10X PBS

[sudheer sangeetham]

Hi People

I have few doubts regarding phosphte buffer saline (PBS). Recently I found
one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
not KCl, am I right?

I have prepared the buffer like this

NaH2PO4 ( 0.038M)
Na2HPO4.2H20 ( 0.162M)
NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
did I do any mistake....

Could any one please tell me did i do any mistake?

Thank you in advance

--
Sudheer Babu.S
Ph.D student
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.

[Hiranya Roychowdhury]

Since you say you have few questions, I am assuming that you are quite certain about your composition. PBS stands for "Phosphate buffered saline" so it is NaCl, not KCl. Usually we use Na-salts for the phosphate combination. Potassium phosphate and -biphosphate may be used, but for all practical purposes it is Na- that is preferred since lot of compounds do not go well with K+


Hiranya S. Roychowdhury, Ph.D.
Associate Professor
Health & Public Services
NMSU-Dona Ana Community College
575 527 7725 (office)

________________________________________
From: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] on behalf of sudheer sangeetham [sudhee...@gmail.com]
Sent: Tuesday, January 03, 2012 9:52 AM
To: Met...@magpie.bio.indiana.edu
Subject: 10X PBS

_______________________________________________
Methods mailing list
Met...@net.bio.net
http://www.bio.net/biomail/listinfo/methods

[Irit Rappley]

The pH of the 10X solution can be slightly different from the pH of the 1X solution (but not usually as big as what you are seeing, I think). So you should check the pH of your 1X solution and adjust accordingly.

When I make PBS 10X stock, the pH is often wrong and I simply adjust it with 5M NaOH. Maybe your calculation did not take into account the pH of the water (usually ~5 or 6, definitely not 7)?

Hope that helps,
Irit

[Jayakumar, R]

You might be referring to the use of PBS in animal systems like mice or rat. When they mean PBS i.p. (was it written this way?), it means administration of PBS into animal was via intraperitoneal injection or i.p (in the abdmomen). Unless otherwise told, PBS contains both KCl and NaCl along with Na2HPO4 and NaH2PO4. Refer the Sambrook manual or the current protocols in Molecular biology or any standard tissue culture manual for the PBS recipe. When the monobasic and dibasic salts are corrected added, the pH will be around 7.5. If it is 6.4, your recipe is not right.
Jay

This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you.

[WS]

Hi, I'm using this recipe for PBS, suitable for many occasions.

Actually, it is a rinsing buffer for cells, prior to trypsinization (D-
PBS from PAA), hence no divalent cations like Calcium and Magnesium.

Depending on your application (when you don't apply it on living
beings e.g.) , you may replace the potassium salts with sodium salts
for simplicity

The mean thing is that one needs to be careful about the actual
molecular weights of the phosphates as they are available with varying
crystal water content, so you might double check the formula and the
molecular weight on the actual box.

1xPBS:

KCl M= 74,6 g/mol 0.2g/l → 2,68 mM
NaCl M= 58.4 g/mol 8g/l → 137 mM
KH2PO4 M= 136.1g/mol 0.2g/l → 1.47 mM
Na2HPO4 M= 142g/mol 1.16g/l → 8.17 mM

Formulation, 10x Concentrate (g/l)
Inorganic Salts

KCl
2.0
KH2PO4
2.0
NaCl
80.0
Na2HPO4 anhydrous
11.58

HTH

Wo

[Jayakumar, R]

Ideally the pH should not change much between 10X and 1X if deionised water was used for dilution. Presnce of NaCl has nothing to do with pH changes since it contributes neither hydroxyl nor hydrogen ions (unless otherwise it is contaminated with something else), the balance of which determines pH (not just ionic strength). The balance between the monobasic and dibasic is thus maintained the same whatever dilution the stock is diluted to. If stock concentrations affected pH, many companies that make 10X buffer stocks will be out of business. During dilutions make sure to use deionised water (Millipore MilliQ for example) or the ions in water can slightly affect the pH, but not that significantly as you observed.
Jay


-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of DK
Sent: Tuesday, January 03, 2012 6:08 PM
To: met...@magpie.bio.indiana.edu
Subject: Re: 10X PBS

In article <mailman.69.132561...@net.bio.net>, sudheer sangeetham <sudhee...@gmail.com> wrote:
>Hi People
>
>I have few doubts regarding phosphte buffer saline (PBS). Recently I found
>one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
>Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
>not KCl, am I right?
>
>I have prepared the buffer like this
>
>NaH2PO4 ( 0.038M)
>Na2HPO4.2H20 ( 0.162M)
>NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
>did I do any mistake....

1. Measurng pH in ~ 2 M NaCl is not going to be very meaningful with
most electrodes.
2. pKa is a function of ionic strenght - no surprise that 10X and 1X have
different pH.
3. Dilute 10X, measure pH and if it's not above 7.2, you either made
an error making the solution or pH electrode is not measuring correctly.
4. The ratio you used above is for 0.2 M sodium phosphate, another
10X away from your 20 mM. (The difference between 200 and 20 will
be small but not negligible).

5. "PBS" can stand for many things. Cell culture PBS solutions
contain not ony some KCl (around 10 mM) but also some divalents,
CaCl2 and/or MgCl2.

DK

[Joshua Silverstein]

You don't need physical chemistry, just general.

If you take 10X PBS and dilute it with deionized water, it will certainly
change the pH. For a 10-fold dilution, it should change the pH by
approximately 1 (log scale).



On Wed, Jan 4, 2012 at 3:38 PM, DK <d...@noemail.thankstospam.net> wrote:

> In article <mailman.75.132570...@net.bio.net>, "Jayakumar,

> R" <R.Jay...@roswellpark.org> wrote:
> >Ideally the pH should not change much between 10X and 1X if deionised
> water was
> > used for dilution. Presnce of NaCl has nothing to do with pH changes
> since it
> > contributes neither hydroxyl nor hydrogen ions
>

> You need to refresh your physical chemistry.
> (Hint: cations compete with protons for binding to anions)

[Jayakumar, R]

DK and others,
This is getting sillier. I cannot believe this. I dilute buffers nearly everyday for the past 20 years. I also check the pH before and after dilution because I am scrupulous. There is a reason why buffers do not change pH upon dilution within its buffering range. I just checked it out again for your sake (check it out yourselves, use a 3 point calibrated pH stick, rarely does anyone calibrate their pH meters before every use, but you should).

High school experiment1: Took 5 ml of 1 M Tris (pH before dilution: 7.81) and diluted it 10 fold (pH after dilution: 7.83). Just to be sure that the "ions" are not misbehaving, I took 10X TBS (I did not have PBS stock presently in the lab), (before dilution pH 7.84) and diluted 10 fold to 50 ml (pH after dilution: 7.85). I used milliQue water of 17.7 Mohm-Cm for the experiment.

I feel that there is no necessity to teach about pH to this group of scientists. But to know why deionised water cannot change the pH of buffers significantly (within a certain buffer range for a particular buffer), and to refurbish you memories, please READ UP ON Lehninger's Principles of biochemistry chapter on Buffers and pKa (very important concept this) and also on the importance of conjugate bases and acids (explains why we use citric acid to adjust citrate buffers and not HCL). Check out the buffer change curves and you will realize why water is different from buffers and the importance of pKa range which is the buffering range for any buffer(THAT IS WHY IT IS CALLED A pH "BUFFER"). MOST IMPORTANT TERM IS pKa. Check it out before replying.
If diluting buffers with deionised water changes pH, then a lot of companies will be out of business selling stock buffers.
Best of luck.

Jay



-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of Joshua Silverstein
Sent: Wednesday, January 04, 2012 4:08 PM
To: DK
Cc: met...@magpie.bio.indiana.edu
Subject: Re: 10X PBS

You don't need physical chemistry, just general.

If you take 10X PBS and dilute it with deionized water, it will certainly
change the pH. For a 10-fold dilution, it should change the pH by
approximately 1 (log scale).



On Wed, Jan 4, 2012 at 3:38 PM, DK <d...@noemail.thankstospam.net> wrote:

> In article <mailman.75.132570...@net.bio.net>, "Jayakumar,
> R" <R.Jay...@roswellpark.org> wrote:

> >Ideally the pH should not change much between 10X and 1X if deionised
> water was
> > used for dilution. Presnce of NaCl has nothing to do with pH changes
> since it
> > contributes neither hydroxyl nor hydrogen ions
>

> You need to refresh your physical chemistry.
> (Hint: cations compete with protons for binding to anions)
>

[Deitiker, Philip R]

There are so many answers to the question for brevities sake I will add mine

1. Phosphate determines the pH, not chloride salts added. These monovalent cation/chloride solutions should have all but no buffering capacity. If the sodium or potassium chlorides show a significant buffering capacity they may have been contaminated by other salts, replace. I've seen worse things happen.

2. Dibasic phosphate is basic and if left exposed to air at room temperature will absorb both moisture and carbon dioxide, this can drop the pH and make the salt more difficult to handle. It is, as basic salts go, pretty resilient to acidification, but conditions in the laboratory may vary (e.g. gas space heaters, storage around areas were flame is used). I seriously doubt this is the issue. The way to know "what's up with this" is keep a record of your buffer salts, for example a 0.01 M solution of dibasic should always be the same pH. So you can record this on the bottle. If the pH of the diluted salt changes over time and it may be time to discard and replace _or_ your pH meter may have a problem. This is a significant problem in the laboratory, DNA hydration buffers at pH8.3 tend to loose about 0.1 pH units per 6 mos, faster if they are frequently opened. Hydroxides are the worst, absorbing both water and carbon dioxide quickly. If the dibasic absorbs enough water it can alter the pH because its molar contribution per weight will be less. This is unlikely to be the cause. For both of these, if you make the solution repeated and repeatedly you get the same deviant pH, then replace.

4. Two salts were confused at weighing, whoops. Seen this one a lot, I've done it and a number of my trainees have done it. The solution remade comes out perfect pH. As my TA used to say 'you make a cook-book buffer, if the pH is wrong, that's a good thing, don't adjust the pH, remake the buffer'.

Here's the recipe for 10X PBS pH 7.2

NaCl 87.7
NaH2PO4 3.05
Na2HPO4 11.1
Initial ddH20 0.9 L (e.g twice distilled water is all that we use, house DI systems cannot be relied upon)
ddH20 Water to 1L

Dibasic will not dissolve quickly, requires about 2-4 hours to dissolve, so keep the vessel covered with parafilm or flat piece of glass. This is not something that you leave overnight on the bench stirring in open air.

Make 100 ml of 1X and test the pH. pH should be 7.2 +/- 0.05 you can lower the monobasic and increase the dibasic to achieve pH 7.4 (See other post). NaOH can be added in small amounts to correct the pH, in most case pH adjustment is not needed.

5. Divalent and trivalent cations are easily trapped by higher ionization states of phosphate, thus heating phosphate can trap calcium and potentially drop the pH. Divalent cation solutions should be added at STP and in diluted form. In addition, one expects as the pH to go up, the stability of calcium phosphate in solution to go down. Sometimes we heat buffers to get them in solutions, but this may not work well with some buffers, or may cause problem with sterilizing solutions. In this case calcium chloride and phosphate buffers need to be sterilized separately, and then combined after they cool down to room temperature.

Unless your water is made from Antarctic ice, it probably has a significant amount of contaminants in it, in our area water needs to be twice distilled (deionized and then steam distilled). Deionization removes hardening solutes, whereas distillation removes volatiles such as gases in the water (such as ammonia, which occurs in areas where chloramines is added to the water supply). All chemistry requires near 18 mega-ohm water, whereas glassware washing, soap solutions, etc, should be done in deionized water. Residue on bottles are frequently carbonates (calcium), at high temperatures (such as in a drying oven) the carbonate can be converted to oxides and hydroxides, which will trap phosphate. A common reason for scale is that someone made a reagent, left it to soak in tap water but forgot about it. The water evaporated leaving a thin coating of calcium. Soaps will not remove this. Bottles that have hard water scale, rust deposits, on them can be cleaned by soaking with 0.01M hydrochloric acid or dilute phosphoric acid. I can go into almost any lab, and if I look at the glassware shelf long enough I will find bottles with scale on the inside of the bottle.

6. Another tip, something people overlook, don't assume that glassware on the shelf is rinsed properly. Frequently, soap residue is found in the glassware. Add some deI and shake, if you see lots of bubbles, it was not clean. This happens much and in almost every lab I have been in. In managing an animal facility for the last 15 years, I found that soaps are not at neutral pH, some soaps are acidic or alkaline enough to damage plastics in the autoclave (yellowing, fissuring and opaqueness), this can lead to trapping of soap and debris. The solution to the problem is that we made our own soap from technical grade SDS (near saturation) that was pHed to neutral with Tris-HCL. In addition use only enough soap to get the job done, and be wary of soap precipitates (premaking soap solution virtually eliminates precipitation of soap). In many cases, you are using an inert aqueous buffer, simply deI rinse the container when done, put upside down on the bench and let set, reuse. How many folks get tenacious tiny bubbles in the melted agarose the first time they microwave in a fresh glass? That's due to glass-ware washing residue on the glass that cannot be rinsed away. For many buffers, simply deI rinse the vessel, invert glass on bench to dry, reuse.

7. Check your pH calibration solutions occasionally. The pH 10 buffer is extremely susceptible to carbonate neutralization. For this reason I calibrate the meter for all pHs below pH 8.5 with the pH 7.0 and pH 4.0 buffers. Before I use pH 10 calibration I first calibrate at pH 4 and 7 and the pH 10 reading should be 'ballpark-ish', if not open an new bottle and compare pHs. To get the best calibrations I rinse the electrode once in each buffer (usually the buffer from the last calibration, sealed in a 20 ml scintillation vial) and replace it with fresh buffer, then set the calibration point. Occasionally one has to deal with an old electrode, the surfaces are coated and equilibration is quite slow, particularly at pH7.0, that's not good at pH 10 if the electrode takes 15 minutes to equilibrate since pH of the buffer has already dropped. In this case you might purge cover electrode in the scint. vial with paraffin. This is not the most common cause of pH difficulties that I see with trainees, but is a reasonably common problem.

8. There are other issues, with specific buffers like Tris/EDTA in that pH can change markedly with dilution and temperature, thus one has to do some buffer tinkering to get the desired pH. Again, one way to know if you have a bad buffer (either because of age or mismaking) is to make a buffer from newly purchased material and determine the pH both concentrated and diluted so that one knows exactly what to expect. It goes without saying that the buffer needs to be pHed at the standard temperature.

I hope this resolves your recurring problem.

Philip

-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of sudheer sangeetham
Sent: Tuesday, January 03, 2012 10:53 AM
To: Met...@magpie.bio.indiana.edu
Subject: 10X PBS

Hi People

I have few doubts regarding phosphte buffer saline (PBS). Recently I found
one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
not KCl, am I right?

I have prepared the buffer like this

NaH2PO4 ( 0.038M)
Na2HPO4.2H20 ( 0.162M)
NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
did I do any mistake....

Could any one please tell me did i do any mistake?

Thank you in advance

--
Sudheer Babu.S
Ph.D student
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.
_______________________________________________
Methods mailing list

http://www.bio.net/biomail/listinfo/methods

[Jayakumar, R]

Dk,
Finally, something we agree on :-))

Jay


-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of DK
Sent: Thursday, January 05, 2012 11:25 AM
To: met...@magpie.bio.indiana.edu
Subject: Re: 10X PBS

In article <mailman.77.132572...@net.bio.net>, Joshua Silverstein <silverste...@gmail.com> wrote:
>You don't need physical chemistry, just general.
>
>If you take 10X PBS and dilute it with deionized water, it will certainly
>change the pH. For a 10-fold dilution, it should change the pH by
>approximately 1 (log scale).

False. We are talking about buffers here...

DK

>
>
>On Wed, Jan 4, 2012 at 3:38 PM, DK <d...@noemail.thankstospam.net> wrote:
>
>> In article <mailman.75.132570...@net.bio.net>, "Jayakumar,
>> R" <R.Jay...@roswellpark.org> wrote:
>> >Ideally the pH should not change much between 10X and 1X if deionised
>> water was
>> > used for dilution. Presnce of NaCl has nothing to do with pH changes
>> since it
>> > contributes neither hydroxyl nor hydrogen ions
>>
>> You need to refresh your physical chemistry.
>> (Hint: cations compete with protons for binding to anions)
>>
>> DK
>>
>> _______________________________________________
>> Methods mailing list
>> Met...@net.bio.net
>> http://www.bio.net/biomail/listinfo/methods
>>
_______________________________________________
Methods mailing list
Met...@net.bio.net
http://www.bio.net/biomail/listinfo/methods

[sudheer sangeetham]

Hi guys

Thanks you very much to each and everyone for giving their invaluable
suggestions preparing 10x PBS buffer.

[Deitiker, Philip R]

I should clarify #8. When you make a stock Tris buffer, the dissolving of Tris is endothermic. As a consequence the temperature will drop. Therefore the pH of the stock should not be taken until the temperature has equilibrated. If the buffer is basic, cover the beaker until the temperature has equilibrated, and while stirring, as this increase the carbonation of the buffer. There is a difference in pH of Tris between concentrated Tris and highly diluted solutions.

At 22'C
- 3M TRIS (1.5M TRIS BASE + 1.5M TRIS HCL) ~ 8.05 Will significantly drop temperature in volumes over a few mls on creation
- 0.3M TRIS (10 fold dilution of the above) ~ 8.25 May significantly drop temperature in volumes over 100 mls on making
- 0.03M TRIS (100 fold dilution of 3M TRIS) ~ 8.35
- 0.003M TRIS (1000 fold dilution of 3M TRIS) ~ 8.45

To answer the question. Diluting TRIS does alter the pH, but it does not alter pH 1 unit per 10 fold dilution. A perfect buffer at pH 7.0 made in degassed pure H20 will not drop on dilution in degassed pure H20. TRIS is an example of a buffer that has strange qualities on dilution.

-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of Deitiker, Philip R
Sent: Thursday, January 05, 2012 11:05 AM
To: 'Met...@net.bio.net'
Subject: pH irregularity in buffers

There are so many answers to the question for brevities sake I will add mine

1. Phosphate determines the pH, not chloride salts added. These monovalent cation/chloride solutions should have all but no buffering capacity. If the sodium or potassium chlorides show a significant buffering capacity they may have been contaminated by other salts, replace. I've seen worse things happen.

2. Dibasic phosphate is basic and if left exposed to air at room temperature will absorb both moisture and carbon dioxide, this can drop the pH and make the salt more difficult to handle. It is, as basic salts go, pretty resilient to acidification, but conditions in the laboratory may vary (e.g. gas space heaters, storage around areas were flame is used). I seriously doubt this is the issue. The way to know "what's up with this" is keep a record of your buffer salts, for example a 0.01 M solution of dibasic should always be the same pH. So you can record this on the bottle. If the pH of the diluted salt changes over time and it may be time to discard and replace _or_ your pH meter may have a problem. This is a significant problem in the laboratory, DNA hydration buffers at pH8.3 tend to loose about 0.1 pH units per 6 mos, faster if they are frequently opened. Hydroxides are the worst, absorbing both water and carbon dioxide quickly. If the dibasic absorbs enough water it can!

alter the pH because its molar contribution per weight will be less. This is unlikely to be the cause. For both of these, if you make the solution repeated and repeatedly you get the same deviant pH, then replace.

4. Two salts were confused at weighing, whoops. Seen this one a lot, I've done it and a number of my trainees have done it. The solution remade comes out perfect pH. As my TA used to say 'you make a cook-book buffer, if the pH is wrong, that's a good thing, don't adjust the pH, remake the buffer'.

Here's the recipe for 10X PBS pH 7.2

NaCl 87.7
NaH2PO4 3.05
Na2HPO4 11.1
Initial ddH20 0.9 L (e.g twice distilled water is all that we use, house DI systems cannot be relied upon)
ddH20 Water to 1L

Dibasic will not dissolve quickly, requires about 2-4 hours to dissolve, so keep the vessel covered with parafilm or flat piece of glass. This is not something that you leave overnight on the bench stirring in open air.

Make 100 ml of 1X and test the pH. pH should be 7.2 +/- 0.05 you can lower the monobasic and increase the dibasic to achieve pH 7.4 (See other post). NaOH can be added in small amounts to correct the pH, in most case pH adjustment is not needed.

5. Divalent and trivalent cations are easily trapped by higher ionization states of phosphate, thus heating phosphate can trap calcium and potentially drop the pH. Divalent cation solutions should be added at STP and in diluted form. In addition, one expects as the pH to go up, the stability of calcium phosphate in solution to go down. Sometimes we heat buffers to get them in solutions, but this may not work well with some buffers, or may cause problem with sterilizing solutions. In this case calcium chloride and phosphate buffers need to be sterilized separately, and then combined after they cool down to room temperature.

Unless your water is made from Antarctic ice, it probably has a significant amount of contaminants in it, in our area water needs to be twice distilled (deionized and then steam distilled). Deionization removes hardening solutes, whereas distillation removes volatiles such as gases in the water (such as ammonia, which occurs in areas where chloramines is added to the water supply). All chemistry requires near 18 mega-ohm water, whereas glassware washing, soap solutions, etc, should be done in deionized water. Residue on bottles are frequently carbonates (calcium), at high temperatures (such as in a drying oven) the carbonate can be converted to oxides and hydroxides, which will trap phosphate. A common reason for scale is that someone made a reagent, left it to soak in tap water but forgot about it. The water evaporated leaving a thin coating of calcium. Soaps will not remove this. Bottles that have hard water scale, rust deposits, on them can be cleaned by soaking with 0.01!

M hydrochloric acid or dilute phosphoric acid. I can go into almost any lab, and if I look at the glassware shelf long enough I will find bottles with scale on the inside of the bottle.

6. Another tip, something people overlook, don't assume that glassware on the shelf is rinsed properly. Frequently, soap residue is found in the glassware. Add some deI and shake, if you see lots of bubbles, it was not clean. This happens much and in almost every lab I have been in. In managing an animal facility for the last 15 years, I found that soaps are not at neutral pH, some soaps are acidic or alkaline enough to damage plastics in the autoclave (yellowing, fissuring and opaqueness), this can lead to trapping of soap and debris. The solution to the problem is that we made our own soap from technical grade SDS (near saturation) that was pHed to neutral with Tris-HCL. In addition use only enough soap to get the job done, and be wary of soap precipitates (premaking soap solution virtually eliminates precipitation of soap). In many cases, you are using an inert aqueous buffer, simply deI rinse the container when done, put upside down on the bench and let set, reuse. How m!

any folks get tenacious tiny bubbles in the melted agarose the first time they microwave in a fresh glass? That's due to glass-ware washing residue on the glass that cannot be rinsed away. For many buffers, simply deI rinse the vessel, invert glass on bench to dry, reuse.

7. Check your pH calibration solutions occasionally. The pH 10 buffer is extremely susceptible to carbonate neutralization. For this reason I calibrate the meter for all pHs below pH 8.5 with the pH 7.0 and pH 4.0 buffers. Before I use pH 10 calibration I first calibrate at pH 4 and 7 and the pH 10 reading should be 'ballpark-ish', if not open an new bottle and compare pHs. To get the best calibrations I rinse the electrode once in each buffer (usually the buffer from the last calibration, sealed in a 20 ml scintillation vial) and replace it with fresh buffer, then set the calibration point. Occasionally one has to deal with an old electrode, the surfaces are coated and equilibration is quite slow, particularly at pH7.0, that's not good at pH 10 if the electrode takes 15 minutes to equilibrate since pH of the buffer has already dropped. In this case you might purge cover electrode in the scint. vial with paraffin. This is not the most common cause of pH difficulties that I !

see with trainees, but is a reasonably common problem.

8. There are other issues, with specific buffers like Tris/EDTA in that pH can change markedly with dilution and temperature, thus one has to do some buffer tinkering to get the desired pH. Again, one way to know if you have a bad buffer (either because of age or mismaking) is to make a buffer from newly purchased material and determine the pH both concentrated and diluted so that one knows exactly what to expect. It goes without saying that the buffer needs to be pHed at the standard temperature.

I hope this resolves your recurring problem.

Philip

-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of sudheer sangeetham
Sent: Tuesday, January 03, 2012 10:53 AM
To: Met...@magpie.bio.indiana.edu
Subject: 10X PBS

Hi People

I have few doubts regarding phosphte buffer saline (PBS). Recently I found
one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
not KCl, am I right?

I have prepared the buffer like this

NaH2PO4 ( 0.038M)
Na2HPO4.2H20 ( 0.162M)
NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
did I do any mistake....

Could any one please tell me did i do any mistake?

Thank you in advance

--
Sudheer Babu.S
Ph.D student
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.

_______________________________________________
Methods mailing list

[Jayakumar, R]

Philip,
Indeed, temperature affects pH a lot, a concept quite forgotten by many researchers. pH should be estimated at the appropriate temperature especially when preparing certain buffers like protein lysis buffers which require a specific pH at much cooler temperatures for cell lysis. Adjusting these buffers to their desired pH at room temperature will not reflect its actual pH at the much cooler temperatures preferred for lysis.
Jay

[Dr Engelbert Buxbaum]

In article <mailman.70.132562...@net.bio.net>,
hroy...@nmsu.edu says...

>
> Since you say you have few questions, I am assuming that you are quite
> certain about your composition. PBS stands for "Phosphate buffered
> saline" so it is NaCl, not KCl. Usually we use Na-salts for the
> phosphate combination. Potassium phosphate and -biphosphate may be
> used, but for all practical purposes it is Na- that is preferred since
> lot of compounds do not go well with K+

Actually no - PBS (in the strict sense) is designd to resemble the
extracellular fluid, and hence contains both Na and K:

mM g/l
NaCl 137 8.01
KCl 2.7 0.20
Na2HPO4 ? 2 H2O 10 1.78
KH2PO4 2.0 0.27

Depending on application, Mg and Ca may also be added (e.g., Dulbecco's
PBS). Even closer to extracellular fluid is the Krebs-Henseleit
solution, which also contains glucose and bicarbonate.

Btw, if one wants to work with intracellular enzymes, PBS should not be
used, and replaced with a high K, low Na medium!

[Dr Engelbert Buxbaum]

In article <mailman.80.132578...@net.bio.net>,
R.Jay...@roswellpark.org says...

>
> I feel that there is no necessity to teach about pH to this group of
> scientists. But to know why deionised water cannot change the pH of
> buffers significantly

Actually, it does, because the pH is determined not by the
concentrations of corresponding base and acid, but by their activities.
And these change upon dilution, especially when you start with highly
concentrated solutions, where non-ideality is more significant.

[Jayakumar, R]

Very true, but the change in pH is really not that much within the pKa range and is deionised water has a lower impact on their activities than tap water or distilled water has. I have not tested this and hence I hope I am right :-) Please correct me if I am not.
Jay

[Pow Joshi]

I like this discussion. A lot of info about old forgotten physical chem :)
Thank you DK, Engelbert, Jayakumar, and others.

On 9 January 2012 03:08, DK <d...@no.email.thankstospam.net> wrote:

> In article <MPG.29740567a...@News.Individual.DE>, Dr Engelbert

> Buxbaum <engelber...@hotmail.com> wrote:
> >
> >Btw, if one wants to work with intracellular enzymes, PBS should not be
> >used, and replaced with a high K, low Na medium!
>

> Well, if we are that strict then chloride salts should not be used either
> :-)
> Intracellular Cl- concentration is pretty low and most free anions are
> large organics. For "pseudo-intracellular" solution, I've isethionate in
> one system and glutamate in another. Some people like using tartrate.
>
> To be sure, it does make a difference. Halide ions bind proteins a lot.
>
> DK
>
> P.S. Recently I came across your book, Engelbert. Pretty good!
> Made me envious :-)

[Duncan Clark]

Historians believe that in newspost
<mailman.105.13261...@net.bio.net> on Mon, 9 Jan 2012,
Pow Joshi <pow....@gmail.com> penned the following literary
masterpiece:

>I like this discussion. A lot of info about old forgotten physical chem :)
>Thank you DK, Engelbert, Jayakumar, and others.

Seconded.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

[Deitiker, Philip R]

I think for most enzymes they give optimal conditions, so you make the buffer they say to use or use the one they supply, or as is the case order the buffer from NEB or the enzyme supplier. The level of K+ or Na+ in most cases is not going to make that much difference. For some proteins, like globins, its best to dissolve them in cold water and then dilute them in the working buffer (usually PBS).

On the issue of pH, if you are using extremely dilute buffers, anything can make a difference, and deionized water is not a clearly defined reagent. Deionized water is produced from RO membranes, the membranes age over time, many institutions add salts to the water to protect the pipes, etc. One common thing that is added in areas where the water is acidic is calcium salts to protect the copper pipes from pitting. Raising the pH past neutral also protects the pipes (calcium and copper interact, and higher pH lowers the rate of oxidation). But raising the pH converts acidic ammonia into dissolved ammonia gas, which has greater mobility. Ammonia is not technically added to water but ammonia adducts are added when the level of chlorine is too high to add more. I don't know any lab, and I have worked in quite a few over the years, that uses RO deionized water directly. IOW it cannot be counted on, therefore go for the ~18 mega-ohm water, purchased water, or double distilled. You would be better off purchasing distilled water from the grocery store than using 'house' deionized water. It is clearly true that more most biochemistry you don't need 18 mega-ohm water as a buffer starting mat, but you do need something more reliable than deionized water.

-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of DK
Sent: Monday, January 09, 2012 2:08 AM
To: met...@magpie.bio.indiana.edu
Subject: RE: 10X PBS

In article <MPG.29740567a...@News.Individual.DE>, Dr Engelbert Buxbaum <engelber...@hotmail.com> wrote:
>

>Btw, if one wants to work with intracellular enzymes, PBS should not be
>used, and replaced with a high K, low Na medium!

Well, if we are that strict then chloride salts should not be used either :-)
Intracellular Cl- concentration is pretty low and most free anions are
large organics. For "pseudo-intracellular" solution, I've isethionate in
one system and glutamate in another. Some people like using tartrate.

To be sure, it does make a difference. Halide ions bind proteins a lot.

DK

P.S. Recently I came across your book, Engelbert. Pretty good!
Made me envious :-)

[Jayakumar, R]

My pleasure Pow :-)
Jay

-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of Pow Joshi
Sent: Monday, January 09, 2012 3:18 PM
To: methods
Subject: Re: 10X PBS

I like this discussion. A lot of info about old forgotten physical chem :)
Thank you DK, Engelbert, Jayakumar, and others.

On 9 January 2012 03:08, DK <d...@no.email.thankstospam.net> wrote:

> In article <MPG.29740567a...@News.Individual.DE>, Dr Engelbert
> Buxbaum <engelber...@hotmail.com> wrote:
> >

> >Btw, if one wants to work with intracellular enzymes, PBS should not be
> >used, and replaced with a high K, low Na medium!
>

> Well, if we are that strict then chloride salts should not be used either
> :-)
> Intracellular Cl- concentration is pretty low and most free anions are
> large organics. For "pseudo-intracellular" solution, I've isethionate in
> one system and glutamate in another. Some people like using tartrate.
>
> To be sure, it does make a difference. Halide ions bind proteins a lot.
>
> DK
>
> P.S. Recently I came across your book, Engelbert. Pretty good!
> Made me envious :-)
>

> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
_______________________________________________
Methods mailing list
Met...@net.bio.net
http://www.bio.net/biomail/listinfo/methods

[Dr Engelbert Buxbaum]

In article <FfxOq.12880$4b4....@newsfe06.iad>,
d...@no.email.thankstospam.net says...

> P.S. Recently I came across your book, Engelbert. Pretty good!
> Made me envious :-)

Thanks -E

--
Car (noun): erratically moving obstacle on the road