[loui223]

Hi,

I will soon try to quantify proline using HPLC and OPA-derivatization. To be able to do this proline has to be pretreated.

This is my protocol:
1. Add 200uL of 250uM proline standard (in 1M NaOH) to 200 uL of 13.3 mM chloramine-T preheated at 60°C for 2 min in a water bath.

2. Incubate 1 min at 60 degrees celcius and add 200 uL of 333 mM NaBH4, incubate at 60°C for 10 min.

So the thing is that i have gotten a bit nervous about this after reading the MSDS of NaBH4. First my plan was to dissolve my proline samples or standard in H2O, but this does not seem to be a good idea. I have looked all over the internet to find information about this and my main question is, if it is safe to weigh NaBH4 (from a 50g container) and dissolve it in the normal lab environment or should i all of this be under inert gas?
And what would in my case be the best thing to dissolve NaBH4 in ? methanol?

For the derivatisation i will be using OPA dissolved in, methanol, mercaptomethanol, sodium borate, and the mobile phase consists of Methanol, 0.1 M sodium acetate and NaN3 ph 7.2

Your help is highly appreciated

[Yakimikku]

Hello,

Is this a known procedure that you got somewhere? If so, what solvent did they use? In any case, the procedure does seem strange as NaBH₄ will no longer be NaBH₄ if it touches water (do not even try). Just think about what the products are for NaBH₄ + H₂O. I doubt any protic solvent would be okay (ie, MeOH, etc.). You'd likely need non-protic, anhydrous solvent which also may create issues for proline solubility.

Don't worry about handling NaBH₄ solid in air. What you need to be careful about it what solution you add it to. Also, 200 uL of 333 mM NaBH₄ is not a lot. Large quantities may require extra precautions however (again, think of the products that might form/heat that might generate). Be sure to use dry solvent. Also, NaN₃ is potentially explosive IMO, but I wouldn't worry about small quantities.

Good luck and be safe!

[Dan]

Sodium borohydride is not nearly as reactive as the MSDS makes it out to be. Be careful, but do not fear it. Wear gloves, specs and a coat.

Sodium borohydride reductions are routinely run in methanol, and can also be run in water. I have carried out sodium borohydride reductions in water and/or alcoholic solvents routinely. There is some generation of hydrogen gas (flammable) - the reaction, and solution preparation, should be carried out in a fume hood.

Despite the fact that sodium borohydride does react with protic solvents, it is generally accepted that the intermediates of these reactions are more active reducing agents than borohydride its self. Generally speaking, you tend to get about 2 hydrides for every equivalent of borohydride you dissolve in a protic solvent. I have reduced aldehydes to alcohols with 0.5 eq NaBH₄ in methanol (or water) many times in 90+ yield.

In your case, your reaction is aqueous so I'd be incluned to use water as the solvent for making your borohydride solution. Add the solid borohydride slowly to cold water - there will be a bit of fizzing and an exotherm.