# About Tris-Cl buffer - (Nov/30/2007 )

Hi~ everyone!

We know that the pH value of Tris-Cl buffer often changes when the temperature is different. Usually, we adjust the pH value at room temperature(25℃). But, my buffer always used at 4 ℃.Then, my question is at which temperature should I fix the final pH value?

Thanks a lot!

-redondoself-

If you use it at 4°C and you need a certain pH, then you have to fix the pH for 4°C. So, cool the buffer and titrate or fix it in the cooling room if you don't have an adjustable temperature on your pH-meter.

-biomaus-

-K.B.-

QUOTE (K.B. @ Nov 30 2007, 02:57 PM)

hii.. sorry.. have no answers but have more questions on this!!

i did a trial calculation for phosphate buffer pH 7, for use at 4 deg C and 25 deg C.

(seee below)
this is surprising..
i thot with increase in temp.. more ionization.. pH wud be higher than at lower temp.. hence u wud adjust it to a higher value at 25 and this wud decrease at 4 deg.. but this seems to be opposite.. can someone explain??

also.. why is there a change in the salt amounts..? i was only expecting a change in the buffer components.. !??
thnksss!!

<H3 style="MARGIN: auto 0in">BUFFER:</H3><H5 style="MARGIN: auto 0in">To make1000 ml of 0.01 M Phosphate(pKa2=7.2) Buffer, pH= 7,
Ionic strength = 0.154 M,
Thermodynamic pKa = 7.2, Apparent pKa' = 6.82
Temperature coefficient = -0.0028 per oC
Prepared at 25oC, used at 4 oC
</H5><H3 style="MARGIN: auto 0in">RECIPE: </H3><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1; tab-stops: list .5in">· Dissolve 0.004 mol of acid component</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1; tab-stops: list .5in">· Dissolve 0.0059 mol of basic component</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1; tab-stops: list .5in">· Add 7.731g NaCl. </H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1; tab-stops: list .5in">· Make up to 1000 ml with pure water</H5><H3 style="MARGIN: auto 0in">Alternatively</H3>

[/size]

<H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l3 level1 lfo2; tab-stops: list .5in">· Dissolve 1.42g of e.g. NaH2PO4 (Mr = 142) in approx. 900ml of pure water.</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l3 level1 lfo2; tab-stops: list .5in">· Add 7.731g NaCl.</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l3 level1 lfo2; tab-stops: list .5in">· Titrate to pH 6.94 at the lab temperature of 25oC with monovalent strong base or acid as needed. </H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l3 level1 lfo2; tab-stops: list .5in">· Make up volume to 1000ml with pure water </H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l3 level1 lfo2; tab-stops: list .5in">· Buffer will, of course, be pH 7.00 at 4oC</H5><H3 style="MARGIN: auto 0in">BUFFER:</H3><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l3 level1 lfo2; tab-stops: list .5in">· To make1000 ml of 0.01 M Phosphate(pKa2=7.2) Buffer, pH= 7,
Ionic strength = 0.154 M,
Thermodynamic pKa = 7.2, Apparent pKa' = 6.81
Temperature coefficient = -0.0028 per oC
Prepared at 25oC, used at 25 oC
</H5><H3 style="MARGIN: auto 0in">RECIPE: </H3><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l2 level1 lfo3; tab-stops: list .5in">· Dissolve 0.0039 mol of acid component</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l2 level1 lfo3; tab-stops: list .5in">· Dissolve 0.006 mol of basic component</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l2 level1 lfo3; tab-stops: list .5in">· Add 7.722g NaCl. </H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l2 level1 lfo3; tab-stops: list .5in">· Make up to 1000 ml with pure water</H5><H3 style="MARGIN: auto 0in">Alternatively</H3>

[size="4"]
<H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4; tab-stops: list .5in">· Dissolve 1.42g of e.g. NaH2PO4 (Mr = 142) in approx. 900ml of pure water.</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4; tab-stops: list .5in">· Add 7.722g NaCl.</H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4; tab-stops: list .5in">· Titrate to pH 7 at the lab temperature of 25oC with monovalent strong base or acid as needed. </H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4; tab-stops: list .5in">· Make up volume to 1000ml with pure water </H5><H5 style="MARGIN: auto 0in auto 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4; tab-stops: list .5in">· Buffer will, of course, be pH 7.00 at 25oC</H5>

-alice!-

QUOTE (alice! @ Nov 30 2007, 11:06 AM)
hii.. sorry.. have no answers but have more questions on this!!

i did a trial calculation for phosphate buffer pH 7, for use at 4 deg C and 25 deg C.

(seee below)
this is surprising..
i thot with increase in temp.. more ionization.. pH wud be higher than at lower temp.. hence u wud adjust it to a higher value at 25 and this wud decrease at 4 deg.. but this seems to be opposite.. can someone explain??

also.. why is there a change in the salt amounts..? i was only expecting a change in the buffer components.. !??
thnksss!!

ionization of the buffer is dependent on ambient pH and the buffer's pK(s) as well as the temperature.

there is changing salt amount shown in the recipes calculated because the program asks if you want to set a constant ionic strength (at one site it was prechecked, at the other it was clear, at least when i looked).

in response to the original question, we always adjust the pH at the temperature we intend to use it at. for example, we have 3 tris pH 7.5 stocks, at 5C, 25C and 37C.

sometimes we determine the pH of a stock at a temperature different from the one at which it was adjusted and use it as that (eg-for pH curve assays).

-mdfenko-