Medical biophysics practices 29. 1 29.

DIFFUSION
SUMMARY:
DIFFUSION: spreading of particles of a substance due to thermal motion.

FICK'S FIRST LAW: the flow of particles per unit time across a unit area (flux) is proportional to the concentration drop,
i.e. J   D  c , where coefficient D is the diffusion coefficient.
x

DIFFUSION COEFFICIENT (D): gives the amount of material diffused across a unit area in a unit time driven by a unit
concentration drop. The unit of the diffusion coefficient is m2/s. It depends on the size and shape of the molecule, on the
interaction with the solvent and on the viscosity of the solvent.

FICK'S SECOND LAW: describes the spatial and temporal changes of the concentration as
 c 
  
  x  c ,
D 
x t
the spatial change of the concentration drop is linked to the temporal change of the concentration.

BROWNIAN MOTION: the random uncorrelated motion of particles due to collisions with the surrounding molecules.

MEAN SQUARE DISPLACEMENT (MSD): a measure of the differences of the positions of particles from a reference
(starting, x0) position:
1 N
MSD    x 0  xi 
2

N i 1

PIXEL: a point in the image. A digital image is a grid of points, each holding the digital value of the light intensity of the
blue, green and red colors in a given spot.

GRAY SCALE: a brightness scale, where the brightness of each pixel is represented by a number between the maximum
(the brightest spot) and the minimum (completely dark spot) value. An example would be 1024 for the maximum and 0 for
the darkness.

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Spreading of the particles due to the arbitrary thermal motion is known as
Further readings:
diffusion. Sugar spreads in the coffee (even without stirring) or the rose scent Damjanovich-Fidy-Szöllősi:
spreads in the room, both by diffusion. This process goes on (it is noticeable) in III /2.1.
case of thermal equilibrium until the distribution of the particles becomes more or 3.1., 3.5., 6.7.4.
less even in the entire volume. Diffusion is extremely important in the living
organism. For example, the exchange of oxygen and carbon dioxide between the diffusion
air in the alveoli of the lungs and the blood within the pulmonary capillaries, and Diffusion
later between the blood and the cells occurs by diffusion. Water, as a very small diffúzió
molecule passes the cell membrane by diffusion as well.
In this practice we will learn the laws of diffusion and we will perform a
measurement of a characteristic parameter of diffusion (diffusion coefficient) on a
free diffusing system.

THEORETICAL OVERVIEW
FICK'S LAWS

The main question according to the diffusion process is what parameters determine diffusion coefficient
the "strength" of diffusion. To characterize this, we define the flow density of Diffusionskoeffizient
particles per second (also called in general: flux): diffúziós együttható

J  , (1)
t  A
that gives the amount of chemical material that passes through a unit area in unit diffusing particle medium D
time. Its unit is mol /(m2∙s). (molecular weight) (m2/s)
H2 (2) air 6.4·10 –5
The answer to the previous question is given by Fick's first law (for stationary O2 (32) air 2·10 –5
diffusion), which sounds in its simplest form as: CO2 (44) air 1.8·10 –5
c H2O (18) water 2.2·10 –9
J   D  , (2)
x O2 (32) water 1.9·10 –9
where c/x is the concentration change along a unit distance (along the x axis), or Glycine (75) water 0.9·10 –9
the concentration drop (concentration gradient). Thus, the flow density of Serum albumine water 6·10 –11
(69 000)
particles per second is proportional to the concentration drop (see Fig. 1). The Tropomyosine water 2.2·10 –11
coefficient of proportionality D is called the diffusion coefficient. D gives the (93 000)
amount of material that diffused through a unit area, in a unit time driven by a unit Tobacco mosaic virus water 4.6·10 –12
concentration drop. The SI unit of the diffusion coefficient is m2/s. The diffusion (40 000 000)
coefficient depends on the size and shape of the diffusing particle and on the
Table 1. Diffusion coefficients of some
viscosity and temperature of the medium. For spherical particles the diffusion
substances at 20 ºC.
coefficient can be calculated from the Einstein-Stokes formula as:
kT
D , (3)
6 r
where r is the radius of the particle,  is the viscosity and T is the temperature of
the medium. The inverse proportionality of D to the size (r) (or molecular weight,
to which the size is proportional) can be seen in the examples of Table 1.
a b c

Fig. 1. Demonstration of Fick's first law. The concentration drop or gradient

(c/x) determines the "strength" of the diffusion in a given system. In panels a
and b, the concentration differences are identical but across different distances.
In panels b and c, the concentration differences are different across the same
distance. Thus, in the a and c panels the identical concentration gradients drive
diffusion of the same "strength".
Medical biophysics practices 29. 3 29. DIFFUSION
 The second important question concerning diffusion is, how fast is the process of
concentration equilibration. Fick's first law does not take into account the
possibility of temporal changes in concentration. It is Fick's second law that
describes the spatial and time changes of concentration (in one dimensional form):

 c 
  
  x  c
D  . (4)
x t
It can be seen that the change of the concentration over the next short time depends
on the actual distribution of the concentration over space at the given time-point.

This is a rather difficult equation, which can not always be solved analytically to
Fig. 2. Agar-agar gel used as a two- give a formula for c(x,t). In general cases numerical (computerized) methods are
dimensional diffusion surface. used, in which we take small Δt time-steps, and step-wise calculate the change of
the concentration over time and space.

DETERMINATION OF THE DIFFUSION COEFFICIENT

We will apply Fick's second law to determine the diffusion coefficients of K + and
colorful (lila) MnO4- ions (together with their hydration shells on a two-
dimensional surface of a gel (Fig.2.). The prepared agarose gel (0.5% m/m%)
provides a hydrated surface, on which the free diffusion of the ions will take place.
t=1 min
The solution of Eq. (4) in a special case when at the beginning of the experiment
the material is concentrated into a very small (practically negligible sized) point
yields a bell-shaped curve, which is broadening over time (See Fig. 3.). The
relative concentration (%)

concentration profile is rotationally symmetric, which means only the distance (r)
from the center is the important spatial parameter:
 r2 
 
 4 Dt 
 
e
c(r , t )  (5)
4Dt
t=1.5 min This equation describes the average spreading of the molecules over time from
their initial position. The broadening of the profile is rapid at the beginning of the
diffusion experiment, and then gradually slows down.
The σ parameter describing the width of the bell-shaped curve is given by:
  2D  t (6)

t=3 min
width at half-maximum (mm)

t=5 min

t=10 min

t=20 min

relative distance from the center

Fig. 3. The concentration profile c(r,t)

as a function of time and space.

elapsed time (minutes)

Fig. 4. The width of the concentration profile over time follows a square-root
function as described by Eq. (6).

We can see from the graph that the width of the bell-shaped curved concentration
profile generally follows a square-root function. From this function it is possible to
determine the diffusion coefficient (D). To make the determination easier, we will
measure the width of the curve as the width at half-maximum, FWHM, (w). This is
the distance of two points where the concentration of the permanganate is half of
the maximal concentration at the center.
Medical biophysics practices 29. 4 29. DIFFUSION
 w  2 ln( 4)  2  D  t , (7)
It is convenient to linearize the equation, by taking the square-root of the time as
the independent variable (x):
w  2 ln( 4)  2  D  x , x t (8)
This is the equation of a straight line, from the slope of the w-x graph the D
diffusion coefficient can be determined. As can be seen, the unit of the diffusion
coefficient is length squared over time (m2/s). It can be seen that the FWHM is
directly proportional to the σ parameter of Eq.(6).
The average distance covered (R) by diffusing particles over time can be calculated
by using the D diffusion coefficient: (in three dimensions)
Raverage  6  D  t (9)

A sample series of images taken over the diffusion can be seen in Fig.5.

Before you start the experiment,

make sure that the agar gel is
fully visible on the camera
image.

If necessary, ask your lab teacher

to help in adjusting the position
of the gel.

Ensure the workplace is evenly

lit, and the image is free from
strong light reflections.

Fig. 5. Sample images taken during the diffusion of potassium permanganate on

an agarose surface. You will take similar images during the lab.
The calibration grid has 5mm
The images taken by a simple usb camera connected to a computer can be used to markings in both directions:
determine the parameters of the concentration profile curve by image analysis.

During the lab we will use the ImageJ open-source scientific image processing 5mm
software to analyze the images. Each image needs a separate calibration, which
can be done by noting that the grids under the petri-dish have a 0.5 cm sized
square.
5mm

Medical biophysics practices 29. 5 29. DIFFUSION

 The steps are the following for the image analysis:
1. Open the image file, and draw a line along one of the lines of the
calibration grid.
Draw an at least 15-
25 mm long
calibration line.

2. Use the “set scale” function to calibrate the camera image to the known
distance (in this example 30mm).

Set the correct scale and unit (here 15 mm) 3. Draw a line through the visible viola spot. Make sure the line crosses at
By ticking the “global” the same calibration the center of the spot.
can be used for all subsequent images.

Since the image is now

calibrated, it is not crucial
that the line you draw is
parallel to any grid line.
The spot can be anywhere
along the line, but
it IS important to cross the
center of the spot.

Medical biophysics practices 29. 6 29. DIFFUSION

 4. Select the „plot profile” menu to get an interactive image of the color
intensity. Use that to read the „x” position of both half-maximum points,
and insert them into the table. A sample gray-scale
(brightness scale) is
shown here:
The brightest pixel has a
value of 255, the
completely dark has a
value of 0. The number
is proportional to the
counted photons in each
pixel.
255 (white)

128 (50% gray)

For consistent results you can pick a gray value corresponding to a grid-
line and use that for every image. The two sample edge points are marked
with a crosshair on the plot.

The „gray value” is the gray-scale value of a pixel (point of the image).
The gray-scale value represents the brightness of the pixel on a numeric
scale. These are recorded by the digital camera for every pixel. (In a
colored image the red, green and blue intensities are separately recorded,
the actual color is a mixture of these values)
0 (black)
5. Use the difference of the „x” data pairs obtained from each image as the 8bit gray scale
width of the curve in the calculations.

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 PLAN OF THE EXPERIMENT
The diffusion will start as soon as the small KMnO4 crystal will be placed on the
surface of the agar-agar gel in the petri-dish.
You will take snapshot images of the gel surface, observing the spread of the viola
color spot of the dissolved and diffusing permanganate ions.
Below is a table draft of the experiment:

Time after Actual time of Left side half- Right side half- Width
crystal is the snapshot maximum “x” maximum “x” (mm)
placed on the (min) position position
surface (min) (mm) (mm)
0 - - - 0
1
2
3
4
5
6
…
…
…

Table 1. Experiment draft.

TASKS
1. Start the image capture software.
2. Align the Petri-dish under the camera.
3. Make sure that the calibration grid is clearly visible on the image on the
computer screen.
4. Take a test-snapshot image.
5. Prepare a timer on your mobile phone or use the stopwatch.
6. Open the Eppendorff tube, and slide the little viola KMnO4 crystal to the
approximate center of the gel surface. Make sure the spot is visible on the
camera image.
7. Start the timer as soon as the crystal is on the surface.
8. At approximately the prescribed time-points take a snapshot with the
camera software and save it on the desktop of the computer.
9. After the last image is acquired, do the image analysis steps and extract
the width data.
10. Make the appropriate graph, fit the theoretical function
11. calculate the diffusion coefficient of KMnO4 on the gel surface.

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