Exercise No. 12
Preparation of 250 ml of NaCI solution with a concentration of 0.15 mol.l-1
Principle
Solution with concentration expressed as the ratio of the amount-of-substance of
solute to the total volume of solution is being prepared. The following formula is used:
C= n/V
n=m/Mr
m= c.m.Mr
The solution is made in a 1000 ml volumetric flask.
Reagents and accessories
NaCl, analytical grade (m. w. = 58)
volumetric flask (1000 ml), pipette, watch glass, funnel
balance, laboratory scoop
Experimental procedure
The mass of NaCl necessary to prepare the solution is calculated and the amount is
weighed on technical balance. The weighted amount of NaCl is transferred with the help of a
funnel into the volumetric flask, and the watch glass is carefully washed with distilled water
into the volumetric flask. NaCl is dissolved in distilled water and the flask is filled with water
to the graduation mark. The flask is equipped with a label stating composition and
concentration of the compound, and date of preparation.
Exercise No. 13
Preparation of 100 ml of NaCl solutions having concentration of 75 and 100 mmol.1 -1 by
dilution of 150 mmol.l-1 NaCl solution
Principle
If the composition of solution is given as amount-of-substance concentration, the
following balance equation- holds for calculation of changes in solution composition by
addition of a solvent: c1 . V1 = c2 . V2 . The equation is derived from balance of the amount of
substance. On dilution of solutions by adding solvent, both volume and concentration of the
solution are changing; the amount of substance stays, however, constant. From the definition
of amount of substance concentration it follows:
n1=c1.V1
n2=c2.V2
n1 = n2
c1 . V1 = c2 . V2
E. g., to prepare 100 ml of a solution with a concentration of 75 mol.l -1 by diluting
physiological saline (150 mmol.l-1 ) it holds that:
c1 = 150 mmol.l –1
V1 = ? ml
c2 = 75mmol.l-1
V2 =100ml
V1 = c2 . V2 / c1 = 75 . 100/150 = 50 ml
The calculated volume of isotonic NaCl solution is measured, poured into a volumetric flask
and filled with distilled waser to the graduation mark.
Reagents and accessories
NaCl solution, c = 0.15 mol.l-1
A set of 100 ml volumetric flasks
Pipettes, graduated cylinder
Experimental procedure
Volumes of isotonic NaCl solution necessary for preparation of the respective
solutions are pipetted (if volume is sufficiently large, they can be measured with a graduated
cylinder) into 100 ml volumetric flasks, the flasks are filled with distilled water to the mark
and closed with a stopper. Flask are equipped with labels stating name of the compound,
solution concentration, and date of preparation.
Exercise No. 14
Examination of hypotonic hemolysis (osmotic fragility) of erythrocytes
Principle
In hypotonic environment, erythrocytes undergo hemolysis. Osmotic resistance (or,
alternatively, osmotic fragility) is examined by monitoring resistance of erythrocytes to
hemolysis in hypotonic environment. The maximal osmotic resistance is determined by that
concentration of NaCl, in which ihe commencement of hemolysis is observed (supernaiant
over erythrocyte sediment is of slight pink color due to the released hemoglobin). Minimal
osmotic resistance is given by that concentration of NaCl, in which hemolysis is maximal (i.
e., erythrocytes are completely hemolyzed, solution has red color and no sediment is observed
at the bottom of the test tube, similarly to the control tube with distilled water).
Examination of osmotic resistance (fragility) is of diagnostic value. It serves diagnostic
purposes LIS well as to differentiate hemolytic diseases.
In clinical practice, as well as in research, osmotic fragility is the more frequently used term
for monitoring hypotonic hemolysis than osmotic resistance. To differentiate between these
two terms, one has to realize that maximal osmotic resistance corresponds to minimal osmotic
fragility, and minimal osmotic resistance corresponds to maximal osmotic fragility. The more
osmotically resistant (more stable against hemolysis) the erythrocyte, the smaller its osmotic
fragility (sensitivity to hemolysis).
Reagents and accessories
Fresh blood sample, or a suspension of washed erythrocytes in an isotonic NaCl solution (20
%, v/v) NaCl solutions with concentrations of 0.05, 0.07, 0.09, 0.11, 0.13 and 0.15 mol.l-1
Experimental procedure
Erythrocytes are isolated from blood by centrifugation (200 rpm for 10 min). After
sucking off the plasma (with a capillary and a water aspirator) erythrocytes are washed three
times with isotonic NaCl solution and subsequently centrifuged. Erythrocytes are resuspended
in five times their volume of physiological solution (20 %, v/v). Erythrocyte suspension
obtained in this way and NaCl solutions .if different concentrations are then pipetted into a set
of centrifuge tubes, according to the table:
1
erythrocyte suspension (ml)
0.1
0.15 mol/l NaCl (ml)
5
0.1 mol/l NaCl (ml)
0.075 mol/l NaCl (ml)
distilled water (ml)
1. incubation 15 min/ 37°C
2. centrifugation 7 min/ 2000 r.p.m.
3. measurement of A540 in supernatant
A 540 (measured against
physiological saline solution)
% of hemolysis
2
0.1
5
-
3
0.1
5
-
4
0.1
5
100
The mixture in tubes is carefully mixed, left standing for two hours at room temperature, or,
more preferably, incubated in a water bath at 37 °C for 15 minutes. After centrifugation (10
min at 2000 rpm) part of supernatant is carefully poured into a cuvette (it is preferable to suck
1 ml off using a doser) and absorbance at 540 nm is measured against water. The values are
recorded into the table.
Evaluation
After centrifugation one can observe in test tubes that the volume of sediment
(erythrocytes) is inversely proportional to NaCl concentration. In the 1, 2, 3, 4 tubes
(hypotonic medium) hemolysis is observed so that supernatant is of red color (hemoglobin
solution). In the 1. tube NaCl solution is isotonic with the inner environment of erythrocytes,
so that under normal conditions (fresh erythrocytes, absence of a hemolytic disease)
hemolysis is not observed. Hemolysis is evaluated quantitatively by calculating percentage of
hemolysis at different NaCl concentrations. The A540 value in the tube with water (tube No. 4)
is taken as 100 %, because in water hemolysis is complete.