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PHC340 - Expt#9
PHC340 - Expt#9
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Lab 9 – Diffusion and Membrane Transport (I) – Permeation Measurement
Created by Maryam Sadat* Mortazavi
Abstract
Introduction
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;
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= 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:
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.
Procedures
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
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
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
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
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
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
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
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
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
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.
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
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
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
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
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
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
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
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
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
biological systems and overall, it can be concluded that the experiment was a successful
one.
References
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