Transcript Universal Calibration - LSU Macromolecular Studies Group

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
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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
4πn
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
2
g
q R
3
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Just add LS detector…and much
complexity.
MALLS
DRI
DRI
degas
pump
injector
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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
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5
4
7
16
15
14
13
12
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10
9
8
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The scattering “envelope” for a single
slice shows how Is decreases with angle.
140000
InterceptMolecular weight
120000
R/Kc
100000
80000
SlopeSize
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
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We add a viscometer in some cases.
DP  h
viscometer
LS90o
DRI
degas
pump
injector
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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”.
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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.
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Universal Calibration says that
whatever comes out at a particular
volume has the same product , [h]M.
[h]AMA = [h]SMS= 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…
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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.
• Columns are expensive, easily damaged.
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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
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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.
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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
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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.
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MALDI-TOF stands for Matrix-Assisted Laser
Desorption Ionization Time-of-Flight Mass
Spectrometry.
http://www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htm
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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
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Patience is a virtue.
You 4010 students will get to practice with a
MALDI dataset, but ….
that’s enough MALDI for now.
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What about separating cows and
elephants? Either will float around the
trees. How do you separate them then?
Moo!
Eeee!
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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.
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The most commonly used field is flow itself: one or
both plates are porous, and a cross-flow is arranged.
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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
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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.
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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)
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Conclusion
GPC is essential in any Nano Lab
GPC may eventually get replaced.
Matrix FPR
FFF
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