How to prepare a phosphate buffered saline solution

Phosphate buffered saline, or PBS solution, is a pH buffer solution commonly used in biology laboratories throughout the world. It is a solution of different salts that has the acid/base pair dihydrogen phosphate/hydrogen phosphate (H ₂ PO ₄ ^-/ HPO ₄ ^– ) as a buffer system. This buffer system is ideal for carrying out experiments of a biological and medical nature, since dihydrogen phosphate (also called diacid phosphate) has a pKa of 7.2, so an equimolar solution of the conjugated pair regulates the pH around 7. .2, adequately simulating the human physiological pH.

In addition to correctly modeling the pH of various body fluids, the PBS solution also has some dissolved salts, such as sodium chloride, potassium chloride, and in some cases calcium and magnesium chloride. Together, the salts and phosphates provide the solution with an osmolarity equivalent to that of blood plasma (around 290 mOsm/L), making it an isotonic solution, ideal for studying cells and other systems under physiological conditions.

Composition of the PBS buffer

There are different formulations that fall under the category of phosphate buffered saline.

As a minimum, this solution must contain sodium chloride as the main salt (from which the term saline solution comes from) and it must also contain at least one salt of one of the two components of the buffer system. It could be sodium or potassium dihydrogen phosphate, or sodium or potassium hydrogen phosphate could also be used.

In the first case, a strong base such as sodium hydroxide must also be added to neutralize half of the acid salt and thus generate the complete buffer system. In the second case, we start in the presence of only the basic salt, so we must neutralize half of it in order to generate enough conjugate acid.

A typical example of the composition of one of these PBS solutions is:

• Sodium chloride (NaCl) 140 mM.
• 12 mM sodium hydrogen phosphate.
• Hydrochloric acid until reaching the desired pH.

However, in the most common case, the PBS solution tries to simulate as much as possible the characteristics of biological fluids, which include ions other than sodium, the various phosphates and chlorides. For this reason, PBS solutions are usually slightly more complex than described above. In the solutions whose preparation is described below, the final concentrations of the relevant ionic species before adjusting the pH are:

_{The particular concentration of hydrogen phosphate (HPO 4} ^2- ) and dihydrogen phosphate (H ₂ PO ₄ ^– ) ions , the dominant species in equilibrium at pH values ​​near 7, will depend on the particular pH to which the solution is adjusted. For example, if the pH is brought to 7.2, the pKa of dihydrogen phosphate, both ions will be in a 1:1 ratio with concentrations around 5.9 mM.

The following is the protocol for the preparation of a typical PBS solution for laboratory use that, in addition to phosphates, chlorides, and sodium ions, also contains potassium, calcium, and magnesium ions.

Preparation of phosphate buffered saline

There are two typical ways to prepare a PBS solution for use in a biology, medicine, or biochemistry laboratory: by weighing and directly dissolving the necessary salts, or by diluting a more concentrated solution.

Both procedures give the same result, but the second is much more practical in laboratories where this solution is used frequently. This is because, by preparing just one concentrated solution, any required dilution can be quickly prepared at the time it is needed, a procedure that is much faster and less error-prone than weighing and dissolving 4 to 6 different salts. each time a solution must be prepared.

Preparation of a 1x PBS solution by direct weighing

The following table contains the masses of the different components that must be weighed to prepare 250 mL, 500 mL, and 1 L of standard PBS solution with a pH between 7.2 and 7.4 and an osmolarity of approximately 290 mOsm/L. This solution is usually called PBS 1x since it is the one with the appropriate concentration for use in experiments.

preparation protocol

The preparation of these solutions is carried out by means of the following steps:

1. All salts are weighed separately using an analytical balance.
2. In a suitable sized beaker or beaker, add water to make up to four fifths of the total volume of the solution to be prepared (200 mL of water for the 250 mL solution, 400 mL for the 500 mL solution, or 800 mL for the 1 L solution). If possible, use ultrapure (deionized) water. It is not necessary to measure this amount of water very accurately.
3. Add the previously weighed salts one by one and shake with a magnetic stirrer or a glass rod until completely dissolved. The resulting solution should have a more alkaline pH of around 7.8 or 7.9.
4. Using a pH-meter or pH meter and under constant stirring, add 0.1 M hydrochloric acid (HCl) solution until the desired pH is reached. To reach a pH of about 7.4 in the 1 L solution, about 30 mL of 0.1 M HCl will be needed. For the other two, proportionately smaller amounts will be needed.
5. Once the desired pH is reached, transfer the solution to a volumetric flask of suitable capacity (250 mL, 500 mL or 1 L, as the case may be).
6. Once in the volumetric flask, add ultrapure water up to the measurement mark.
7. With the balloon stopper in place, shake the solution to ensure the solution is homogeneous.

Preparation of a 10x PBS solution by direct weighing

The 10x PBS solution is a ten times more concentrated version of the standard solution. This solution is not used to carry out experiments, but as a concentrated solution to prepare the standard solution by dilution.

The following table contains the masses of the different components that must be weighed to prepare 250 mL, 500 mL, and 1 L of 10x PBS solution with a pH between 7.2 and 7.4 and an osmolarity of approximately 2.90 Osm/ L.

preparation protocol

The preparation of any of the concentrated solutions is carried out by following the same steps as in the previous case. The only important changes are the masses of the solutes, which are ten times greater.

On the other hand, since it is a much more concentrated solution, the pH adjustment should also be carried out with a more concentrated HCl solution, preferably 1 M. It should not be carried out with the same 0.1 M solution, since the volume that will be needed to reach a pH between 7.2 and 7.4 will end up exceeding the final total volume of the solution that you want to prepare.

Preparation of 1x PBS solution by dilution of 10x solution

The 10x concentrated solution described in the previous section is used as a stock or mother solution for the preparation on the spot of small amounts of 1x PBS solution. The following table indicates the amounts of concentrated solution that must be measured according to the amount of 1x PBS solution that you want to prepare.

preparation protocol

In all cases, the preparation of the dilutions requires the use of a volumetric pipet whose capacity is equal to the volume of the 10x solution to be measured, as well as a volumetric flask whose volume is equal to the amount of solution that is desired. prepare.

The steps are the following:

1. Verify that the concentrated solution has not precipitated. If so, shake it until completely dissolved.
2. Pour a slightly larger amount of the 10x PBS solution to be measured into a suitable sized beaker.
3. Using a suitable volume volumetric pipet (as described above) measure the corresponding volume of the concentrated solution and add it to the appropriate volumetric flask.
4. Add water up to the capacity mark.
5. With the balloon stopper in place, shake the solution to ensure the solution is homogeneous.

Sterilization and storage of PBS solutions

Although these solutions can almost always be used immediately after preparation, some applications of phosphate-buffered saline require that it be sterilized first. In these cases, a 20 minute sterilization in an autoclave at 15 psi is sufficient.

Regarding storage, both 1x and 10x PBS solutions can be stored at room temperature.

If necessary, they can also be stored under refrigeration to extend their shelf life. In the case of the standard solution, cooling does not cause any problem. However, in the case of the 10x solution, being a relatively concentrated solution, cooling can cause the precipitation of some salts, especially if the solution contains calcium and magnesium.

Precipitation is normally not a problem, as long as steps are taken to redissolve all solutes before using this solution to prepare the respective dilutions. To achieve this, it is sufficient to allow the solution to reach room temperature and then shake it until all the solids formed are dissolved. If necessary, the solution can also be heated slightly to help dissolve the solutes more quickly.

References

AAT Bioquest, Inc. (2021, October 20). PBS (Phosphate Buffered Saline) (1X, pH 7.4).» . Retrieved from https://www.aatbio.com/resources/buffer-preparations-and-recipes/pbs-phosphate-buffered-saline

Checa R., A. (2017, December 13). Method: Phosphate buffer solution (PBS) . know. http://conogasi.org/articulos/metodo-disolucion-amortiguadora-de-fosfastos-pbs/

Gil, M. (2019, April 17). Phosphate buffer (PBS): rationale, preparation and uses . lifer. https://www.lifeder.com/buffer-phosphates/