Re: [ccp4bb] preparation of citrate buffer pH3-6.5
Dear All, Thank you for your replies: 2 different methods were suggested for the citrate buffer preparation over a small range of pH: 1. mixing citric acid with sodium citrate salts 2. mixing citric acid with NaOH we found the latter method easier for our purpose.
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
The easiest way to produce repeatable conditions is to titrate a stock solution (say 1M) of citric acid with NaOH to the desired pH and use that to mix your screen. That's what Hampton does anyway. If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH won't be linear with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in wrote: Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
It’s a pain but I usually just make each pH of whatever buffer I’m using (if you make it concentrated then you’ll only have to do it once). Also, if you haven’t already found it, Hampton has a nice link to calculate volume of components while designing a tray as long as you tell it the concentrations. http://hamptonresearch.com/make_tray.aspx Nick From: Roger Rowlett rrowl...@colgate.edumailto:rrowl...@colgate.edu Reply-To: Roger Rowlett rrowl...@colgate.edumailto:rrowl...@colgate.edu Date: Thursday, January 30, 2014 at 7:23 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5 The easiest way to produce repeatable conditions is to titrate a stock solution (say 1M) of citric acid with NaOH to the desired pH and use that to mix your screen. That's what Hampton does anyway. If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH won't be linear with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.inmailto:somon_...@yahoo.co.in wrote: Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
But you have to be aware that pH depends on the concentration of the buffer. This is especially the case for phosphate and citrate buffer. Daniel Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit : It’s a pain but I usually just make each pH of whatever buffer I’m using (if you make it concentrated then you’ll only have to do it once). Also, if you haven’t already found it, Hampton has a nice link to calculate volume of components while designing a tray as long as you tell it the concentrations. http://hamptonresearch.com/make_tray.aspx Nick From: Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu Reply-To: Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu Date: Thursday, January 30, 2014 at 7:23 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5 The easiest way to produce repeatable conditions is to titrate a stock solution (say 1M) of citric acid with NaOH to the desired pH and use that to mix your screen. That's what Hampton does anyway. If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH won't be linear with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in mailto:somon_...@yahoo.co.in wrote: Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
Alternatively you could make a stock solution of citric acid (say 1 M for example) and stock solution of sodium citrate (also 1 M). Mix them in the appropriate ratio to ballpark the right pH and just adjust up or down with the stock solution. The concentration of citrate will be the same no matter the final volume. You can then dilute that down to whatever your final concentration of citrate needs to be. If you are looking for the actual method to do the calculations I would suggest finding a chemistry textbook and looking at the chapter on buffering and the Henderson-Hasselbalch equation. Cheers, Katherine On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot daniel.pi...@ibpc.fr wrote: But you have to be aware that pH depends on the concentration of the buffer. This is especially the case for phosphate and citrate buffer. Daniel Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit : It's a pain but I usually just make each pH of whatever buffer I'm using (if you make it concentrated then you'll only have to do it once). Also, if you haven't already found it, Hampton has a nice link to calculate volume of components while designing a tray as long as you tell it the concentrations. http://hamptonresearch.com/make_tray.aspx Nick From: Roger Rowlett rrowl...@colgate.edu Reply-To: Roger Rowlett rrowl...@colgate.edu Date: Thursday, January 30, 2014 at 7:23 AM To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5 The easiest way to produce repeatable conditions is to titrate a stock solution (say 1M) of citric acid with NaOH to the desired pH and use that to mix your screen. That's what Hampton does anyway. If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH won't be linear with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in wrote: Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama -- Nil illegitimo carborundum* - *Didactylos
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
RE citrate buffer preparation The Calbiochem buffers has some generally useful information about buffers; pKa and such. http://www.antibodybeyond.com/books/Calbiochem_Buffers_Booklet_CB0052_E.pdf http://wolfson.huji.ac.il/purification/PDF/Buffers/Calbiochem_Buffers_Booklet.pdf -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
Hi Sreetama, For most buffers, I use Katherine's method, but in the case of citrate I'd recommend just titrating citric acid with NaOH. I've made a pH series from citric acid and Na3Cit before, and it's a huge pain. It's very difficult to calculate how much of each you'll need, because citrate is triprotic and the pKas overlap. When I made a pH series this way, I ended up using much more stock than I anticipated, and just had an overall unpleasant afternoon. One other point - high citrate concentrations can cause your pH meter to drift, so don't leave the probe in the citrate solution any longer than needed, and calibrate it frequently. I'm told this is because citrate chelates metals and that throws off the electrode, though admittedly I don't know the mechanism and can't find any references to back me up - it's just been lab folklore for me. Either way it might be worth testing the stability of your electrode over time to get an idea. Shane Caldwell McGill University On Thu, Jan 30, 2014 at 10:40 AM, Katherine Sippel katherine.sip...@gmail.com wrote: Alternatively you could make a stock solution of citric acid (say 1 M for example) and stock solution of sodium citrate (also 1 M). Mix them in the appropriate ratio to ballpark the right pH and just adjust up or down with the stock solution. The concentration of citrate will be the same no matter the final volume. You can then dilute that down to whatever your final concentration of citrate needs to be. If you are looking for the actual method to do the calculations I would suggest finding a chemistry textbook and looking at the chapter on buffering and the Henderson-Hasselbalch equation. Cheers, Katherine On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot daniel.pi...@ibpc.frwrote: But you have to be aware that pH depends on the concentration of the buffer. This is especially the case for phosphate and citrate buffer. Daniel Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit : It's a pain but I usually just make each pH of whatever buffer I'm using (if you make it concentrated then you'll only have to do it once). Also, if you haven't already found it, Hampton has a nice link to calculate volume of components while designing a tray as long as you tell it the concentrations. http://hamptonresearch.com/make_tray.aspx Nick From: Roger Rowlett rrowl...@colgate.edu Reply-To: Roger Rowlett rrowl...@colgate.edu Date: Thursday, January 30, 2014 at 7:23 AM To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5 The easiest way to produce repeatable conditions is to titrate a stock solution (say 1M) of citric acid with NaOH to the desired pH and use that to mix your screen. That's what Hampton does anyway. If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH won't be linear with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in wrote: Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama -- Nil illegitimo carborundum* - *Didactylos
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
You are correct about certain buffers as interferents. Certain buffer species will coordinate with or precipitate silver or mercurous ions that are present in the reference electrode compartment of the combination pH electrodes. Tris is notorious for clogging the little porous frit on the reference electrode, and this, along with reference solution cation ion depletion, will drive the electrode crazy until the frit is cleared and/or the reference electrode filling solution is replenished. Making 1M stock Tris solutions is enough to knock out a pH electrode for several hours if you overexpose it to the solution. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 1/30/2014 12:23 PM, Shane Caldwell wrote: Hi Sreetama, For most buffers, I use Katherine's method, but in the case of citrate I'd recommend just titrating citric acid with NaOH. I've made a pH series from citric acid and Na3Cit before, and it's a huge pain. It's very difficult to calculate how much of each you'll need, because citrate is triprotic and the pKas overlap. When I made a pH series this way, I ended up using much more stock than I anticipated, and just had an overall unpleasant afternoon. One other point - high citrate concentrations can cause your pH meter to drift, so don't leave the probe in the citrate solution any longer than needed, and calibrate it frequently. I'm told this is because citrate chelates metals and that throws off the electrode, though admittedly I don't know the mechanism and can't find any references to back me up - it's just been lab folklore for me. Either way it might be worth testing the stability of your electrode over time to get an idea. Shane Caldwell McGill University On Thu, Jan 30, 2014 at 10:40 AM, Katherine Sippel katherine.sip...@gmail.com mailto:katherine.sip...@gmail.com wrote: Alternatively you could make a stock solution of citric acid (say 1 M for example) and stock solution of sodium citrate (also 1 M). Mix them in the appropriate ratio to ballpark the right pH and just adjust up or down with the stock solution. The concentration of citrate will be the same no matter the final volume. You can then dilute that down to whatever your final concentration of citrate needs to be. If you are looking for the actual method to do the calculations I would suggest finding a chemistry textbook and looking at the chapter on buffering and the Henderson-Hasselbalch equation. Cheers, Katherine On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot daniel.pi...@ibpc.fr mailto:daniel.pi...@ibpc.fr wrote: But you have to be aware that pH depends on the concentration of the buffer. This is especially the case for phosphate and citrate buffer. Daniel Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit : It's a pain but I usually just make each pH of whatever buffer I'm using (if you make it concentrated then you'll only have to do it once). Also, if you haven't already found it, Hampton has a nice link to calculate volume of components while designing a tray as long as you tell it the concentrations. http://hamptonresearch.com/make_tray.aspx Nick From: Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu Reply-To: Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu Date: Thursday, January 30, 2014 at 7:23 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5 The easiest way to produce repeatable conditions is to titrate a stock solution (say 1M) of citric acid with NaOH to the desired pH and use that to mix your screen. That's what Hampton does anyway. If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH won't be linear with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in mailto:somon_...@yahoo.co.in wrote: Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
If the pK's are well separated, so that only one is titrating at a time ( assume only two species exist at a time), the number of equiv of NaOH to add would be : 1/(1+10^(pK1-pH)) + 1/(1+10^(pK2-pH)) + 1/(1+10^(pK3-pH)) where each term transitions from 0 to 1 as pH passes through it's pKa In principle the calculated buffers should be more accurate, especially in the strong buffering regions where a small error in weighing out doesn't affect pH much. They depend on accurate pKa values, but these have probably been determined by a physical chemist with a hydrogen electrode (not glass electrode) in a junctionless system with temperature control. They are probably more reproducible, since analytical balances don't drift as much or show as much temperature dependence as pH electrodes. However if you publish just giving pH and someone else tries to reproduce your results by adjusting a 1M stock solution at room temperature to the pH and then diluting it to 10 mM in the assay at 4C, they are likely to get a very different pH. Also check the label on your NaOH bottle to see the water content and adjust the MW accordingly- NaOH is often less than 90% NaOH by weight. Finally if the reason for using a tribasic buffer is to get similar conditions over a wide pH range, this is a delusion because different buffer species are present at different pH's. MES^- is a lot more like MOPS^- than citrate^- is like citrate^-2, ionically speaking. And the ionic strength will vary greatly from one end of the range to the other, as you go from mostly neutral citric acid to mostly Na2- or Na3-citrate. eab Katherine Sippel wrote: Alternatively you could make a stock solution of citric acid (say 1 M for example) and stock solution of sodium citrate (also 1 M). Mix them in the appropriate ratio to ballpark the right pH and just adjust up or down with the stock solution. The concentration of citrate will be the same no matter the final volume. You can then dilute that down to whatever your final concentration of citrate needs to be. If you are looking for the actual method to do the calculations I would suggest finding a chemistry textbook and looking at the chapter on buffering and the Henderson-Hasselbalch equation. Cheers, Katherine On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot daniel.pi...@ibpc.fr mailto:daniel.pi...@ibpc.fr wrote: But you have to be aware that pH depends on the concentration of the buffer. This is especially the case for phosphate and citrate buffer. Daniel Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit : It’s a pain but I usually just make each pH of whatever buffer I’m using (if you make it concentrated then you’ll only have to do it once). Also, if you haven’t already found it, Hampton has a nice link to calculate volume of components while designing a tray as long as you tell it the concentrations. http://hamptonresearch.com/make_tray.aspx Nick From: Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu Reply-To: Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu Date: Thursday, January 30, 2014 at 7:23 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5 The easiest way to produce repeatable conditions is to titrate a stock solution (say 1M) of citric acid with NaOH to the desired pH and use that to mix your screen. That's what Hampton does anyway. If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH won't be linear with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in mailto:somon_...@yahoo.co.in wrote: Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama -- Nil illegitimo carborundum/- /Didactylos
[ccp4bb] preparation of citrate buffer pH3-6.5
Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama
