Physiological Solutions

Physiological Solutions for Biochemical investigations

The body's fluids are all electrolyte solutions, meaning they contain ions (charged particles) such as Na⁺, K⁺, and Cl^- dissolved in water. Electrolyte solutions are necessary to maintain water balance, pH of body fluids, nervous transmission, muscle contraction, etc.

Physiologists divide the solutions of the body into intracellular fluid (ICF) which is the solution found inside cells and extracellular fluid (ECF) which is the solution in which cells are bathed. The ECF can be further divided into a) plasma volume and b) interstitial volume. They both function to provide the cell with what it needs and protect it from changes in the outer environment. Physiological solutions may be different for different purposes. Some solutions contain higher solutes while some contain less solute in the solution. There is certain terminology that is used when trying to predict whether osmosis will occur. These terms are used when comparing two solutions separated by a semi-permeable membrane.

·         A hypertonic solution has a greater solute concentration than an adjacent solution after accounting for the permeability of the membrane (after considering whether the solute will diffuse).  Cells get shrinkage in hypertonic solutions.

·         A hypotonic solution has a lesser solute concentration than the adjacent solution after accounting for the permeability of the membrane. Cells get turgid  and may burst in hypotonic solutions (e.g. RBC get hemolyzed in distilled water)

·         An isotonic solution is when the two solutions have the same solute concentration. Again, the permeability of the membrane has to be accounted for first. There are no changes in cell activity in isotonic solution. These types of solutions are best for biochemical investigations.

In biochemical investigations physiological solutions play vital roles in the protection of biomolecules under the study. Without use of physiological solutions, it is very difficult to achieve the active form of cellular molecules. Besides protecting biomolecules, it also protects whole cell. So in order to study biomolecules, suitable physiological solutions must be used during extraction and purification of such molecules. Some of the important physiological solutions are normal saline and buffers. In case of biochemical investigations, buffers are most commonly used.

Buffers

Proteins have a pH dependent charge and many of the properties of proteins change with pH. Consequently, in working with proteins, it is important to control the pH. This is achieved by the use of buffers, and so at the outset it is important to have some insight into buffers, to know which buffer to use for any particular purpose, and how to make up the buffer.

Buffers are solutions of weak acids or bases and their salt(s), which resist changes in pH. Weak acids and bases are distinguished from strong acids and bases by their incomplete dissociation. In the case of a weak acid the dissociation is:-

HA      ®        H⁺ + A^-

 and the dissociation constant is:-        Ka = [H+][A-]/[HA]

From above Equations i and ii are forms of the Henderson-Hasselbalch equation, which can be written in a general form as:-
Pka = pH - log [basic species]/[acidic species]

                                                           

From which it can be seen that, when [basic species] = [acidic species],

then,                                                   

It will be noticed that when pH = pKa, the solution resists changes in pH, i.e. it functions best as a buffer in the range pH = pKa ± 0.5.

In acetate buffer, CH3COOH is the acidic species in this buffer and CH3COO- is the basic species. It may be observed that a solution of acetic acid itself (CH3COOH) will have a pH less than the pKa of acetic acid. Conversely, a solution containing only sodium acetate will have a pH greater than the pKa of acetic acid. It is important to understand this point in order to appreciate how to make an acetate buffer using the approach described in figure 1.

Figure 1: Schematic titration curve of acetic acid

A tri-protic acid, such as phosphoric acid will yield a titration curve having three inflexion points (Figure 2), corresponding to the three pKa values of phosphoric acid.

Figure 2: Schematic titration curve of phosphoric acid

For most biochemical purposes, pKa2 is of greatest interest, since it is closest to the pH of the extracellular fluid of animals.

Note that:-

At pKa₂,                     [NaH₂PO₄] = [Na₂HPO₄]

At pH< pKa₂              [NaH₂PO₄] > [Na₂HPO₄]

At pH> pKa₂              [NaH₂PO₄] < [Na₂HPO₄]

Put another way, a solution __ NaH₂PO₄ will have a pH less than pKa₂ and a solution of Na₂HPO₄ will have a pH greater than pKa₂. It is important to understand this point in order to appreciate how to make a phosphate buffer using the approach described below.

Making a buffer

A simpler method for preparing is as follows:-

1. Choose the buffer:

A buffer works best at its pKa, so the first step is to choose a buffer with a pKa as close as possible to the desired pH.

2. Identify the buffering species:

As described above, a buffer consists of two components: a weak acid and its salt or a weak base and its salt. The second step is thus to identify the species which will constitute the buffer. For example, in the case of an acetate buffer, the buffering species are CH₃COOH and CH₃COONa.

In a phosphate buffer at pKa₂, the buffer species are NaH₂PO₄ and Na₂HPO₄.

3. Identify whether the buffer is made from an acid or a base:

The two buffer examples given above are made from acids, acetic acid or phosphoric acid. In the case of phosphate buffer at pKa₂, the acid is NaH₂PO₄. An example of a buffer made from a base is Tris/Tris- HCl, which buffers best at pH 8.1, the pKa of Tris.

4. Choose the species that gives no by-products when titrated:

Almost all buffers can be made up by weighing out one component, dissolving in a volume just short of the final volume, titrating to the right pH, and making up to volume. It is not necessary to make up separate solutions of the two buffer constituents - the required salt can be generated in situ by titrating the acid with an appropriate base - or vice versa in the case of a buffer made from a base. [Remember: Titrate an acid “up” (i.e. with a strong base) and titrate a base “down” (i.e. with a strong acid)].

Remember,                  acid + base = salt + water

and,                             a buffer = (acid + its salt ) or (base + its salt)

                                                                                              

The term “its salt” is important. For example, if we wanted to make an acetate buffer, it is easy to

identify that this buffer is made from acetic acid and its salt, say, sodium acetate. But,

Q: Could the required mixture of CH₃COOH and CH₃COONa be made by titrating a solution of CH₃COONa to the correct pH with HCl?

A: No! Because the reaction in this case is:-

CH₃COONa + HCl    ®        CH₃COOH + NaCl   

and the resultant solution contains NaCl, which is an unwanted byproduct and which is not a salt of acetic acid (i.e. it is not “its salt”).

On the other hand,

Q: Could the required mixture be made by titrating a solution of CH₃COOH with NaOH?

A: Yes! The reaction in this case is:-

            CH₃COOH + NaOH  ®        CH₃COONa + H₂O

Which yields only the salt of acetic acid and water, i.e. there are no byproducts. Similarly, in the case of a phosphate buffer, if one chooses Na₂HPO₄, the pH of a solution of this salt will be higher than pKa, and this will require titration with an acid. If one chooses HCl, the reaction will be:-

            Na₂HPO₄ + HCl         ®        NaH₂PO₄ + NaCl

Which yields NaCl as an unwanted by-product. (And if one chooses NaH₂PO₄, this will change the phosphate molarity.) However, if one starts with NaH₂PO₄, the pH of a solution of this salt will be lower than pKa, and this will require titration with a base. If one chooses NaOH, the reaction will be:-

            NaH₂PO₄ + NaOH     ®        Na₂HPO₄ + H₂O

Which yields only the desired salt (Na₂HPO₄) and water.

For a Tris buffer, one should start with the free base and titrate this with HCl to yield the salt of Tris, Tris-HCl.

5. Calculate the mass required to give the required molarity:

Having settled on the single buffer component to be weighed out, calculate the mass required to give the required molarity, when finally made up to volume. For example, the molarity of a phosphate buffer is determined by the molarity of the phosphate moiety (-PO₄^3-), which does not change when NaH₂PO₄ is titrated to Na₂HPO₄. If a liter of a 0.1 M buffer is required, then 0.1 moles of NaH₂PO₄ can be weighed out.

6. Add all other components titrate and make up to volume

Buffers often contain ingredients other than the two buffering species. For ion-exchange elution the buffer might contain extra NaCl, and buffers often contain preservatives such as NaN₃ or chelating agents such as EDTA. Except for NaN₃, these should all be added before the titration. All constituents should be dissolved in the same solution to just less than the final volume, i.e. a volume must be left for the titration but the final dilution after titration should be as small as possible. (The Henderson-Hasselbalch equation predicts that the pH of a buffer should not change with dilution, but this is only true over a small range, due to non-ideal behavior of ions in solution.) Finally the solution is titrated to the desired pH and made up to volume. NaN₃ should be added after titration as it liberates the toxic gas, HN₃, when exposed to acid. Manganese salts should also be added after adjustment of the pH as these may form irreversibly insoluble salts at pH extremes.

Buffers of constant ionic strength

Besides pH, which influences the sign and magnitude of the charge on a protein, proteins are also influenced by the specific ions present in solution and by the solution ionic strength. In a buffer, the pH and the ionic strength are related. The Henderson-Hasselbalch equation, for a buffer made from an acid, is:-                        

The ionic strength of the buffer is a function of the [salt]. Therefore, in this case as the pH rises, the buffer ionic strength also rises. Ionic strength is also a function of the molarity of the buffer.

For a buffer made from a weak base, the relevant form of the Henderson-Hasselbalch equation is:-                                                          

In this case, therefore, the ionic strength increases as the pH decreases and the relationship.

Applications and uses:

1. Provide constant pH: Physiological solutions such as buffers are used to maintain the constant pH environment surrounding the cell mass protecting pH sensitive molecules. Hence it is also used in cell culture and microbial cultivation. E.g. It protect pH sensitive molecules such as  enzymes, proteins, nucleic acids (DNA and RNA) etc.

2. Provide constant ionic strength: Buffer solutions also maintain the constant ionic strength of solution or environment. The pH of the buffers is the function of ionic strength of specific molecules (Phosphate moiety in phosphate buffer) and they are not utilized by microbes so it maintains constant ionic strength of solution and protects the denaturation of biomolecules.

3. Naturally present: Buffers are not only used in biochemical investigations but also found in many biological systems like in soil, blood, cytoplasm etc. The clay and humus particles act as buffer in soil while bicarbonates and free amino acids are buffering components in blood.

4. Preserve energy loss: By protecting denaturation of biomolecules, it minimizes the energy loss for synthesis of same biomolecules.

5. It protects cell organelles and vital organs in higher organisms: E. g. uptake of physiological solutions prevents the loss of electrolytes and dehydrations hence protecting kidney and liver.

6. It is required for effective activity for biomolecules:

Phosphate buffer for               ®        Proteins

Tris buffer for                                     ®        Nucleic acids (DNA and RNA)

Bi-carbonate buffer                 ®        Blood cells and Blood molecules

Acetate buffer with glucose o            r sucrose          ®        tissue homogenization-etc.