Putting a speed gun on macromolecules: what can we learn from how fast they go, and can we do someth

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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 31Cleveland State University
National Science Foundation
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Generic Talk Outline

• Thank hosts
• Tell joke, story or limerick
• Explain what were trying to do
• Explain what we actually did
• Today, that will lead naturally to applied
  things
• Thank accomplices

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There once was a theorist from France who
wondered how molecules dance. Theyre 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
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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

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DLS for Molecular Rheology of Complex
FluidsProspects Problems

• Studied a lot

Barely studied

• Wide-ranging autocorrelators
• gt 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.

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Translational Diffusion Leads to Intensity
Fluctuations
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Rotational Diffusion Between Polarizers Leads to
Intensity Fluctuations
Polarizer
Looking into the laser, vertically polarized
Crystalline inclusion
Analyzer
bright
dim
dim
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Dynamic Light Scattering
Uv Geometry (Polarized)
?
V
?Uv q2Dtrans
Hv Geometry (Depolarized)
?
V
H
?Hv q2Dtrans 6Drot
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• DLS can be used for sizing if viscosity is known,
  for viscosity if size is known

Is
DLS diffusion coefficient, inversely proportional
to size.
Stokes-Einstein Law
Dtransh constant
Also Droth constant
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Correlation Functions etc.
Where G(g) cMP(qRg) g q2D
q2kT/(6phRh) Rh XRg
ILT
MAP
CALIBRATE
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Strategy

• Find polymer that should (???) entangle

• Random coil
• Polysaccharide
• Invisible in HvDLS

Dextran

• Find polymer that should not entangle

• Highly-branched
• Polysaccharide
• Invisible in HvDLS

Ficoll

• Find a rodlike probe that is visible in DDLS

• Rigid rod
• Virus
• Visible in HvDLS

TMV

• Measure its diffusion in solutions of
• each polymer separately

BARELY
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As expected, viscosity rises with c
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DIY farming--keeping the A in LSU AM
Seedlings? Sick Plants ? And close-up of mosaic
pattern.
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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)

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All measurements made at low TMV
concentrationsno self-entanglement

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Matrix is invisible
Hv correlation functions for 14.5 dextran and
28 ficoll with and without added 0.5 mg/mL
TMV
The dilute TMV easily outscatters either matrix
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Hey, it works!

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I didnt thinkI experimented.

• ---Wilhelm Conrad Roentgen

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Early resultsvery slight errors
rotation
translation
Macromolecules 1997,30, 4920-6.
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Stokes-Einstein Plots if SE works, these would
be flat. Instead, apparent deviations in
different directions for Drot and Dtrans
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Macromolecules 1997,30, 4920-6.
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rotation
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!
translation
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Huh? Drot still diving in Ficoll?
translation
rotation
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Uh-oh, maybe we should think now.
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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
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Mixing in Polarized TMV Light
Uv light from misalign
True Hv light
Drot too low
G
G
G
6Drot
6Drot
q2
q2
q2
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Even at the highest concentrations, only a few
degrees out of alignment.
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Slight, but important, improvement.
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Improved Drot/Dtrans Ratio Plots
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Improved Stokes-Einstein PlotsBlack TMV
TranslationBlue TMV Rotation
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Hydrodynamic RatioEffect of Matrix M at High
Matrix Concentration
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Effect of Dextran Molecular WeightHigh Dextran
Concentration ( 15)
TMV Translation
TMV Rotation
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Summary Depolarized DLS new opportunities in
nanometer-scale rheology.
I cannot tell you the coolest part of this, but
postdoc Grigor Bantchev found a trick that is
definitely a treat!
Randy Cush David Neau Ding Shih Holly
Ricks Jonathan Strange Amanda Brown Zimei
Bu Grigor Bantchev Zuhal Savas
Kucukyavuz--METU Seth FradenBrandeis Nancy
ThompsonChapel Hill
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Too much dancing and not nearly enough
prancing!
Can probe diffusion actually do something?
C. Montgomery Burns, The Simpsons
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Matrix Fluorescence Photobleaching Recovery for
Macromolecular Characterization
Garrett Doucet, Rongjuan Cong, David Neau,
Others Louisiana State University Funding NSF,
NIH, Dow
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Blue input light
Fluorescent Sample
Fluorescence Photobleaching
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Blue input light
Fluorescent Sample With Fluorescence Hole in
Middle
Recovery of Fluorescence
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Modulation FPR Device Lanni Ware, Rev. Sci.
Instrum. 1982
SCOPE
5-10 bleach depth
PA
IF
c

TA/PVD
PMT
D
S

X?

M
DM
RR
OBJ

M
ARGON ION LASER
AOM
computer link
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Cue The Movie
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Dextran Diffusionin Hydroxy-propylcellulose, a
probe diffusion study the more HPC, the more
nonlinearity in semilog plots.Hmmm.
Bu Russo, Macromolecules, 27, 1187 (1994)
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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?

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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.

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They were young when GPC was.
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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
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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.

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Must we separate em to size em?Your local
constabulary probably doesnt think so.
Atlanta, GA I-85N at Shallowford Rd. A Saturday
at 4 pm
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Molecular Weight Distribution by DLS/Inverse
Laplace Transform--B.Chu, C. Wu, c.
Where G(g) cMP(qRg) g q2D
q2kT/(6phRh) Rh XRg
ILT
MAP
CALIBRATE
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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.

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Matrix Diffusion/Inverse Laplace
Transformation Goal Increase magnitude of
bthis will improve resolution.

• Difficult in DLS because matrix 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

Solution b -1/2
log10D

• Replace DLS with FPR.
• Selectivity supplied by dye.
• Matrix same polymer as analyzed, except no
  label.
• No compatibility problems.
• G(g) c (sidechain labeling)
• G(g) n (end-labeling)

log10M
Stretching b
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The Plan to Measure M Using FPR
Sample
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Labeling is Often Easy
Pullulan
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
5-(4,6-dichlorotriazinyl)amino fluorescein
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Matrix FPR for Pullulan (a polysaccharide)
Probe Diffusion Polymer physics
Calibration polymer analysis
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GPC vs. FPR for a Nontrivial Case
User-chosen CONTIN 25 Matrix b only 1
PL Aquagel 40A 50A
20,000 70,000 Dextran
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How Good COULD it Be? Simulation of FPR Results
for b 2(Most Desirable Situation)
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What could we separate from 10K, according to b
2 simulations?
Shazamm!
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Using an HPC Matrix

• Indicates targeted M.

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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 thatjust
  replaces Ve with D.
• We havent yet addressed two questions
• --Is it worth setting this up?
• --Miniaturization/Automation?

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Macromolecules for The Dementedand methods for
their study
Help from Keunok Yu, Jirun Sun, Bethany Lyles,
George Newkome and LSUs Alz-Hammers Research
Team Krispy Kreme Donut Day, September
2003 Supported by National Institutes of
Health-AG, NSF-DMR and NSF-IGERT

• How Alzheimers happens
• Attempts to prevent or reverse it
• Characterization challenges
• Alzheimers model systems with materials
  implications

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Positron emission tomography Age 20 -- 80
Normal -- 80 AD
Postmortem Coronal Sections Normal Alzheimers
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• APP Amyloid Precursor Protein
• 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.

http//www.bmb.leeds.ac.uk/staff/nmh/amy.html
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Amyloid hypothesis fibrils or protofibrils cause
cell death, possibly as the bodys 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
Ca2 metabolism
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Two FPR Contrast Decay Modes are Often Observed
Fast small Slow large.
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Doing More Experiments Faster with Less Precious
Amyloid Dialysis FPR
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Reversing Amyloid Aggregationby pH
Diffusion from in situ FPR of 5-carboxyfluorescein
-Ab1-40 (25 mixed with unlabeled 75 Ab1-40)
starting at pH 11, then alternately dialyzed
between 50 mM phosphate (pH 2.7) and 50 mM
phosphate (pH 7.4).
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Probe diffusion works at fundamental and
practical levels.Happy Halloween!
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Examples of Separation Results from Simulation
Data

• Indicates targeted M.

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Matrix FPR Chromatogram
Sure this is easy. Also easy for GPC. But not
for DLS or DOSY!

• Indicates targeted M.

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Making the M vs. D calibration is fast easy

6 fractions from analytical scale GPC Enough for
100s of FPR runs in ½ hour Mw/Mns as now as
good as anionically polymerized, patchy
standards.
Cong, Turksen Russo Macromolecules 37(12),
4731-4735 (2004)
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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.
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Why is the cup half empty?
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Half empty, continued
dextran (?), and pullulan probes (?).
Pullulan (destran similar)
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No wonder the cup is half emptyno plateau
modulus!
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Correlationssuggests soft-sphere like behavior
from branching of matrix.