On-line Detector Methods…and a peak at possibly better things to come. Theme: being average (and averages generally) suck. Reference: Study of Rodlike Homopolypeptides by Gel Permeation Chromatography with Light Scattering Detection: Validity of Universal Calibration and Stiffness Assessment E. Temyanko, P. S. Russo and H. Ricks, Macromolecules, 34, 582-586 (2001)
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?
c c log10M DRI degas c log10M Simple SEC log10M Ve pump injector
pV = nRT n = g/M c = g/V p = cRT h Osmometry: Real Science Semipermeable membrane: stops polymers, passes solvent.
c Light Scattering: Osmometer without the membrane 100,000 x q
q = 0 in phase Is maximum q > 0 out of phase, Is goes down LS adds optical effects Size
MALLS DRI degas SEC/MALLS DRI pump injector
SEC/MALLS Scattered intensity Scattering angle Ve
DP h viscometer DRI degas SEC/RALS/VIS LS90o pump injector
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.
Universal Calibration Equations AMA = SMS= 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!
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?
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.
[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
Mark-Houwink Relations  = 0.011·Mw0.725for PS  = 1.26·10-5·Mw1.29for PSLG = 1.58 10-5·Mw1.35for PBLG
Polypeptide Samples Were Reasonably Monodisperse NCA-ring opening was used to make these samples. Most were just isolated and used; a few were fractionated.
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
2nd Virial Coefficient (Excluded Volume Limit) is Another Universal Descriptor
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.
What the biggest polymers in our sample would look like at this ap Persistence Length of Helical Polypeptides is “Very High”
SEC/MALLS in the Hands of a Real Expert Macromolecules, 29, 7323-7328 (1996) ap 15 nm Much less than PBLG
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?
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 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.
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
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.
Examples of Separation Results from Simulation Data • Indicates targeted M.