1. Isotonicity Pre-reading
Isotonicity Pre-reading

2. Before our next class you need to be able to*:
Define a) hypertonic, and b) hypotonic
Explain why a solution containing 2 mM glucose (impermeable) and 2 mM boric acid (permeable) is hypotonic to a solution containing 3 mM sucrose (impermeable).
Explain what happens to red blood cells when they are immersed in a hypotonic solution or a hypertonic solution
Calculate Ciso for each of the classes of L
* not all the answers are in these notes, you might need to look elsewhere

3. For more information
Martin, “Physical Pharmacy,” second half of Chapter 8, i.e., ignore the section on buffered solutions (eg in 4th Ed, read from p. 180 on)
Florence and Attwood, “Physicochemical Principles of Pharmacy” Ed 4, pp 69-75

4. remember osmotic pressure?
patm h
posm = p
Pure
solvent
Solution
it’s a colligative property
depends on the number of components in the solution, not their type or chemistry

5. Osmolarity- a measure of solute concentration (osmotic molarity)
Definition: the number of osmoles (Osm) of solute per litre of solution (osmol/L or Osm/L) Glucose does not ionise and thus 1 mole forms 1 osmol of solute. Whereas, NH4Cl will dissociate to form 2 ions and thus 1 mole of NH4Cl forms 2 osmoles. A 3 Osm solution might consist of: 3 moles glucose, or 1.5 moles NH4Cl, or 1 mole glucose + 1 mole NH4Cl, or 2 moles glucose mole NH4Cl, or any other such combination

6. but volume changes with temperature??
The osmolality (m) is the mass of solute dissolved in 1 kg of water which exerts an osmotic pressure ´equal to that exerted by 1 mole of an ideal unionised substance dissolved in 1 kg of water.
The osmolarity may be calculated from measured osmolality
In dilute solution osmolality and osmolarity are ~ the same. In concentrated solution the amount of solute becomes significant, it might change the volume of the solution. remember: osmolarity is per litre of solution.

7. Iso-osmotic - have the same osmolarity
solutions are iso-osmotic when they have the same osmolarity
it doesn’t matter what the components are. eg. one soln with 3 moles glucose, and another with 1.5 moles NH4Cl are iso-osmotic
3 moles
glucose
1.5 moles
NH4Cl

8. Real membranes are leaky
3 moles
glucose +
0.75 moles
NH4Cl
0.75 moles
NH4Cl
leaky membrane:
ammonium chloride but not
glucose can get through

9. You need to be careful about what can and what can’t get through
TONICITY is a measure of osmotic pressure for two solutions separated by a real membrane (eg a red blood cell membrane)
is influenced only by solutes that cannot cross the membrane, as only these exert osmotic pressure.
Solutes able to freely cross the membrane do not affect tonicity because they will always be in equal concentrations on both sides of the membrane. (ref: Wikipedia)

10. Only the impermeable solutes count for tonicity
Solution A contains 2 mM of sucrose (impermeable) and 5 mM of urea (permeable); Solution B contains 2 mM of sucrose (impermeable).
A is hyperosmotic to B because it contains more total solutes.
But A is isotonic to B because the amount of impermeable solutes is equal. The urea will diffuse until equal conc on each side. Therefore, there is no osmotic pressure or water flow.

11. Effect of tonicity on FP
For example, aqueous solutions are iso-tonic with blood serum and lachrymal secretions (tears) when they have a freezing point depression of 0.52º C or contain about 308* mOsmoles of solute.
Question: A hypotonic solution will have a freezing point
lower than -0.52º C.
between -0.52º C and 0º C
greater than 0º C
Question: A hypertonic solution will have a freezing point
* an approximate value

12. We need to be able to measure tonicity
a colligative property
Freezing point depression instrument
Measures crystallization microscopically
As it is a colligative property, the change in freezing point is proportional to the concentration of the solution:
DTf  c
as our drug could be an electrolyte, we need to take into account dissociation with a constant L:
DTf = Lc

13. Well’s Liso values
From: Wells, J.M., J.Am.Pharm. Assoc. Prac. Ph. Ed., 5, 99 (1944)
_________________________________________________________________
TYPE EXAMPLE Liso
1A Sucrose
Non‑ Glycerin
electrolyte Urea
Camphor
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ 1B
Weak Boric Acid
electrolyte Cocaine
Phenobarbitone 2A
Di‑valent MgSO4
electrolyte ZnSO
2B NaCl
Uni‑univalent KCl
electrolytes Ephedrine HCl
Pilocarpine NO3 3A
Uni‑di‑valent Na2SO
electrolyte Atropine SO4 3B
Di‑uni‑valent ZnCl2
electrolyte CaBr
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
etc etc

14. Liso So from before ΔTf = Lc
So under isotonic conditions ΔTf = Lisociso
Rearranging Ciso = 0.52/ Liso
In general, Liso may be grouped as
1.86 for non-electrolytes
2.0 for weak electrolytes
3.4 for uni-univalent electrolytes, eg NaCl
Higher values for electrolytes of higher valency
So if know Liso for class of additive can calculate isotonic concentration

15. Example – Calculation of isotonic composition for NaCl
A 1% solution of sodium chloride in water has a measured freezing point depression of 0.576º C, i.e, FD1% = 0.576º C
What is the percentage composition of NaCl required in an aqueous solution to be isotonic with tears?
We must calculate the % of NaCl in water that will create a freezing point depression of 0.52º C (called Ciso)
Using cross-multiplication
Ciso NaCl = (0.52 /0.576) x 1% from 1% NaCl = °C
= 0.90%. ΔTf(iso) = 0.52 °C
This is the accepted value (w/v) of a sodium chloride solution that is isotonic to body fluids.
deltaTf = Kfm

16. Ciso
The isotonic concentration of a substance is given by the following equation:
Ciso = (0.52 / FD1%) %
where FD1% is the freezing point depression of a 1 per cent solution.
Why 0.52? Because the FD required for all body fluids is 0.52º C.

17. example
A 1% solution of apomorphine hydrochloride has a freezing point depression of 0.08° C.
Ciso = (0.52 / FD1%) %
= (0.52/0.08) %
= 6.5%

18. Measurement of Isotonicity
Several methods – most involve the use of red blood cells.
Two most commonly used methods are:
Haemolytic method of Husa.
Haematocrit (proportion of blood volume occupied by red blood cells) method of Setnikar and Temelcou.

19. Haemolytic Method of Husa
Biological measure
Use red blood cells
Haemolysis detected colorimetrically (detected by a measure of its colour, either analytically or by comparison to standards)
Sodium chloride used as a reference (0.6% to 0.3%)
Remember: haemolysis occurred for hypotonic solutions where there was a low concentration of NaCl outside the cell.

20. Haematocrit Method of Setnikar and Temelcou
biological method
red blood cells as membrane
suspend measured quantity of washed red blood cells in test solution
centrifuge and measure size of pellet
Packed Cell Volume gives a measure of cell volume and thus tonicity.
This technique works as the PCV is well known and if it is higher or lower than usual it indicates hypotonicity or hypertonicity, respectively.
Packed Cell Volume

21. Learning Objectives for these classes*
Define the term ‘iso-osmotic’
Define isotonic and differentiate from iso-osmotic
Explain the mechanism of action of hypertonic and hypotonic solutions on RBCs
Explain what strategies are used to make hypertonic and hypotonic solutions isotonic
Define osmolarity and osmolality
Be able to use equations involving Ciso%, FD1%, and SCE.
Describe how one can measure isotonicity of biological fluids
*in addition to: Before our next class you need to be able to

24. Hypotonic solutions
there is a lower concentration or ions outside the RBC, higher conc inside
On administration, liquid passes from
the solution into the red blood cells to
achieve osmotic equilibrium and they
eventually burst if the difference is large
enough.
This process is called haemolysis and is irreversible.
Na+ Cl-
H2O
Na+ Cl-
Na+ Cl-

25. Hypertonic solutions A higher concentration of ions outside
Na+ Cl-
A higher concentration of ions outside
the RBC causes a flow of solvent out of the RBC until there is no longer an osmotic pressure imbalance
The RBC’s shrink
The process is called crenation and
is reversible
H2O
Na+ Cl-
Na+ Cl-

26. Preparation of Isotonic Solutions
Hypotonic solutions are usually made isotonic with body fluids by adding some therapeutically inactive solute.
e.g., dextrose or NaCl for intravenous use
boric acid or NaCl for ophthalmic use
Hypertonic solutions are usually made isotonic with body fluids by dilution with water.

27. Conditions for isotonicity
Solutions which are iso-osmotic with body fluids are only considered to be isotonic if :
membranes are impermeable to the solute (urea, boric acid, hexamine can pass thru RBC membranes)
the solute does not alter the permeability of membranes (ethanol, some anaesthetics, and propylene glycol can increase the permeability)
no chemical reaction leads to a change in the total concentration of dissolved ions or molecules.