BETTER THAN SEC’s

1. BETTER THAN SEC’s Paul S. Russo Louisiana State University Texas Polymer Center Freeport, TX October 31, 2001

2. Obligatory Equation SEC = GPC = GFC Size Exclusion Chromatography Gel Permeation Chromatography Gel Filtration Chromatography

3. 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?

4. c c log10M DRI degas c log10M Simple SEC log10M Ve pump injector

5. pV = nRT n = g/M c = g/V p = cRT h Osmometry: Real Science Semipermeable membrane: stops polymers, passes solvent.

6. c Light Scattering: Osmometer without the membrane 100,000  x q

7. q = 0 in phase Is maximum q > 0 out of phase, Is goes down LS adds optical effects  Size

8. MALLS DRI degas SEC/MALLS DRI pump injector

9. SEC/MALLS Scattered intensity Scattering angle Ve

10. Scattering Envelope for a Single Slice

11. DP  h viscometer DRI degas SEC/RALS/VIS LS90o pump injector

12. Universal CalibrationGrubisic, 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.

13. Universal Calibration Equations []AMA = []SMS= f (Ve) Universal Calibration A = analyte; S = standard [h] = KMa Mark-Houwink Relation Combine to get these two equations, useful only if universal calibration works!

14. 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?

15. Hydrodynamic volume Strategy d L Severe test of universal calibration: compare rods & coils Combine M’s from GPC/MALLS with [h]’s from literature Mark-Houwink relations.

16. [CH-CH]x [NH-CHR-C]x O R = (CH2)2COCH2 Polymers Used Polystyrene (expanded random coil) Solvent: THF = tetrahydrofuran Homopolypeptides (semiflexible rods) PBLG = poly(benzylglutamate) Solvent: DMF=dimethylformamide R = (CH2)2CO(CH2)CH3 PBLG = poly(stearylglutamate) Solvent: THF = tetrahydrofuran

17. Mark-Houwink Relations [] = 0.011·Mw0.725for PS [] = 1.26·10-5·Mw1.29for PSLG []= 1.58 10-5·Mw1.35for PBLG

18. Polystyrene Standards: the Usual

19. Polypeptide Samples Were Reasonably Monodisperse NCA-ring opening was used to make these samples. Most were just isolated and used; a few were fractionated.

20. Universal Calibration Works for These Rods and Coils

21. 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

22. 2nd Virial Coefficient (Excluded Volume Limit) is Another Universal Descriptor

23. 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.

24. What the biggest polymers in our sample would look like at this ap Persistence Length of Helical Polypeptides is “Very High”

25. SEC/MALLS in the Hands of a Real Expert Macromolecules, 29, 7323-7328 (1996) ap 15 nm Much less than PBLG

26. 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?

27. They were young when GPC was.

28. 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…

29. 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.

30. Must we separate ‘em to size ‘em?Your local constabulary probably doesn’t think so. I-85N at Shallowford Rd. Sat. 1/27/01 4 pm

31. Large, slow molecules t Small, fast molecules Sizing by Dynamic Light Scattering—a 1970’s advance in measuring motion, driven by need to measure sizes, esp. for small particles. Is It’s fluctuations again, but now fluctuations over time! DLS diffusion coefficient, inversely proportional to size.

32. Molecular Weight Distribution by DLS/Inverse Laplace Transform--B.Chu, C. Wu, &c. G(g) g(t) log10t g=q2D b » -1/2 c log10D M log10M Where: G(g) ~ cMP(qRg) g = q2D » q2kT/(6phRh) Rh = XRg ILT MAP CALIBRATE

33. 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.

34. deGennes D ~ M-2 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."* Reptation: inspired enormous advances in measuring polymer speed…and predicts More favorable results for polymers in a matrix. More generally, we could write D ~ M-b where b increases as entanglements strengthen *With apologies to Walter Stockmayer

35. Matrix Diffusion/Inverse Laplace Transformation Goal: Increase magnitude of b D D Matrix: 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

36. Small Angle Neutron Scattering Forced Rayleigh Scattering Fluorescence Photobleaching Recovery Index - matched DLS match solvent & polymer refractive index can't do in aqueous systems Depolarized DLS works for optically anisotropic probes works for most matrix polymers Painting Molecules* Makes Life Easier *R. S. Stein

37. 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.

38. FPR for Pullulan (a polysaccharide) Probe Diffusion: Polymer physics Calibration: polymer analysis

39. FPR Chromatogram Sure this is easy. Also easy for GPC. But not for DLS or DOSY! • Indicates targeted M.

40. Separation ResultsPullulan M = 50/50 mix of 11,800 and 380,000

41. 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.

42. Simulation of FPR Results(Most Desirable Situation)

43. Examples of Separation Results from Simulation Data • Indicates targeted M.

44. 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

45. 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 that—just replaces Ve with D.

46. L S U Thank you!

47. Physical Info from SEC Elena Temyanko Holly Ricks N$F Replacing SEC Garrett Doucet David Neau Wieslaw Stryjewski Better than SEC’sMonday, January 29, 2001

48. 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