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.
Chapter 4
THE OXYGEN CONTENT IN WATER
The concentration of oxygen determines the value of the redox potential and largely direction and rate of processes of chemical and biochemical oxidation of organic and inorganic compounds.
Oxygen regime has a deep impact on the life of the reservoir. The minimum content of dissolved oxygen that ensures the normal development of fishes is about 5 mg О2/l. If this measure decreases until 2 mg/l, it will cause massive lethality of fishes. Supersaturation of the water with oxygen also adversely affects its condition. Threshold limited value of dissolved oxygen for the water objects of fishery purposes in the winter period is 4 mg/dm3, in the summer – 6 mg/dm3.
Determination of oxygen in surface waters is included in the program of observations in order to assess the living conditions of aquatic organisms, including fish, indirect water quality characteristics, intensity of processes of producing and destruction of organic matter, self-purification of reservoirs, and so on.
Concentration of oxygen is expressed either in milligrams per liter or as a percentage of saturation, the calculation of oxygen is made according to the formula
where, Сх – concentration of oxygen found experimentally, mg/l;°C - normal concentration at a given temperature, normal and atmospheric pressure 760 mm (found from Table. 4.1); p – atmospheric pressure at the time of analysis.
The principle of measuring the concentration of oxygen.
Amperometric method of analysis forms the basis for measuring the concentration of dissolved oxygen. The oxygen concentration is determined by current intensity, flowing in the circuit of the electrode system of the sensor О2.
Electrodes, cathode and anode, of О2 sensor are located in the electrolyte solution and separated from the analyzed liquid by gas permeable membrane. The oxygen freely diffuses through the membrane and electrolyte to the electrodes, which are under the constant voltage coming from the source of polarizing voltage of the device. The current, which is caused by a reduction reaction of molecular oxygen, generates in the electrode circuit and flows by the following scheme:
The current is converted into voltage, which is measured and, in turn, is converted into a value of concentration of oxygen.
Preparation and conducting of measurements.
Measurements of the concentration of dissolved oxygen are produced by special sensors. Sensors «generate» the current that is proportional to the oxygen concentration in the environment. The conversion coefficient depends on several factors and therefore requires periodic calibration of the sensor.
Calibration – put of parameters of the measuring channel sensor → converter, current-voltage of the device obtained in solutions with known concentrations of oxygen into the memory of the device.
Such relatively easy reproducible and available solutions are solutions with zero (0 %) and one hundred percent (100 %) of the dissolved oxygen concentration.
Calibration