Red Blood Cell Membrane Permeability.
Lab #2
I. Introduction.
Materials are continually being exchanged between living organisms and their environments.
At the cellular level this depends on several physiological properties collectively
referred to as the permeability of the cell or plasma membrane. Membrane
permeability is based on properties of surface membranes and driving forces. Cell
membranes are differentially permeable to various materials and show alterations in
permeability under (Fig. 1) different physiological and environmental conditions.
Driving forces are those physical forces that depend on the concentration of a substance
across a cell membrane and on the electrical potential of the cell. These experiments
consider cell permeability in relation to diffusion gradients and the structure of the cell
membrane as revealed by the penetration of solutes with various characteristics.
Phospholipid
Bilayers
Ions
Integral
Protein
Figure 1. Ion channel located in a cell’s membrane.
Hemolysis (Fig. 7), indicated by the sudden appearance of a clear red solution in place of the
previously murky suspension of red blood cells (Fig. 2), results from the swelling and
bursting of the red blood cells because of the penetration of solute and water through
the membrane. RBCs can increase in volume as much as 30-40% before bursting.
Thus, the plasma membrane of red cells is freely permeable to water, relatively
impermeable to salts, and varies in permeability with respect to organic substances.
Figure 2. Normal Red Blood Cells (RBCs).
MCB 403
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Red Blood Cell Membrane Permeability.
Lab #2
Red blood cells placed in an iotonic (Fig. 3B) saline solution retain their normal size. If
placed in a hypotonic (Fig. 3A) salt solution, the influx of water is greater than the
efflux, thus, the red cell increases in volume. The plasma membrane is relatively
inelastic and ruptures (hemolysis) following only a very slight volume increase, leaving
the cell membrane (ghost) behind. If a RBC is placed in a hypertonic (Fig. 3C)
medium the efflux of water is greater than the influx, causing a shrinking of the cell
that results in a crenated RBC (Fig. 4).
A. Hypotonic
B. Isotonic
C. Hypertonic
Figure 3. RBCs in various osmotic conditions. A. Hypotonic. B. Isotonic. C.
Hypertonic. The size of the arrows indicates the relative efflux (on
the right) and influx (on the left) of water into/out of the cell.
Solutes that are permeable or nearly as permeable as water (for example urea) do not
contribute to osmotic pressure. Thus a cell will swell in a urea solution much as it will
in distilled water. Subsequently the plasma membrane may rupture due to excessive
internal pressure.
Figure 4. Scanning micrograph of both normal and crenate RBCs.
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Red Blood Cell Membrane Permeability.
I. The effect of electrolyte
membrane permeability.
and
non-electrolyte
Lab #2
concentration on
The molar concentrations of non-electrolytes that prevent hemolysis are approximately equal.
Osmotic pressure depends on the number of particles in solution. Since electrolytes
dissociate in aqueous solution, hemolysis will occur at lower molar concentrations for
electrolytes than for non-electrolytes. For example, NaCl1 isotonic molar concentration
should be roughly one-half that of Sucrose.
IA. Procedure. (There are 2 methods to perform this procedure. Read both before
proceeding.)
Traditional technique.
1. Set up two rows of 5 test tubes each. One row for Sucrose and the other for NaCl.
(2 Drops or
100 ul)
(2 Drops or
100 ul)
Figure 6. Sequence of solution additions to the series of five (5) test tubes.
2. To each appropriate tube add 2 ml of one of the various molar solutions of Sucrose
or NaCl:
NaCl
Sucrose
0.16,
0.31,
0.12
0.25,
0.08,
0.15,
0.04,
0.08,
0.02,
0.04
0.00
0.00
**Note one of your group should check that these values are correct with the osmometer and flame photometer. ***
1 Physiological saline is 0.9% NaCl or 150 mM.
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Red Blood Cell Membrane Permeability.
3.
4.
5.
6.
Lab #2
Add 2 drops (or 100 ul) of red blood cell suspension.
Mix immediately, but gently.
Set tubes aside for 20 minutes. Remember to set a timer!!
Record your data in the spreadsheet for this lab.
Alternative Technique. With spectrophotometer attached to the data acquisition
system on the Macintosh. Ask the TAs how to operate and Calibrate.
1. Set the spec. to 700 nm (Why ?)
2. Start the acquisition.
3. Type in “Added 1 drop of blood to 0.08 M NaCl” now but DO NOT hit <Return>.
4. Just when you partner adds the blood to the cuvette hit <Return>.
5. Monitor the absorbance over time.
6. Repeat for all six (6) solutions of Sucrose and NaCl.
Does the hemolysis occur slowly and steadily or does it occur quickly? Is there a latency
or does the hemolysis seem to start immediately?
Determine which solutions produced hemolysis (Fig. 7) by holding each tube against this
printed page. If the printing clear, hemolysis has occurred.
When blood is added
Lines on a sheet of white paper held
up behind the test tubes
+
Hemolytic
Compound
=
RBC Intact
RBC Lysed
Figure 7. Example of non-lysed and lysed blood.
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Red Blood Cell Membrane Permeability.
Lab #2
7. From the tubes in which hemolysis did not occur, remove a drop with a disposable
pipette and place on a hemocytometer slide and a cover slip. Using the
microscope compare the size and shape of the cells in the several solutions and
in whole blood (Figs. 2 - 4). It may be necessary to first concentrate the cells by
centrifuging the tubes.
?
?
1
2
?
3
?
?
4
5
Remember: Check each solution
in di vi du al l y !!
No
Hemolysis
?
Microscope
Slide
6
crenated
hypertonic
normal
isotonic
enlarged
lysis
hypotonic
osmolarity
Figure 8. Schematic for determining the status of RBCs in test tubes in which NO
hemolysis occurred.
8. For both NaCl and Sucrose take a sample of:
a. Lowest concentrations adequate to produce hemolysis
b. 0.16 M NaCl.
c. 0.32 M Sucrose.
Determine the concentrations of [Na+] and the osmolarity of these solutions.
MCB 403
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Red Blood Cell Membrane Permeability.
Lab #2
IB. Analysis.
Two familiar formulae are associated with the concepts of osmotic pressure and
depression of freezing point of a solution by addition of solute:
! = cRT
where:
! = Osmotic Pressure (atm. or osmolarity)
c = Concentration (moles/liter)
R = 0.0821 atm./°K mole, Gas Constant
T = temperature (°K)
And
∆Tf (°C) = -1.86 °C * c
where
∆Tf - Freezing point depression.
c - concentration in moles/liter H20.
The equations only apply, however, to an ideal, non-dissociating, non-associating molecule.
Because of this, several factors need to be taken into account. First, most nonelectrolytes do not behave "ideally", in that their osmotic or colligative effect is either
greater or less than would be predicted from their concentration. For example, the
freezing point of an aqueous solution of 0.1 M sucrose is -0.188 °C rather than -0.186
°C as expected, and at 1 M it is 2.06 °C, rather than -1.86 °C. Hence, an "Activity
Coefficient" (i) is needed which is defined as the observed colligative effect divided by
the theoretical colligative effect.
Activity Coefficient =
Observed colligative property
Theoretical colligative property
or
Activity Coefficient=
Observed osmolarity
Theoretical
osmolarity
In the above example at 0.1 M, the osmotic coefficient
is O.188/0.186 = 1.010.
A second consideration, which is fairly obvious, but nevertheless one which students and
others often contrive to forget, is that the osmotic effect of electrolytes depends upon
the number of ions yielded by dissociation in solution. Thus a 0.1 M solution of NaCl
should behave approximately as a 0.2 M sucrose solution, and a 0.1 M Na2S04 should
behave as a 0.3 M sucrose solution. Hence, an appropriate multiplier should be
introduced in the above equation when dealing with dissociating electrolytes.
A third consideration which follows directly from the second is that many electrolytes do not
dissociate completely. Even though strong electrolytes like NaCl, KBr, and Na2S04
have an osmotic effect slightly less than predicted, as if they were not totally
dissociated (even though they are). Some authorities treat the correction for this
separately, but here there seems to be no practical reason for not including it as part of
the previously defined Activity Coefficient. A 0.1 M NaCl solution would be expected
to have a freezing point of -0.372°C and in fact has ∆Tf of -0.347, thus an i of 0.95. At
0.01 M and 1 M the i for NaCl is 0.968 and 0.905 respectively.
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Red Blood Cell Membrane Permeability.
Lab #2
Because of such anomalies, D. A. T. Dick (Water and Living Cells) has proposed that the term
"osmole" be defined as that concentration of any specific solute which has the effect of
one mole of an ideal non-electrolyte in aqueous solution (i.e. the concentration which, for
example, causes a ∆Tf of -1.86 °C). Thus, solutions of two different solutes, each of
which was 1 osM would very likely have different molar concentrations. This term, and
Dick's definition of it, are widely used.
IC. Discussion.
1. At what concentrations of NaCl and Sucrose was hemolysis produced?
2. At what concentrations would the cells be isotonic (i.e. undergo no volume change)?
3. In what way does the shape of the cell bring about a difference in the answers between
Questions (l) and (2)?
4. What would be the osmotic pressure exerted at room temperature by the solute in
previously unmolested red blood cells, if the cells were placed in pure water, if its
volume did not change, and if the membrane allowed no diffusion out?
Use
! = cRT
where:
! = osmotic pressure (atm.)
c = concentration (moles/liter)
R = 0.0821 atm./°mole
T = temperature (°K)
&
Osmotic pressure = cRT
Where
c=0.29M (an RBC has an osmolarity of 290 mosM)
T=298°K (25° C)
R=0.0821 atm/°Kmole
Thus:
OP=.29*0.0821*298
OP=7.09atm
Recall that one mole of an ideal non-electrolyte in 1 liter H20 exerts 22.4 atm. at
STP.
5. Which of the conditions stated or implied in Question 4 (i.e. constraint on volume,
permeability, etc.) would not be met in the real situation?
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Red Blood Cell Membrane Permeability.
Lab #2
II.The effects of molecular size upon permeability.
Generally, molecular weight of a substance gives some idea of the approximate diameter
of its molecules -- the larger the molecule the greater the molecular weight. Steric
configuration and symmetry, or its lack, also influence the effective diameter of a
molecule. Very large molecules (e.g. proteins) have great difficulty entering a cell,
while small molecules (e.g. amino acids) may enter cells more freely.
** Note - other factors, such as partition coefficients, along with size must **
be considered in evaluating the rate at which a given substance enters a cell.
IIA. Procedure.
1. Add 2 drops (or 100 ul) of red blood cell suspension to each of four test tubes containing
respectively 2 ml of 0.3 M solutions of:
Compound
Molecular Weight
Urea
60
Ethylene glycol
62
Glycerol
92
180
Glucose.
(2 Drops or
100 ul)
Figure 9. Sequence of solution additions to the series of four (4) test tubes.
2. Mix each tube gently and hold it against a printed page.
3. Determine the time (seconds) required for hemolysis to occur for each tube.
MCB 403
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Red Blood Cell Membrane Permeability.
Lab #2
IIB. Results and Discussion.
1. What correlation2 exists between hemolysis time and molecular weights of the substances
tested? Is there a correlation between the observed (time to hemolysis) and the known
(molecular weight)? If so what is the correlation coefficient (R2)3?
Time to
Hemolysis.
Molecular weight.
Figure. 10. Plot of the time (in seconds) that it took solutions of various molecular weight to
cause hemolysis.
2. Since all the solutions used in this part of the experiment are hyperosmotic with respect to
the intracellular fluid, why did hemolysis occur? What is the difference between
isotonic and isosmotic? Is it possible for a solution to be isosmotic, but not isotonic?
How and Why?
2 Use the curve fitting option in your plotting program.
3 From the curve fitting option.
MCB 403
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Red Blood Cell Membrane Permeability.
Lab #2
III. Correlation of permeability with partition coefficients.
Nonpolar compounds are compounds in which electrons are equally shared by the two atoms
forming a bond. They have a high solubility in fats or fat solvents, but a low solubility
in water. The solubility ratio of a compound in oil or fat to its solubility in water is
called the Partition Coefficient.
Solubility in oil/lipids
Partition Coefficient =
Solubility in water
The ability of substances to penetrate cells has been related to their solubility in lipid
substances. Increasing lipid solubility generally parallels increasing the length of
carbon chain and higher molecular weight.
*** Note - All three factors affectpenetration rates of compounds into cells ***.
IIIA. Procedure.
1. Add 2 drops (or 100 ul) of red blood cell suspension to each of 4 test tubes containing
respectively 2 ml of 0.6 M solutions of:
Compound
Methyl alcohol
Structure
CH3OH
Ethyl alcohol
CH3CH2OH
0.0337
Propyl alcohol
CH3CH2CH2OH
n-Butyl alcohol.
CH3CH2CH2CH2OH
0.156
0.588
Partition Coefficient
0.0097
(2 Drops or
100 ul)
Figure 11. Sequence of solution additions to the series of four (4) test tubes.
2. Mix each of the tubes and hold them against a printed page.
3. Determine the time required for hemolysis to occur for each tube. If the hemolysis is too
fast to allow an accurate estimate of time, try chilling the cells in ice4 water.
4 Be sure to record in the data the temperature of the "ice water" and the Temp of your solutione when the
experiment was run
MCB 403
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Page 10 of 13
Red Blood Cell Membrane Permeability.
Lab #2
IIIB. Results and Discussion.
1. Plot hemolysis time against partition coefficient and comparative length of carbon
chain.
Time to
Hemolysis
Time to
Hemolysis
Carbon Length
Partition
took Coefficient
solutions of various
Figure. 12. Plot the time (in seconds) that it
partition coefficients to cause hemolysis.
carbon length or
2. What can be concluded about the effect of carbon chain length on red blood cell membrane
permeability?
MCB 403
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Red Blood Cell Membrane Permeability.
Lab #2
IV. The effect of polar groups on membrane permeability.
In non-ionic polar compounds the electrons of a bond between two atoms are attached more to
one atom than to the other. As a consequence, the molecule acts as if it had a negative
end and a positive end. Because of this polarity, it orients itself in an electric field. In
general, uncharged or non polar substances enter a cell more readily than charged or
polar substances.
IVA. Procedure.
1. Add 2 drops (or 100 ul) of red blood cell suspension to each of 4 test tubes containing
respectively 2 ml of 0.3 M solutions of:
Compound
Formamide
Acetamide
Ethylene glycol
Propyl alcohol
Dielectric Constant
109.0
59.0
37.1
20.1
(2 Drops or
100 ul)
Figure 13. Sequence of solution additions to the series of four (4) test tubes.
2. Mix each of the tubes and hold them against a printed page.
3. Determine the time required for hemolysis to occur for each tube.
MCB 403
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Red Blood Cell Membrane Permeability.
Lab #2
IVB. Results and Discussion.
1. Plot hemolysis time against molecular weight and dielectric constants.
Time to
Hemolysis
Time to
Hemolysis
Molecular Weight
Dielectric
Constants
the time (in seconds) that it took solutions
of various dielectric constants
Figure. 14. Plot
or molecular weight to cause hemolysis.
2. What do these data suggest about the electrical condition of the membrane?
3. From the above experiment (Sections I through IV) what can be concluded about the
chemical nature of the cell membrane? Discuss your answer in terms of evidence
collected from these experiments.
MCB 403
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Group Alpha
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Procedure I. Electrolytes/Nonelectrolytes.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
E
Compound
0.16
0.12
0.08
0.04
0.02
0.00
0.32
0.25
0.15
0.08
0.04
M
M
M
M
M
Lysis(Y/N)
M
M
M
M
M
M
Osmolarity (mOsm)
Stock
Solution
[Na] (mM)
Stock
Solution
Notes
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
[Na]
Osmolarity
Lowest [NaCl] to cause hemolysis
0.16 M NaCl
Lowest [Sucrose] to cause hemolysis
0.32 M Sucrose
Procedure II. Molecular Size.
Compound
Molec. Wt.
Lysis (sec)
0.3 M Urea
0.3 M Ethylene Glycol
0.3 M Glycerol
0.3 M Glucose
32
33
34
35
36
37
38
Compound
0.6 M Methyl
0.6 M Ethyl
0.6 M Propyl
0.6 M n-Butyl
39
40
41
42
43
44
Compound
0.3 M Formamide
0.3 M Acetamide
0.3 M Ethylene Glycol
45
0.3 M Propyl Alcohol
Procedure III. Partition Coefficients.
Molec. Wt.
Part Coef.
Carbon Chain Lgth
Notes
Lysis (sec)
Notes
Alcohol
Alcohol
Alcohol
Alcohol
Procedure IV. Polar groups.
Molec. Wt.
Dielectric Coef
Lysis (sec)
Page 1 of 5
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Group Beta
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Procedure I. Electrolytes/Nonelectrolytes.
7
8
9
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12
13
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16
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23
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Compound
0.16
0.12
0.08
0.04
0.02
0.00
0.32
0.25
0.15
0.08
0.04
M
M
M
M
M
Lysis(Y/N)
M
M
M
M
M
M
Osmolarity (mOsm)
Stock
Solution
[Na] (mM)
Stock
Solution
Notes
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
[Na]
Osmolarity
Lowest [NaCl] to cause hemolysis
0.16 M NaCl
Lowest [Sucrose] to cause hemolysis
0.32 M Sucrose
Procedure II. Molecular Size.
Compound
Molec. Wt.
Lysis (sec)
0.3 M Urea
0.3 M Ethylene Glycol
0.3 M Glycerol
0.3 M Glucose
32
33
34
35
36
37
38
Compound
0.6 M Methyl
0.6 M Ethyl
0.6 M Propyl
0.6 M n-Butyl
39
40
41
42
43
44
Compound
0.3 M Formamide
0.3 M Acetamide
0.3 M Ethylene Glycol
45
0.3 M Propyl Alcohol
Procedure III. Partition Coefficients.
Molec. Wt.
Part Coef.
Carbon Chain Lgth
Notes
Lysis (sec)
Notes
Alcohol
Alcohol
Alcohol
Alcohol
Procedure IV. Polar groups.
Molec. Wt.
Dielectric Coef
Lysis (sec)
Page 2 of 5
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Group Gamma
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Procedure I. Electrolytes/Nonelectrolytes.
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8
9
10
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12
13
14
15
16
17
18
19
20
21
22
23
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S
Compound
0.16
0.12
0.08
0.04
0.02
0.00
0.32
0.25
0.15
0.08
0.04
M
M
M
M
M
Lysis(Y/N)
M
M
M
M
M
M
Osmolarity (mOsm)
Stock
Solution
[Na] (mM)
Stock
Solution
Notes
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
[Na]
Osmolarity
Lowest [NaCl] to cause hemolysis
0.16 M NaCl
Lowest [Sucrose] to cause hemolysis
0.32 M Sucrose
Procedure II. Molecular Size.
Compound
Molec. Wt.
Lysis (sec)
0.3 M Urea
0.3 M Ethylene Glycol
0.3 M Glycerol
0.3 M Glucose
32
33
34
35
36
37
38
Compound
0.6 M Methyl
0.6 M Ethyl
0.6 M Propyl
0.6 M n-Butyl
39
40
41
42
43
44
Compound
0.3 M Formamide
0.3 M Acetamide
0.3 M Ethylene Glycol
45
0.3 M Propyl Alcohol
Procedure III. Partition Coefficients.
Molec. Wt.
Part Coef.
Carbon Chain Lgth
Notes
Lysis (sec)
Notes
Alcohol
Alcohol
Alcohol
Alcohol
Procedure IV. Polar groups.
Molec. Wt.
Dielectric Coef
Lysis (sec)
Page 3 of 5
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Group Delta
6
Procedure I. Electrolytes/Nonelectrolytes.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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Z
Compound
0.16
0.12
0.08
0.04
0.02
0.00
0.32
0.25
0.15
0.08
0.04
M
M
M
M
M
Lysis(Y/N)
M
M
M
M
M
M
Osmolarity (mOsm)
Stock
Solution
[Na] (mM)
Stock
Solution
Notes
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
[Na]
Osmolarity
Lowest [NaCl] to cause hemolysis
0.16 M NaCl
Lowest [Sucrose] to cause hemolysis
0.32 M Sucrose
Procedure II. Molecular Size.
Compound
Molec. Wt.
Lysis (sec)
0.3 M Urea
0.3 M Ethylene Glycol
0.3 M Glycerol
0.3 M Glucose
32
33
34
35
36
37
38
Compound
0.6 M Methyl
0.6 M Ethyl
0.6 M Propyl
0.6 M n-Butyl
39
40
41
42
43
44
Compound
0.3 M Formamide
0.3 M Acetamide
0.3 M Ethylene Glycol
45
0.3 M Propyl Alcohol
Procedure III. Partition Coefficients.
Molec. Wt.
Part Coef.
Carbon Chain Lgth
Notes
Lysis (sec)
Notes
Alcohol
Alcohol
Alcohol
Alcohol
Procedure IV. Polar groups.
Molec. Wt.
Dielectric Coef
Lysis (sec)
Page 4 of 5
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Time
(min)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Time
(min)
1
32
33
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7
8
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15
Alternative procedure I. Electrolytes/Nonelectrolytes.
Absorbance
0.16 M
0.12 M
0.08 M
0.04 M
0.02 M
NaCl
NaCl
NaCl
NaCl
NaCl
0.32 M
Sucrose
025 M
Sucrose
Absorbance
.015 M
Sucrose
45
Page 5 of 5
0.08 M
Sucrose
0.04 M
Sucrose
0.00 M
NaCl
AJ