Category Archives: Water Analysis

The Biophysical Effects of Heavy Water – My Defense Presentation

Defense Outline

Just over a week away now…

  1. Introduction
    1. What is D2O?
    2. The history of D2O
      1. Gilbert Lewis:
        1. purification
        2. biological effects
        3. The hypothesis
      2. Joseph Katz
        1. various experiments
    3. Uses of D2O
      1. NMR, mass spec
      2. The need for a D2O adapted organism
    4. Experiments in DDW
      1. use for space travel
      2. cure for cancer?
  2. The effects on life
    1. Tobacco Seeds
      1. The Crumley experiment and repeating the experiment
      2. Tobacco seed germination rate
      3. tobacco seed growth rate in low deuterium concentration
    2. Arabidopsis
      1. arabidopsis growth rate
      2. arabidopsis morphology
    3. E. coli
      1. growth rates
      2. adaptation and adapted growth
      3. morphology
    4. Yeast
      1. growth rates
      2. adaptation – can’t adapt
      3. morphology
        1. stall during cell division
        2. microtubule stabilization in D2O
  3. Molecular effects
    1. Stabilization of biomacromolecules
      1. DLS experiments
        1. Catalase
        2. Ovalbumin
      2. YPD longevity
    2. Investigation of HD exchange
      1. mechanism and exploitation for protein struture studies
      2. FT-IR analysis
      3. Cavity ring-down analysis
        1. low cost measurement of local atmosphere isotopic composition
    3. Effect on DNA
      1. The pursuit of shotgun DNA mapping
      2. optical tweezers
      3. methods
      4. overstretching data
  4. Future Work
    1. Arabidopsis
      1. adaptation
      2. seed growth in low deuterium
    2. Tobacco growth in low D2O
    3. Yeast morphology in taxol
    4. E coli protein expression in D2O and protein structure analysis
    5. DNA
      1. overstretching in D2O with intercalators

Well there is my idea of how to present my dissertation. I’m not sure if/where I should put my discussion on open notebook science. Also there are a couple things that I could see going elsewhere. I could describe the yeast and e. coli stuff in parallel instead of one after another. Also the HD exchange stuff could easily go right after the yeast, e. coli, or even the tobacco seed stuff. What to do…

Otherwise I think the story is pretty compelling: history of D2O and the unanswered question by Lewis. Investigations into D2O effects and trying to understand low D2O concentration effects, effects on macromolecules, and the understanding of large volume/long-term HD exchange.

Any feedback you may have would be GREATLY appreciated. I’ll send you a figshare t-shirt, or if you are XL, I’ll send you a hoodie (but I only have one).

Preliminary Results of YPD deterioration

Absorbance of DI YPD (pink), D2O YPD (green), and blank (red)
Absorbance of DI YPD (pink), D2O YPD (green), and blank (red)

These are the results of the experiment I stated a couple weeks ago. I have been tracking the deterioration (previously called aggregation, but I’m not entirely sure aggregation is the correct terminology) of YPD in both solvents. Today they looked pretty well degraded so I thought I’d share the results. Between the two, the DI YPD is more absorbent than the D2O YPD at nearly every wavelength measure (major uncertainty below 350nm).
I’m associating degradation with absorbance since the blank (which is also DI YPD) has an absorbance of zero at all the same frequencies.

D2O YPD also records 0 for absorbance at 600nm, which is the wavelength used for cell count studies, so there would be no interference from the solution. Whether or not the media is still usable by cells is undetermined.

I’m beginning a second experiment that would track the absorbance every few days via the same mechanism. If you recall, I began this experiment taking pictures and eventually moved toward using the nanodrop. This probe seems to do a good job so its continued use is reasonable.

Man I’ve been writing my dissertation for too long…

D-exchange over time

This is a follow up from the experiment on Thursday with results posted on Friday. At my request, Scott measured the exposed DDW sample over time on Friday and Saturday (I believe). He also made a concoction by mixing the DDW I gave him with some other water he labels as “1.28 at. % water.” I’m not sure of the ratios of DDW to mystery water but I’ll find out.

Either way, here are some preliminary results. Plotted above is the ratio of Deuterium to Oxygen-18 for each water sample. The pure DDW sample is to the left. As you can see, the amount of D and O-18 increases each day and is approaching natural water readings. I’d say that is to be expected. The mystery water that Scott made seems to have D and O-18 added to the water and those amounts are dropping back to the normal water range. If I’m understanding this correctly, I’d say that is to be expected as well. DI water is being measured as well and that sits right in the natural water range.

Some notes:

  • I’d say it would be pretty interesting to watch the D/O-18 ratio change daily for an extended period of time. I’d assume the fluctuations may be random daily.
  • It would be interesting to have some natural NM rain water and tap water compared with the DI water and watch those fluctuations.
  • I should find out what volume of water we’re dealing with. With regards to the Crumley experiments, I used 6mL of water. And for the yeast and e.coli experiments I ¬†used 10mL of water. I think we are dealing with less in these experiments, so D-exchange might not be as prevalent as I assumed. The microbe experiments last a day, and the Crumley experiments were for two weeks, but were as isolated from the environment as possible.
  • We should track the evaporation rate.

There is a lot to consider in these experiments and right now, my brain is swirling with ideas and results. A planning meeting seems to be in order.

Deuterium exchange analysis experimental planning

The future direction of the experiment using the ring down cavity spectroscopy device will be planned here. This should be a fun collaboration between Scott Jasechko (Earth and Planetary Sciences) and myself (Physics). Join the conversation!


An alternative analysis to FT-IR to study deuterium exchange

Via figshare:

Deuterium Content of Deuterium Depleted Water: 1st Trial. Anthony Salvagno, Scott Jasechko. figshare.
Retrieved 21:24, Jul 20, 2012 (GMT)

A few days ago I made a new friend named Scott Jasechko. We met to discuss the possibility of creating a TED branded forum to UNM. We got to talking about our research and it turned out that our research interests are very much aligned and he studies water isotope amounts with relation to natural water around the planet. He’s about to publish his findings and once that goes through I’ll link to that.

It turned out that his lab has a device that can very accurately measure small concentrations of deuterium and oxygen-18 in water samples. I told him about my DDW experiments and how deuterium exchange may affect my experiments, but that I can’t quantitatively measure it’s affect or the process in general. So we got to talking and he wanted to help me out.

The data linked above is the results of the mini-collaboration that I predict will turn into more. Scott used his Picarro cavity ring down spectroscope, which means very little to me right now, to analyze the water samples I gave him. The water was used in these two experiments (each word is a separate link). And was then stored in our desiccator (with drierite to reduce moisture exchange) until yesterday (July 19, 2012).

Surprisingly, the data shows very little change from what Sigma claims (less than 1 part per million D to H) to yesterday, showing very little exchange. There are two things to consider here: (1) the machine wasn’t calibrated for such low levels of D, which skew the readings (since they actually give us negative ratios), and (2) volume may play a part in exchange.

I don’t understand the mechanism very well but I suspect that surface interactions play the largest role in deuterium exchange. Once deuterium is introduced into the sample, then I would guess diffusion takes over, but this is probably slower in nature than evaporation/other mechanisms that are involved on the surface. Basically this is a thermodynamics problem that I would need to spend 3 months thinking about to compare with experimental analysis (since that is how long the Thermo class is, 1 semester).

The follow up to this experiment should be better organized. Obviously we’ll need to redo this experiment. Scott and I are also performing another experiment where Scott has left the lid off the samples I gave him so he can see if there is a new value the spectroscope provides. From here we may want to do some longer time analysis and some other studies that we’ll have to plan. I’ll start a new thread for that, in the mean-time I hope he’ll introduce himself in the comments of this post.

Some other notes:

  • I geeked out when I realized the power of this sort of collaboration. If I hadn’t been affiliated with TEDxABQ and thus this new idea of TEDxUNM (not officially licensed), I would have never met Scott. These are the sorts of collaborations that I hope can be introduced because of the Open Research IGERT proposal.
  • I mega-geeked out when I realized I can do a project planning thread with someone here at the university in another lab that can also participate in the project! Open science at the core all the way.
  • I super-mega-geeked out when I realized that I implemented a crucial aspect of ONS. As I wrote this post I wondered what experiments I had used the water for. Then I realized that it’s all documented and that I can show everyone what those experiments are. It’s not enough that I have it recorded by date, but also that you all saw those experiments in real-time and their use in future experiments has been realized. That’s a major win for ONS.

Project planning notes via Handrite

Here are some notes I took using handrite for android regarding my project plans. I don’t mean to advertise for anything, but I use this app ALL THE TIME and it is super helpful. Typing on my phone is annoying sometimes and Handrite allows me to use my finger to write (albeit sloppily). These notes were taken yesterday while I was meeting with Steve (on my phone) and now I have time to paste them in my notebook. So here is some background on my ramblings:

  • I would like to effectively analyze the amount of hydrogen-deuterium exchange in my samples. This is a phenomenon I’ve talked about a lot, know very little about, and stress a lot over. We tried FT-IR of water samples to see if we can determine differences in the water types. We’ve had mixed results in this regard. It boils down to the fact, that I don’t trust anything. How do I know that deuterium isn’t sneaking into my unopened bottles of DDW, or that hydrogen isn’t getting into my D2O? What about after it’s been opened? What about in my samples? If I’m going to get reliable results with yeast and e. coli I’ll need to know if I can trust commercial products.
  • NMR (nuclear magnetic resonance) seems to be a promising route to try and there may be some equipment either in the Chemistry department or Biomedical Engineering here at UNM that I’ll need to look into.
  • Mass spectrometry may be another avenue to determine precise amounts of deuterium in water.
  • Something not written but that I talked with Steve about yesterday and just remembered is that I need to talk with Sigma to find out how they measure the purity of the DDW and how reliable those measurements are long term. If something sits on a shelf for a year, how do I know it isn’t really just pure natural water?
  • I also have some stuff in my notes about the Repeating Crumley experiment. I just setup a D2O only experiment to show definitively that tobacco seeds do not grow in D2O. Previously I would only track this data for 10-15 days and Crumley went up to a month (if I remember correctly). I’ll write the setup in a minute, but I want to do 30 days so I have some pictures for my open access, self published RC paper (spoiler alert!).
  • Also to show that I’m no slouch, I want to try and repeat Crumley’s original experiment with paper towels to show that the growth of the 100% D2O seeds was due to H-D exchange and other environmental factors and not because they eventually grow in D2O. Yea I’m a badass.
  • The rest of the notes are some ramblings that are not really private but thoughts I would prefer not to elaborate on now.

Tata for now!