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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement

Created by Maryam Sadat* Mortazavi

Maryam Sadat* Mortazavi
Stu#: 995000742
PHC340- Formal Report
Nov. 24/ 09

Expt 9 – Diffusion and Membrane Transport (I)
Permeation Measurement

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

Abstract

Membrane diffusion is a very important concept in formulation of a drug. The
diffusion of a drug plays a key role in regulating cellular transport and gastrointestinal
absorption in biological systems. Distribution of solutes and solvents takes place as a
result of their respective chemical potential gradient usually at constant temperature and
pressure. The mathematical measurement of the rate of diffusion of a given drug across a
membrane is given by the term flux as the rate of change of moles per unit time
multiplied by surface area of the membrane (mol/cm2sec) where a larger flux value
corresponds to faster diffusion. The diffusion across a membrane quantified by the term
flux is dependent upon a variety of parameters such as the concentration gradient across
membrane(C1-C2), membrane thickness(h), membrane partitioning(K), and membrane
diffusion coefficient(D). The membrane permeability parameter Pm = DK/h is calculated
in this experiment, the value of which can be further used to calculate the original drug
concentration in the first compartment once equilibrium is reached. The findings of this
experiment can be used in a variety of applications such as controlled-release drug
delivery systems design, industrial separations, and hemodialysis.

Introduction

Membrane diffusion is an important characteristic of a drug to be measured
during the pre-formulation process as it has important applications in a variety of areas
ranging from industrial separations, hemodialysis to controlled-release drug delivery
systems. The movement of solutes and solvents across a thin membrane is based on their
respective concentration gradients at constant temperature and pressure. Diffusion is a
redistribution of molecules towards concentration equilibrium brought upon by random
Brownian motion of the dissolved molecules.

A measure of how fast the drug diffuses through a given surface area such as a
membrane is expressed mathematically by the term flux, J, which is the rate of change of
moles per unit time multiplied by surface area through which diffusion occurs. In other
words, flux is the speed of drug movement. A larger flux implies faster diffusion;

J(t) = 1/A * (dn/dt) (1)
in which J= molar flux per unit area (mol/(cm2*sec)), A= the total surface area of the
membrane (1/cm2), and dn/dt is the rate of diffusion of the drug in moles per unit time
(mol/sec).

Flux can be related to the concentration gradient dC/dx by the Fick’s law:

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

J(t) = -D * dC(t)/dx (2)
where D is the diffusion coefficient which regulates flux of molecules through the
membrane. The negative sign in the above equation denotes a decrease in the initial
concentration.

The simplest arrangement for a model of membrane transport consists of two
finite-volumes (V1 & V2) compartments containing aqueous solutions of different solute
concentrations (C1 & C2) separated by a membrane of thickness h. In this model, several
assumptions are made such as absence of a boundary layer and concentration/hydrostatic
pressure gradient within each compartment. Also, it is assumed that C1º > C2º and the
volume change due to osmotic water flow from compartment 1 to compartment 2 is
negligible. It is also important to note that due to the membrane’s different polar
environment than the surrounding fluids, concentrations in the membrane are different
than the concentrations in the fluids.
Due to the fact that known concentration of drug is added to compartment 1 and hence, at
any point in time, the total number of moles in the system is equal to the initial number of
moles added, writing a mass balance equation of the drug and assuming nmembrane=0,
followed by integration of Fick’s law, and making simplifying assumptions such as the
membranes are well stirred and rapid equilibration takes place, one can obtain:

J= Pm (C1-C2) , (3)
Where Pm = DK/h is the bulk membrane permeability constant, and K is the membrane
partition coefficient which is constant for both compartments.

Subsequent derivations and calculations will yield the most useful form of the
above formula which expresses C1 in terms of C2:

ln ( 1 – C2/Cº1 * (V2/V1 +1)) = -Pm*A (1/V1 + 1/V2)* t (4)
in which slope = -Pm*A*(1/V1 +1/V2)

Subsequently, one can derive expressions for the compartment 2 concentration as
a function of time:

C2(t) = Cº1 * (V1/V1+V2) * (1- e –Pm*A*(1/V1+1/V2)*t) (5)

Therefore, as the time approaches infinity, C2 approaches the value it would have
been if the two volumes were mixed together with no membrane present and mass

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

balance expression (eq. 3) allows solving for C1 even though it was never measured as a
function of time.

Considering what was mentioned above, this membrane diffusion experiment is
designed to calculate the membrane permeability constant of salicylate through a dialysis
membrane by investigating the change of salicylate concentration diffused through the
membrane as a function of time.

Procedures

I. Standard Curve: Salicylate

A 1M sodium salicylate stock solution is diluted by a factor of 100 to make a
10mM solution of sodium salicylate.
The following concentrations of sodium salicylate were made from the 10mM
solution in a 50mL volumetric flask: 0.5, 1.0, 1.5, 2.0, and 2.5 mM. Then, 1mL of each
solution was pipetted in a test tube followed by addition of 5mL distilled water and 2
drops of ferric chloride indicator. The blank solution was prepared by using 6mL of
distilled water + 2 drops of FeCl2. The wavelength of UV/Visible spectrophotometer used
for the subsequent absorbance measurements of the entire experiment is set to λ=525 nm.
The spectrophotometer was zeroed using the blank solution and the absorbance of each
solution was measured which lead to construction of calibration curve by plotting
absorbance vs. molar [SA].

II. Preparation of the Diffusion Cell

The appropriate length of the dialysis tubing for 5mL of solution was calculated
and the dialysis tubing was pre-soaked in a beaker with distilled water. One end of the
tubing was shut with a closure, 5 mL of the 1M sodium salicylate solution was pipetted
into the open end and the open end was closed by squeezing out the entrapped air and
using another closure to snap the end shut avoiding leakage or bulging by placing the

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

closure at an appropriate distance to the open end. The weight of the dialysis tubing
assembly was measured. Then, 1.5 L of distilled water was transferred to a 2L beaker.
The time at which the filled dialysis tubing was placed inside the beaker was recorded as
t=0 min. The submergence of the dialysis tubing assembly at all times was ensured by
using a magnetic stir bar in the 2L beaker with the stirring speed set to an appropriate
value. This step is necessary to ensure that the whole membrane surface area can be
available for diffusion. 1mL of sample from the 2L beaker was withdrawn at time=0 and
diluted with 5mL distilled water in a test tube. 2 drops of ferric chloride indicator were
added and the OD of sample was measured at λ=525 nm. Subsequent sampling and OD
measurements were performed at the same wavelength every 15 minutes for 1.5 hours.
The dialysis tubing was removed at the end of the experiment, blotted dry and the final
weight of assembly was measured using an analytical balance. Finally, using a ruler, the
width of the flattened dialysis tube near the closure and the length of tubing between the
two closures were measured and the measurements were subsequently used in the
calculation of surface area.

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

Discussion

The experiment starts with performance of absorption assay on solutions with

known concentration of sodium salicylate. The data points provide the calibration curve

of Absorbance vs. [SA] (ug/mL). The line equation obtained from the calibration curve is

y = 0.2814x – 0.0014 and shows that the two variables are directly proportional to one

another. The R2= 0.9956 which represents good correlation between the two parameters.

In the next part of the experiment, the calibration line equation obtained from first

part along with absorbance measurements of subsequent 5 mL samples taken from the 2L

beaker at 15 minute intervals for a total of 1.5 hours, were used to calculate the C 2=[SA]

that was diffused through the membrane into the 2L beaker. Then, using this data, along

with initial sample concentration and the volume ratio of beaker over dialysis tube, a plot

of ln[1-(C2/Cº1) * (1+(V2/V1))] vs. time was obtained for each calculated [SA] diffused.

The graph gives a straight line of equation: y= -0.0596x + 0.1213 with R 2 = 0.9346 which

shows relatively good linearity of the data. This line is what had been predicted by eq.# 4

in the introduction section of this report in which the slope = -Pm * A * (1/V1 +1/V2)

which was used along with calculated area of dialysis tubing of A = 14.88 cm 2 and the

corresponding volumes of the dialysis tubing and the beaker to determine the value of

permeability constant of sodium salicylate, Pm = 0.02 ml/(cm2*min).

As mentioned in the introduction section earlier, several assumptions were made

for ease of formula derivation and in order to provide a simple model of membrane

diffusion. One of these assumptions was that neither the volume of dialysis tubing, nor

the volume of the beaker change during the course of the experiment. In other words,

partitioning of water into the dialysis tubing caused by osmosis was neglected. The

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

validation of this assumption is further investigated by estimation of volume change in

the dialysis tubing compartment due to osmotic water flow. By using the difference

between the initial and final weights of the dialysis tube, along with calculating the total

mass (g) of salicylate diffused out of the dialysis tube at 90 minutes which marks the end

of the experiment, it was calculated that the total volume of water that entered the dialysis

tube due to osmosis during the course of 90 minutes equaled 1.35 mL which in turn

corresponds to a 27% increase in the volume of dialysis tube, and a mutual decrease in

the volume of beaker, marking a decrease in the ratio V2/V1 as more time elapses. This

calculation proves that the initial assumption of constant V2/V1 ratio is to be rejected.

Therefore, as time goes by, the ratio of V2/V1 is decreased and therefore, the C2

concentration calculated does not correspond to the actual [SA] released in the beaker, in

fact, as water penetrates through the semi-permeable membrane into the dialysis tubing,

the volume of beaker decreases and since in our calculations the decrease in volume of

the beaker was not accounted for, calculated C2 value is higher than the actual value it is

supposed to be if the volume change is taken into consideration. The decrease in ratio

V2/V1 along with the increased calculated C2/Cº1, reaches a point that starts affecting the

experimental results in a negative manner. Meaning that a negative value of

1-[(C2/Cº1)*(1+V2/V1)] is obtained from which a ln value cannot be obtained. This results

in the omission of 2 data points corresponding to t=75 & t=90 mins from the graph of

Fig2 for which calculated values of C2 result in negative value of the above underlined

term. Also note, that the constant volume ratio was assumed because even though

accounting for the volume change would give more accurate results, however, it would

cause further complexity and time constraint to the experimental design and technique.

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

Also, using the Henderson-Hasselbalch equation: pH= pKa + log[acid]/[base] and

pKa,SA = 2.97, the percent ionization of sodium salicylate at pH = 5.5 was calculated to be

99.70% which essentially means sodium salicylate predominantly exists in the ionized

state. This finding is fascinating as we have conducted the experiment by adding sodium

salicylate to unbuffered water. We did not buffer the water because the membrane of the

dialysis tubing used in this experiment is permeable to both the ionized and the unionized

forms of the salicylate, meaning that even if under certain circumstances, the pH of the

solution is changed to values such as 5.5 that would cause the sodium salicylate to be

predominantly ionized, it will still diffuse through the membrane and therefore, usage of

a buffered water system seems unnecessary in this experiment which is incompatible with

models mimicking the biological drug delivery systems that consist mostly of

hydrophobic cellular membranes that are absolutely impermeable to ionized forms of the

compounds.

According to eq. #3, J= DK/h (C1-C2), flux of a system depends on the change in a

few parameters including concentration gradient between the two membranes. As it can

be observed from figure 3, at the beginning of the experiment, the concentration gradient

between the two compartments is at its larger value causing a faster flux (J) which can be

determined by the proportional increase in concentration of SA in the second

compartment. Diffusion and flux decrease with time as the value C1-C2 becomes smaller.

Finally, at equilibrium, the value of flux = 0 because the concentration gradient between

the two compartments is zero, i.e. CSA,1=CSA,2.

Flux also is dependent on membrane thickness h, which is inversely proportional

to rate of diffusion, value of membrane partitioning, K, the small amounts of which will

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

cause a slower rate of diffusion and conversely if K>=1, diffusion will be promoted

(value of K is in turn affected by drug HLB, drug ionization, and membrane HLB). The

last parameter affecting flux is the diffusivity constant, D. A low value of D for the drug

slows down drug diffusion. D is in turn affected by membrane pore size/molecular weight

cut-off, drug size, shape, molecular weight. At given values f Km=1 and h=2.5x10-3 cm,

by using the calculated value of membrane permeability constant, Pm= 0.02 ml/cm2*min

the diffusion coefficient of sodium salicylate, D in the membrane phase is calculated as

5 * 10-5 ml/cm*min. It is expected that the calculated diffusion coefficient produced here

be lower than the diffusion coefficient of salicylate in water because usually the diffusion

coefficient of a substance in liquid is higher than in solid because as it can be inferred

from the formula of the permeability constant, the h factor is omitted in a liquid, causing

an increase in the value of the diffusion coefficient calculated from the corresponding

membrane permeability.

This experiment was conducted at room temperature usually within a range

22-25ºC. However, if the experiment were conducted at 37ºC, one would expect an

increase in the value of the diffusion coefficient. The reason is that as the temperature is

increased, the kinetic energy of the molecules in the system will also increase which

implies greater random Brownian motion of the molecules, i.e. faster mixing of the solute

and solvent, which in turn translates to higher diffusion rate.

In the last part of the experiment, it can be seen that the model that we used for

mimicking membrane diffusion fits the data that was obtained during the lab period. The

concentrations of SA obtained from the calibration curve corresponding to absorbance at

each different time interval are listed in table 3 and it can be seen that these values are

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

very close to the re-calculated value of [SA] (mM) diffused through the membrane using

the obtained value of Pm, A, and Volumes. For example at t=15min, Table3: C2(at

15min)=[SA] diffused = 1.682302772 mM which closely resembles the C2 value

obtained from the permeability constant and listed in Table4: (C2 at 15min)= 1.96581182

mM. The little variation in the value can be due to the fact that for the values listed in

table 4, the Pm, volumes, etc. where all approximated to the significant digits which leads

to different values than if the complete decimal places had been considered in the

calculations. Also, on graph#3 it can be seen that both values of C2 plotted as a function

of time have the same data trend which generally shows that the rate of diffusion

increases as time goes by until the point of equilibrium where C1=C2 and flux is stopped.

This last graph shows that the model that we used for calculating the membrane

permeability model is indeed a valid one.

Results and observations gathered from this experiment, provide basic

understanding of the process of membrane diffusion mimicking over-simplified models

of drug absorption and distribution, however, experimental protocol still has room for

improvement. One of the important improvements in this protocol can be accounted for

by somehow accounting for the change in volume of the beaker and the dialysis tubing

caused by osmosis of water, with the calculated correction factor used in the Pm

calculations to yield a more precise membrane permeability constant.

Conclusion

The experiment starts with the construction of calibration curve by plotting absorbance at

λ=525 nm vs. [SA] (mM). The observed line equation is subsequently used to calculate

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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi

the concentration of SA released in the 2 L beaker at 15 minute time intervals. This C2

value is subsequently used to obtain a plot of ln(1-(C2/C1)*(1+V2/V1)) vs. time, the slope of

which can be used for calculation of Pm= 0.02 ml/(m2min). Also, during the course of the

experiment, the dependence of flux on the concentration gradient was observed by

plotting the C2 vs. time which gives an exponential decay, implying that at the beginning

of the experiment, the flux/membrane diffusion is faster, however, as time is elapsed, the

concentration gradient of SA between the two compartments becomes smaller and the

curve on figure 3 levels off at a certain point, where the value of flux and as a result the

value of membrane diffusion correspond to zero. It is important to note that based on

graph 3, It should also be noted that the amount of membrane diffusion is also dependent

on the ionization state of the drug in biological systems, however, in our over-simplified

model of drug diffusion, it is not accounted for as the membrane of dialysis tubing used

in this experiment is permeable to both ionized and unionized states of sodium salicylate.

The findings of this experiment can provide further insight into mimicking drug delivery

through biological membranes and the information gathered can be used in diverse areas

such as design of controlled-release drug delivery systems and hemodialysis. The data

obtained very closely resembles an oversimplified model of membrane diffusion in

biological systems and overall, it can be concluded that the experiment was a successful

one.

References

1. PHC340Y lab manual (2009-2010)
2. CRC Handbook
3. Wikipedia: www.wikipedia.com

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