For Journal of Fluorescence
October 12, 2015
Supporting Information
Colorful Polyelectrolytes: an Atom Transfer Radical Polymerization
Route to Fluorescent Polystyrene Sulfonate
Wayne Huberty#, Xiaowei Tong#, Sreelatha Balamurugan, Kyle Deville, Paul S. Russo* and
Donghui Zhang
Department of Chemistry and Macromolecular Studies Group
Louisiana State University
Baton Rouge, Louisiana 70803
# These authors contributed equally to the work
* To whom communications should be sent at this current address:
School of Materials Science & Engineering
Georgia Institute of Technology
MRDC Building
801 Ferst Drive
Atlanta, Georgia 30332-0245
paul.russo@mse.gatech.edu
1
GPC/MALS Details
Figure 5 from the main article is reproduced as Figure S1-A for convenience. Such a plot has
become routine in the age of online light scattering detectors. As noted in the main text, the
peaks tail towards the high-elution-volume side of the chromatogram. Figure S1-B shows the
same datasets in a concentration vs molecular weight representation. Only for the largest
polymer does the shape match expectation. The loops and arcs seen for the smaller two polymers
indicate that the same molecular weight was measured more than once while collecting the
elugram. Such pathological traces can occur when separation is not based entirely on size
exclusion but instead relies partly on interactions with the stationary phase. Light scattering
would still report the molecular weight correctly, as long as the composition (and with it, dn/dc)
has not changed during elution. Branching or other architectural defects, such as cyclization, that
may cause unusual c vs M profiles are not expected from the synthetic protocol used here.
Another possible explanation for seemingly poor resolution is column overload; the low
molecular weights of these two polymers necessitated more polymer. As an exercise, the
concentrations within a given molecular weight range were summed into bins to give a more
meaningful representation of the concentration vs molecular weight, Figure S1-C. These results
demonstrate that simple and common representations such as Figure S1-A can be misleading.
2
B)
3.0
1.0
2.5
-1
1.2
concentration/10 g·mL
0.8
100:1
2.0
300:1
-4
ILS( =90) /a.u.
A)
0.6
100:1
200:1
0.4
300:1
0.2
0.0
6
8
10
1.5
200:1
1.0
0.5
0.0
12
0
Elution Volume/mL
1
2
3
4
5
5
Mw/10 g·mol
Figure S1
6
7
8
-1
C)
A) Same as Figure 4 in main document. Light
140
traces are of LNaPSS in buffer (200 mM
120
g·mL
-1
scattering signal from GPC chromatograph. The
100
concentration/10
-4
NaNO3, 10 mM H2PO4, 2 mM NaN3 at pH 7.5)
with monomer:initiator ratio of 100:1 [M]:[I]
(blue line), 200 [M]:[I] (red line), and 300
[M]:[I] (black line).
100:1
80
60
40
20
B) Concentration vs molecular weight
0
0
representation.
C) Concentrations collected into bins for 100
[M]:[I] sample.
3
5
-1
Mw/10 g·mol
1
Fluorescence Photobleaching Recovery of LNaPSS
For a more through description of fluorescence photobleaching recovery (also known as
fluorescence recovery after photobleaching) see other literature.2-5 In the modulation detection
FPR scheme used here, a fluorescent sample is placed in an epifluorescence microscope and
illuminated by a weak laser beam. A Ronchi ruling in a rear image plane of the microscope casts
a striped pattern on the sample and a DC intensity signal is measured. Briefly, an intense laser
illuminates the sample, destroying the fluorescent molecules not protected by the dark stripes of
the Ronchi ruling. An AC signal is created by a tuned amplifier/peak voltage detection circuit
when the striped pattern goes into reciprocal motion, causing the illumination pattern to go into
and out of phase with the pattern bleached into the sample. The destroyed fluorophores produce
no signal but the persevering fluorescent molecules do. Over time, the AC signal decreases due
to the diffusion of the fluorescent molecules, decreasing the sharpness of the bleached line
produced and the signal loss is fit to Equation 1
𝐴𝐶 𝑠𝑖𝑔𝑛𝑎𝑙 = 𝐴1 𝑒 (−𝐷𝐾
2 𝑡)
Equation 1
where A1 is the amplitude, t is time, D is the diffusion coefficient, 𝐾 = 2𝜋⁄𝐿 and 𝐿 = spatial
period of the Ronchi ruling. The fit provides a tracer self-diffusion coefficient, different than the
mutual diffusion coefficient sensed in DLS; otherwise, the fitting regimen will be very familiar
to a DLS practitioner.
4
0.8
10
0.25
0.20
8
0.15
-2
/10 Hz
0.4
0.10
6
0.05
4
0.00
0.0
0.1
2
0.2
5
-2
0.3
0.4
DC / volt
Peak AC / volt
0.6
K /10 cm
0.2
2
0.0
-10
0
10
20
30
0
40
t/s
Figure S2. Recovery for 4% wt. FNaPSS (Mw= 88,500 and PDI = 1.3) in 0.15 M
NaCl. The red line is a single exponential fit to the data and the blue line is the
measured intensity. The inset shows a linear dependence on squared spatial
frequency.
Figure S2 shows a typical decay for ATRP-produced LNaPSS with ample AC signal at
4% w/w of LNaPSS in water. The blue line in Figure S2 shows the measured intensity of
fluorescent signal. The drop to zero volts at time zero reflects closure of a shutter to protect the
photomultiplier tube during the bright bleaching pulse. A period of almost 10 seconds was
required before stable DC signal was again observed after re-opening the shutter, and the postbleach DC signal is just a few percent lower than the initial. Such shallow photobleaching is
desirable in FPR measurements because it reduces heating and the production of potentially
damaging byproducts of the photobleaching reaction. Ignoring AC signal points during this
settling time, a single exponential fits the AC data well, providing the decay rate.
5
Additional Images
6
A
B
Figure S3. Images of LNaPSS (5 μL drop of 5 g/L LNaPSS, 0.1 M NaCl,
dried in 42 % relative humidity). A: Differential interference contrast
image overlaid with a fluorescence pseudo-colored image using nearest
neighbors constrained iterative deconvolution. B: Pseudo-colored laser
confocal compilation image of 16 images of 2 μm difference in the zplane. The image was deconvolved using a nearest neighbors algorithm.1
7
Movies
Movie 1
This movie is a video of a differential interference contrast image overlaid with a fluorescence
image using nearest neighbors constrained iterative deconvolution and false color added.
movie 3d volume_4.wmv
8
Movie 2
This movie is the same as Movie 1 but it is a closer look at the salt crystal.
movie 3d volume_2.wmv
9
References
1.
Monck, J. R. O., A. F.; Keating, T.J.; Fernandez, J.M. The Journal of Cell Biology 1992,
116, 745-759.
2.
Huberty, W. Synthesis of Flourescent Poly(styrene sulfonate). Louisiana State
University, 2012.
3.
Russo, P. Q., J.; Edwin, N.; Choi, Y.W.; Doucet, G. J.; Sohn, D., Fluorescence
Photobleaching Recovery. In Soft Matter: Scattering Imaging and Manipulation,
Pecora, R. and Borsali, R., eds. Springer, New York, 2008.
4.
Lanni, F.; Ware, B. R. Rev. Sci. Instrum. 1982, 53, (6), 905-908.
5.
Zero, K.; Ware, B. R. Journal of Chemical Physics 1984, 80, (4), 1610-1616.
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