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

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