Читать книгу Manual for laboratory classes in biological physics - Коллектив авторов - Страница 5

The constancy of the hydrogen ion concentration of blood and other body fluids is provided by a number of mechanisms. Despite the many processes that result can change the internal environment of the reaction, the pH is constant. In warm-blooded animals, constancy of pH of the internal environment is maintained during the life of a very narrow range, the deviation does not exceed 0,03-0,04 units. (in human blood pH is 7,35±0,02).

The shift reaction medium leads to a change in many physical and chemical indicators of protein nuclein, and protein lipid complexes – the magnitude of the charges, the extent of hydration, stability of colloids, viscosity, electrical conductivity, etc. Changes in biochemical reactions lead to disruption of conjugation of many biochemical processes that may be the cause of death of the organism.

However, in a normally functioning blood in the living body continuously it receives the acid products of working organs and the gastrointestinal tract. Alkaline reaction products enter the body mainly in food. To maintain a constant internal environment include buffers which neutralize both the acid and alkali in the received blood products stably maintain the reaction medium (hydrogen ion concentration) at a predetermined level. Buffer systems are presented with proteins or systems consisting of a weak acid and a strong base salt of this acid (Н2СО3 and NaHCO₃; H₃PO₄ and Na₂HPO₄; H₃PO₄ and Na₃PO₄ etc.).

The main role of proteins in the body member which are amphoteric compounds, and proteins from the blood hemoglobin plays a specific role, in which human blood contains up to 14 % of its weight. In second place is the carbonate system in the third – a phosphate buffer system.

Of course, each buffer system may be kept constant pH within certain limits, which depend on its buffer capacity. Under the buffering capacity to understand the amount of 0.1N acid or alkali which is needed to shift the pH unit. An alkaline buffer is contained in the serum is about 5 times more than the acid, i.e. the acid can be neutralized to 5 times greater than the alkali. This means that the body is better protected from disturbances in blood entering the acidic products.

Laboratory work № 5
Definition of alkaline and acid buffers of serum blood

Objective: Buffer properties of serum

Tasks:

1. Carry out calibration of the device on standard solutions.

2. Measure рН of blood

4. Draw graphs of pH values

5. Make conclusions on the observed phenomena and do the report.

Equipment and Materials: pH-meter burette 1 and 50 ml, 50 ml cups, 0.1N solutions of hydrochloric acid and potassium hydroxide or sodium, 0.02 % methyl orange solution, 0.1 % ethyl alcohol solution phenolphthalein, serum.

Task 1. Acid buffer.

Procedure. Pour into a glass 5 ml of the distilled water and add 2-3 drops of methyl orange indicator, which has a transition zone pH = 3,1-4,4. Titrate drop by drop from a burette 0.1 N hydrochloric acid solution until a slight red coloration (1 drop of acid).

Pour into another glass 5 ml of the distilled water, add 1 ml of serum and 2-3 drops of methyl orange and titrated to the same stain; count the number of drops, or determine the amount of 0.1N hydrochloric acid solution that spent to the titration.

Calculation: for acidification 5 ml of water was spent A ml of 0.1 N hydrochloric acid (1 drop – 0.03 ml of 0.1N hydrochloric acid or alkali). By adding 1 ml of serum was spent B ml of hydrochloric acid. Thus, for 5 ml serum would be spent: (B-A × 5ml 0.1N Hydrochloric acid). In the calculations of the buffer capacity of the water can be ignored. Make a calculation for 100 ml of serum, which would be its buffer capacity. The results bring to the table 3.4.1.

Normally, for serum it is necessary to add 250-300 times more hydrochloric acid than for water.

Task 2. Alkaline buffer.

Procedure.

Pour into a glass 10 ml of the distilled water and add 2-3 drops of a phenolphthalein; transition zone pH =8,9-9,8. To titrate 0,1 N solution of caustic sodium before appearing weak-violet coloring (usually 1-2 drops).

In the second glass to pour 10 ml a dist. waters to add 1 ml of serum and 2-3 drops of a phenolphthalein. To titrate 0,1 N solution of caustic sodium before the same coloring, as in the first case, counting quantity of drops or measuring volume. Write the results in table 3.4.1.

Table 3.4.1

Acidic and alkaline buffers serum

Calculation. For change (shift) reaction of 10 ml distilled water is necessary D drops of alkali. At addition of 1 ml of serum for neutralization of the acid buffer is required E ml alkali, so for 10 ml of serum will be required 10×(Е-0,03D) ml alkali. Usually to serum it is necessary to add at 50-60 times more alkalis to cause identical change of reaction.

Report design. Write results in the table, make calculations and draw figures (histogram). Calculate standard error of measurements and make conclusions.

Laboratory work № 6
Buffer capacities of biological liquids

Objective: the buffer capacity of serum of blood or urine

Tasks:

1. To carry out calibration of the device on standard solutions.

2. To take pH measurement of blood.

3. Draw figures of pH values.

4. Make conclusions on the observed phenomena.

5. Make conclusions and write report.

Equipment and materials: pH-meter, burette 1 and 50 ml, 50 ml cups, 0.1N solutions of hydrochloric acid and potassium hydroxide or sodium, 0.02 % methyl orange solution, 0.1 % ethyl alcohol solution phenolphthalein, serum.

Task 1. Determination of buffer capacity of serum or urine.

Procedure: measure рН of biological liquid, then titrate the studied solution (volume of 100 ml) in the small portions of acid or alkali and write down that quantity of a reactant which is necessary for change рН on 0,1 units of a scale. Titrate until reaction will change on 2 units of рН (concentration of ions of hydrogen changed in relation to initial by 100 times). For calculations of own buffer capacity take that quantity of a reactant which is necessary for change рН on unit, i.e. concentration of ions of hydrogen has to change by 10 times.

Change рН to 7,2 and 7,55 is a sign of the most serious condition which is usually coming to death of an organism. It is necessary to know not only the size of buffer capacity at a concentration deviation by 10 times from normal value, but it is much more important to know that amount of acid and alkali which is necessary for a deviation рН to value 7,2 and 7,5. Besides, it is important to know the course of curve dependence рН from amount of titrable substance (acid and alkali). For this purpose it is necessary to remove dependence of change рН solutions (protein, serum of blood, milk, urine) from amount of the added decinormal acid or alkali. Titration should be carried out before achievement of pH values 10-11,0 in alkaline area and to 2,0-1,5 in the sour.

According to the obtained data to construct the schedule of dependence of size рН from quantity of a reactant (fig. 3.5.1)

Determination of buffer capacity of blood

Figure 3.6.1. Designations: on abscissa axis – amount of the NaOH or HCl solution, on ordinate axis – pH value

Task 2. Buffering capacity of hemoglobin.

Procedure: Take 1 ml of packed red blood cells (blood centrifuged 20 minutes at 5000 rpm), wash twice with saline or 5.4 % glucose, and 10.3 % aqueous solution of sucrose and add to 10 ml of distilled premeasured water with pH. Using a pH meter to measure the concentration of hydrogen ions after the full osmotic erythrocyte hemolysis (appears quite intense staining in the red, and the solution becomes completely transparent). If hemolysis occurs slowly, increase the temperature in the beaker to 40-50 °C. Then produce titration with 0.1N. HCl solution. Take a new batch of packed red blood cells, hemolyze and titrate with 0.1 N NaOH solution to pH shift by 1. Perform per 100 ml of packed red blood cells. This will be the quantity of alkaline and acidic buffer capacity respectively.

Report design. Write these results in the table to make calculations and plot graphs (bar graphs). Define measurement error and draw conclusions.

Chapter 3 «Buffering properties and buffer capacity of biological fluids» questions:

1. What is the pH of the solutions and how to determine its value?

2. Why does the pH change?

3. What instruments allow us to estimate the pH

4. How big limits of pH changes in living objects?

5. What substances are buffers?

6. Describe the dissociation of the peptide.

7. Describe the dissociation of sulfuric acid, for some reason it does not have buffer properties?

8. Explain biological significance for constancy of pH within each cell, and in all body fluids.