1. Analytical biochemistry laboratory (BIOC 343) L. Nouf Aalshareef KAU- Biochemistry department Second semester 2013

2. What is pH? Is potential of hydrogen or amount of hydrogen (conc.) of H + in solution It measures acidity or alkalinity of a solution Conc. of H + ion in any solution ranges between 10 -14 M to 1 M To makes these numbers easy and more practical there is an equation that turns these conc. to numbers between 1 to 14 pH= - log 10 [H + ] ; [H + ] by M molar pH: is the negative power of 10 of [H + ] conc. or is the reciprocal of [H + ] conc.

3. If pH of a given solution < 7 … solution is acidic If pH of a given solution > 7 … solution is alkaline If pH of a given solution = 7 … solution is neutral

4. pH Measurements It is difficult to determine the accurate value of pH of solution. Usually pH value of a given solution is compared with the pH of other known solution. Three methods to measure pH: 1- using pH indicators 2- using pH strips or papers 3- pH meter

5. 1- Using pH indicators Indicators are weak acids or bases that turns their color when pH of solution is changed. Usually used in titration Example: Bromophenol blue, phenol phethalen

6. 2- Using pH strips or papers Papers contain indicators or mixture of indicators There are different types: - Universal: check pH from 1-14 - Narrow range: check pH from 3-7 or 7-14 Example litmus paper

7. 3- Using pH meter Is the most convenient and reliable method (trusted, accurate) Depends on measuring the electro-chemical potential between two solutions: one with a known pH (liquid inside glass electrode) and one with unknown pH

8. pH and life In biological system, pH should be maintained constant, to do this there are many buffering systems that keep pH unchanged. Example of biological buffering system: 1. Proteins 2. Bicarbonate 3. Phosphate

9. Buffers Are solutions which resists change in pH if acid or base is added. This definition is true if limited amount of acid or base is added to the buffer. Suppose that one letter of acid or base is added; the pH should be changed in this case, since each buffer has a certain capacity to maintain the pH. Therefore it is important to define the term buffer capacity. Buffer capacity: Is number of moles of H + or OH - which should be added to one Liter of buffer to change its pH by one unit.

10. Buffer composition Buffers usually consist of: weak acid and its conjugated base or weak base and its conjugated base  weak acids: acids which are incompletely dissociated  strong acids: acids which are completely dissociated Example: Weak acid: Conjugated base

11. pH of Buffers Buffers may be acidic or alkaline or neutral so it can takes any pH it should not be neutral all the time. Buffer pH can be calculated by Henderson-Hasselblach equation: pH= P ka + log [slat]/[acid] conc. By molar

12. How buffer works?

13. If a strong base is added the weak acid in a buffer, will give up its H + in order to transform the base (OH - ) into water (H 2 O) and the conjugate base: HA + OH - → A - + H 2 O. So, added OH - is absorbed by this reaction, the pH will change only slightly.

14. If a strong acid is added the weak base in the buffer will react with the H + from the strong acid to form the weak acid HA: H + + A - → HA. The H + is absorbed by the A - instead of reacting with water to form H 3 O + (H + ), so the pH changes only slightly.

15. Buffer preparation By mixing certain amount of weak acid with certain amount of weak base, theses amounts are obtained from certain tables depending on the pH that you wish to prepare. (See table on the next slide) Note: usually these tables give you the theoretical pH, the real pH (practical) that you will get during your preparation may be slightly differ from the theoretical, therefore you have to adjust this pH by adding NaOH or HCl

16. Lab work You are going to learn: 1. Solutions preparations: Prepare solution from liquid material Prepare solution from solid material 2. Buffer preparation.