putting a speed gun on macromolecules: what can we learn from how fast they go, and can we do...
TRANSCRIPT
Putting a speed gun on Putting a speed gun on macromolecules: what can we learn macromolecules: what can we learn from how fast they go, and can we from how fast they go, and can we do something useful with that do something useful with that information?information?
Monday, October 31Monday, October 31Cleveland State UniversityCleveland 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 Francewho wondered how molecules dance.“They’re like snakes,” he observed, “As they follow a curve, the large onescan hardly advance.”
D ~ M -2
P.G. de GennesScaling Concepts in Polymer Physics
Cornell University Press, 1979
P. G. de GennesNobel Prize
PhysicsTons per mole!
Diffusion
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
+ + + 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.
Studied a lot
Barely studied
Translational Diffusion Leads to Intensity Fluctuations
t
Intensity
Rotational Diffusion Between Polarizers Leads to Intensity Fluctuations
Crystalline inclusion
Looking into the laser,vertically polarized
dim dim bright
Analyzer
Polarizer
Dynamic Light ScatteringDynamic Light Scattering
Hv = q2Dtrans + 6Drot
LASER
VV HH
PMT
Hv Geometry Hv Geometry (Depolarized)(Depolarized)
Uv Geometry Uv Geometry (Polarized)(Polarized)
VV
Uv = q2Dtrans
o
nq
2/sin4
PMT
LASER
DLS can be used for sizing if viscosity is known, for viscosity if size is known
transoπη6 D
kTRh
t
Is
DLS diffusion coefficient, inversely proportional to size.
Large, slow moleculesSmall, fast molecules
Stokes-Einstein Law
Dtrans= constant
Also Drot= constant
Correlation Functions etc.
dtGtg )exp()()(Where: G() ~ cMP(qRg)
= q2D
q2kT/(6Rh)
Rh = XRgg(t)
log10t
ILT
q2
D
G()
CALIBRATE MAP
M
c
log10M
log10D
StrategyStrategy
•Find polymer that should not “entangle”
•Find a rodlike probe that is visible in DDLS
•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
Dextran
Ficoll
TMV
•Find polymer that should (???) “entangle”
BARELYBARELY
0 5 10 15 20 25 30 35 400
1
2
3
4
5
6
7
8
9
10
11
BothViscosity
sp/
c /d
L-g
-1
c/g-dL-1
Dextran 670,000 Ficoll 420,000
As expected, viscosity rises with c
Seedlings
Sick Plants And close-up of mosaic pattern.
DIY farming--keeping the “A” in LSU A&M
TMV CharacterizationTMV 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)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
200
300
400
500D
t /10-8cm
2s-1
Dr /
s-110 L3
c/mg-mL-1
0
1
2
3
4
5
6
Rotation
Translation
Experiments are in dilute regime. TMV overlap (1/L3)
All measurements made at low TMV concentrations—no self-entanglement
Hv correlation Hv correlation functions for 14.5% functions for 14.5% dextran and 28% dextran and 28% ficoll with and ficoll with and without added without added 0.5 mg/mL TMV0.5 mg/mL TMV
The dilute TMV The dilute TMV easily “outscatters” easily “outscatters” either matrixeither matrix
1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100
1.0
1.2
1.4
Ficoll >6000 s acquisition
TMV + Ficoll 600s aquisition
g(2
)
t/s
1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 1000.9
1.0
1.1
1.2
1.3
Dextran >6000 s acquisition
TMV + Dextran 215 s acquisition
g(2
)
t/s
Matrix is invisible
0 1 2 3 4 5
0
500
1000
1500
2000
2500
3000
3500
4000
Hv TMV / Dextran / Buffer
Uv TMV / Buffer
Hv TMV / Buffer
/s-1
q2/1010 cm-2
Hey, it works!
I didn’t think—I experimented.
---Wilhelm Conrad Roentgen
0 2 4 6 8 10 12 14 160
1
2
3
4
5
6
Dtr
ans/1
0-8 c
m2
s-1
wt% dextran0 2 4 6 8 10 12 14 16
0
50
100
150
200
250
300
350
Dro
t/ s-
1
wt% dextran
Early results—very slight errors
rotation translationMacromolecules 1997,30, 4920-6.
Stokes-Einstein Plots: if SE works, thesewould be flat. Instead, apparent deviations in
different directions for Drot and Dtrans
0 2 4 6 8 10 12 14 16
0.0
0.5
1.0
1.5
Dt /10
-9g-cm
-s-2
Dr /
g-cm
-1-s
-1
wt% Dextran
0
2
4
0 2 4 6 8 10 12 14 16
0 5 10 15 20
0
2
4
6
8 /cP
Dr/D
t /1
09 cm-2
wt % dextran
0 5 10 15 20
0
20
40
60
80
Dextran overlap
Macromolecules 1997,30, 4920-6.
At the sudden transition: L/c.m. ~ 13 and L/ ~ 120
L
cm
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 300
1
2
3
4
5
6
Dtr
an
s/10-
8 cm
2 s-
1
wt% ficoll
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0
50
100
150
200
250
300
350
Dro
t/ s-
1
wt% ficoll
rotation
translation
We believed that the transition represented topological constraints.
It was suggested that more systems be studied.
BEGIN FICOLL
When we did Ficoll, many more points were added!
0 5 10 15 20 25 300.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Dtra
ns /10
-9g-cm-1-s
-1
Dro
t /g-
cm-1-s
-1
wt% ficoll
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Huh? Drot still diving in Ficoll?
rotationtranslation
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…
TMV = 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
q2 q2 q2
Drot too low
6Drot6Drot
Even at the highest concentrations, only a few degrees out of alignment.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 360
50
100
150
200
250
300
Op
tica
l Ro
tatio
n /
arc
-min
ute
s
wt %
Dextran Ficoll
0 5 10 15 20 25 30 35
0
50
100
150
200
250
300
350
NewFicollRatio_PR
Right way Wrong way
Dro
t / s
-1
wt% ficoll
Slight, but important, improvement.
Improved Drot/Dtrans Ratio Plots
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160
1
2
3
4
5
6
7
8
NewDexConcStudy_PR
Dro
t/Dtr
ans/
109 cm
-2
wt% dextran0 5 10 15 20 25 30 35 40
0
1
2
3
4
5
6
7
8
NewFicollRatio_PR
Dro
t/Dtr
ans/
109 c
m-2
wt% ficoll
Improved Stokes-Einstein PlotsBlack = TMV Translation
Blue = TMV Rotation
0 2 4 6 8 10 12 14 160.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.0
0.2
0.4
0.6
0.8
NewDexConcStudy_PR
Dro
t/g-c
m-1s-1
wt% dextranD
trans /10-9g-cm
-s-2
0 5 10 15 20 25 30 350.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.0
0.2
0.4
0.6
0.8
NewFicollRatio_PR
Dro
t/g-c
m-1 s
-1
wt% ficoll
Dtrans / 10
-9g-cm-s
-2
Hydrodynamic Ratio—Effect of Matrix M at High Matrix Concentration
0 2 4 6 8 10 12 14 16 18 200
1
2
3
4
5
6
7
8
9DextranMWStudy_PR
Dro
t/Dtr
ans/
109 cm
-2
dextran MW/ 105 daltons
Effect of Dextran Molecular Weight—High Dextran Concentration (~ 15%)
10000 100000 1000000 1E71
10
100
DextranMWStudy_PR
-0.62 ± 0.04
Dro
t / s
-1
Dextran MW10000 100000 1000000 1E7
0.1
1
10
DextranMWStudy_PR
-0.72 ± 0.01
Dtr
ans/
10-9
cm
2 s-1
Dextran MW
TMV Translation TMV Rotation
Randy CushDavid Neau
Ding Shih
Holly Ricks
Jonathan Strange
Amanda Brown
Zimei Bu
Grigor Bantchev
Zuhal & Savas Kucukyavuz--METU
Seth Fraden—Brandeis
Nancy Thompson—Chapel Hill
Summary: Depolarized DLS = new opportunities in nanometer-scale rheology.
I cannot tellyou the coolest part of this, but postdocGrigor Bantchev found a trick that is definitely a treat!
“Too much dancing and not nearly enough prancing!”
C. Montgomery Burns, “The Simpsons”
Can probe diffusion actually do something?
Matrix Fluorescence Photobleaching Recovery for Macromolecular
Characterization
Garrett Doucet, Rongjuan Cong, David Neau, OthersLouisiana State UniversityFunding: NSF, NIH, Dow
Fluorescence & Photobleaching
Blue input light
FluorescentSample
Green Detected
Light
Recovery of Fluorescence
Blue input light
FluorescentSample
With FluorescenceHole in Middle
Green Detected
LightSlowly Recovers
Modulation FPR Device Lanni & Ware, Rev. Sci. Instrum. 1982
*
*
*
*
AOM
M
M
D
RR
DM
OBJ
S
PMT
PA
SCOPE
TA/PVD
ARGON ION LASER
* = computer link
IF
X
c
5-10% bleach depth
Cue The Movie
Dextran Diffusionin Hydroxy-propylcellulose, 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, GAI-85N at Shallowford Rd.A Saturday at 4 pm
Molecular Weight Distribution byDLS/Inverse Laplace Transform--B.Chu, C. Wu, &c.
dtGtg )exp()()(Where: G() ~ cMP(qRg)
= q2D
q2kT/(6Rh)
Rh = XRgg(t)
log10t
ILT
q2
D
G()
CALIBRATE MAP
M
c
log10M
log10D
Hot Ben Chu / Chi Wu Example
MWD of PTFESpecial solvents at ~330oC
Macromolecules, 21, 397-402 (1988)
Problems: •Only “works” because MWD is broad•Poor resolution.•Low M part goofy. •Some assumptions required.
Matrix Diffusion/Inverse Laplace TransformationGoal: Increase magnitude of —this will improve
resolutionDifficult in DLS because matrix scatters, except special cases.Difficult anyway: dust-free matrix not fun!Still nothing you can do about visibility of small scatterersDOSY not much betterReplace 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)log10M
log10D
Stretching
Solution:
Matrix:
The Plan to Measure M Using FPR
Sample
Analyze Using ILT
10-3 10-2 10-1 100
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Arb
itrar
y A
mpl
itude
/ s-1
Collect Data Using FPR
0 200 400 600 800 10000
1
2
3
4
5
6
C(t
)
t / s
Convert to Molar Mass by Mapping onto Calibration
Plot
103 104 105
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Arb
itrar
y A
mpl
itude
M / Da
Labeling is Often Easy
H O
O
OH
OH
OH
OH
n
Dextran M = 2 Million Da as the matrix at different concentrations in 5 mM
NaN3 solution
Pullulans of different M labeled with 5-DTAF as probes
O
O
O
O
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH OH
n
Pullulan
O
COOH
O OH
NH
N
N
N
Cl
Cl
5-(4,6-dichlorotriazinyl)amino fluorescein
Matrix FPR for Pullulan Matrix FPR for Pullulan (a polysaccharide)(a polysaccharide)
104 105
0.01
0.1
1
10
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)
Dap
p / 1
0-7 c
m2 s
-1
M
0.1 1 1010
4
105
MD
app / 10
-7 cm
2 s
-1
Probe Diffusion: Polymer physics Calibration: polymer analysis
GPC vs. FPR for a Nontrivial Case
104 105
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Rel
ativ
e C
once
ntra
tion
M / g mol-1
20,000 & 70,000 Dextran
PL Aquagel 40A & 50A User-chosen CONTIN 25% Matrix only ~1
104 105
0
2
4
6
8
10
12
Am
plit
ud
e/A
rbitr
ary
M
How Good COULD it Be? How Good COULD it Be? Simulation of FPR Results for Simulation of FPR Results for
= 2= 2(Most Desirable Situation)(Most Desirable Situation)
y = -0.4998x + 1.1518
0
1
2
3
4
5
6
-10 -8 -6 -4 -2 0
log D
log M
y = -2.0009x + 2.3045
-12
-10
-8
-6
-4
-2
0
2
4
0 2 4 6 8
log M
log
D
What could we separate from 10K, according to = 2
simulations?
10000 100000
2000040000
5700080000
113000160000
MDetected
Shazamm!
Using an HPC Matrix
Indicates targeted M.
1000 10000 100000 10000000.0
0.2
0.4
0.6
0.8
1.0
Pullulan, 8% HPC Solution, M=12,200 and 48,000
CONTIN Exponential Exponential
F Arb
itra
ry U
nits
M
MFPR ConclusionsMFPR ConclusionsWe 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 studyHelp from Keunok Yu, Jirun Sun, Bethany Lyles, George Newkome and LSU’s Alz-Hammer’s Research Team
Krispy Kreme Donut Day, September 2003Supported 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
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
Positron emission tomographyAge: 20 -- 80 Normal -- 80 AD
Postmortem Coronal Sections
NormalAlzheimer’s
http://www.bmb.leeds.ac.uk/staff/nmh/amy.html
APP = Amyloid Precursor Protein
APP = the larger, lighter pink one
•Transmembrane protein•Normal function not known•Educated guesses
May help stem cells develop identityOr help relocate cells to final locationMay “mature” cells into structural typeMay protect brain cells from injurySynaptic actionCopper 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 Grouphttp://focus.hms.harvard.edu/1998/June4_1998/neuro.html
Competing hypothesis: channel formation disrupts Ca+2 metabolism
1 10 100 1000
1E-3
0.01
0.1
1
Co
ntr
ast
/ A
rbitr
ary
t/s
pH 2.7 pH 6.9 pH 11
Two FPR Contrast Decay Modes are Often Observed: Fast = small; Slow = large.
Doing More Experiments Faster with Less Precious Amyloid:
Dialysis FPR
Cover slip
PTFE spacer Dialysis membraneO-ring
Sample
Exchange Fluid
Pump
Diffusion from in situ FPR of 5-carboxyfluorescein-A1-40 (25% mixed with unlabeled 75% A1-40) starting at pH 11, then alternately dialyzed between 50 mM phosphate (pH 2.7) and 50 mM phosphate (pH 7.4).
0 200 400 600 800 1000 1200 1400 1600 1800
1E-8
1E-7
1E-6
FPR Study: Reversibility of -Amyloid Aggregation100M 5-CF--Amyloid
1-40+ -Amyloid
1-40 pH 11
dialysis against 50mM PB pH 7.4
dialysis against 50mM PB pH 2.7
D/1
0-6 c
m2 s-1
Time/min
Reversing Amyloid Aggregation…by pH
Probe diffusion works at fundamental and practical
levels.
Happy Halloween!
1000 10000 1000000.0
0.5
1.0
1.5
2.0
M = 10,000 and 20,000
CONTIN 2 Exponential
F Arb
itrar
y U
nits
M
1000 10000 100000 10000000.0
0.5
1.0
1.5
2.0
M = 10,000 and 160,000
CONTIN 2 Exponential
F Arb
itra
ry U
nits
M
1000 10000 1000000.0
0.5
1.0
1.5
2.0
M = 10,000 and 57,000
CONTIN 2 Exponential
F Arb
itrar
y U
nits
M
Examples of Examples of Separation Results Separation Results
from Simulation from Simulation DataData
Indicates targeted M.
Matrix FPR ChromatogramMatrix FPR Chromatogram
1000 10000 100000 10000000
5
10
15
20
25
30
35
40
45 CONTIN Analysis Exponential Analysis Exponential Analysis
Pullulan, 5%w/w Dextran Matrix, 50/50 mix of 380K and 11.8K
FA
rbit
rary
Uni
ts
M Indicates targeted M.
Sure this is easy. Also easy for GPC.But not for DLS or DOSY!
Cong, Turksen & Russo Macromolecules 37(12), 4731-4735 (2004)
}6 fractions from analytical scale GPCEnough for 100’s of FPR runs in ½ hourMw/Mn’s as now as good as anionicallypolymerized, patchy standards.
Making the M vs. D calibration is fast & easy
“Cleanup on Aisle 1”
Millipore Centricon --http://www.millipore.com/userguides.nsf/docs/p99259
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.
105 106
1
10
Matrix D
extran
FD500s
FD150
FD70
FD40
Rg ~ M (0.158 ± 0.002)
Rg ~ M (0.410 ± 0.005)
R g /
nm
M
Why is the cup half empty?
Half empty, continued
0.11
10
104 105
1
10
/ n
m
w
M / g mol-1
Rh / nm
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
D /
D0
Rh /
Pullulan (destran similar)dextran (●), and pullulan probes (○).
No wonder the cup is half empty—no plateau modulus!
1 10 1000.01
0.1
1
10
100
G' /
Pa-
s
/ Hz
Correlations—suggests soft-sphere like behavior from
branching of matrix.
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00
5
10
15
20
25
30
35
40
45
50Sc
atte
ring
/ 10
-5 A
rbitr
ary
Uni
ts
q2 / nm-2