BETTER THAN SECs

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BETTER THAN SECs
Paul S. Russo Louisiana State University Texas
Polymer Center Freeport, TX October 31, 2001
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Obligatory Equation
SEC GPC GFC
Size Exclusion Chromatography Gel Permeation
Chromatography Gel Filtration Chromatography
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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.
• It is thought that particle volume controls the
  order of elution.
• But what about shape?

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Simple SEC
log10M
Ve
pump
injector
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Osmometry Real Science
Semipermeable membrane stops polymers, passes
solvent.
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Light Scattering Osmometer without the membrane
100,000 ?
x
q
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LS adds optical effects ? Size
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SEC/MALLS
DRI
pump
injector
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SEC/MALLS
Scattered intensity
Scattering angle
Ve
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Scattering Envelope for a Single Slice
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SEC/RALS/VIS
LS90o
pump
injector
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Universal Calibration Grubisic, Rempp Benoit,
JPS Pt. B, 5, 753 (1967)
One of of the most important Papers in polymer
science. Imagine the work involved! 6 pages long
w/ 2 figures. Selected for JPS 50th Anniv. Issue.
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Universal Calibration Equations
  ?A?MA ?S?MS f (Ve)
Universal Calibration A analyte S standard
h KM a
Mark-Houwink Relation
Combine to get these two equations, useful only
if universal calibration works!
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Objectives

• Use a-helical rodlike homopolypeptides to test
  validity of universal calibration in GPC.
• Can GPC/Multi-angle Light Scattering arbitrate
  between disparate estimates of stiffness from
  dozens of previous attempts by other methods?

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Strategy
L
d
Severe test of universal calibration compare
rods coils Combine Ms from GPC/MALLS with
hs from literature Mark-Houwink relations.
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Polymers Used
Polystyrene (expanded random coil) Solvent THF
tetrahydrofuran
Homopolypeptides (semiflexible rods)
PBLG poly(benzylglutamate) Solvent
DMFdimethylformamide
R (CH2)2CO(CH2)CH3
PBLG poly(stearylglutamate) Solvent THF
tetrahydrofuran
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Mark-Houwink Relations
? 0.011Mw0.725 for PS   ?
1.2610-5Mw1.29 for PSLG   ? 1.58
10-5Mw1.35 for PBLG
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Polystyrene Standards the Usual
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Polypeptide Samples Were Reasonably Monodisperse
NCA-ring opening was used to make these samples.
Most were just isolated and used a few were
fractionated.
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Universal Calibration Works for These Rods and
Coils
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2nd Virial Coefficient Equations
Osmotic pressure in number density concentration
(n) units
p nkT(1 nA2,n )
Relationship to the normal 2nd virial
coefficient for conc. in mass per volume units.
A2,n M 2A2 /Na
Onsager 2nd virial coefficient for rods (L
length, d dia.)
A2,n dL2/4
Rg for rods
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2nd Virial Coefficient (Excluded Volume Limit) is
Another Universal Descriptor
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Persistence Length ap from Rg
Persistence length is the projection of an
infinitely long chain on a tangent line drawn
from one end. ap ? for true rod.
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Persistence Length of Helical Polypeptides is
Very High
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SEC/MALLS in the Hands of a Real Expert
Macromolecules, 29, 7323-7328 (1996)
ap ? 15 nm Much less than PBLG
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Conclusions
The new power of SEC/Something Else experiments
is very real. SEC is now a method that even the
most jaded physical chemist should embrace. For
example, our results favor higher rather than
lower values for PBLG persistence length. This
helps to settle about 30 years of uncertainty.
Universal calibration works well for
semiflexible rods spanning the usual size range,
even when the rods are quite rigid. So, SEC is
good enough for physical measurements, but is it
still good enough for polymer analysis?
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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 10 am, 0.1 M salt at 2 pm?
• 45oC at 8 am and 50oC at noon?
• Non-size exclusion mechanisms binding.
• Big, bulky and slow (typically 30
  minutes/sample).
• Temperature/harsh solvents no fun.
• You learn nothing by calibrating.

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Must we separate em to size em?Your local
constabulary probably doesnt think so.
I-85N at Shallowford Rd. Sat. 1/27/01 4 pm
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• Sizing by Dynamic Light Scatteringa 1970s
  advance in measuring motion, driven by need to
  measure sizes, esp. for small particles.

Is
Its fluctuations again, but now fluctuations
over time! DLS diffusion coefficient, inversely
proportional to size.
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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
This only works because of that wide, wide M
distribution. Main problem with DLS/Laplace
inversion is poor resolution. Things kinda go to
pot at low M, too. Some assumptions have to be
made to do this.
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• Reptation inspired enormous advances in
  measuring polymer speedand predicts
• More favorable results for polymers in a matrix.

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."
More generally, we could write D M-b where b
increases as entanglements strengthen
With apologies to Walter Stockmayer
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Matrix Diffusion/Inverse Laplace
Transformation Goal Increase magnitude of b

• 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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Painting Molecules Makes Life Easier
R. S. Stein
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Fluorescence Photobleaching Recovery
3. An exponential decay is produced by
monitoring the amplitude of the decaying sine
wave. Fitting this curve produces G, from which
D can be calculated.
2. A decaying sine wave is produced by moving
the illumination pattern over the pattern written
into the solution.
1. An intense laser pulse photobleaches a
striped pattern in the fluorescently tagged
sample.
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FPR for Pullulan (a polysaccharide)
Probe Diffusion Polymer physics
Calibration polymer analysis
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FPR Chromatogram
Sure this is easy. Also easy for GPC. But not
for DLS or DOSY!

• Indicates targeted M.

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Separation ResultsPullulan M 50/50 mix of
11,800 and 380,000
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Better Resolution Soon?
Improvement in resolution is observed at lower
concentrations due to a more viscous
characteristic. A compatibility problem is seen
though at higher concentrations.

• Indicates targeted M.

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Simulation of FPR Results(Most Desirable
Situation)
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Examples of Separation Results from Simulation
Data

• Indicates targeted M.

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Ultimate Goal A Black Box for MWD
Press for MWD

• Matrix FPR
• Easily Maintained
• Accurate
• Precise
• Simple Concept
• Expedient
• Easy System Switch
• Basic Info Obtained
• Miniaturizable
• Detect Large Masses
• Labeling Required

• GPC
• Accurate
• Simple Concept
• Miniaturizable
• No Labeling Required
• Broad Distributions
• Pumps
• Parts

DOSY Easy System Switch Precise Accurate Obtain
Basic Info Labeling Required
DLS Form Factor Index Matching Long Acquisition
for Multiangle Experiments Precise Accurate
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Conclusions

• For a limited number of cases, this could really
  work.
• We may not always need leaking pumps and large
  parts bins for polymer characterization.
• What is good about GPC (straight GPC) is the
  simple concept Matrix FPR keeps thatjust
  replaces Ve with D.

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Thank you!
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Better than SECsMonday, January 29, 2001

• Physical Info from SEC
• Elena Temyanko
• Holly Ricks
• NF

• Replacing SEC
• Garrett Doucet
• David Neau
• Wieslaw Stryjewski

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History of this Talk

• Used first at Georgia Tech, mods made after
• Same modifications to the USC talk, which is
  designed to be a little shorter simpler
• The changes affect mostly the early parts of the
  diffusion part, near deGennes and Chu
• Used at Dow--Freeport