Dear Jinsha Koroth, - apologize for longness -

personally I would not be surprised that you made this experience (who else is measuring pH that correctly during set up and mixing of stock solutions!?).

I guess that your experiment calls for an EDTA solution of pH 8 (correct?) or is it only in your recipe to do so?

Will you have to dilute the stock solution further? e.g. (1) 50 mM EDTA (pH 8.0) and/or (2) EDTA 0.125 M, pH 7.0….(see below)

So first you will have to adjust your EDTA solution to pH 8.0, independently of its incomplete/complete dissolution at either pH – say -7.5 oder 7.65 or 8.0. right?

There are some recipes to be found in various sources, some of which in my opinion are not very helpful for beginners or even for the expert, for example:

i) STOCK solution EDTA 0.5M pH8.0: add 186,1 g disodium EDTA to approx. 800ml of deionized water (NB: for sophisticated experiments I would suggest to use rather “true” A.dest, if not A. bidest.) ,

STIR VIGOROUSLY and adjust pH with sodium hydroxide (NaOH) pellets (approx. 20 g) to pH 8.0 (Cave: no advice for personal saftey can be found herein!!)

ii) for 50 mM [(1) above]: by dilution from 0.5 M Stock solution with (deionized) water(better at least: A. dest.)

iii) for 0.125M EDTA, pH 7.0: “dilute from 0.5 M stock solution to pH 7.0 using (deionized) water (A.dest) and adding concentrated HCl….(this one I believe is a real crude method in terms of “chemistry”: no amounts of which concentration of HCl given! which means that you very easily do overtitration!...back and forth, etc...)

You tell us that you use 10 N NaOH for adjusting the pH.

This is approx. 40 g NaOH pellets in 100 ml A.dest..

According to

i) you perhaps need approx. 50 ml of 10N NaOH to get close to pH 8.0. Usually nobody will measure the true turning point where all of the EDTA has been dissolved (concentrated NaOH addition being a little bit “hot”, I guess…(concentrated NaOH reacts exothermic up to 50/60 degr.C, depending on volumes of solution to be adjusted and NaOH-volume added).

You have NOT said which method you used to measure that pH-turning point (point of “full dissolution / dissociation of EDTA”).

If it was measured "real-time"= concomitantly) by a pH-Electrode/pH-Meter or an other instrument with an electrode (considering the ): keep in mind that pH varies (can differ) significantly with temperature of the solution. For that reason a pH-meter should be equipped with a thermosensor / thermocouple recording the actual temperature in the solution. Adjusting the system with "control buffers" at pH 4.0, 7.0 and 10.0 commercially are available if not delivered with the system (Follow instructions, especially concerning temperature of the calibration solutions!) Have you measured the actual temperature at the (your) magic “turning point” (i.e. pH 7.65, versus solution at room temperature at that point)?

And as second parameter: it might be "seen by your eyes" the solution is “clear” (means: completely dissolved) but perhaps in reality (in terms of chemistry) has not completely dissociated: I guess by naked eye you won’t see existing micro- and nanocrystals of EDTA in your solution.

The pH ( @25degr.C) of a 1% Dinatrium-EDTA-solution will be between 4.0 and 5.0 and solves at pH > 7.0. Working concentrations usually are between 0.5 mM to 0.125 M.

All recipes I have in files and have seen for now state “adjust to pH 8.0”, none of those sources says:

“crystals will dissolve at pH 7.65 or 7.7”, but (see below): " ..as the pH of the solution nears 8.0”, whatever “near” in this context might mean…

Using NaOH pellets in my eyes certainly is conceivable at least at the beginning of titration (Cave: burnings/cauterization, prevent splashing, use protective glasses and suited gloves and consider the exothermic reaction in your lab flask!), but a 10N NaOH solution is much better, especially using both of the methods one should be really careful when coming to the endpoint of the titration: cf. also:

2) http://chemistry.about.com/od/labrecipes/a/Edta-Solution.htm

citation: “Add NaOH solution to adjust the pH to 8.0. If you use solid NaOH pellets, you'll need about 18-20 grams of NaOH. Add the last of the NaOH slowly so that you don't overshoot the pH. You may wish to switch from solid NaOH to a solution toward the end, for more precise control.......

3) another recipe (see section ) from the web at

http://bioinfoweb.com/Protocol-preparation-of-0.5M-EDTA-stock-solution.htm describes also the varying amount of EDTA using either or .

I therefore won’t mind to use a 0.5 M Stock solution of EDTA at pH 8.0 (which has been seen or been measured to have dissolved completely at pH 7.65)

Meanwhile, as I have seen there was also the post of Jan Piwowarski, the principle of “trivial” but important advice I second 150%....().

best wishes and good luck, Wolfgang

Calibrate your pH-meter please, check your EDTA on another one. By the not calibrated pH-meter I have lost 3 months of my work. Could be by your observation you avoided future disasters...

Dear Jinsha Koroth, - apologize for longness -

personally I would not be surprised that you made this experience (who else is measuring pH that correctly during set up and mixing of stock solutions!?).

I guess that your experiment calls for an EDTA solution of pH 8 (correct?) or is it only in your recipe to do so?

Will you have to dilute the stock solution further? e.g. (1) 50 mM EDTA (pH 8.0) and/or (2) EDTA 0.125 M, pH 7.0….(see below)

So first you will have to adjust your EDTA solution to pH 8.0, independently of its incomplete/complete dissolution at either pH – say -7.5 oder 7.65 or 8.0. right?

There are some recipes to be found in various sources, some of which in my opinion are not very helpful for beginners or even for the expert, for example:

i) STOCK solution EDTA 0.5M pH8.0: add 186,1 g disodium EDTA to approx. 800ml of deionized water (NB: for sophisticated experiments I would suggest to use rather “true” A.dest, if not A. bidest.) ,

STIR VIGOROUSLY and adjust pH with sodium hydroxide (NaOH) pellets (approx. 20 g) to pH 8.0 (Cave: no advice for personal saftey can be found herein!!)

ii) for 50 mM [(1) above]: by dilution from 0.5 M Stock solution with (deionized) water(better at least: A. dest.)

iii) for 0.125M EDTA, pH 7.0: “dilute from 0.5 M stock solution to pH 7.0 using (deionized) water (A.dest) and adding concentrated HCl….(this one I believe is a real crude method in terms of “chemistry”: no amounts of which concentration of HCl given! which means that you very easily do overtitration!...back and forth, etc...)

You tell us that you use 10 N NaOH for adjusting the pH.

This is approx. 40 g NaOH pellets in 100 ml A.dest..

According to

i) you perhaps need approx. 50 ml of 10N NaOH to get close to pH 8.0. Usually nobody will measure the true turning point where all of the EDTA has been dissolved (concentrated NaOH addition being a little bit “hot”, I guess…(concentrated NaOH reacts exothermic up to 50/60 degr.C, depending on volumes of solution to be adjusted and NaOH-volume added).

You have NOT said which method you used to measure that pH-turning point (point of “full dissolution / dissociation of EDTA”).

If it was measured "real-time"= concomitantly) by a pH-Electrode/pH-Meter or an other instrument with an electrode (considering the ): keep in mind that pH varies (can differ) significantly with temperature of the solution. For that reason a pH-meter should be equipped with a thermosensor / thermocouple recording the actual temperature in the solution. Adjusting the system with "control buffers" at pH 4.0, 7.0 and 10.0 commercially are available if not delivered with the system (Follow instructions, especially concerning temperature of the calibration solutions!) Have you measured the actual temperature at the (your) magic “turning point” (i.e. pH 7.65, versus solution at room temperature at that point)?

And as second parameter: it might be "seen by your eyes" the solution is “clear” (means: completely dissolved) but perhaps in reality (in terms of chemistry) has not completely dissociated: I guess by naked eye you won’t see existing micro- and nanocrystals of EDTA in your solution.

The pH ( @25degr.C) of a 1% Dinatrium-EDTA-solution will be between 4.0 and 5.0 and solves at pH > 7.0. Working concentrations usually are between 0.5 mM to 0.125 M.

All recipes I have in files and have seen for now state “adjust to pH 8.0”, none of those sources says:

“crystals will dissolve at pH 7.65 or 7.7”, but (see below): " ..as the pH of the solution nears 8.0”, whatever “near” in this context might mean…

Using NaOH pellets in my eyes certainly is conceivable at least at the beginning of titration (Cave: burnings/cauterization, prevent splashing, use protective glasses and suited gloves and consider the exothermic reaction in your lab flask!), but a 10N NaOH solution is much better, especially using both of the methods one should be really careful when coming to the endpoint of the titration: cf. also:

2) http://chemistry.about.com/od/labrecipes/a/Edta-Solution.htm

citation: “Add NaOH solution to adjust the pH to 8.0. If you use solid NaOH pellets, you'll need about 18-20 grams of NaOH. Add the last of the NaOH slowly so that you don't overshoot the pH. You may wish to switch from solid NaOH to a solution toward the end, for more precise control.......

3) another recipe (see section ) from the web at

http://bioinfoweb.com/Protocol-preparation-of-0.5M-EDTA-stock-solution.htm describes also the varying amount of EDTA using either or .

I therefore won’t mind to use a 0.5 M Stock solution of EDTA at pH 8.0 (which has been seen or been measured to have dissolved completely at pH 7.65)

Meanwhile, as I have seen there was also the post of Jan Piwowarski, the principle of “trivial” but important advice I second 150%....().

best wishes and good luck, Wolfgang

I advice you too to use 1N NaOH solution and as Jan said check first your pH-meter.

good luck

yeah ! your disodium EDTA (0.5M) dissolved at ph 7.65,so it is fine. finally you adjust your EDTA PH at 8 and use this.

Because EDTA dissolve at slightly different-different PH it may be depend on EDTA (solute)concentration in Solvent.

Dear Mohamed,

thank you for your post and advice for Jinsha.

This reminds me I have forgotten to say it clearly in my "long" post above:

using (only) 1N NaOH will not suffice to get the correct & desired solution

(calculating for resulting in 0.5 M EDTA - stock solution:

800 ml A.dest. for dissolving the EDTA-powder + approx. 50 ml 10 N NaOH (for pH 8.0) =

ml for adjusting pH + ca 150ml A.dest to equilibrate and finish 1000 mL

?equals (? ask yourself?)

800 ml A.dest. for dissolving the EDTA-powder + approx. ml 1 N NaOH =

END VOLUME for adjusting pH ??? solution will not be 0.5 M )

So if you advise how to proceed for a successful titration, one way could be:

for 1L of 0.5 M Di-Na-EDTA

1) use Erlenmayer flask (2000mL),

2) - add186,1 g of disodium EDTA in 800 ml A. dest. (deionized water),

3) - stir moderately to vigorously with a magnetic rotator / stirring device,

[(i) add slowly add approx. 19 g NaOH pellets, cave: strong exothermic reaction! and finish following iii and iv ) ] or - better -

ii) add 40 ml of 10N NaOH (monitoring/testing actual pH if desired),

iii) + 10-16 ml 5N NaOH solution (monitoring/testing actual pH),

iv)+ approx. 10 ml 1N NaOH (monitoring/testing actual pH at roomTemp necessary)

finally:

fill up to end volume 1000 mL with A. dest.

(monitoring/testing actual pH desireable).

Best wishes and regards,

Wolfgang

I have a question, can I use TAE for acrylamide gels too ???

di sodium salt of EDTA, is readily soluble in water no need of NaOH. if you are using EDTA then only you have to adjust the pH to 8 with NaOH.

http://www.sigmaaldrich.com/catalog/product/sial/ed2ss?lang=en®ion=US

Dear Diana,

usually TAE (Tris-Acetat-EDTA-) buffers are used with the agarose-gel-electrophoresis techniques.

Not familiar with this specifically requested matter I only can point you to a ResearchGate posting from

Jun 13, 2013 by Pardis FAZLI, How long may a polyacrylamide gel be stored in TAE 1% buffer?

stating that TAE 1% buffer has been used in/for polyacrylamide gels:

cf. http://www.researchgate.net/post/How_long_may_a_polyacrylamide_gel_be_stored_in_TAE_1_buffer

Best wishes and good luck

Hi, as Sharad has indicated, disodium EDTA is soluble in water (just it s better to dissolve first in case a-mix of-solution). Also, when you start to increase pH (which is around what you stated) it is becoming soluble early. Good Luck!

Thank you all for your suggestions,,,

After caliberating the ph meter my EDTA showed the ph 7.77. But before making TE (for dissolving primers,DNA,RNA) with this EDTA, should I bring the EDTA ph to 8? or this ph 7.77 is ok?

And dear Diana, you can use TAE for making polyacrylamide gel which i usually use for running small sized DNA fragments(around 100-300bp) after PCR.

For standard TE you use 1mM EDTA, 10mM TRIS, thus mainly TRIS will buffer your TE. I would use it for this purpose.