Annals of Biomedical Engineering, Vol. 33, No. 2, February 2005 (©2005) pp.
214–222
DOI: 10.1007/s10439-005-8980-9
Diffusivity and Solubility of Nitric Oxide in Water and Saline
IAN G. ZACHARIA1 and WILLIAM M. DEEN1,2
1
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA and 2 Biological Engineering Division,
Massachusetts Institute of Technology, Cambridge, MA
(Received 5 April 2004; accepted 13 September 2004)
Abstract—Aqueous diffusivities and solubilities of NO were
determined by using a chemiluminescence detector to measure
time-dependent fluxes across stagnant liquid films confined be-
tween polydimethylsiloxane membranes. The NO diffusivities in
pure water and PBS at 25◦ C were found to be (2.21 ± 0.02) ×
10−5 cm2 s−1 and (2.21 ± 0.04) × 10−5 cm2 s−1 , respectively. Al-
though lower than most previous values for NO at room tem-
perature, these diffusivities are very similar to those for O2 ,
as one would expect. Extrapolation to 37◦ C yielded a value of
3.0 × 10−5 cm2 s−1 . The solubility of NO in water at 25◦ C was
(1.94 ± 0.03) × 10−6 mol cm−3 atm−1 , in agreement with the liter-
ature. This agreement, along with the excellent fits obtained to the
transient flux data (<4% rms error in each experiment), supports
the validity of the simultaneously measured diffusivity. The solu-
bility of NO in PBS at 25◦ C was (1.75 ± 0.02) × 10−6 mol cm−3
atm−1 . The modest (10%) reduction in solubility relative to that
in pure water is consistent with the usual effects of salts on gas
solubilities.
Keywords—Diffusion of dissolved gases, Silastic, Chemilumi-
nescence detection.
INTRODUCTION
The physiological importance of nitric oxide (NO),
both as a signaling molecule and in the nonspecific
immune response, has motivated numerous efforts to
predict NO concentrations in vivo and in experimental
systems.3–6,17,24,35,39 A key parameter in such calculations
is the NO diffusivity (DNO ) in the liquid or liquid-solid
mixture of interest (tissues, cells, or culture media). How-
ever, even in water or buffer solutions, the value of DNO
is surprisingly uncertain. Wise and Houghton38 used mea-
surements of the rate of collapse of NO bubbles in water to
obtain DNO = 3.0 × 10−5 cm2 s−1 at 25◦ C. Denicola et al.8
used the ability of NO to quench the fluorescence of pyrene
derivatives to infer that DNO = (4.5 ± 0.3) × 10−5 cm2 s−1
in buffer solutions at 20◦ C. A value of 2.6 × 10−5 cm2 s−1
Address correspondence to William M. Deen, Department of
Chemical Engineering, 66-572, Massachusetts Institute of Technology,
77 Massachusetts Avenue, Cambridge, MA 02139. Electronic mail:
wmdeen@mit.edu
214
0090-6964/05/0200-0214/1 
C 2005 Biomedical Engineering Society
(in water at 25◦ C) appeared in a handbook,20 but was undoc-
umented and was omitted from recent editions. Vanderkooi
et al.34 used a phosphorescence quenching technique to ob-
tain DNO = 8.7 × 10−7 cm2 s−1 in water at 20◦ C. That value
is far too low to be reasonable for a molecule as small as NO,
and potential errors in the methodology have been pointed
out.8 Overall, the existence of at least a two-fold range in
the reported DNO values suggests that additional data are
needed.
There are many well-established techniques for measur-
ing the diffusivities of dissolved gases in liquids.12 Monitor-
ing the flux of the gas across a stagnant film of the test fluid is
one of the most direct approaches. This type of measurement
has been used, for example, to obtain aqueous diffusivities
for O2 9,13,16,32 and CO2 ,10,23,33 and a diffusivity for NO in
formamide.37 The simplest variation of this method relies
on the fact that the steady-state flux will be proportional
to the permeability, which is the product of the solubility
and diffusivity. With the film thickness and external par-
tial pressures set by the experiment, and a solubility that is
obtained independently, the diffusivity is readily calculated
from the steady state flux.10,23,32,33 In practice, the liquid
must be confined by one or two gas-permeable membranes,
and corrections made for their diffusional resistances.
Another way to use liquid film measurements is to focus
on the time-dependent fluxes obtained after a sudden change
in the gas concentration on one side of the liquid. The ad-
vantage in using transient data, either by extracting the lag
time before a flux is observed13,16,37 or fitting the full set
of time-dependent data,9 is that the calculated diffusivity is
independent of solubility. In a few instances, transient and
steady state information have been combined to determine
both the diffusivity and the solubility.13,16
In the work reported here, we used a chemiluminescence
detector to measure transient and steady fluxes of NO across
aqueous films bounded by polydimethylsiloxane (Silastic)
membranes. Oxygen was purged from the system to ensure
that reactions would not influence the measured fluxes, and
separate measurements of NO permeation through the Silas-
tic membranes were made to account for their contributions
to the overall resistance. By simultaneously adjusting the
 Diffusivity of Nitric Oxide
FIGURE 1. Scale drawing of cross-section of diffusion cell
used to measure diffusivity and solubility of NO.
aqueous NO solubility and diffusivity to obtain a best fit
to the flux data over an entire run, both properties could
be determined. A comparison of the solubility values with
well-established results in the literature provided a check on
the validity of the NO diffusivities. Results were obtained
for both pure water and saline.
MATERIALS AND METHODS
Apparatus
The diffusion cell used is shown in Fig. 1. It consisted
mainly of two acrylic plates (64 mm × 64 mm × 15 mm)
adapted from a dialysis cell (Spectra/Por MacroDialyzer,
Spectrum Laboratories, Rancho Dominguez, CA). Each
plate had three ports fitted with gas-tight Luer fittings. The
test fluid was bounded by a neoprene gasket (51 mm o.d.,
∼32 mm i.d.) and a pair of Silastic membranes sandwiched
between the plates. The Silastic membranes of 47 mm di-
ameter were punched from 0.25 mm thick sheets (Specialty
Manufacturing Inc, Saginaw, MI). Silastic was selected be-
cause of its high permeability to NO and O2 , and neoprene
because of its low gas permeabilities.21 Stainless steel sup-
port screens (Millipore Corp., Bedford, MA, part number
XX4004704) kept the Silastic membranes flat, while being
porous enough to have a negligible diffusional resistance.
Four cap screws were used to press the acrylic plates down
onto the gasket until a desired liquid thickness was achieved,
with silicone vacuum grease applied around the edges to en-
sure a gas-tight assembly. The final liquid thickness, which
was typically 2.7 mm, was set by Teflon spacers placed
outside the gasket. The actual thickness was determined for
each run by dividing the volume of liquid injected by the
open area of the gasket.
Water and PBS were used as test fluids. The water
was deionized and sterilized (Milli-Q RG, Millipore Corp.,
Bedford, MA) and the PBS, purchased sterile (AccuGENE
1xPBS, Cambrex Corp., East Rutherford, NJ), consisted of
150 mM NaCl, 5 mM Na2 HPO4 , and 1.7 mM KH2 PO4 . To
load the diffusion cell, 27G syringe needles were inserted
into opposite sides of the gasket, and a syringe attached to
215
one. To minimize air bubbles, the cell was rotated to put
the syringe needle at the bottom, and liquid injected until it
exited via the top needle.
Gas connections to the diffusion cell allowed either an
NO mixture (1% NO in N2 , BOC, Edison, NJ) or pure
N2 (99.999% pure, low oxygen grade, BOC, Edison, NJ)
to flow past the “upstream” membrane, while N2 flowed
past the “downstream” membrane. The NO gas was first
passed through a column of 4–8 mesh soda lime pellets to
remove higher nitrogen oxides. The downstream gas exited
to a chemiluminescence detector (Sievers NOA 280, Ionics
Instrument Business Group, Boulder, CO) for continuous
monitoring of the NO concentration. The N2 carrier gas
kept the downstream NO concentration small but still eas-
ily measurable; the detection limit of the instrument was
about 1 ppb. The detector response time was much shorter
than the diffusion time scale in the test liquid (∼2 s as com-
pared to ∼20 min). The chemiluminescence detector was
calibrated according to the manufacturer’s specifications
using an NO gas mixture (45 ppm NO in N2 , Scott Medical
Products, Plumsteadville, PA) and a gas with negligible NO
concentration (filtered air). The concentration signal from
the detector was outputted to a computer for later analysis.
The flow rate of the carrier gas was also measured (Gilmont
Accucal flowmeter, Barnant Company, Barrington, IL), so
that it could be multiplied by the NO concentration to deter-
mine the molar flow rate through the test fluid as a function
of time.
Aqueous Diffusivity and Solubility Procedure
After assembly of the diffusion cell, N2 was flowed past
both membranes for 60 min to remove dissolved O2 from
the Silastic and test fluid (The basis for that purge time is
discussed later). The diffusion experiment was begun by
switching the upstream gas to the NO mixture. The NO
molar flow rate across the test fluid was then monitored
typically for 90 min, which was sufficient to reach a steady
state. These and other measurements were done at room
temperature (23–27◦ C). The resistances in the gas lines
were such that the total pressure in each half-cell was very
close to the ambient value. Pressures in the lab were mea-
sured each day for calculation of the NO partial pressure in
the upstream chamber and total pressure in the downstream
chamber.
Evaporation Measurements
Evaporation of the liquid film occurred to a small extent
during the experiments, because all gases used were dry.
The evaporation rate was determined in separate experi-
ments by weighing the filled diffusion cell as a function of
time while N2 flowed through both chambers. After 60 min
(the duration of the O2 purge), ∼0.6% of the liquid had
evaporated, and after 150 min (the total time for a typical
216 I. G. ZACHARIA and W. M. DEEN

run), the evaporative loss was ∼1.5%. To correct for this, tified by the fact that the downstream NO gas concentration
the thickness of the liquid film measured prior to the exper- (<200 ppb) was always negligible relative to the upstream
iment was reduced by 1% in all of the aqueous solubility concentration (10,000 ppm).
and diffusivity calculations. Equality of partial pressures and fluxes at the aqueous-
Silastic interfaces required that
Silastic Diffusivity and Solubility Procedure
CNO (0, t) C̄NO (0, t)
= (5)
The experimental setup used to measure the NO diffusiv- αNO ᾱNO
ity and solubility in Silastic was identical to that for the test
∂CNO ∂ C̄NO
fluids, except that the upstream Silastic layer was removed, DNO (0, t) = D̄NO (0, t) (6)
2–3 Silastic sheets were placed together on the downstream ∂x ∂x
side, and the test fluid was absent. CNO (L f , t) C̄NO (L f , t)
= (7)
αNO ᾱNO
Calculation of Diffusivity and Solubility
∂CNO ∂ C̄NO
The NO flux measurements for the full system or for DNO (L f , t) = D̄NO (L f , t) (8)
∂x ∂x
Silastic alone were analyzed using one-dimensional tran- With t = 0 being the moment when the O2 purge was ended
sient diffusion models. As shown in Fig. 2, the model for the and NO introduced, the initial conditions were
complete system included a layer of test fluid of thickness
L f in series with two Silastic membranes, each of thickness CNO (x, 0) = 0 (9)
L s . The aqueous and Silastic NO concentrations in molar C̄NO (x, 0) = 0 (10)
units are denoted as CNO (x, t) and C̄NO (x, t), respectively,
where the position coordinate (x) is defined in Fig. 2 and The model for the experiments with Silastic alone was
t is time. (Overbars are used throughout to denote quanti- similar, except that the aqueous layer was omitted and the
ties in Silastic.) The O2 concentration following the purge Silastic membranes combined.
was assumed to be low enough to make NO oxidation rates The steady-state problem for the three-layer system can
negligible (see below). With reactions absent, the species be solved analytically to give
conservation equations applied to the aqueous and Silastic ∂ C̄NO
layers were m NO (∞) = −A D̄NO (L s + L f , ∞)
∂x
∂CNO ∂ 2 CNO A PNO
= DNO (1) = (11)
∂t ∂x2 L f /(αNO DNO ) + 2 L s /(ᾱNO D̄NO )
∂ C̄NO ∂ 2 C̄NO where mNO (t) is the molar flow rate of NO to the detector
= D̄NO (2)
∂t ∂x2 and A is the open area of the test cell. As shown in the right-
The boundary conditions at the external membrane sur- hand expression, the steady molar flow rate depends on the
faces were solubility-diffusivity products in the respective phases, so
that neither solubilities nor diffusivities can be determined
C̄NO (−L s , t) = ᾱNO PNO (3) separately from such data. The same is true for two Silastic
C̄NO (L s + L f , t) = 0 (4) membranes alone, where the solution is obtained by setting
L f = 0 in Eq. 11. To extract more information from our
where ᾱNO is the NO solubility in Silastic and PNO is the experiments, we used the time-dependent molar flows from
NO partial pressure in the upstream gas. Equation 4 is jus- each run. After solving Eqs. 1–10 to determine CNO (x, t)
and C̄NO (x, t), mNO (t) was computed by evaluating the flux
out of the downstream Silastic membrane, as in the first
equality in Eq. 11.
Solutions to the transient diffusion problems (with or
without an aqueous layer) were obtained using a com-
mercial finite element package (FEMLAB 2.2, Comsol
Inc., Burlington, MA). The implicit solver “fldaspk” was
used in all simulations. The key time-stepping parameters
were the relative tolerance (0.001), the absolute tolerance
(1 × 10−5 ), and the maximum time step (0.01, with time
FIGURE 2. Schematic for transient diffusion model of com- scaled by L 2f /DNO ). Reducing any of these parameters by
plete system. CNO (x, t) and C̄ NO (x, t) are the concentrations of a factor of 10 led to changes of less than 0.01% in the
NO in the aqueous and Silastic phases, respectively; DNO and
D̄ NO are the respective diffusivities; and αNO and ᾱNO are the transient NO flux at the downstream membrane, demon-
respective solubilities. strating that they were all sufficiently small. The finite
 Diffusivity of Nitric Oxide
element mesh used was the default mesh refined twice us-
ing the refinement tool, and further refinement also pro-
duced less than a 0.01% change in the fluxes. Second-
order Lagrange (quadratic) elements were used in all
simulations.
The experiments using Silastic alone were used to eval-
uate D̄NO and ᾱNO . With the Silastic properties determined,
DNO and α NO for the test fluid could be found from the
aqueous-Silastic experiments. In either case, the two un-
known parameters were calculated simultaneously by fitting
the full set of data for mNO (t). A least-squares fit based on
relative errors was used to determine the final DNO and α NO
values. The MATLAB minimization function “fmins” was
used to minimize the rms error between the simulations and
the experimental data. This function used the Nelder–Mead
simplex (direct search) method to choose the best fit. The
key option for the fmins function is the second parameter,
which is the termination tolerance of the fitting parameters;
after first fitting the results with a less stringent value of
2 × 10−2 , it was set to 5 × 10−3 . The final parameter val-
ues were confirmed to be independent of the initial guesses,
within a two-fold range.
The evaluation of both parameters from each set of data
was facilitated by their very different effects on mNO (t):
whereas both affected the steady state molar flow rate
(Eq. 11), the time to reach the steady-state value was deter-
mined by the diffusivity alone. The individual effects of the
two parameters are illustrated later.
The measured diffusivities and solubilities are expressed
as mean ± standard error, and Student’s t-test was used to
evaluate the significance of differences between mean val-
ues in water and PBS. Each experimental run provided a
value for the diffusivity and solubility in water or PBS by
extracting the best-fit parameters. Mean values and stan-
dard errors were determined from 8 runs in water and
5 runs in PBS. The aqueous diffusivities at room temper-
ature were adjusted to 25◦ C by assuming that DNO µ/T is
constant, where µ is the viscosity of water and T is absolute
temperature.26 The aqueous solubilities were temperature-
corrected using an expression of the form log αNO = a +
b/T ,29 with the temperature-dependence parameter evalu-
ated from previous data as b = 1512 K.7
Simulation of Oxygen Purge
The experimental design was guided by an analysis of
O2 removal during the purge period and the effects of any
residual O2 during the experimental period. The objective
was to predict how long it would take to achieve O2 con-
centrations that were low enough to ensure that chemical
reactions would not influence the calculated diffusivity and
solubility for NO. Simulating the effects of residual O2 on
the measured flux of NO required that transient diffusion-
reaction problems be solved simultaneously for the NO and
O2 concentrations.
217
Included in these simulations were the well-known oxi-
dation of NO by O2 in water.19 Neglecting trace intermedi-
ates (NO2 and N2 O3 ), the overall reaction between O2 and
NO in water can be written as
k1
4NO + O2 + 2H2 O −→ 4NO−
2 + 4H
+
(12)
Using Ri to denote the volumetric rate of formation of
species i (e.g., mol m−3 s−1 ), the aqueous reaction rates
for NO and O2 were
RNO = −4k1 CNO
2
CO2 = 2RO2 (13)
where CO2 is the oxygen concentration and k1 = 2.1 ×
106 M−2 s−1 at 23◦ C.19 The partial oxidation of NO that has
been proposed to occur in Silastic was neglected,36 because
preliminary calculations suggested that its effects would be
negligible here.
In general, the conservation equations for species i (NO
or O2 ) in the aqueous and Silastic phases were rewritten as
∂Ci ∂ 2 Ci
= Di + Ri (14)
∂t ∂x2
∂ C̄i ∂ 2 C̄i
= D̄i + R̄i (15)
∂t ∂x2
with reaction rates as given by Eq. 13. For the purge period
(prior to introduction of NO), only the O2 problem had
to be solved and the reaction terms were absent. The O2
concentrations in both gas streams were negligible, and the
system was saturated initially. Accordingly, the boundary
and initial conditions for the purge period were
C̄O2 (−L s , t) = 0 = C̄O2 (L f + L s , t) (16)
CO2 (x, 0) = αO2 PO2 (17)
C̄O2 (x, 0) = ᾱO2 PO2 (18)
where PO2 is the ambient partial pressure of O2 . The match-
ing conditions at the internal interfaces were analogous to
Eqs. 5–8. At the start of the experimental period (the mo-
ment NO was introduced), the new initial condition for O2
corresponded to the concentration profile obtained at the
end of the purge. Thereafter, the coupled reaction-diffusion
problems were solved for both NO and O2 .
The parameter values used in the purge simulations are
given in Table 1. The diffusivity and solubility for O2 in
Silastic were equated with the NO values measured by us
(as presented later), based on previous reports that the O2
and NO diffusivity and solubility in Silastic are similar.18,27
For simplicity, DNO (obtained from this work) and DO2 9
were each rounded here to 2 × 10−5 cm2 s−1 .
RESULTS
Adequacy of Deoxygenation
To avoid artifacts in the diffusivity and solubility deter-
minations caused by chemical reactions, it was necessary
218 I. G. ZACHARIA and W. M. DEEN

TABLE 1. Parameter values used to simulate the ef- the achievement of a steady state. However, once O2 re-
fects of varying purge times. moval was sufficiently complete, further increases in the
Parameter Units Value Source purge time had no effect. Curves for a 60 min purge and
for a diffusion run with no O2 present (not shown) both
DNO cm2 s−1 2 × 10−5 This work differed by <0.2% from that for a 40 min purge. The maxi-
DO2 cm2 s−1 2 × 10−5 9
mum O2 concentrations calculated for purge times of 5, 20,
D NO cm2 s−1 1 × 10−5 This work
D O2 cm2 s−1 1 × 10−5 See text
40, and 60 min were 150, 11, 0.34, and 0.010 µM, respec-
α NO PNO µM 20 This work tively. The steady state molar flow rate of NO was found to
αO2 PO2 µM 260 7 be unaffected by the purge time, because any residual O2
ᾱNO PNO µM 320 This work was always calculated to be gone within ∼60 min, removed
ᾱO2 PO2 mM 4.2 See text both by diffusion and by its reaction with NO. Overall, the
k1 M−2 s−1 2.1 × 10−6 19
results in Fig. 3 suggested that a 60 min purge would be
Lf mm 2.54 —
Ls mm 0.254 — conservative.
A cm2 7.67 — An experimental assessment of the adequacy of O2 re-
moval is shown in Fig. 4. Here, data are shown for purge
times of 0, 60, and 120 min. The three runs involved slightly
to thoroughly deoxygenate the test cell prior to introduc- different values of the liquid film thickness Lf , partial pres-
ing NO. Combining Eq. 14 for NO with the reaction rate sure PNO , and temperature (which affects DNO and α NO ).
given in Eq. 13 reveals that the Damkohler number (Da) Accordingly, to allow the results to be compared more pre-
that measures the importance of NO oxidation relative to cisely, the mass flow rates were made dimensionless as de-
diffusion in the liquid film is Da = 4k1 CNO
0
CO0 2 L 2f /DNO , scribed in the figure legend, and t was scaled by the char-
where CNO 0
and CO0 2 are the aqueous concentration scales acteristic time for diffusion across the liquid (L 2f /DNO ). As
for NO and O2 , respectively. For water in contact with 1% expected from the simulations, there was a large difference
NO gas at 25◦ C, CNO
0
= 19 µM. Using the parameter values in the mNO (t) curves between purge times of 0 and 60 min,
in Table 1, we estimated that CO0 2 < 0.2 µM is needed to but the results for 60 and 120 min were indistinguishable.
make Da < 0.1. In comparison, the aqueous O2 concentra- This further supports the use of a purge time of 60 min in
tion under ambient conditions is 260 µM. Thus, >99.9% all subsequent experiments. When plotted on the same axes,
of the O2 needed to be removed during the purge. the data with no purge in Fig. 4 agreed fairly well with the
The results of simulations performed to estimate the re- analogous simulation in Fig. 3. This confirms that no large
quired purge time are given in Fig. 3, in which mNO (t) is error was introduced by neglecting the reaction of NO with
plotted for hypothetical diffusion runs preceded by purge O2 in the Silastic.
times ranging from 0–40 min. In general, increasing the
purge time, and therefore reducing the consumption of NO
at early times in the diffusion run, was predicted to speed

FIGURE 4. Experimental data showing effect of purge

FIGURE 3. Simulations showing effect of purge time on NO
molar flow rate, with water as test fluid. The system was as-
sumed to be purged initially of O2 by flowing N2 over both
membranes for the four times indicated. The parameter values
used are given in Table 1.
time on NO molar flow rate, with water as test fluid. As
shown, time was scaled by dividing the actual time by
L2f /DNO ; one unit is ∼60 min. The scaled molar flow rate is
mNO (t)Lf /(ADNO αNO PNO ). For no purge (“0 min”), where a valid
DNO and αNO could not be obtained from the data, the DNO and
αNO values used for the plot were based on the mean results
for water (Table 2). The data for purge times of 60 and 120 min
are indistinguishable.
 Diffusivity of Nitric Oxide 219

NO in Silastic
The apparatus was designed so that, based on literature
values for the NO properties, the steady state diffusional
resistance of the two Silastic layers would be small. (The
aqueous and Silastic resistances appear in the right-hand
denominator of Eq. 11.) Nonetheless, separate experiments
were performed to check the Silastic properties. The NO
permeability measured from the steady-state fluxes in three
experiments was ᾱNO D̄NO = (3.11 ± 0.02) × 10−10 mol
cm−1 s−1 atm−1 at 25◦ C. From this and the permeability
results that will be presented shortly for water and PBS, the
resistance of the two Silastic membranes was calculated
to be <3% of the total. The full time-dependent data
for Silastic yielded best-fit values of D̄NO = 1.0 × 10−5 FIGURE 5. Comparison of representative data for water with
simulations. The best-fit curve is nearly indistinguishable from
cm2 s−1 and ᾱNO = 3.1 × 10−5 mol cm−3 atm−1 , with the data. The other curves are based on selective changes in
estimated uncertainties in each case of about 20%. DNO , with the permeability maintained at its best-fit value of
The much greater uncertainty in D̄NO than in ᾱNO D̄NO DNO αNO = 4.29 × 10−11 mol cm−1 s−1 atm−1 . The deviations
between those curves and the best-fit curve (or data) show the
was caused by the thinness and high permeability of the sensitivity of the transient NO molar flow rate to DNO .
Silastic membranes. The rise to a steady state was very
rapid in these experiments (∼1 min), and the high fluxes
caused the detector to switch sensitivity modes during the
The results for water and PBS at 25◦ C are sum-
runs. Accordingly, it was difficult to accurately resolve the
marized in Table 2. The mean NO diffusivity in wa-
time-dependent mass flow rate, as is needed in calculating
ter (2.21 × 10−5 cm2 s−1 ) was the same as that in PBS
a precise value of D̄NO . However, simulations performed
(2.21 × 10−5 cm2 s−1 ). These values are 36% lower than
by varying D̄NO (with ᾱNO D̄NO fixed) indicated that a 20%
the diffusivity of Wise and Houghton38 and less than half
uncertainty in D̄NO would lead to a <5% uncertainty for
that of Denicola et al.8 The NO solubility in PBS was
DNO in water or PBS. Although contributing little to the
slightly (10%) but significantly lower than that in pure wa-
steady state resistance, the Silastic membranes had a very
ter (1.75 × 10−6 mol cm−3 atm−1 in PBS as compared with
noticeable effect on the dynamics; ignoring them would
1.94 × 10−6 mol cm−3 atm−1 in water).
have decreased the fitted DNO by 22%.

NO in Water and PBS DISCUSSION

The time-dependent molar flow rates from a represen-
tative run with water are shown by the discrete symbols
in Fig. 5. As indicated by the solid curve, adjusting DNO
and α NO produced an excellent fit to the data over the full
range of times. To demonstrate the sensitivity of the results
to DNO , the dashed and dotted curves show simulations us-
ing aqueous diffusivities 20% above or below the best-fit
value. In these calculations the product α NO DNO was held
constant, which fixed the steady state molar flow rate. The
sizable deviations of the dashed and dotted curves from the
solid (best-fit) curve indicate that, even in a single experi-
ment, the NO diffusivity could be resolved to within a few
percent accuracy. All other data for water and PBS had a
similar appearance to that in Fig. 5, and the fit to the entire
curve was excellent in each case, with rms errors of <3.6%.
For transient diffusion across a liquid film, the time scale
should vary as L 2f and the steady flux as 1/Lf . This was tested
by reducing the film thickness in one experiment from the
usual 0.27 to 0.22 mm. As expected, the ∼20% reduction
in thickness reduced the rise time by ∼40%. The diffusivity
calculated from the data for the thinner film was within 6%
Measuring time-dependent molar flow rates of NO
across deoxygenated aqueous films provided a relatively
direct way to determine DNO . As already mentioned, sim-
ilar methods have been used numerous times to measure
the diffusivities of gases in liquids.9,10,13,16,23,32,33,37 The
main complication in applying this approach to NO was
that, to avoid artifacts due to chemical reactions, great care
was needed to remove O2 from the system. Simulations
of O2 removal during a preliminary purge period (Fig. 3)
guided the design of the experiments, and the purge time
TABLE 2. Diffusivity and solubility of NO in water and PBS at
25◦ C.
Liquid DNO (10−5 cm2 s−1 ) α NO (10−6 mol cm−3 atm−1 )
Water 2.21 ± 0.02 1.94 ± 0.03
PBS 2.21 ± 0.04 1.75 ± 0.02a
Values are mean ± s.e. for 8 experiments with water and 5 with
PBS.
a Significantly different from value in water, by Student’s t-test ( p =

of the mean results for the thicker films. 0.0003).

220 I. G. ZACHARIA and W. M. DEEN
that was adopted was confirmed to be adequate (Fig. 4).
Fitting the full set of transient data permitted calculation
of an aqueous solubility, as well as a diffusivity, from each
experimental run. Because the solubility of NO in water is
well known, comparing the value of α NO so obtained to that
in the literature provides an independent check on the data.
Our value of αNO = (1.94 ± 0.03) × 10−6 mol cm−3 atm−1
at 25◦ C matches the literature solubility1,7 of 1.93 − 1.95 ×
10−6 mol cm−3 atm−1 . This suggests that the diffusivities
were not affected by systematic errors. Moreover, the aque-
ous diffusivities and solubilities were highly reproducible
(standard errors of about 1–2%, as shown in Table 2).
A potential source of artifacts when using flux measure-
ments to obtain diffusivities is convection in the liquid film,
which would tend to enhance the flux. Indeed, problems
with forced or free convection have been identified in some
previous studies. Convection effects in the O2 experiments
of Goldstick and Fatt,9 which appear to have been due
mainly to direct contact between the liquid and upstream
gas (an upstream membrane was not used), were elimi-
nated by reducing the gas velocity and liquid film thickness.
Stroeve31 and Meldon et al.23 inferred that step changes in
total pressure associated with their methods of introduc-
ing the test gas (O2 and CO2 , respectively) induced some
initial fluid motion that caused the diffusivities calculated
from their transient data to differ from those obtained at
steady state. Our experimental design avoided these sources
of convection by employing an upstream membrane and by
maintaining constant (atmospheric) gas pressures. Another
problem seen in experiments with CO2 , which has a partic-
ularly high aqueous solubility, was buoyancy-driven flow.22
That is, density gradients created by the imposed concen-
tration difference could induce free convection. Meldon22
showed experimentally that these effects were negligible
for Ra = ( ρ/ρ)g L 3f /(ν D) < 2000, where Ra is the mass-
transfer Rayleigh number, ρ/ρ is the fractional change in
liquid density, g the gravitational acceleration, ν the kine-
matic viscosity, and D the diffusivity of the solute (in that
case, CO2 ). For our experiments with NO, we estimated
that the fractional density change was ρ/ρ = 4 × 10−7
and that Ra < 40. Together with the consistency of our tran-
sient and steady-state results, this indicates that convection
was negligible in our experiments.
The short diffusional transients and high fluxes obtained
when using Silastic membranes alone (without a liquid film)
made our apparatus less well suited for measuring Silastic
properties than aqueous properties. Nonetheless, our diffu-
sivity value of D̄NO = 1.0 × 10−5 cm2 s−1 at 25◦ C com-
pares well with a previous report of 1.2 × 10−5 cm2 s−1 .18
Our permeability value of ᾱNO D̄NO = 3.1 × 10−10 mol
cm−1 s−1 atm−1 falls between those reported for untreated
Silastic (1.7 × 10−10 mol cm−1 s−1 atm−1 ) and Silastic af-
ter autoclaving (3.4 × 10−10 mol cm−1 s−1 atm−1 ).18 Other
variables affecting the properties of Silastic and silicone
rubbers, aside from their thermal history, are the amounts
of crosslinking and the fillers used,2,25 information which
is usually proprietary. Thus, measurements must be made
with the specific material of interest if accurate diffusiv-
ity and/or permeability values are needed. As noted earlier,
the diffusional resistance of our Silastic membranes was
small relative to that of the aqueous film. Thus, a mod-
erately accurate value of D̄NO was sufficient to give ac-
ceptably small errors in the desired aqueous properties for
NO.
Under unreactive conditions, the diffusion behavior of
NO closely parallels that of O2 . That is, our NO diffusiv-
ity in pure water of 2.21 × 10−5 cm2 s−1 at 25◦ C is nearly
identical to that for O2 . Based on time-dependent flux mea-
surements across aqueous films, Goldstick and Fatt9 found
that DO2 = 2.13 × 10−5 cm2 s−1 at 25◦ C. (Although their
methods were unusually rigorous, their result did not differ
appreciably from the average O2 diffusivity in 18 other stud-
ies they reviewed.) Nearly identical diffusivities for NO and
O2 are expected on the basis of their similar molecular sizes,
as shown by standard correlations used to predict gas dif-
fusivities in liquids. The results obtained by applying three
such correlations to O2 and NO (all of which use the liquid
molar volume of the diffusing species at its normal boiling
point) are shown in Table 3. The correlations in Table 3
have been found to predict diffusivities in many systems
to within an average error of ∼10%.26 It is seen that each
correlation predicts an O2 diffusivity slightly lower than the
NO value, the differences being ≤10%. The absolute values
predicted by the correlations were reasonably accurate for
both gases, the maximum error being a 14% underestimate
of DNO from the Tyn and Calus correlation.
The comparisons between O2 and NO properties can be
extended to PBS. Our average value for DNO in PBS of
2.21 × 10−5 cm2 s−1 was the same as that in pure water.
Similarly, the diffusivity of O2 in PBS has been reported
to be only 3% lower than in water.9 Our NO solubility in
PBS was 10% lower than in water (a statistically signifi-
cant difference), as compared with a reported reduction of
5% for O2 .28 Those solubility changes are consistent with
expectations; a correlation that describes the effects of salts
TABLE 3. Comparison of measured and predicted diffusivities
for O2 and NO in water at 25◦ C.
Source DO2 (10−5 cm2 s−1 ) DNO (10−5 cm2 s−1 )
Wilke–Chang 2.24 2.50
correlationa
Tyn and Calus 1.90 1.91
correlationa
Hayduk and Minhas 1.85 2.00
correlationa
Measured value 2.13 ± 0.01b 2.21 ± 0.02c
a As given in Reid et al.26
b From Goldstick and Fatt.9
c From Table 2.
 Diffusivity of Nitric Oxide
on gas solubilities predicts that the NO (and O2 ) solubility
in PBS should be 6% lower than in water.11
What is of greatest physiological interest, of course, is
the diffusivity of NO in various media at 37◦ C. Measure-
ments are reported here for room temperature only, because
we were unable to obtain reliable data when the diffusion
cell was placed in a 37◦ C oven. Although the temperature
control in the oven was good, the fits to the transient data
were much less accurate than at room temperature. The ap-
proach to a steady state tended to be too slow, and that led
to unrealistically high values of the aqueous solubility. Be-
cause residual O2 would have slowed the rise of the NO flux
(Figs. 3 and 4), the most likely explanation seemed to be
outgassing of O2 from the various components. However,
preheating the apparatus and/or extending the purge time
failed to resolve the problem.
Because of the experimental difficulties just described,
diffusivities at body temperature were obtained by extrapo-
lation of the room temperature data. To do this we assumed
that µDNO /T is independent of temperature, a relation-
ship which has its theoretical basis in the Stokes–Einstein
equation26 and which is embedded also in the Wilke–Chang
and Tyn and Calus correlations used in Table 3. Aqueous
diffusivities of O2 from 17 to 33◦ C have been reported to
closely follow this relationship.31 It predicts that diffusiv-
ities at 37◦ C should be 35% larger than at 25◦ C, which is
similar to the 42% increase that has been found for O2 .14,15
When used in conjunction with our data for NO at 25◦ C, the
predicted diffusivity at 37◦ C is DNO = 3.0 × 10−5 cm2 s−1 .
It should be noted that the data of Wise and Houghton38
suggest a much larger (73%) increase in DNO over this tem-
perature range. In support of our assumption that the tem-
perature dependence of DNO is similar to that of DO2 , the
mass transfer coefficient for the delivery of NO from Silas-
tic tubing into stirred PBS, under unreactive conditions, was
found to be indistinguishable from that for O2 both at 23
and 37◦ C.36 Approximately half of the overall mass transfer
resistance in those experiments was in the aqueous phase,
so that any large difference between DNO and DO2 , at either
temperature, should have been noticeable.
The present results are restricted to water and saline so-
lutions, but it should be possible to use the same approach to
measure NO diffusivities and solubilities in more complex
media, such as protein solutions and cell suspensions. Mea-
surements of DNO in protein solutions would be helpful in
better characterizing NO transport in blood plasma, for ex-
ample. The reported reactivity of NO with serum albumin30
is one factor that might distinguish its transport in plasma
from that of O2 .
ACKNOWLEDGMENT
This work was supported by a grant from the National
Cancer Institute (PO1-CA26731).
221
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