This presentation exemplifies the latest Power Point dogma. dogma, n. A settled or established opinion, belief, or principle A full sentence at the top tells the main point. Vertically aligned lists are avoided in favor of diagonal arrangement to create visual tension. Tasteful use of animation helps. A page number helps reader ask questions. Occasionally, put in something strange or fun to keep professors and other tired listeners awake. 1 “You cannot measure absolute molecular weights” Dow manager, 1983 Wrong! Correct, even in 1983: you NEED NOT always measure ABSOLUTE molecular weights…but you could have. Correct in 2005: it is almost always essential to measure absolute M s. 2 So, how would you analyze polymers and nanoparticles for polydispersity? Here is the easiest equation in this talk. SEC = GPC = GFC Size Exclusion Chromatography Gel Permeation Chromatography Gel Filtration Chromatography 4 A riddle: After a hurricane, many trees fall over and bend into a river. Also, a cow and a dog fall into that flooded river. Which one reaches the ocean first, cow or dog? Moo! Woof! 5 In GPC, as when throwing a cat through a tree, the big things come out first.. •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? 6 c log10M Simple SEC is only simple when you don’t have to do it yourself. c DRI Ve c degas pump injector log10M 7 In simple GPC, you first make a map (calibrate), then follow it. logM DRI log M A DRIA MA Vo VeA VeVe 8 Are you straight? GPC Calibration 120 8 log(Mw ) = 0.0009V e3 + 0.6303V e + 6.8161 R = 0.9996 7 6 80 5 60 4 log(Mw) Detector Response 100 - 0.0534V e2 2 3 40 2 20 1 0 0 14 16 18 20 22 24 26 28 Ve [mL] 9 Use the right tool (column) for the job. 10 OK, but how did you get the M values for the standards? • Osmometry Scattering MALDI Analytical ultracentrifugation End group analysis Goal in this class: make it seem reasonable that it IS possible to measure M values. 11 Osmometry is Real Science because it is tied closely to thermodynamics. h pV = n R T n = g/M c = g/V 1 p = c R T ( A2c ...) M Semipermeable membrane: stops polymers, passes solvent. 12 Light scattering operates on thermodynamics; think of it as an osmometer without the membrane. 100,000 c x 2p q 1 p Is cRT ( 2 A2c ...) M c T , p 1 q 4 πn o sin( / 2) 13 Teaching Light Scattering to Exemplify and Reinforce Basic Principles, D. S. Poche', P. S. Russo, B. Fong, E. Temyanko and H. Ricks, J. Chem. Ed., 1999, 76 (November), 1534-1538. LS adds optical effects Size. q = 0 in phase Is maximum 2 q > 0 out of phase, Is goes down Is 1 q R 2 g 3 14 Just add LS detector…and much complexity. MALLS DRI DRI degas pump injector 15 This waterfall plot shows many “slices” of a chromatogram: 13 angles were recorded ~8 times per second as the sample flowed by the MALS detector. 3D Plot - PBLG Scattered intensity 6 5 4 7 16 15 14 13 12 11 10 9 8 16 The scattering “envelope” for a single slice shows how Is decreases with angle. 140000 InterceptMolecular weight 120000 R/Kc 100000 80000 SlopeSize 60000 c = 0.044 mg/mL M = 130000 g/mol 40000 20000 0 0.0 0.2 0.4 0.6 sin ( /2) 0.8 1.0 2 17 We add a viscometer in some cases. DP h viscometer LS90o DRI degas pump injector 18 Intrinsic viscosity is a secondary molecular weight method so good it’s almost like the real thing. Mark-Houwink-Sakurada relationship between the intrinsic viscosity and the molecular weight. [h ] KM a These words have special meaning in polymer science. M ([h ] / K ) 1/ a K and a are constants for a given polymer, not strongly dependent on solvent or temperature, as long as we’re talking about a “good solvent”. 19 Universal Calibration lets you get the molecular weight of one kind of polymer knowing only the Mark-HouwinkSakurada values of a standard (look it up) and your unknown (uh-oh). 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. 20 Universal Calibration says that whatever comes out at a particular volume has the same product , [h]M. [h]AMA = [h]SMS= f (Ve) Universal Calibration A = analyte; S = standard [h] = KM a Mark-Houwink Relation K A M Aa A 1 K S M SaS 1 K S M SaS 1 MA KA 1 a A 1 Combine to get these two equations, useful only if universal calibration works! 21 They were young when GPC was. They were young when GPC hit middle age. Here is a 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… 24 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. • Columns are expensive, easily damaged. 25 Must we separate ‘em to size ‘em? Your local constabulary probably doesn’t think so. Atlanta, GA I-85N at Shallowford Rd. Sat. 1/27/01 4 pm 26 Matrix Fluorescence Photobleaching Recovery* is an LSU-invented method designed to compete with GPC for certain systems (aqueous commodity polymers). Pullulan, 8% HPC Solution, M=12,200 and 48,000 1.0 FArbitrary Units 0.8 CONTIN Exponential Exponential 0.6 0.4 0.2 0.0 1000 10000 100000 M *G. J. Doucet, D. Dorman, R. Cueto, D. Neau, P. S. Russo, D. DeKee, J. Pople Macromolecules 2006, 39(26), 9446 – 9455. 1000000 Indicates targeted M. 27 Ultimate Goal: A Black Box for MWD Matrix FPR GPC DOSY Easily Maintained Accurate Precise Simple Concept Expedient Easy System Switch Basic Info Obtained Miniaturizable Detect Large Masses Labeling Required Accurate Simple Concept Miniaturizable No Labeling Required Broad Distributions Pumps Parts Easy System Switch Precise Accurate Obtain Basic Info Labeling Required DLS Form Factor Index Matching Long Acquisition for Multiangle Experiments Precise Accurate 28 In other words, the search continues. Two promising contenders are discussed next. MALDI-TOF: most effective when the molecules are small, biological and not very polydisperse. Can be coupled to GPC! FFF: like GPC only a flowing field replaces the stationary phase, stuff comes out backwards, and big stuff can be handled as well as small. 29 MALDI-TOF stands for Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry. http://www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htm 30 These data obtained at LSU: click the figure to analyze these results. a.i. 400 File will download from the website Compute Mn,Mw,Mz MANUALLY Compute Mn,Mw,Mz in Excel or something else You can subtract the mass of silver from each peak We hope Dr. Tracy McCarley will give a tour of the MALDI Counts 300 200 Guess what Mw/Mn is. 100 0 0 2000 4000 6000 8000 10000 12000 Mass 31 Patience is a virtue. You 4010 students will get to practice with a MALDI dataset, but …. that’s enough MALDI for now. 32 What about separating cows and elephants? Either will float around the trees. How do you separate them then? Moo! Eeee! 33 Field Flow Fractionation, that’s how! In FFF, large nanoparticles are made to flow between plates; a field is applied to separate them by size. 34 The most commonly used field is flow itself: one or both plates are porous, and a cross-flow is arranged. 35 What happens because of the cross-flow? Little nanoparticles come out first! At LSU, only one plate is porous. 1. Everyone calls it AF4 = Asymmetric Flow Field Flow Fractionation 36 2. How to you get a crossflow then? Potential Advantages of FFF Handles a wider range of particles. May be easier for some aggressive solvents. 37 AF4 can even separate large PTFE latex particles. RMS radius vs Time 4 tm AF separation of algoflon 180 1.0 o LS 90 160 140 0.6 120 100 0.4 80 0.2 relative intensity rms radius (nm) 0.8 60 0.0 40 30 35 40 time (min) 38 Conclusion GPC is essential in any Nano Lab GPC may eventually get replaced. Matrix FPR FFF 39