2 Before our next class you need to be able to*: Define a) hypertonic, and b) hypotonicExplain 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 solutionCalculate 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 informationMartin, “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? patmhposm = pPuresolventSolutionit’s a colligative propertydepends 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 osmolalityIn 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 osmolarityit doesn’t matter what the components are. eg. one soln with 3 moles glucose, and another with 1.5 moles NH4Cl are iso-osmotic3 molesglucose1.5 molesNH4Cl
8 Real membranes are leaky 3 molesglucose +0.75 molesNH4Cl0.75 molesNH4Clleaky membrane:ammonium chloride but notglucose 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 pointlower than -0.52º C.between -0.52º C and 0º Cgreater than 0º CQuestion: A hypertonic solution will have a freezing point* an approximate value
12 We need to be able to measure tonicity a colligative propertyFreezing point depression instrumentMeasures crystallization microscopicallyAs it is a colligative property, the change in freezing point is proportional to the concentration of the solution:DTf cas our drug could be an electrolyte, we need to take into account dissociation with a constant L:DTf = Lc
14 Liso So from before ΔTf = Lc So under isotonic conditions ΔTf = LisocisoRearranging Ciso = 0.52/ LisoIn general, Liso may be grouped as1.86 for non-electrolytes2.0 for weak electrolytes3.4 for uni-univalent electrolytes, eg NaClHigher values for electrolytes of higher valencySo 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 CWhat 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-multiplicationCiso NaCl = (0.52 /0.576) x 1% from 1% NaCl = °C= 0.90%. ΔTf(iso) = 0.52 °CThis is the accepted value (w/v) of a sodium chloride solution that is isotonic to body fluids.deltaTf = Kfm
16 CisoThe 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 exampleA 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 measureUse red blood cellsHaemolysis 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 methodred blood cells as membranesuspend measured quantity of washed red blood cells in test solutioncentrifuge and measure size of pelletPacked 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-osmoticExplain the mechanism of action of hypertonic and hypotonic solutions on RBCsExplain what strategies are used to make hypertonic and hypotonic solutions isotonicDefine osmolarity and osmolalityBe 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 solutionsthere is a lower concentration or ions outside the RBC, higher conc insideOn administration, liquid passes fromthe solution into the red blood cells toachieve osmotic equilibrium and theyeventually burst if the difference is largeenough.This process is called haemolysis and is irreversible.Na+ Cl-H2ONa+ Cl-Na+ Cl-
25 Hypertonic solutions A higher concentration of ions outside Na+ Cl-A higher concentration of ions outsidethe RBC causes a flow of solvent out of the RBC until there is no longer an osmotic pressure imbalanceThe RBC’s shrinkThe process is called crenation andis reversibleH2ONa+ 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 useboric acid or NaCl for ophthalmic useHypertonic 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.