# How can I prepare a PBS (Phosphate Buffer Solution)? pH=7.4 [closed]

Phosphate-buffered saline (abbreviated PBS) is a buffer solution commonly used in biological research. It is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate.

• You want $1\times$PBS or $10\times$PBS? Or just a protocol for prep? – Mathew Mahindaratne Apr 3 '19 at 18:18

The preparation of pH 7.4 PBS (with KCl and KH2PO4) is described on this site here. Preparation of many other buffer systems are also described.

Here are the conditions (lifted from the linked site)

Prepare 800 mL of distilled water in a suitable container.
Add 8 g of NaCl to the solution.
Add 200 mg of KCl to the solution.
Add 1.44 g of Na2HPO4 to the solution.
Add 240 mg of KH2PO4 to the solution.
Adjust solution to desired pH (typically pH ≈ 7.4).
Add distilled water until volume is 1 L.

Since $$\mathrm{p}K_\mathrm{a}$$ of monosodium phosphate ($$\ce{NaH2PO4}$$) is 7.21, for biological applications, $$\ce{NaH2PO4}$$ and its conjugate base, disodium phosphate ($$\ce{Na2HPO4}$$), can be used to generate buffers of $$\ce{pH}$$ values around 7. Usually, biochemists use the Henderson-Hasselbalch equation, $$\left(\ce{pH} = \mathrm{p}K_\mathrm{a} + \log \frac{[\text{Base}]}{[\text{Acid}]}\right)$$, to calculate what ratio of acid to base is required to make a buffer of the desired $$\ce{pH}$$. Technically, this buffer is called phosphate buffer (PB).

Thus, to prepare buffer with $$\ce{pH} = 7.4$$, let's calculate the $$\frac{[\text{Base}]}{[\text{Acid}]}$$ from above equation: $$\frac{[\text{Base}]}{[\text{Acid}]} = 10^{\left(\ce{pH} - \mathrm{p}K_\mathrm{a} \right)} = 10^{\left(7.4 - 7.21 \right)} = 10^{0.19}=1.549$$

Technically, if you want $$\pu{1.0 M}$$ buffer, that means $$[\text{Acid}] + [\text{Base}] = 1.0$$. So, you can calculate $$[\text{Acid}]$$ and $$[\text{Base}]$$ using these two relationships to get $$\pu{1.0 M}$$ buffer of $$\ce{pH} = 7.4$$: $$[\text{Base}] = 1.549 \times [\text{Acid}]$$, and $$[\text{Base}] = 1.0 - [\text{Acid}]$$. Once calculated, use that amount to prepare your buffer with procedure given by Waylander in his answer. You may use phosphoric acid and $$\ce{NaOH}$$ solutions to adjust $$\ce{pH}$$ to desired value (7.4 in this case), if you want to have only phosphate components in your solution. Also, final make up to the &\pu{1.0 L}\$, you may use a volumetric flask if desired.

However, phosphate buffer saline (PBS) closely mimics the $$\ce{pH}$$, osmolarity, and ion concentrations of the human body. Therefore, it is usually, $$\pu{0.01 M}$$ (or $$\pu{10 mM}$$) in $$\ce{Na2HPO4}$$ (base), $$\pu{0.0018 M}$$ (or $$\pu{1.8 mM}$$) in $$\ce{KH2PO4}$$ (acid), $$\pu{0.137 M}$$ (or $$\pu{137 mM}$$) in $$\ce{NaCl}$$, and $$\pu{0.0027 M}$$ (or $$\pu{2.7 mM}$$) in $$\ce{KCl}$$. This is called $$1 \times$$PBS. If you want $$10 \times$$PBS, you may multiply each component by 10. Once, all components are weighted into large enough container, use enough deionized (or distilled) water to dissolve them, and ensure to adjust $$\ce{pH}$$ to $$7.4$$ using phosphoric acid and $$\ce{NaOH}$$ solutions before make it up to 1.0 L using deionized (or distilled) water.

The internet is replete with recipes of all kinds and many tables; so, I will respond on the practical aspect of the question. In reality, many people just buy a premixed packet, mix it with the correct volume of water, and get on with their research. Additionally some buy it as a liquid ready to go. So, depending on the volume needed, your resources, and the stringency of the experiments, mixing up buffer and manually adjusting the pH may just not be worth the effort.