Field of Science

Making the hemin stock/solving a chemical puzzle

Yesterday I tried, and failed, to make a new lab stock of the hemin that Haemophilus influenzae needs as an iron source. 

Hemin comes as a fine black powder; it's not really soluble in aqueous solutions or even miscible in water.   For our culture media it's prepared as a sterile suspension by mixing 100 mg of hemin with 100 ml of a solution of 4% triethanolamine (a surfactant), and incubating this at 65°C in a waterbath for 30 min.  The hemin forms a black suspension that's, rather surprisingly, both sterile and chemically stable.  Sterility is surprising because 30 min at 65°C isn't expected to kill spores, and stability is surprising because, once the hemin is added to culture media it goes off within 48 hr.


Anyway, we're down to our last 100 ml bottle of hemin stock, and it's my turn to make up lab stock solutions, so yesterday I tried to make more.  We've always used triethanolamine that came as a viscous liquid, but recently we accidentally purchased triethanolamine hydrochloride, which comes as a white powder.  So I made up a 4% solution (nicely soluble) and mixed it with the hemin.  NO SUSPENSION, even after heating.  No different than hemin in pure water.

What to do?  Was the problem the triethanolamine or the hemin, which was also a new bottle (well, a bottle of uncertain age that had never been opened).  The new and old hemins were both from Sigma and had identical labels (the old one in a brown glass bottle, the new one in a white plastic bottle), so that's probably not the problem.  Our ancient CRC handbook (the 'rubber bible') recently disappeared, so I looked triethanolamine up in the Merck Index.  Not much help, so I just left the problem for the next day, thinking I'd email the building to see if anyone had any liquid triethanolamine.

But this morning I've done some Google searching and discovered that liquid triethanolamine is mildly alkaline, but solutions of the hydrochloride are quite acidic.  Triethanolamine hydrochloride is used in various molecular biology and microscopy protocols, always with the pH adjusted to 8.0.  So this morning I'm going to try adjusting the pH of the 4% triethanolamine before I add it to the hemin.

Later:  And here's the result!  Raising the pH caused the hemin to form a nice evenly black suspension (bottle on left).  The bottle on right has its original low pH and all the hemin has settled to the bottom of the bottle.

8 comments:

  1. For Xylella fastidiosa, we dissolve Hemin Chloride in 0.05N NaOH. Now you've got me wondering what the difference is between using NaOH and using triethanolamine pH8.

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  2. Huh! How do you sterilize it? How do you store it? How long is it stable for?

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  3. We use DMSO (dimethylsulfoxide). Filter-sterilise through a 0.2um nylon membrane, store frozen at -20C in small or single-use aliquots.

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    Replies
    1. What is the maximum solubility in DMSO?

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  4. IANAChemist, but it seems pretty clear to me. Hemin contains two carboxylic acid groups. At low pH, these will be protonated, and thus uncharged. Large uncharged organic molecules aren't usually very water soluble.

    At high pH, those groups will deprotonate, giving the molecule a charge of -2. That will dramatically increase water solubility. That's consistent with Adam's report of simply using dilute NaOH.

    Triethanolamine is also basic, due to the tertiary amine, so adding TEA to the water should have the same effect. TEA-HCl, the conjugate acid, would not raise the pH, but adjusting the pH to 8.0 would do the trick.

    I'm not chemically savvy enough to comment on the possibility that TEA helps due to surfactant activity, but my money is on the simple pH effect. (I do seem to recall that TEA may have mild antimicrobial properties, though, so that might be a reason to use it over NaOH.)

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  5. Rosie, this is something I'll have to look into in more detail next time I use it. So far, I've just been following received protocols with respect to Hemin.

    One important issue is that we are just using it as a delivery vehicle for iron. The hemin itself is not necessary for Xylella (I had forgotten that it was "factor X" for Haemophilus). We always autoclave it once it is mixed with the main medium. Our stock solution is stable (for our purposes) for over a month at 4 degrees... but I've never looked into the details of its behavior.

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  6. how do you store the stock solution and for how long?

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    Replies
    1. In the fridge, in a glass bottle. It keeps for years.

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