16 replies to this topic

[jiajia1987]

Hello peeps,

I am using the disodium version of EDTA (that is the only one available in my lab right now) and the molecular weight is 372.24g/mol. I tried to make a 1M stock by dissolving 186.1g of EDTA in 500mL of water, but it REFUSES to dissolve. I went on to check that it is necessary to change the pH of the solution to 8.0 before the powder will go into the solution, but still, it DOESNT dissolve.

Next, I had a look at sambrook and russell's molecular cloning book appendix and it says that I should make 186.1g of EDTA in 800mL of water and adjust the pH to 8.0. In the book, it says that will give me 0.5M, but this isn't true! To get 0.5M, I have to dissolve 186.1g of EDTA in 1L of water, isn't it? I left my 186.1g of EDTA and 800mL of water to stir overnight with a magnetic stirrer and I am hoping to see it dissolved tomorrow but I dun have much hopes.  I can't afford to make another solution because the EDTA is running out. Any help? I should add 200mL water to increase the volume to 1L, right? That SHOULD give me 0.5M, right? I don't get why the book says it will give 0.5M

ANy help will be appreciated!!

Thanks in advance.

[jiajia1987]

Sorry to add on to this.

I realized from one website that NaOH PELLETS are used to adjust the pH to 8.0. The thing I did was to make 5M NaOH stock and added it to the solution to adjust the pH... does this make a difference? ALmost all the websites I went to says that I add NaOH to adjust the pH... I didnt know that it should be pellets...

[mad]

nope you dont really need it to be in pellet.

by the way you need to add really really a lot of NaOH. I too have just tried to make EDTA just last week but on a smaller scale of only 100ml (cause i only needed to make TBE with them). I was following the Sambrook's formula which stated for 1L i needed to add abount 20g NaOH pellet. So I thought..ok...so for 100ml I should only need to add 2g....big mistake.  

Not sure how many g i added but it was way above the calculated 2g. Just add them slowly and you should be fine.


MAD

[little mouse]

To prepare EDTA, I take time, reading papers in the same time.
(I prepare only 100 mL of 100 mM).
I put the solution on a magnetic stirrer, and add NaOH from time to time until it dissolves. If I'm hurry the pH will be to high. That's why I take time and I don't need to check the pH, I add NaOH, and wait, and add again and wait, when it is dissolved, the pH is close to 8.0.

[GeorgeWolff]

DiSodium EDTA has a soloublity limit of about 10% (MSDS at: http://www.jtbaker.c...hhtml/e0170.htm )

[jiajia1987]

hey peeps,

thanks for replying fast.

I went back to the lab just a while ago to follow your suggestions of adding the NaOH slowly. I also increased the volume to a little less than 1L with dH20 so I can still add NaOH to adjust the pH at the same time. It is horrifying how much NaOH I had to add to adjust the pH. It took forever but at least it is dissolving now. I am letting it stir overnight.

By the way.. when you guys made NaOH, ever noticed how the solution seem to feel very very very warm? I felt uncomfortable handling it.

[jiajia1987]

mad, on Jul 22 2009, 04:39 PM, said:

nope you dont really need it to be in pellet.

by the way you need to add really really a lot of NaOH. I too have just tried to make EDTA just last week but on a smaller scale of only 100ml (cause i only needed to make TBE with them). I was following the Sambrook's formula which stated for 1L i needed to add abount 20g NaOH pellet. So I thought..ok...so for 100ml I should only need to add 2g....big mistake.  

Not sure how many g i added but it was way above the calculated 2g. Just add them slowly and you should be fine.


MAD

And...

The sambrook formula I saw stated 800mL instead of 1L. I calculated it as 1L and that was why I was confused about the book. But anyway, I added more water to a little before 1L and fill it up with NaOH and the pH was 8.03 to 8.05. It is dissolving now. Finally.

[little mouse]

jiajia1987, on Jul 22 2009, 11:28 AM, said:

hey peeps,

thanks for replying fast.

I went back to the lab just a while ago to follow your suggestions of adding the NaOH slowly. I also increased the volume to a little less than 1L with dH20 so I can still add NaOH to adjust the pH at the same time. It is horrifying how much NaOH I had to add to adjust the pH. It took forever but at least it is dissolving now. I am letting it stir overnight.

By the way.. when you guys made NaOH, ever noticed how the solution seem to feel very very very warm? I felt uncomfortable handling it.

yes it's normal.  be careful when you manipulate NaOH.

[jiajia1987]

hmmmm okay. I hope I don't have to handle so much of hydrochloric acid and sodium hydroxide next time round. Im always getting into some sorta minor spills.

Anyway, thank you all!!!

[bochum]

Hi If i am right, it is not possible to prepare 1M EDTA,ie what even sambrook and russell's molecular cloning book suggest to prepare 0.5M EDTA. And regarding calculation when the molecular weight is 372.24g/mol,then to prepare 1M solution you have to take 372.24g in 1litre and to prepare 0.5M you have to take 186.1g ie what sambrook and russell's molecular cloning book suggesting you.

But if you prepare it by adding excess of NaOH or HCL by any means it is wrong...and it has to go directly to dust bin

So point is you cannot or should not prepare 1M EDTA and there is nothing wrong in the book...

Edited by bochum, 22 July 2009 - 07:11 AM.

[mdfenko]

one more thing. you start with 800ml water, add 186.1gm edta and then about 20gm naoh. the final adjustment to 1 liter should be minimal.

you neither make adjusted solutions at the final volume (before adjustment) nor do you start with the final volume and then add to it.

Edited by mdfenko, 22 July 2009 - 01:25 PM.

[swanny]

Sambrook et al wrote their protocols because they worked, so it's not wise to decide they are wrong and just do your own thing before trying things their way. EDTA has very low solubility at low to neutral pH, which is why you need to throw in large amounts of NaOH to get it to dissolve. Remember it is an acid, so you shouldn't have to worry about adding extra acid.
Why are you wanting 1M EDTA? Most of the time, you only need 1 - 5 mM, so a 1M stock is not really necessary.

[jiajia1987]

bochum, on Jul 22 2009, 10:49 PM, said:

Hi If i am right, it is not possible to prepare 1M EDTA,ie what even sambrook and russell's molecular cloning book suggest to prepare 0.5M EDTA. And regarding calculation when the molecular weight is 372.24g/mol,then to prepare 1M solution you have to take 372.24g in 1litre and to prepare 0.5M you have to take 186.1g ie what sambrook and russell's molecular cloning book suggesting you.

But if you prepare it by adding excess of NaOH or HCL by any means it is wrong...and it has to go directly to dust bin

So point is you cannot or should not prepare 1M EDTA and there is nothing wrong in the book...

Hi bochum,

Yes, it is not possible to prepare 1M EDTA, but I did not know that prior to doing it until I read the sambrook molecular cloning book.

What I did was to add 186.1g and added 800mL of water. What I am trying to tell you is that after adding NaOH to adjust the pH to 8.0, it was right at 1L. So there was nothing wrong with what I was doing. I still get 0.5M EDTA stock because 186.1g of EDTA was dissolved in a final volume of 1L. It took quite a lot of NaOH before the solution adjusted to pH8.0

[jiajia1987]

mdfenko, on Jul 23 2009, 05:23 AM, said:

one more thing. you start with 800ml water, add 186.1gm edta and then about 20gm naoh. the final adjustment to 1 liter should be minimal.

you neither make adjusted solutions at the final volume (before adjustment) nor do you start with the final volume and then add to it.

Dear mdfenko,

That was what I did.

In my previous entry, I stated
"I went back to the lab just a while ago to follow your suggestions of adding the NaOH slowly. I also increased the volume to a little less than 1L with dH20 so I can still add NaOH to adjust the pH at the same time. It is horrifying how much NaOH I had to add to adjust the pH. It took forever but at least it is dissolving now. I am letting it stir overnight."

I left my solution of 186.1g EDTA and 800mL water in the lab while I posted here for help, after reading a few replies, I went back to the lab again and added the NaOH slowly. Water was also added because to get 0.5M, it is necessary to hit a total of 1L.

The reason why I added water was because pH8.0 was hit and the EDTA started to dissolve and I have to add water to hit it to 1L. As it dissolved, the pH went down again due to the reaction and I added NaOH to adjust the pH to 8.0 again before everything dissolved.

[jiajia1987]

swanny, on Jul 23 2009, 09:31 AM, said:

Sambrook et al wrote their protocols because they worked, so it's not wise to decide they are wrong and just do your own thing before trying things their way. EDTA has very low solubility at low to neutral pH, which is why you need to throw in large amounts of NaOH to get it to dissolve. Remember it is an acid, so you shouldn't have to worry about adding extra acid.
Why are you wanting 1M EDTA? Most of the time, you only need 1 - 5 mM, so a 1M stock is not really necessary.

Dear swanny,

THanks for your comment.

Well... I made 1M EDTA initially because I wanted to have a stock solution and it was my first time making an EDTA stock solution. So naturally.. I did not know that 1M EDTA was impossible.