Putting a speed gun on macromolecules:
what can we learn from how fast they go,
and can we do something useful with that
information?
Monday, October 31
Cleveland State University
National Science Foundation
Generic Talk Outline
• Thank hosts
• Tell joke, story or limerick
• Explain what we’re trying to do
• Explain what we actually did
• Today, that will lead naturally to applied things
• Thank accomplices
This is what I mainly came to say!
There once was a theorist from France
who wondered how molecules dance.
“They’re like snakes,” he observed,
“As they follow a curve, the large ones
can hardly advance.”
D ~ M -2
P. G. de Gennes
Nobel Prize
Physics
Tons per mole!
Diffusion
P.G. de Gennes
Scaling Concepts in Polymer Physics
Cornell University Press, 1979
When does the speed of polymers
(and stuff dispersed in them) matter?
•
•
•
•
•
How fast can it dissolve?
How fast can we process it?
How long until the additives ooze out?
How long does it take to weld polymers together?
How fast do chain termination steps occur during
polymeriztion?
• How fast will phase separation destroy the polymer?
• Will an image on film (remember film?) stay sharp?
• Speed  Viscosity
DLS for Molecular Rheology of Complex Fluids:
Prospects & Problems
Studied a lot
Barely studied
+ + + Wide-ranging autocorrelators
> 10 decades of time in one measurement!
– – – Contrast stinks: everything scatters, esp.
in aqueous systems or most supercritical
fluids, where refractive index matching
cannot hide the matrix.
Translational Diffusion Leads to
Intensity Fluctuations
Intensity
t
Rotational Diffusion Between Polarizers Leads to
Intensity Fluctuations
Looking into the laser,
vertically polarized
Polarizer
Crystalline inclusion
Analyzer
dim
dim
bright
Dynamic Light Scattering
LASER
Uv = q2Dtrans

V
LASER

V
H
Hv = q2Dtrans + 6Drot
q
4n sin / 2 
o
Uv Geometry
(Polarized)
Hv Geometry
(Depolarized)
DLS can be used for sizing if viscosity is known,
for viscosity if size is known
Large, slow molecules
Small, fast molecules
Is
t
DLS diffusion coefficient, inversely proportional to size.
Stokes-Einstein Law


kT

Rh  
 6πηo Dtrans 
Dtransh = constant
Also Droth = constant
Correlation Functions etc.
Where:
G() ~ cMP(qRg)
 = q2D  q2kT/(6hRh)
Rh = XRg
g (t )   G (  ) exp(  t )d
g(t)
ILT
G()
log10t
log10D
q2D
  1/2
c
MAP
CALIBRATE
log10M
M
Strategy
•Find polymer that should (???) “entangle”
Dextran
•Find polymer that should not “entangle”
Ficoll
•Find a rodlike probe that is visible in DDLS
TMV
•Measure its diffusion in solutions of
each polymer separately
•Random coil
•Polysaccharide
•Invisible in HvDLS
•Highly-branched
•Polysaccharide
•Invisible in HvDLS
•Rigid rod
•Virus
•Visible in HvDLS
BARELY
As expected, viscosity rises with c
BothViscosity
11
Dextran 670,000
Ficoll 420,000
10
9
8
hsp/c /dL-g
-1
7
6
5
4
3
2
1
0
0
5
10
15
20
c/g-dL
25
-1
30
35
40
DIY farming--keeping the “A” in LSU A&M
Seedlings
Sick Plants 
And close-up of
mosaic pattern.
TMV Characterization
Sedimentation, Electron Microscopy and DLS
•Most TMV is intact.
•Some TMV is fragmented
–(weaker, faster mode in CONTIN)
•Intact TMV is easy to identify
–(stronger, slower mode in CONTIN)
All measurements made at low TMV
concentrations—no self-entanglement
nL
0
1
3
6
5
-8
Dr /s
Translation
Dt /10 cm s
-1
500
400
Rotation
3
300
2
Experiments are
in dilute regime.
3
TMV overlap (1/L )
1
200
0
0.0
0.5
1.0
1.5
2.0
c/mg-mL
-1
2.5
3.0
2 -1
4
Matrix is invisible
TMV + Dextran 215 s acquisition
1.3
g
(2)
1.2
1.1
1.0
Dextran >6000 s acquisition
0.9
1E-6
1E-5
1E-4
1E-3
0.01
0.1
1
10
100
Hv correlation
functions for 14.5%
dextran and 28%
ficoll with and
without added
0.5 mg/mL TMV
t/s
g(2)
1.4
TMV + Ficoll 600s aquisition
The dilute TMV
easily “outscatters”
either matrix
1.2
Ficoll >6000 s acquisition
1.0
1E-6
1E-5
1E-4
1E-3
0.01
t/s
0.1
1
10
100
Hey, it works!
4000
3500
/s
-1
3000
Hv TMV / Buffer
2500
2000
Uv TMV / Buffer
1500
1000
Hv TMV / Dextran / Buffer
500
0
0
1
2
3
2
10
4
-2
q /10 cm
5
I didn’t think—I experimented.
---Wilhelm Conrad Roentgen
Early results—very slight errors
350
6
/10-8cm2 s-1
300
200
trans
150
100
5
4
3
2
D
rot
D / s-1
250
50
1
0
0
0
2
4
6
8
10
wt% dextran
rotation
12
14
16
0
2
4
6
8
10
12
14
16
wt% dextran
translation
Macromolecules 1997,30, 4920-6.
Stokes-Einstein Plots: if SE works, these
would be flat. Instead, apparent deviations in
different directions for Drot and Dtrans
2
4
6
8
10
12
14
16
1.5
-1
4
-9
hDt /10 g-cm-s
hDr /g-cm -s
-1
0
1.0
-2
2
0.5
0
0.0
0
2
4
6
8
wt% Dextran
10
12
14
16
At the sudden transition: L/xc.m. ~ 13 and L/x ~ 120
x
Dextran overlap
5
10
15
20
9
8
80
6
60
4
40
2
20
h/cP
Dr/Dt /10 cm
-2
0
xcm
L
0
0
0
5
10
15
20
wt % dextran
Macromolecules 1997,30, 4920-6.
350
300
rotation
250
D / s-1
200
rot
We believed that the
transition represented
topological constraints.
150
100
It was suggested that more
systems be studied.
50
0
0
2
4
6
8
10
BEGIN FICOLL
12
14
16
18
20
22
24
26
28
30
wt% ficoll
trans
D
When we did Ficoll, many
more points were added!
/10-8cm2 s-1
6
translation
5
4
3
2
1
0
0
2
4
6
8
10
12
14
16
18
wt% ficoll
20
22
24
26
28
30
Huh? Drot still diving in Ficoll?
3.5
0.8
2.5
0.6
-1
rotation
0.5
2.0
0.4
1.5
0.3
1.0
0.2
0.0
0.0
0
5
10
15
20
wt% ficoll
25
30
-1
0.1
-1
0.5
-9
hDrot /g-cm -s
-1
0.7
hDtrans /10 g-cm -s
3.0
translation
Uh-oh,
maybe we should think now.
The chiral dextran and ficoll alter
polarization slightly before and
after the scattering center.
With a strongly depolarizing probe,
this would not matter, but…
rTMV = IHv/IUv ~ 0.003
While matrix scattering is minimal,
polarized scattering from TMV
itself leaks through a “twisted” Hv
setup.
Most damaging at low angles
Mixing in Polarized TMV Light
Uv light from misalign True Hv light



6Drot
q2
Drot too low
q2
6Drot
q2
Even at the highest concentrations,
only a few degrees out of alignment.
300
Optical Rotation / arc-minutes
250
Dextran
Ficoll
200
150
100
50
0
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
wt %
Slight, but important, improvement.
NewFicollRatio_PR
350
Right way
Wrong way
300
Drot / s
-1
250
200
150
100
50
0
0
5
10
15
20
wt% ficoll
25
30
35
Improved Drot/Dtrans Ratio Plots
NewDexConcStudy_PR
8
7
7
6
6
Drot/Dtrans/ 10 cm
-2
-2
8
5
5
9
9
Drot/Dtrans/ 10 cm
NewFic
4
3
2
1
4
3
2
1
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
wt% dextran
0
0
5
10
15
20
25
wt% ficoll
30
35
40
Improved Stokes-Einstein Plots
Black = TMV Translation
Blue = TMV Rotation
NewFicollRatio_PR
NewDexConcStudy_PR
5.0
5.0
0.8
4.5
2.0
1.5
0.5
0.6
3.0
2.5
0.4
2.0
1.5
0.2
1.0
0.5
0.0
0
2
4
6
8
10
wt% dextran
12
14
0.0
16
0.0
0.0
0
5
10
15
20
wt% ficoll
25
30
35
-2
1.0
-2
0.2
3.5
-9
0.4
-9
2.5
-1
3.0
hDrot/g-cm s
0.6
hDtrans/ 10 g-cm-s
3.5
-1
4.0
hDtrans/10 g-cm-s
-1 -1
4.0
hDrot/g-cm s
0.8
4.5
Hydrodynamic Ratio—Effect of Matrix M
at High Matrix Concentration
DextranMWStudy_PR
9
8
-2
5
Drot/Dtrans/10 cm
6
9
7
4
3
2
1
0
0
2
4
6
8
10
12
5
14
16
dextran MW/ 10 daltons
18
20
Effect of Dextran Molecular Weight—
High Dextran Concentration (~ 15%)
TMV Translation
TMV Rotation
DextranMWStudy_PR
DextranMWStudy_PR
100
10
-9
Drot / s
-1
2
Dtrans/ 10 cm s
-1
-0.62 ± 0.04
-0.72 ± 0.01
1
0.1
10000
100000
1000000
Dextran MW
1E7
10
1
10000
100000
1000000
Dextran MW
1E7
Summary: Depolarized DLS = new
opportunities in nanometer-scale rheology.
Randy Cush
David Neau
Ding Shih
Holly Ricks
Jonathan Strange
Amanda Brown
Zimei Bu
Grigor Bantchev
Zuhal & Savas Kucukyavuz--METU
Seth Fraden—Brandeis
Nancy Thompson—Chapel Hill
I cannot tell
you the coolest
part of this, but
postdoc
Grigor Bantchev
found a trick that is
definitely a treat!
“Too much dancing and not nearly
enough prancing!”
Can probe diffusion
actually do something?
C. Montgomery Burns, “The Simpsons”
Matrix Fluorescence Photobleaching
Recovery for Macromolecular
Characterization
Garrett Doucet, Rongjuan Cong, David Neau, Others
Louisiana State University
Funding: NSF, NIH, Dow
Blue input light
Green
Detected
Light
Fluorescent
Sample
Fluorescence & Photobleaching
Blue input light
Green
Detected
Light
Slowly Recovers
Fluorescent
Sample
With Fluorescence
Hole in Middle
Recovery of Fluorescence
Modulation FPR Device
Lanni & Ware, Rev. Sci. Instrum. 1982
SCOPE
5-10% bleach depth
IF
PA
c
X
TA/PVD *
PMT
*
D
S
*
DM
OBJ
M
RR
*
M
ARGON ION LASER
AOM
* = computer link
Cue The Movie
Dextran Diffusion
in Hydroxypropylcellulose, a
probe diffusion
study: the more
HPC, the more
nonlinearity in
semilog plots.
Hmmm….
Bu & Russo, Macromolecules, 27, 1187 (1994)
Can FPR be used for
MWD characterization?
Questions bearing on this
• Need: are new analytical methods needed
in a GPC/AFFF multidetector world?
• Ease of labeling the analyte?
• How hard to calibrate?
• Worth the price of setup?
• Miniaturization?
GPC
•Solvent flow carries molecules from left to right; big ones come out first
while small ones get caught in the pores.
•Non-size mechanisms of separation complicate regular GPC, are much
less of a problem for multidetector methods, but they correspondingly
more complicated.
They were young when GPC was.
Small Subset of GPC Spare Parts
To say nothing of unions, adapters, ferrules, tubing (low pressure and
high pressure), filters and their internal parts, frits, degassers, injector
spare parts, solvent inlet manifold parts, columns, pre-columns,
pressure transducers, sapphire plunger, and on it goes…
Other SEC Deficiencies
•
•
•
•
•
•
•
0.05 M salt at 11 am, 0.1 M phosphate pH 6.5 at 1 pm?
45oC at 8 am and 80oC at noon?
Non-size exclusion mechanisms: binding.
Big, bulky and slow (typically 30 minutes/sample).
Temperature/harsh solvents no fun.
You learn nothing fundamental by calibrating.
For straight GPC, what you measure is not what you
calibrated. Good for qualitative work, otherwise
problematic.
Must we separate ‘em to size ‘em?
Your local constabulary probably
doesn’t think so.
Atlanta, GA
I-85N at
Shallowford Rd.
A Saturday at 4 pm
Molecular Weight Distribution by
DLS/Inverse Laplace Transform--B.Chu, C. Wu, &c.
Where:
G() ~ cMP(qRg)
 = q2D  q2kT/(6hRh)
Rh = XRg
g (t )   G (  ) exp(  t )d
g(t)
ILT
G()
log10t
log10D
q2D
  1/2
c
MAP
CALIBRATE
log10M
M
Hot Ben Chu / Chi Wu Example
Macromolecules, 21, 397-402 (1988)
MWD of PTFE
Special solvents
at ~330oC
Problems:
•Only “works” because MWD is broad
•Poor resolution.
•Low M part goofy.
•Some assumptions required.
Matrix Diffusion/Inverse Laplace Transformation
Goal: Increase magnitude of —this will improve resolution.
Difficult in DLS because matrix
log10D
Solution:   1/2
D
D
Matrix:   
log10M
Stretching 
scatters, except special cases.
Difficult anyway: dust-free matrix
not fun!
Still nothing you can do about
visibility of small scatterers
DOSY not much better
Replace DLS with FPR.
Selectivity supplied by dye.
Matrix = same polymer as
analyzed, except no label.
No compatibility problems.
G() ~ c (sidechain labeling)
G() ~ n (end-labeling)
Sample
Arbitrary Amplitude
The Plan to Measure M Using FPR
Collect Data
Using FPR
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
Convert to Molar
Mass by Mapping
onto Calibration Plot
-3
10
-2
10
-1
 /s
-1
10
0
10
Analyze Using ILT
5
C(t)
4
3
2
1
0
0
200
400
t/s
600
800
1000
Arbitrary Amplitude
6
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
10
3
10
4
M / Da
10
5
Labeling is Often Easy
Pullulan
H O
H
O
OH
O
O
OH
OH
OH
O
O
OH
O
O
OH
n
OH
OH
OH
OH
OH
OH
OH
OH
n
Dextran M = 2 Million Da as the
matrix at different concentrations
in 5 mM NaN3 solution
O
O
OH
COOH
Pullulans of different M labeled with
5-DTAF as probes
Cl
N
N
NH
N
Cl
5-(4,6-dichlorotriazinyl)amino fluorescein
Matrix FPR for Pullulan
(a polysaccharide)
1
10
5
10
4
M
-7
Dapp / 10 cm s
2 -1
10
0.1
NaN3(aq) solution ( = 0.537 ± 0.035)
5% Matrix solution ( = 0.822 ± 0.018)
10% Matrix solution ( = 0.907 ± 0.038)
15% Matrix solution ( = 0.922 ± 0.037)
0.01
4
10
10
5
0.1
1
10
-7
M
Probe Diffusion: Polymer physics
2
Dapp / 10 cm s
-1
Calibration: polymer analysis
GPC vs. FPR for a Nontrivial Case
1.0
12
0.9
10
0.7
Amplitude/Arbitrary
Relative Concentration
0.8
0.6
0.5
0.4
0.3
8
6
4
2
0.2
0.1
0
0.0
4
10
M / g mol
-1
5
10
PL Aquagel 40A & 50A
10
4
M
10
5
User-chosen CONTIN
25% Matrix
 only ~1
20,000 & 70,000 Dextran
How Good COULD it Be? Simulation
of FPR Results for  = 2
(Most Desirable Situation)
6
5
y = -0.4998x + 1.1518
log D
0
-2 0
-4
-6
log M
4
2
4
y = -2.0009x + 2.3045
3
2
4
6
8
2
-8
-10
-12
log M
1
0
-10
-8
-6
-4
log D
-2
0
What could we separate from 10K,
according to  = 2 simulations?
Shazamm!
10000
MDetected
100000
Sim
ula
ted
M
20000
40000
57000
80000
113000
160000
Using an HPC Matrix
Pullulan, 8% HPC Solution, M=12,200 and 48,000
1.0
FArbitrary Units
0.8
CONTIN
Exponential
Exponential
0.6
0.4
0.2
0.0
1000
10000
100000
M
1000000
 Indicates targeted M.
MFPR Conclusions
We are entitled to expect something better than GPC.
For some situations, MFPR could really work.
What is good about GPC (straight GPC) is the simple
concept; Matrix FPR keeps that—just replaces Ve with D.
We haven’t yet addressed two questions
--Is it worth setting this up?
--Miniaturization/Automation?
Macromolecules for The Demented
and methods for their study
Help from Keunok Yu, Jirun Sun, Bethany Lyles, George Newkome and
LSU’s Alz-Hammer’s Research Team
Krispy Kreme Donut Day, September 2003
Supported by National Institutes of Health-AG, NSF-DMR and NSF-IGERT
•
•
•
•
How Alzheimer’s happens
Attempts to prevent or reverse it
Characterization challenges
Alzheimer’s model systems with materials implications
Positron emission tomography
Age: 20 -- 80 Normal -- 80 AD
Postmortem Coronal Sections
Normal
Alzheimer’s
PET images courtesy of the Alzheimer's Disease Education and Referral Center/National Institute on Aging; Postmortem images
courtesy of Edward C. Klatt, Florida State University College of Medicine
APP = Amyloid Precursor Protein
http://www.bmb.leeds.ac.uk/staff/nmh/amy.html
APP = the larger, lighter pink one
•Transmembrane protein
•Normal function not known
•Educated guesses
May help stem cells develop identity
Or help relocate cells to final location
May “mature” cells into structural type
May protect brain cells from injury
Synaptic action
Copper homeostasis
•Anyway, you need it.
•Normal “clipping” of APP by a “secretase” enzyme (in red, and also assumed
to be a transmembrane protein) is shown.
•There are several secretases, also associated proteins, and they seem to
mutate easily: there is a genetic link.
•It is not exactly clear why things go awry with advanced age.
Amyloid hypothesis: fibrils or protofibrils
cause cell death, possibly as the body’s own
defenses tries to clear such “foreign” matter.
Peter Lansbury Group
http://focus.hms.harvard.edu/1998/June4_1998/neuro.html
Competing hypothesis: channel formation disrupts Ca+2 metabolism
Two FPR Contrast Decay Modes are Often
Observed: Fast = small; Slow = large.
Contrast / Arbitrary
1
pH 2.7
pH 6.9
pH 11
0.1
0.01
1E-3
1
10
100
t/s
1000
Doing More Experiments Faster with Less
Precious Amyloid: Dialysis FPR
Pump
Exchange Fluid
Cover slip
Sample
PTFE spacer
Dialysis membrane
O-ring
Reversing Amyloid Aggregation…by pH
FPR Study: Reversibility of -Amyloid Aggregation
100M 5-CF--Amyloid1-40+ -Amyloid1-40 pH 11
dialysis against 50mM PB pH 7.4
-6
D/10 cm s
2 -1
1E-6
1E-7
dialysis against 50mM PB pH 2.7
1E-8
0
200
400
600
800
1000
1200
1400
1600
1800
Time/min
Diffusion from in situ FPR of 5-carboxyfluorescein-A1-40 (25% mixed with
unlabeled 75% A1-40) starting at pH 11, then alternately dialyzed between 50
mM phosphate (pH 2.7) and 50 mM phosphate (pH 7.4).
Probe diffusion works at fundamental
and practical levels.
Happy Halloween!
M = 10,000 and 20,000
Examples of
Separation Results
from Simulation Data
2.0
FArbitrary Units
1.5
CONTIN
2 Exponential
1.0
0.5
0.0
1000
10000
M = 10,000 and 160,000
100000
M
2.0
M = 10,000 and 57,000
CONTIN
2 Exponential
1.5
FArbitrary Units
1.5
FArbitrary Units
2.0
CONTIN
2 Exponential
1.0
0.5
1.0
0.0
1000
0.5
10000
100000
1000000
M
0.0
1000
10000
M
100000
 Indicates targeted M.
Matrix FPR Chromatogram
Pullulan, 5%w/w Dextran Matrix, 50/50 mix of 380K and 11.8K
45
40
Sure this is easy.
Also easy for GPC.
But not for DLS or
DOSY!
CONTIN Analysis
Exponential Analysis
Exponential Analysis
35
FArbitrary Units
30
25
20
15
10
5
0
1000
10000
100000
M
1000000
 Indicates targeted M.
Making the M vs. D calibration is fast & easy
}
6 fractions from analytical scale GPC
Enough for 100’s of FPR runs in ½ hour
Mw/Mn’s as now as good as anionically
polymerized, patchy standards.
Cong, Turksen & Russo Macromolecules 37(12), 4731-4735 (2004)
“Cleanup on Aisle 1”
Millipore Centricon Device
Pre-poured gel filtration
columns are also very
useful.
Analytical scale GPC itself
is a great way to clean
up unreacted dye.
Millipore Centricon -http://www.millipore.com/userguides.nsf/docs/p99259
Why is the cup half empty?
Rg ~ M
(0.158 ± 0.002)
Rg ~ M
R g / nm
10
5
10
Matrix Dextran
FD500s
FD70
FD40
FD150
1
6
M
(0.410 ± 0.005)
10
Half empty, continued
4
M / g mol
10
-1
5
10
1.0
0.9
0.8
0.7
10
D/D
10
Rh / nm
x / nm
0
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1
1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Rh / x
0.1
w
dextran (●), and pullulan probes (○).
Pullulan (destran similar)
0.7
No wonder the cup is half empty—
no plateau modulus!
100
G' / Pa-s
10
1
0.1
0.01
1
10
 / Hz
100
Correlations—suggests soft-sphere
like behavior from branching
of matrix.
50
-5
Scattering / 10 Arbitrary Units
45
40
35
30
25
20
15
10
5
0
0.0
0.1
0.2
0.3
0.4
0.5
2
0.6
-2
q / nm
0.7
0.8
0.9
1.0