pH Responsive Energy Transfer Systems

In order to gather more data before the conference I spent most of my finals weeks running acid trials.  I ran into some problems using a 50:50 (volume) mixture of DMF to Water.  After a dilution the absorbance would go blank.

Some of the professors I discussed my work with in Florida suggested an alcohol based solvent system.  So my first week back I decided to test some different systems.

However, methanol and ethanol, in 1:2, 1:3 and 1:4 ratios with water, both showed remarkably slow opening, none were faster than 7 times as slow.  Some systems even displayed kinetics that were not first order, and are not something I want to attempt explain.  Instead I will continue using my original solvent system.

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Christina and I had a great time at the conference.  We got to attend a variety of talks. They make up the bulk of the conference.  I really was interested in two particular talks. Kevin Belfield explained how he is using photochemistry to develop 3-Dimensional optical data storage; it was fascinating.  And Rober Liu talked about his observations on photoisomerization when larger molecules (like enzymes) are involved or around.  He named a certain molecular movement the hula twist, and he did a great job describing it so that even some of us first-timers could see what was going on.

Sunday, we had a poster session where people walked around and we go to talk about our research.  Everyone had some great suggestions for my research (and on my Graduate school choices, ha).  Many suggested that I switch to an alcohol based solvent system.  It was nice to be able to talk about my research and get feedback from other researchers with new eyes to my project.

Here are pictures of me with Christina and me with Dr. Harbron in front of our little corner of poster at the conference!

We had such a blast!

Harbron is such an amazing advisor and mentor.

There was even a little of time left to walk and hang out on the beach, but not much! (Which was probably alright considering the chilly temperatures…)

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Tomorrow I leave for the Inter-American Photochemical Society winter conference in St Pete’s Beach (outside of Tampa).  Professor Harbron and my lab mate, Christina, are also attending the conference.  Christina and I are presenting posters on our current research.  I will be talking all about my dyes, my successes and my struggles with a wide variety of other photochemists.  This is my first conference and I am actually at a loss for what to expect overall, but I know it will be a wonderful experience.  I am really excited to talk about my research, meet chemists from other schools with various research interests, and  see other current research being done around the world.

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Dry solvents and glassware have worked!  I must be more patient with my new process because of the boiling off of the solvent: I have to watch for bumping and am constantly regulating the vacuum… there is a reason they invented the rotovap, or a few.  My major roadblock was the solid mass of lipstick consistancy (and color, in my case) after the solvent was removed.  I first tried to just add the next solvent and crossed my fingers that the stir bar would break it up…whoops.  I then turned to sonicating and vigorous shaking of my own.  This improved my reaction, but is still not perfect.

The new step I added was a column to purify the product.  Because this was a new, messy step that involved a lot of preparation and extra knowledge I had avoided it.  I saw the synthetic chemists around me struggling with their columns and choosing eluents and all sorts of problems. I was even encouraged by Prof. Harbron to attempt recrystallizing instead of following some of the papers because it had worked for earlier R6G derivatives.

But with the product and reactant having very different properties (which encouraged the first separatory funnel followed by recrystallization method…) a column was a good idea.  In EtOAc, R6G barely moves down the column and never makes it to the end of the column (at least not in the short time I run the column).  I’m really happy we switched to the column.  I have learned a lot about thin-layer chromatography and columing in general.  Plus I retrieved my product without hassel.  I wish I had given in to the “new” technique weeks ago!

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My acyl chloride reactions have had no success, I am starting to wonder if I am even making the acyl chloride.  This is a long 2 to 3 day reaction and it has been frustrating.  But now I am changing my game plan, mixing up my lineup, and using far more sources to lead me in the right direction.  And considering the one paper I was following as if it were canonical used the “solvent” 1,2-Dichlormethane, I’m starting to wonder about that paper.  [In case you are unaware of the humor, the numbers in front refer to which element (usually carbon) the chlorines are connected to, a methane molecule only has 1 carbon, so there is no #2 carbon >.<]  At first I just assumed it was dichloroethane, but my guessing days are over.  I am now switching solvents in my amination reaction, to acetonitrile. 

Another problem that potentially could have negatively affected my reaction is water.  Water will react with my acyl chloride more readily than the amine, thereby reforming the acid rather than moving forward to the amide.  Although I knew this, I was not taking the proper steps to avoid such a reaction.  Now I will dry my solvents with calcium hydride and distillation, and I am going to thoroughly dry all my glassware.  I will also switch the rotovaping, which creates an excessively humid environment, to an oil bath and vacuum distillation set-up.  These steps may be the required improvements that will lead my reaction to the desired product.  Cross your fingers for me.

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For the past two weeks I have been struggling with synthesis. It really isn’t my gig, but it is exciting to have made something and be able to analyze it.

Here is a table of the various substituents we have tried and their accompanying pKas.  It looks as if the success of the reaction (displacement of the OEt by an amide) has something to do with the pKa of the amine.  The reactions involving amines with a low pKa did not go to completion.  I always had a very pink product, usually of a sludgy consistency.  However, the reactions with higher pKa amines were easier. The prime window appears to be between 8 and 9.5; even higher pKa amines were harder to retrieve but did actually react.  However, there is not a clear line as we expected, or phenethylamine would have worked.  I am disappointed in the 33% success rate I have had.  So, to open the doors of opportunity, and to understand more fully the relationship between the pKa of the R6G derivative and the substituent, I am now trying to go through the route of the acyl chloride of R6G.  This will give me a larger group of amines to use, as it will not be competing with the –OEt for leaving group power.  However, this derivative synthesis route involves two more steps.  The first is a 2 hour reflux of R6G with NaOH to hydrolyze the ester to the carboxylic acid.  The second step is a 3 hour reflux with POCl₃, which creates the acyl chloride.  Then I add my amine and just stir for at least 6 hours, I usually leave it overnight. 
My first attempts at this were quite unsuccessful, blast. I believe it was because I added triethylamine, which the original literature called for when making the –OH derivative. But perhaps that is not required when the added amine does not have a chlorine atom needing to be sopped up, which the hydroxyl amine does have and others do not.

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I have decided to start on this second road.  Harbron synthesized the two R6G derivatives I have already used and experimented with, R6G-Hydrazone and R6G-Octyl, both of which are spyroamides.   The blue triangles represent where my data points are when I did an acid trial of R6G-Hydrazone, and the red circles are the data points from my R6G-Oct acid trial.  We had hoped they would not be the same, and they are not! This means that each different compound starts turning pink at a different pH.

So what makes one dye’s pKa more acidic than another’s? I have no idea!! It could be electronics or protonation sites or a myriad of things.  However, it does not look like the latter because the protonated species (Hydrazone) has a more basic pKa than the non-protonated one.    But it is too early to really know.  So these past few days I have been synthesizing and purifying a new derivative: R6G-Ethyl Amine or R6G-EtA.  Its pH trial is shown in green.  It has quite a different pKa and even a different shape than the earlier two.  I am so excited about this project.  I really loved taking the project from start to finish, from the bottle to the fluorimeter, etc.  I did have several failures this week, including attempts to react R6G with Hydroxylamine and with p-Nitrophenylaniline.  Both of which were solids rather than the liquid form of the successful amines. Now I need to pick some more amines, maybe a liquid aniline?

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This past week I have determined that extremely low pH, about 3.3, irreversibly alters my nanoparticles.  When the PFPV nanos, which are in a solution of water, are treated with acid, fluorescence resonance energy transfer (FRET) does not occur, and the absorbance shifts, as is depicted by the two following graphs.  Here in this first one, the red line shows R6G doped PFPV nanoparticle fluorescence before it is treated with any acid.  Amazingly, the R6G peak at 560 nm is more excited by the polymer fluorescence than when I try to excite it directly at 530 nm (not shown).   One explanation of this could include that the polymer PFPV protects or shields the dyes from direct excitation but the close contact of the nanoparticle offers plenty of opportunity for FRET.  The Blue line is the same nanoparticle solution with some hydrochloric acid added.  At a pH of 2.34, there is no FRET.  Dye can still be excited directly but the polymer is not exciting it for some reason.

This reason may have to do with this next graph.  Below one sees three lines; these are the normalized (highest point set to 1) absorbance spectra of PFPV control (no dye) samples.  The green line is the absorbance of the film.  And the red line is the absorbance of the nanoparticles before they are treated with acid.  The blue line is obviously neither. It is actually the same nanoparticle as the red line, but in an acidic environment.  See how film-like the spectrum is?  This shows a significant change in either conformation or chemistry of the particles.

Both my research adviser and I are puzzled by this change.  There are two routes to take now: 1) to perform more nanoparticle experiments to see exactly where the change becomes irreversible, to see if base has the same effect, etc, or, 2) to change the pKas of our dye to reduce the need for extreme pH.

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I am working with a new dye, Rhodamine-6G (R6G). The R6G we bought is functionalized with an ethyl ester.  This is easily converted to an amide of various forms.  One thing I am currently investigating is the unique characteristics of the rhodamines with various substituents.  Do the Octyl and Hydrazone groups off the amide alter the pKa of the molecule or the lambda max of fluorescence?  I am hoping that they are different pKas because this would give opportunity for various sensors. If the pKas are different, then alternate substituents could provide a more basic pKa, for instance in the biologically relevant range of pH 4-5. So this week has been spent formulating acid curves, pH vs fluorescent intensity.  This is a tedious procedure consisting of taking absorbance on the UV-Vis spectrometer and fluorescence on the fluorimeter, removing the liquid from the cuvette, adding a known amount of acid via a pipetteman, taking the pH reading, reloading the cuvette for spectral analysis.  As I am the first in the lab to be working with pH there is quite a learning curve as to settings, acids, tools, and handling.  I’m very excited though, to have the chance to pioneer such a course for our lab.

Along with perfecting my knowledge of the dye, I am also attempting to create a new system that will hopefully end in color change with pH change.  When the fluorescence of a polymer overlaps with the absorbance of a dye, which is responsive to an external stimulus, color change is possible with the variation of that stimulus.  I have tested the interactions between both R6G with a polyfluorene (PF) polymer and also with another polymer we call PPE.  The overlap in spectra was very slight, and I was unable to observe any energy transfer. We ordered a new copolymer to create a system with the rhodamine.  This new copolymer that we call PFPV has a redder fluorescence than the PF or PPE, however not as red as PPV which has been used in the lab most recently.

There are several avenues to test the interactions between dye and polymer, including films, solutions and nanoparticles.  I am very comfortable with films because of my extensive use of the microscope.  That is where I started my research in Professor Harbron’s lab.  However, altering the environment with acid proved difficult.  Even using the constantly “open” or conjugated form R6Gthe studies were challenging because the fluorimeter has not been properly aligned for films, this made it difficult to get decent and consistent spectra.  To address the environment change problem, I switched to solutions for my studies.  With such a set up pH can be quickly altered and measured.  All of the polymers I am working with are very hydrophobic, so studying the polymers in solution with the dye is quite impractical because of the aqueous acid being added.  Therefore, nanoparticles are the perfect way in which to study them.  This is my next step!

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