[merlinthewitch]

Hey all!

I'm a university student employed in a lab for the summer doing a bunch of immunohistochemistry. PBS (phosphate buffer) is a common solution used in the procedures so I've made it up several times using a recipe already present in the lab. The recipe is for a 10X PBS solution but no where does it say what the molarity is. I'm embarrassed to say that I've actually forgotten how to calculate this. I was hoping someone could point me in the right direction or give me a refresher. Most of the molarity calculation help on the internet seems to be for simpler problems, like adding one compound in grams to water and then figuring out the molarity. I can do that but I'm having problems remembering how to do more complicated problems.

The ingredients for the 10X buffer solution I use in the lab is as follows:

80 g sodium chloride (NaCl)
2g potassium chloride (KCl)
21.7 g Sodium phosphate heptahydrate (Na2HPO4.7H2O)
2g potassium phosphate monohydride (KH2PO4)
Total volume of water will be 1L

Any help with this would be greatly appreciated!

Thank you,
V

[sjb]

The ingredients for the 10X buffer solution I use in the lab is as follows:

80 g sodium chloride (NaCl)
2g potassium chloride (KCl)
21.7 g Sodium phosphate heptahydrate (Na2HPO4.7H2O)
2g potassium phosphate monohydride (KH2PO4)
Total volume of water will be 1L

Any help with this would be greatly appreciated!

Thank you,
V

If you're certain that this is the recipe, then things may be slightly indeterminate, as to make, e.g. 1 litre of a 1 M solution of KCl, you take 1 M of KCl, and then make it up to 1 litre of solution, rather than simply adding 1 litre of solvent. It might be that this gives an error that is acceptable in this case, but I am not over familiar with the needs of 10X PBS standards

You can probably calculate each of the components individually, then add the sodiums from the NaCl and Na₂HPO₄.7H₂O and similar..?
S

[merlinthewitch]

If you're certain that this is the recipe, then things may be slightly indeterminate, as to make, e.g. 1 litre of a 1 M solution of KCl, you take 1 M of KCl, and then make it up to 1 litre of solution, rather than simply adding 1 litre of solvent. It might be that this gives an error that is acceptable in this case, but I am not over familiar with the needs of 10X PBS standards

You can probably calculate each of the components individually, then add the sodiums from the NaCl and Na₂HPO₄.7H₂O and similar..?
S

Well, this is the recipe that's in our "experimental protocols" binder that I had been told to use, which had apparently been made by a previous laboratory manager. No one in the lab seems to know what molarity of it is which surprised me since... what if we need to change it? Hah ha. So I have set to find out and then realized I couldn't remember the calculations.

In preparing it I generally dissolve all of the ingredients in less than 1L of water and then I make it up to 1L when everything is nicely dissolved.

To clarify the last part, do you mean I should calculate the molarity of each of the ions individually? I can do that I'm just having trouble remembering how the molarity of the individual parts would translate to the molarity of the entire solution. It's probably something simple that I'm forgetting.

[Borek]

You have mixture of different salts, there is no such thing of "molarity of the mixture". You can calculate separate molarities of each salt, or - even better - molarity of each individual ion.

[merlinthewitch]

You have mixture of different salts, there is no such thing of "molarity of the mixture". You can calculate separate molarities of each salt, or - even better - molarity of each individual ion.

Okay, that definitely makes sense. And just now I calculated the molarity of the ions (Na⁺, Cl^-, K⁺, HPO₄^2-, H₂PO₄^-).

So thanks to you both for your help.

I think I was confused since sites like this (http://www.ihcworld.com/_protocols/washing_buffers/pbs.htm) would state that something is a 0.1 M PBS solution or a 0.2 M solution. However, they must be referring to just one of the ions... So I'll calculate the theoretical values for those and take a look at which one matches. Thanks again.

[rats79]

I routinely run immunos in my lab.   Your 10X PBS recipe is a 0.1M PBS solution. For most immuno, for washes etc, you would use a 0.01 M (in other words 10 mM) 1X PBS solution which contains the following
  1X PBS:
  137 mM NaCl
    2.7  mM KCl
    10  mM Na2HPO4
     2     mM KH2PO4
therefore, 10X PBS STOCK solution would be:
  1.37 M NaCl
    27  mM KCl
    100  mM Na2HPO4
     20     mM KH2PO4

You calculate this in the following manner:
For example 10X PBS
Given: MW NaCl 58.44, that is 58.44 g in 1 L = 1 M (58.44 g/L), your desired molarity 1.37 M in 1 L
58.44 g/L x 1.37 M =  80 g in 1 L
Given MW KCl 74.55, desired molarity 27 mM (or in other words 0.027 M) in 1 L
74.55 g/l x 0.027 M =2 g in 1 L
etc....

Hope this helps!

[merlinthewitch]

Wow! That's really helpful, rats79. Thank you so much. Although I understand your sample calculations, I think I'm still a little confused about how it has been determined that those amounts will lead to the 10x PBS being 0.1 M. What exactly is being referenced here since each of the salts/ions has a different molarity?

[Phlogiston]

I routinely run immunos in my lab.   Your 10X PBS recipe is a 0.1M PBS solution. For most immuno, for washes etc, you would use a 0.01 M (in other words 10 mM) 1X PBS solution which contains the following
  1X PBS:
  137 mM NaCl
    2.7  mM KCl
    10  mM Na2HPO4
     2     mM KH2PO4
therefore, 10X PBS STOCK solution would be:
  1.37 M NaCl
    27  mM KCl
    100  mM Na2HPO4
     20     mM KH2PO4

You calculate this in the following manner:
For example 10X PBS
Given: MW NaCl 58.44, that is 58.44 g in 1 L = 1 M (58.44 g/L), your desired molarity 1.37 M in 1 L
58.44 g/L x 1.37 M =  80 g in 1 L
Given MW KCl 74.55, desired molarity 27 mM (or in other words 0.027 M) in 1 L
74.55 g/l x 0.027 M =2 g in 1 L
etc....

Hope this helps!

Where do the desired molarities come from that you've listed- is it for the ionic strength, or perhaps the osmotic pressure of the solution?

[baseball07]

I have a question.  Can NaH2PO4 be substituted for KH2PO4 (which you are using in this thread)?  I have seen both and I am wondering what the difference is.  Thank you.