”
…. the Dionex Micromembrane
Suppressor (MMS) Unit !
….. A model for post-column derivatization
chemistry to enhance detection
Applying MMS to Anions
IEC Column anion
separation
P
NaOH
m.p.
MMS (No retention)
Na+
CD
Inj
H+
Cation
exchange
membrane
MMS function: acid/base
chemistry is driving force
H2SO4
Pump regenerant
~0.5 M H2SO4
Mobile Phase suppression:
NaOH + H2SO4
H2O + NaHSO4
No Reaction
with Analyte!
Result: low background “signal” and noise, positive DS(t),
good S/N, good Limit of Detection (LOD).
Conductivity Signal S(t) for Chloride
S(t), absolute conductance
Before MMS:
NaOH
Poor S/N
Cl- peak
as NaCl
Cl- peak
as NaCl
After MMS:
Good S/N and
Good LOD
H2O
0
0
Time
Keq,rel on CATEX, E+ = H+
Cation
Keq,rel
H+
1
Li+
0.76
Na+
1.2
K+
1.72
Rb+
1.86
Cs+
2.02
Co2+
2.45
Ni2+
2.61
Cu2+
2.49
Zn2+
2.37
R-E+
+
Mobile Phase, E+
Easy to Separate
Difficult to Separate
based upon K’s
alone…too similar
C+
K
R-C+ + E+
R- = -SO3- stationary phase fixed anion
Optimizing Selectivity and Sensitivity for Transition
Metal Separation and Detection
Ion exchange equilibria for two metals, Mm+,
Nn+ with eluent E+ and counter-ion L-.
Resulting k’ expressions:
f = fraction of metal in free form. 0 < f < 1 so log(f) < 0,
reducing retention, and providing selectivity since f is metal dependent
Added Selectivity with Side-Equilibria
Additional complexation with eluent counter-anion L (ligand)
Selectivity, defined by a:
Fundamental
difference of
equilibrium
constants
Complexation
with ligand L-
Different
charges,
separate
easily
Sensitive Mn+ detection: Absorbance of
PAR Complexes at 520 nm
Using membrane-based post-column derivatization…..
PAR
O-
n
N
+ Mn+
N N
OH
ANEX: transfer PAR- to mobile phase
side using MMS-type device
• Coordination number = 2
• emax 
to
very sensitive
absorbance, A = ebC
•Kf very large, 1025 to 1030
104
105,
- H+
Kf
O-
N
N N
O
M
Detected Complex M(PAR)2
IEC of Cations using Counter-Ligand Selectivity
and PAR Detection
Determination of Transition Metal Ions
Mobile Phase: L- = oxalate
E+ = H+, Na+
ppb (injected)
Time, minutes
Lecture 20
Mixed Mode RP-LC: mixed mode separation
mechanism – ion exchange and hydrophobic
interactions, mobile phase chemistry for cation
and anion separations, use of micelles and
surfactants in separations
Mixed-Mode Separations
On Ion-Exchange Columns
C
NR3+OH-
CH2
PS-DVB
Substrate
C
NR3+
CH2
C
H
Analyte = RSO3-
CH2
Mobile Phase = OH- in H2O
with Na+ and organic modifier
C
NR3+OH-
CH2
C
Anion exchanger with
hydrophobic character
bound to a cross-linked
PS-DVB substrate
H
Mixed-Mode Separations
On Ion-Exchange Columns
C
NR3+OH-
CH2
PS-DVB
Substrate
C
NR3+
CH2
C
Anion exchanger with
hydrophobic character
bound to a cross-linked
PS-DVB substrate
IEC mechanism
H
Analyte = RSO3-
CH2 RP-LC mechanism
Mobile Phase = OH- in H2O
with Na+ and organic modifier
C
NR3+OH-
CH2
C
H
Head group AND tail group of
Analyte both contribute to retention!
Mixed-Mode Separation: RP-LC and IEC
Gradient Separation of Aromatic Acids
RP-LC of Catecholamines Ion-Paired with Heptane Sulfate
Adsorbosphere HS C18, 7mm,
150 x 4.6 mm
Mobile Phase: Methanol: 0.001 M Sodium
Heptane Sulfate in 0.07M
KH2PO4, pH 3.0 (13:87)
Flow Rate: 2.0 mL/min
Detector: UV at 280 nm
Column:
Micelle Formation
Surfactants in solution will tend toward the formation of
oriented aggregates with increasing concentration
Hydrophobic tail
Charged or
polar head
group
Micelle
Organic interior, 3D sphere
Micelle formation occurs when the concentration of the
surfactant exceeds its critical micelle concentration (CMC).
= sodium dodecyl sulfate, CMC = 10 mM
Concentration of “free” surfactant
Surfactant Behavior
CMC
More micelles
Micelle techniques
(free surfactant and
micelles present)
Ion-pair techniques (free
surfactant only)
Concentration of surfactant added to H2O
What are micelles good for…………
Solubility of Arenes in Water and Micellar Solutions
Data from: M. Almgren et. al. J. Am. Chem. Soc. 1979, 101, 279.
Example: Anthracene is 30,000 times more soluble in SDS micelles
in water than in water alone.
Use of Micellar Mobile Phases for RP-HPLC
• Adjust strength of mobile phase without using organic solvents
• Decrease retention times of neutral analytes
• Increase solubility of analytes in mobile phase
Stationary Phase
micelle
KM
KD
Stationary Phase
analyte
“Soap Chromatography”: RP-HPLC with SDS
1.8
2-naphthol
Log k’
1.4
Effect of SDS concentration
(in water) on retention using
C18 stationary phase.
CMC for SDS in water is 10
mM or log[SDS] = -2.0
1.0
0.6
phenol
Log [SDS] (M)
Dorsey, J.G. Adv. Chromatography 1987, 27, 167.
Lecture 21
Electrophoresis: electrophoretic migration,
capillary electrophoresis (CE), electro-osmotic flow,
band broadening behavior in open tubular CE,
and separation optimization.
Electrophoresis: Chemical separation based upon
differential migration in the presence of an electric field
Electrophoretic Migration
Applied Force (E-field):
(along arbitrary axis-x)
Biological Applications
q = solute
(analyte)
net charge +/-
= f/L
Opposing Frictional Force:
= solute radius (spherical approximation)
At steady-state (quickly obtained):
Ion mobility
….electrophoretic mobility
Solute migration in the separation is dependent upon v (velocity):
(spherical approximation)
Open Tubular CE Instrumentation
Separation Capillary
(no stationary phase!)
Cathode (-)
Anode (+)
Detector
Electrolyte
Power Supply
HV
HV ~ 30,000 V / meter capillary
Electrolyte
Capillary Electrophoresis for Standard Proteins (3 runs)
12 4 5
3
Signal (V)
1.0
6
1. a-Lactalbumin
2. -Lactoglobulin
3. Trypsin inhibitor
4. Carbonic anhydrase
5. Ovalbumin
6. Bovine serum
albumin
7. Conalbumin
8. -Galactosidase
7
8
0.5
MW: 14400 -116000
Separation buffer:
0.1MTris+0.1MCHES
+0.1%SDS+8%pullulan
0.0
10
15
20
25
Time (min)
30
35
40
Capillary Electrophoresis (CE)
Typical Conditions:
•
•
•
•
Sample Injection?!
HV = 30,000 V, f
Cap. Dia = 100 mm or less
Cap. L = 1 m
Vol. Reservoir  Vm
(neglect transfer)
No Pump: E-field driven flow
Flow Effects:
1. Electrophoretic Migration (analyte): m (+/-)
2. Electro-osmotic Flow (mobile phase): mosm (+ only)
High Speed CE Experiment
gate
probe
0
Time, sec
Diagram of capillary mount showing the relative position of the
capillary, the “gating” and the “probe” laser beams.
C.A. Monnig and J.W. Jorgenson, Analytical Chemistry, 63, (1991) 802- 807.
10
High Speed CE Electropherogram
Arginine
174 g/mol, 5% more mass than GA
FITC = Fluorescein
isothiocyanate
 400 g/mol
1% changes in mass
easily separated including
FITC contribution.
Phenylalanine
165 g/mol, 3% more mass than GA
Glutamic Acid
147 g/mol
3 seconds !
Electropherogram of a mixture of FITC-labeled amino acids. Sample
introduction time was 40 ms. The electric field in the small-diameter capillary
was maintained at 3.3 kV/cm. Column length was 1.2 cm.
C.A. Monnig and J.W. Jorgenson, Analytical Chemistry, 63, (1991) 802- 807.
Mobile Phase Velocity Profile Across Capillary Diameter
Laminar as in
chromatography
(pushed flow)
Plug flow as in CE  constant uMP , the
velocity of mobile phase (pulled at each
volume element)
Good for reducing band
broadening: no C-term
as in chromatography
uMP
Chromatography:
Capillary Electrophoresis:
0
Capillary diameter
dc
Capillary Electrophoresis (CE)
Separation method based upon differential
migration in an electric field
Capillary
-
-
-
EOF
 Separation is performed in
+
+
+
narrow-bore (25 – 75 mm id),
fused silica capillary
 Electroosmotic flow (EOF) is
Intensity
superimposed on electrophoretic
+
-
flow and carries all analytes to the
cathode
 Separation is based on q/r,
Migration time
the analyte charge-to-size ratio
 Well suited for biological samples
Open Tubular CE Equations
Velocity
Efficiency
Analysis Time
Resolution
Lecture 22
State-of-the-art CE: Theory vs. Experiment,
CE-on-a-chip.
Integrated Capillary Electrophoresis Devices with
an Efficient Post-column Reactor in Planar Quartz
and Glass Chips: Microfabricated Systems
Layout scheme of a device
used for post-column
derivatization reactions with ophthaldialdehyde (OPA). Thin
lines indicate channels with
the nominal width of 45 mm for
PCRD1, while thick lines show
regions 240 mm wide.
Potentials were applied and
solutions introduced through
reservoirs at the end of each
channel.
Fluri, K., et. al., Anal. Chem. 1996, 68, 4285 - 4290.
Control and modulate voltages to inject 60 pL samples
Layout of the double-T
injector design. Injected
plugs were about 120 mm
long (about 60 pL volume
in PCRD1).
LOAD step (shown): Arrows
indicate direction of fluid flow
with applied voltages.
INJECT step (not shown):
Buffer (GND), Sample and
Injection Waste both floating,
end of Separation Channel is –
1000V.
Fluri, K., et. al., Anal. Chem. 1996, 68, 4285-4290.
“Zero Dead Volume” Mixing Junction
Amino Acid + OPA
Fluorescent Product
Electron micrographs showing the mixing junction
of devices etched in quartz for layout (A) PCRD1,
13 mm deep, and (B) PCRD2 9 mm deep.
Fluri, K., et. al., Anal. Chem. 1996, 68, 4285-4290.
Electropherogram: CE-on-a-chip
B
A
Electropherogram of amino acids
and hydrolyzed dansyl chloride
with postcolumn derivatization with
OPA in PCRD1.
Expansion of region in A showing the
separation and peak widths of valine,
phenylalanine, and hydrolyzed dansyl
chloride following postcolumn mixing
with OPA within device PCRD1.
Fluri, K., et. al., Anal. Chem. 1996, 68, 4285-4290.