EDTA precipitates out of EDTA-oxalate buffer - (Apr/02/2013 )
Dear all,
I'm trying to make buffers for redissolving iron-virus flocculate.
The recipes are taken up from the paper: John et al. (2011): A simple and efficient method for concentration of ocean viruses by chemical flocculation, Environmental Microbiology reports,3, 195-202.
Ascorbate and oxalate are in the buffers to dissolute Fe-virus, and EDTA chelates iron ions to prevent re –precipitation if iron-virus complex.
Ascorbate -EDTA buffer (0.25M ascorbate, 0.25M Mg2EDTA, 0.25M Tris, pH 6,7 or 8)
10mL 2M Mg2EDTA
10mL 2.5M Tris HCl
25mL 1M ascorbic acid
Mix components and adjust to pH 6 with 10M NaOH. A precipitate may form before the solution pH is adjusted.
Oxalate-EDTA buffer (0.25M oxalate, 0.25M Mg2EDTA, 0.25M Tris, pH 6,7 or 8)
10mL 2M Mg2EDTA
10mL 2.5M Tris HCl
25mL 1M oxalic acid
Mix components and adjust to pH 6 with 10M NaOH. A precipitate may form before the solution pH is adjusted to pH 6-8.
Problem is that we couldn't buy Mg2EDTA in our country, so we bought Na2MgEDTA. I added MgCl2 x 6H20 to have the same molarity of Mg2+ ions as in original recipe (the buffer has to contain excess of Mg2+ ions, so EDTA can chelate Mg2+, and not viruses). I thought that excess of natrium ions (from EDTA salt), and chloride ions (from magnesium chloride hexahydrate) won't interfere with buffer contents because we already have chloride ions from Tris HCl, and we add natrium ions when we adjust pH.
I can post the modified recipes if there is any need (I checked the calculus many time, and I made new stocks few times to ensure that I didn’t make mistake while making stocks)
But EDTA precipites out when I add oxalate or ascorbate, and it does not dissolve when I adjust pH. In the paper, they used buffers in pH range 6-8, but EDTA didn’t dissolve in that pH range to me.
I also tried to make more diluted buffers in hope that it will help, but it didn’t.
Can you please tell me what the problem is? Is it because I’m using a different EDTA salt?
Thank you very much in advance.
Sandra
EDTA is a metal ion chelator, so it won't touch the Cl- ions. However, I suspect that the concentration of the metal ions is not what is causing the precipitation, rather it is likely to be the pH of the water, try adding the components in a different order to what you have listed.
For how long did you stir it? Can you post the modified recipe? Maybe is not an issue but you will increase the Cl quite a lot. Do you use Trizma base + HCl or Tris-HCl try to calculate the correct Cl molarity. That may help a little with the increased salinity.
Do you add the components in the order they appear? Try adding TrisHCl + oxalic first, they will have more effect in the pH than the other components, check the pH and maybe you will need to raise it a little bit before adding the remaining components.
EDTA is essentially insoluble at pH less than about 7.5, and is insoluble above 500 mM even at pH 8.0. I think you will find it very difficult or impossible to make a 2.5 M solution of MgEDTA, especially since the Magnesium hydroxide is also essentially insoluble. I suspect that you need to make this up at near final dilution, and make it with magnesium hydroxide rather than magnesium chloride. The Tris sounds as if it was calculated to neutralize the acid, but it won't do a good job of this. I would mix EDTA and Mg(OH)2 and acid at near final dilution, then neutralize the acid with NaOH. I would not expect anything to go into solution until the pH was above 7. Heating with a stir bar, and patience will also probably be required.
Hi,
Thank you all for the suggestions. I will try them all this week and inform you about results!
I noticed I made a typo – final EDTA concentration is 0.2M.
Here’ my modified recipes:
Ascorbate -EDTA buffer (0.25M ascorbate, 0.2M Mg2EDTA, 0.25M Tris, pH 6,7 or 8)
20mL 1M Mg2EDTA (I couldn’t make 2M solution, so I made 1M and double the volume added)
4,0662g MgCl2x6H20, dissolved in minimum water
Mix components and stir.
Add 10mL 2.5M Tris HCl
Stir.
Add 25mL 1M ascorbic acid
Stir.
Adjust pH with 10MNaOH.
Oxalate-EDTA buffer (0.25M oxalate, 0.2M Mg2EDTA, 0.25M Tris, pH 6,7 or 8)
20mL 1M Mg2EDTA (I couldn’t make 2M solution, so I made 1M and double the volume added)
4,0662g MgCl2x6H20, dissolved in minimum water
Mix components and stir.
Add 10mL 2.5M Tris HCl
Stir
25mL 1M oxalic acid
Stir.
Adjust pH with 10MNaOH.
I used Trizma base +HCl. I left "the buffer" stirring overnight once in hope it will dissolve, but it didn't.
First I will calculate Cl molarity and try making the buffer by adding the components in different order. We have Tris-HCl also in the lab; I can try to make the buffer with it if you think it will make a difference.
Phage434,
You’re right, I couldn’t make 2M solution of EDTA (I used 1M). I regularly use stir bar, and I tried heating too, but it didn’t help.
I think that substitution of magnesium chloride with magnesium hydroxide is a very good idea!
But I’m not sure if I understood you well: you propose that I don’t use Tris at all?
Can you please send me a link with data about EDTA solubility, for the future?
How do you mean “would not expect anything to go into solution until the pH was above 7“? You think it’s impossible? How did the authors of the paper succeed to dissolve components at pH6 then?
Thank you all once again!
Please try to make the buffer with this order below, I followed typical Oxalate-EDTA buffer protocol except replacing Mg2EDTA to Na2EDTA dihydrate and MgCl2 hexahydrate.
Add autoclaved-distilled water 30 ml.
1. Tris base 0.755g (0.125M)
2. Na2EDTA dihydrate 1.86g (0.1M)
3. MgCl2 hexahydrate 2.035g (0.2M)
Until here, the pH should be 4.2-4.3
4. add 4 ml of 10N NaOH
then the pH rises to 11.8~ , and the solution turns to be opaque.
5. add oxalic acid 1.76g (0.2M)
The solution becomes clear and pH decreases to 6.0~6.1. Finally add ADW up to 50 ml.
Caution:
1. Completely dissolve each component and go to the next step.
2. You may not adjust pH using HCl or NaOH after adding oxalic acid. (I tried it but again EDTA precipitated out...)
I do not know well about chemistry, but tried it many times with different orders and finally succeeded making it as following this order.
Please don't ask me for details... ;) Good luck!
Somebody please help regarding the preparation of the oxalate EDTA buffer,,Thank you Cetoine for the modified recipe you shared. I tried on with Ascorbic acid as oxalic acid did not worked for me.
Actually what is the colour of the buffer and what might be the colour when it is added to the filter paper..Please help me..
make sure the autoclaved dw is fresh or it will be more acidic and may cause the pH to be too low to maintain solubility.
you can adjust the pH of an edta solution to a little below 7 after it is completely solubilized at higher pH. be careful not to "shock" the solution while reducing the pH (ie add the oxalic acid slowly) or the edta will crash out of solution.
When done try adjusting pH with .02N Sulfuric