Published on January 30, 2008
On-line Detector Methods…and a peak at possibly better things to come.: On-line Detector Methods…and a peak at possibly better things to come. 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) Theme: being average (and averages generally) suck. GPC: 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? Simple SEC: Simple SEC pump injector Ve log10M Osmometry: Real Science: Osmometry: Real Science Semipermeable membrane: stops polymers, passes solvent. Light Scattering: Osmometer without the membrane: Light Scattering: Osmometer without the membrane 100,000 x q LS adds optical effects Size: LS adds optical effects Size SEC/MALLS: SEC/MALLS pump injector DRI SEC/MALLS: SEC/MALLS Scattering angle Ve Scattered intensity Scattering Envelope for a Single Slice: Scattering Envelope for a Single Slice Slide10: SEC/RALS/VIS pump injector LS90o Universal Calibration Grubisic, Rempp & Benoit, JPS Pt. B, 5, 753 (1967): 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. Universal Calibration Equations: Universal Calibration Equations [h] = KM a AMA = SMS= f (Ve) Mark-Houwink Relation Universal Calibration A = analyte; S = standard Combine to get these two equations, useful only if universal calibration works! Objectives: 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? Strategy: Severe test of universal calibration: compare rods & coils Combine M’s from GPC/MALLS with [h]’s from literature Mark-Houwink relations. Strategy L d Polymers Used: Polymers Used R = (CH2)2CO(CH2)CH3 Polystyrene (expanded random coil) Solvent: THF = tetrahydrofuran Homopolypeptides (semiflexible rods) PBLG = poly(benzylglutamate) Solvent: DMF=dimethylformamide PBLG = poly(stearylglutamate) Solvent: THF = tetrahydrofuran Mark-Houwink Relations: Mark-Houwink Relations  = 0.011·Mw0.725 for PS  = 1.26·10-5·Mw1.29 for PSLG  = 1.58 10-5·Mw1.35 for PBLG Polystyrene Standards: the Usual: Polystyrene Standards: the Usual Slide18: Polypeptide Samples Were Reasonably Monodisperse NCA-ring opening was used to make these samples. Most were just isolated and used; a few were fractionated. Universal Calibration Works for These Rods and Coils: Universal Calibration Works for These Rods and Coils 2nd Virial Coefficient Equations: 2nd Virial Coefficient Equations A2,n = M 2A2 /Na p = nkT(1 + nA2,n + …) A2,n = dL2/4 Osmotic pressure in number density concentration (n) units Relationship to the “normal” 2nd virial coefficient for conc. in mass per volume units. Onsager 2nd virial coefficient for rods (L= length, d = dia.) Rg for rods 2nd Virial Coefficient (Excluded Volume Limit) is Another Universal Descriptor: 2nd Virial Coefficient (Excluded Volume Limit) is Another Universal Descriptor Persistence Length ap from Rg: 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. Persistence Length of Helical Polypeptides is “Very High”: Persistence Length of Helical Polypeptides is “Very High” SEC/MALLS in the Hands of a Real Expert: SEC/MALLS in the Hands of a Real Expert ap 15 nm Much less than PBLG Macromolecules, 29, 7323-7328 (1996) Slide25: 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? They were young when GPC was.: They were young when GPC was. Small Subset of GPC Spare Parts: 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: 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. : 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”?: Better Resolution “Soon”? Indicates targeted M. Improvement in resolution is observed at lower concentrations due to a more viscous characteristic. A compatibility problem is seen though at higher concentrations. Slide31: Examples of Separation Results from Simulation Data Indicates targeted M.