Isotonicity Pre-reading Ian.Larson@monash.edu Isotonicity Pre-reading

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

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

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

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 + 0.5 mole NH4Cl, or any other such combination

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.

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

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

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)

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.

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

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

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 1.9 electrolyte Urea Camphor ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑----------------------------- 1B Weak Boric Acid electrolyte Cocaine 2.0 Phenobarbitone 2A Di‑valent MgSO4 electrolyte ZnSO4 2.0 2B NaCl Uni‑univalent KCl electrolytes Ephedrine HCl 3.4 Pilocarpine NO3 3A Uni‑di‑valent Na2SO4 4.3 electrolyte Atropine SO4 3B Di‑uni‑valent ZnCl2 electrolyte CaBr2 4.8 ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ etc etc

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

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 = 0.576 °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

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.

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%

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.

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.

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

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

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-

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-

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.

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.