In this article we will discuss about the Determination of pH of a Biochemical Solution:- 1. Methods for Determination of pH of a Biochemical Solution 2. Principle of Determination of pH 3. Determination of a Colourless Solution 4. Determination of Coloured Solution.
Methods for Determination of pH of a Biochemical Solution:
The electrometric method is the most accurate method for the determination of pH of a solution. This method measures the electromotive force set up due to the existence of H+ and OH– in the solution. The instrument which measures pH in this manner is known as pH-meter.
The other method is the colorimetric method which is also fairly accurate and can be used in the absence of a pH-meter. The basis of it is that certain organic dyes (indicators) change colour with change in pH.
Each indicator in its ionised state gives one colour and in unionized state gives another colour. Each indicator has its useful range of pH within which it can indicate change in pH by change of colour and beyond which there is no further change in colour.
It is possible to prepare an indicator whose pH range covers almost the entire pH scale. Such an indicator is known as ‘Universal indicator’. The universal indicator enables us to determine the approximate pH of a solution and thus helps us to choose an appropriate indicator and buffers for subsequent more exact determination of pH of the solution.
Electrometric Method of pH Determination:
The switch of the instrument is put on and after a few minutes when the instrument is warm enough, the pointer is adjusted to 0 mV or 7.0 pH position by set zero control. A standard buffer solution is taken in a clean glass or polythene beaker. Electrodes are lowered so that they are immersed in the solution to a depth of about one inch.
The temperature of the buffer solution is measured by a thermometer and the instrument is again adjusted by temperature compensate control to this value. The pointer is again set at 7.0 pH by means of set control.
The Selector switch is now turned to proper range, i.e. 0-7 or 7-14. Pointer will now move to show the pH of the buffer and by the help set buffer control, the pointer is set to the exact pH value of the buffer. Now the pH meter is standardized and is ready for determination of pH of any given solution.
To measure the pH of a given solution, the selector is brought back to Zero position and the electrodes are cleaned by distilled water. The given solution is taken in a clean beaker and the electrodes are immersed in it. Selector switch is now put on expected pH range and the reading of the pointer on the pH scale is noted. This value is the pH of the given solution.
Principle of Determination of pH:
When equal volumes of the unknown solution and a series of standard buffer solutions with closely varying pH values are treated with a suitable indicator, the colour of the unknown solution will be found to match with one of the standard buffer solutions. The pH of the unknown is, therefore, the same as the pH of the buffer with which its colour matches.
Permanent standard made from glass discs of the appropriate colour and shade for each indicator may also be used in place of the standard buffer and indicator. Hellige’s comparator with its permanent standard discs made of coloured glass is a very convenient instrument for this purpose.
pH Determination of a Colourless Solution:
First determine the approximate pH of the solution with the help of a universal indicator and colour chart. Take a small piece of universal indicator paper and just dip its one end in the given solution.
After about 30 seconds compare the colour of the paper with the standard colour chart provided and know the approximate pH. If universal indicator solution is to be used in place of paper, put a drop of it in about 2 ml of the solution and compare the colour of the solution with that of the colour chart.
After having known the approximate pH of the solution, choose from the set of indicators an indicator which more closely covers the pH range of the solution under examination. Then add a drop of this indicator to approximately 2 ml of the solution, and match the shade of the colour produced against the series of coloured standard buffers.
The standard colour solutions are prepared by adding one drop of the same indicator to 2 ml of each of the buffer solutions (chosen in the pH range of the unknown). The pH of the unknown solution is that pH of the buffer with which the colour matches.
In case, the colour of the unknown is intermediate between two colours produced by two consecutive buffers, the pH of the unknown lies between the two known standards. The accuracy of this method can be increased by employing set of buffer solutions with closely differing pH values.
pH Determination of Coloured Solution:
Place a test tube containing plain urine of coloured solution in front of the coloured buffer standards. View the two test tubes together and then match this against the urine or coloured solution containing the same indicator in front of which a test tube containing water is to be placed. The use of a comparator makes the matching easier, otherwise, a double rowed test tube rack is also convenient.