J. Biomedical Science and Engineering, 2009, 2, 594-605
doi: 10.4236/jbise.2009.28086 Published Online December 2009 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online December 2009 in SciRes. http://www.scirp.org/journal/jbise
Water activity and glass transition temperatures of disaccharide
based buffers for desiccation preservation of biologics
Justin Reis1, Ranjan Sitaula2, Sankha Bhowmick1,2,3
1Department of Mechanical Engineering, University of Massachusetts Dartmouth, Massachusetts, USA;
2Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, Massachusetts, USA;
3285 Old Westport Road Room # Textile 210 N. Dartmouth, MA 02747, USA.
Received 22 August 2009, revised 27 September 2009; accepted 28 September 2009.
ABSTRACT
Studying the thermophysical properties of disaccha-
ride based ternary solutions are gaining increasing
importance because of their role as excepients in pres-
ervation protocols for biologics in general and mam-
malian cells in particular. Preservation strategies in-
volve not only cryopreservation, but novel approaches
like room temperature vitrification and lyophilization.
In this study we investigate the water activity and glass
transition temperature of citrate and tris buffers
(widely used in the gamete preservation industry) with
trehalose or sucrose after partial desiccation. After
obtaining the water activity (aw) through equilibration
at different relative humidity environments, we
measured the glass transition temperature (Tg) of these
partially desiccated solutions using a differential
scanning calorimetry (DSC). The experimental data
was used in conjunction with the Gordon-Taylor
equation to obtain 3-D contours of Tg as a function of
water content and relative salt/sugar concentration.
Results indicate that the glass transition behavior is a
strong function of the excepient combination. Overall,
that trehalose solutions yielded larger values for Tg
than sucrose counterparts at low moisture contents in
combination with the same buffer. We also saw that
citrate solutions yielded larger glass transitions than
their tris counterparts. Based on these results, a tre-
halose-citrate mixture can be picked as the preferred
composition for storage applications. The 3-D contours
which show a wide variation in slope depending on the
salt-sugar concentration constitute important infor-
mation for the desiccation preservation of biologics.
Keywords: Trehalose; Sucrose; TRIS; Citrate; DSC;
Glass; Transition; Tenperature
1. INTRODUCTION
Desiccation preservation offers an attractive alternative
to cryopreservation for the long term storage of mam-
malian cells and gametes. While cryopreservation has a
stringent requirement of storage in liquid nitrogen at a
temperature in the vicinity of-196°C, desiccation pres-
ervation offers the ability to store cells at or near ambi-
ent conditions. At the same time it eliminates the usage
of toxic cryoprotectants such as glycerol and DMSO
which require removal upon returning cells to ambient
temperatures, severely affecting cell survival in the
process [1].
One of the hypotheses behind the mechanism of des-
iccation preservation is the formation of glassy structure,
a highly viscous state that minimizes molecular mobility
of the matrix thereby suspending metabolic activities in
the cells. Sugars, particularly disaccharides, have been
effective in imparting cellular protection in the desic-
cated state. A number of studies have demonstrated the
ability of different sugars such as trehalose, sucrose,
raffinose and maltose to sustain a stable glassy state at
low moisture content [2,3,4,5,6,8]. Such sugars form
glasses at ambient temperature, thereby reducing mo-
lecular mobility and allowing a prolonged stable storage
of biomaterials and cellular components [3,8,9,10,11].
The survival of mammalian cells in vitro requires a
buffer or culture media generally consisting of various
salt mixtures. In our study we chose to study ternary
sugar-salt-water solutions. The interactions of ternary
solutions can often be extremely difficult to predict
without proper experimental studies of their thermo-
physics [6]. These interactions can produce results that
may vary significantly even from similar studies of bi-
nary solutions [1,12].
Two key thermophysical parameters that will deter-
mine a desiccation preservation protocol include water
activity (aw) and glass transition temperature (Tg)
[6,8,9,13,14]. Water activity (aw) is defined as the ratio
of the vapor pressure of water in a material (p) to the
vapor pressure of pure water (po) at the same tem-
perature [15]. It is an equilibrium state that is greatly
responsible for a solution’s ability to participate in
physical, chemical and microbiological reactions [2,

J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605 595
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16]. The glassy state is a non-equilibrium state at
which substances exhibit an amorphous glass structure.
Glass transition (Tg) is the temperature at which
amorphous solids transition from solid to a less vis-
cous state. The glass transition temperature is a func-
tion of the solution constituents and the moisture con-
tent, as well as a function of the water activity of the
storage condition [17].
The objective of the current experimental study was
to investigate the effect of water activity (given by the
equilibrium relative humidity of the storage environ-
ment for room temperature conditions) on moisture
contents and the subsequent effect of moisture content
on Tg of various sugar-buffer-water ternary system.
The particular buffers chosen for this study were the
Tris and Citrate buffers whose composition can be
seen in Table 1. These buffers are widely used bovine
sperm extenders under a wide range of temperatures
[18,19,20,21,22]. These buffers have an excellent
buffering characteristics for biochemical studies, are
non-toxic to living cells, and effective for maintaining
osmotic pressure in cells. Trehalose and sucrose were
chosen as the excipient sugars owing to their superior
glass forming ability and their effective role in desic-
cation preservation which have been well documented
in the studies of various biologics [4,5,8,11,23,24, 25].
The goal of our study was to create water activity ta-
bles for different concentrations of the sugars in each
of the buffers. The Tg of samples were then plotted in
as a 3D surface plot as a function of sugar concentra-
tion as well as moisture content. These 3D plots are
extremely important for references in future work in
determining which solutions will produce glass transi-
tions at appropriate temperature levels.
2. MATERIALS AND METHODS
2.1. Sample Preparation
Trehalose dihydrate (Sigma Aldrich assay > 99%) and
sucrose (Sigma Aldrich assay > 99.5%) were both pur-
chased from Sigma Aldrich. The tris and citrate buffers
were obtained from ABS global, Deforest, WI in con-
centrated forms and diluted with distilled water to 1X
concentrations, which corresponds to an isotonic solu-
tion of 325 mosm. The composition of the tris and cit-
rate buffers are presented in Table 1. Molar calcula-
tions were carried out to determine the appropriate
quantity of trehalose or sucrose to be added to each
individual volume of the buffer. Vials were then mixed
thoroughly to ensure homogeneity. The range of each
sugar in combination with respect to the buffer for the
tris buffer were 0.713, 1.426, 2.139, 2.85, 5.705, 11.41
g sugar/g tris while solutions utilizing the citrate buffer
used the range of 1.485, 2.97, 4.455, 5.94, 11.72, 23.76
g sugar/g citrate.
2.2. Generation of Humidity Environment and
Drying Kinetics Curves
Stable relative humidity (RH) environments were gener-
ated by equilibrating samples in humidity boxes at room
temperature (20C). Humidity boxes consisted of a
sealed plastic food container with a chosen desiccant
inside, that was placed inside larger stackable desicca-
tion cabinets (Sanplatec Polystyrene Mini Desiccator,
Osaka, Japan) [26,27]. Our chosen desiccants were su-
persaturated solutions of magnesium nitrate, potassium
acetate, lithium chloride, and lithium bromide that pro-
vided us with 53%, 21%, 11%, and 6% RH respectively.
Drierite salts (Drierite Aldrich Chemical Company, St
Louis, MO) was used to obtain 1.5% RH environment. A
digital hygrometer (Oakton Thermohygrometer, Vernon
Hills, IL) was used to determine the equilibrium RH
values generated by the various desiccants. Dry samples
(0% RH) were obtained by baking in a natural convec-
tive drying oven (Quincy Labs Model 10 Lab Oven,
Chicago, IL) at 75°C for a minimum of 14 days or till no
detectable variation in weight was observed. A 30μL
volume of sample solution was carefully placed in a
standard aluminum Differential Scanning Calorimeter
(DSC) pan from TA Instruments (New Castle, DE). Pan
weight and the sample weight measured using a digital
scale (Mettler Toledo AB265S FACT, Columbus, OH)
and recorded for gravimetric analysis and for the DSC
experiment to determine the glass transition temperature
(Tg). Pans were then carefully transported with tweezers
to the appropriate relative humidity (RH) box where they
were allowed to equilibrate. The samples were weighed
periodically after they had been placed into the humidity
box. Based on these weight measurements, a drying ki-
netics chart was generated. These charts were used to
ascertain the time at which the samples had reached
equilibrium moisture content.
2.3. DSC Experiments
After equilibration, the samples in the DSC pans were
promptly hermetically crimped and sealed using a crimp
from TA instruments to reduce any exposure to the room
RH conditions. Samples were then ready for appropriate
DSC experiments.
Table 1. Shows a breakdown of components of both the par-
ticular tris and citrate buffers which were used in this study. gm
%=grams/100mL water.
Tris Buffer Citrate buffer
2.42 gm % tris
(hydroxymethyl aminomethane)
2.12 gm % sodium
citrate dihydrate
1.38 gm % citric acid
mono-hydrate
0.183 gm % citric acid
monohydrate
1.0 gm % fructose

596 J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605
SciRes Copyright © 2009 JBiSE
Table 2. Provides a generalized walkthrough of each portion of a DSC run. Each step is given a general description and the explana-
tion for its use is found in the same row. Experiments were taken to at least 30˚C above the expected glass transition temperature
while also considering degradation of samples.
Step # Function Description
1 low temperature equilibration The low temperature equilibration is used to view for any crystallization. It also pro-
vides a constant starting point for each of the runs to begin for consistency.
2 First Heating Cycle
The purpose of this heating run is to erase any thermal history of the sample. During
this run we want release any of the non-equilibrium properties of the sample such as
a buildup of entropy and enthalpy which occurs due to the non equilibrium glassy state.
3 Holding Isothermal
The isothermal run is to equilibrate the sample at a temperature above the glass tem-
perature. This makes sure that all we will have a sample in the equilibrium for our
run.
4 Cooling Now we need to cool our sample back down to our initial baseline. We have erased
all the thermal history of the sample and are now ready to begin our actual experiment.
5 Second Heating Cycle It is during this heating cycle where we will be able to determine our glass transition
temperature. This is the cycle which we analyze and is the one we are interested in.
6 Cooling to ambient This is merely to return the sample to ambient conditions where it can be safely re-
placed into the auto sampler.
Note:
All heating and cooling runs were performed at a rate of 5 degrees Celsius per minute
from -40˚C to 180˚C except for final cooling to ambient which was performed at 15
degrees Celsius per minute to ambient 25˚C.
Table 3. Shows the k values used in the gordon taylor equation
when it was used for solutions containing moisture. k values
were dependent upon sugar concentration in the buffer as well
as the combination of sugar-buffer being modeled.
Table of k
Trehalose-
Tris solutions
Trehalose-
Citrate
solutions
Sucrose-
Tris solutions
Sucrose-
Citrate
solutions
Lowest sugar
Concentration k=1.4 k=0.37 k=0.5 k=0.57
Largest sugar
concentration k=0.17 k=0.34 k=0.35 k=0.4
A typical DSC run with a heat-cool-heat cycle is
show-n in Table 2. All experiments for evaluating Tg
were performed using a Q1000 DSC (TA Instruments,
New Castle, DE) which is also equipped with a refriger-
ated cooling system (RCS). High purity nitrogen gas was
used to purge at a flow rate of 50 mL/min for each run to
ensure an inert experimental environment. Sample pans
were placed in the auto sampler alongside a reference
pan of known weight for comparison during the actual
running of the DSC. After the DSC run had been con-
cluded, the TA Universal Analysis software was used to
analyze the graph of heat flux as a function of tempera-
ture. The glass transition was then located on the graph
and calculated using the Tg software function.
2.4. Data Analysis
Initially large quantity of our sugar buffer concentration
range samples were baked to determine a wet to dry
weight ratio by weighing samples before entering the
oven and then again after being baked at 75°C for at
least 2 weeks. This ratio would then be multiplied to the
initial weights of other samples prior to entering equili-
bration in humidity chambers in order to provide a
weight for the sample if all moisture were removed
which is required for the calculation of Dry Basis Mois-
ture Content (DBMC). DBMC is a measure of residual
moisture in samples in relation to their dry weight which
is calculated to be void of moisture.
EB
B
W - W
% Dry basis moisture content (DBMC) =*100
W
(1)
where WE is the equilibrated weight and WB is the baked
weight.
All experimental Tg’s were plotted as a function of
DBMC to see the plasticizing effect of moisture on our
ternary solutions. All experiments consisted of at least 3
repeats (n=3). The error bars in the figures represented
the standard deviations of the repeats. The statistical
significance of the experiment data were evaluated using
the analysis of variance. Moisture contents as well as
Tg’s were tested for significance using Microsoft Excel’s
ANOVA Single Factor variance test. Statistical signifi-
cance was assessed as p<0.05.
2.5. Modeling of Tg
Gordon and Taylor first developed a model for the pre-
diction of glass transition in 1952 in their study of syn-
thetic rubbers based upon individual components con-
tribution to the glass transition of the overall homoge-
nous uniformly packed mixture [10]. This Eq.1 was used

J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605 597
SciRes Copyright © 2009 JBiSE
for modeling our desiccated ternary solutions.
112 2
12
**
g
g
g
wTkw T
wkw
T



(2)
where, w1 represents the weight fraction and the sub-
script 2 designates the component with larger Tg. k is a
model specific parameter. For baked samples (without
any moisture) the value of k was given as the ratio of the
smaller Tg over that of the larger Tg (k= Tg1/Tg2) com-
monly referred to as the Fox equation. However, sam-
ples containing moisture required the determination of a
different k value than the dried samples. This was ac-
complished by using the Tg of water (136K) as Tg1 in
Eq.2 and the baked salt-sugar mixture as Tg2. Best fit
analysis using a minimization function for percent dif-
ference of analytical and experimental data was used in
order to determine the most accurate value for k. All the
values for k were then used for the creation of 3D plots.
3. RESULTS
3.1. Drying Kinetics of Solutions
Figure 1 is a representative plot of the drying kinetics
of different weight fraction sugar-buffer solution sam-
ples equilibrated at room temperature. Trehalose-tris
solutions dried in a 7% RH environment and measured
on a weekly basis for 12 weeks. Figure 1 shows that
most of the drying takes place within the first week of
storage. The samples attain almost constant moisture
contents after a period of three weeks. Figure 1(b)
suggests a lower trehalose concentration resulted in a
greater retention of moisture in the equilibrated state.
While the equilibrium value of DBMC averaged
16.46% for samples with a trehalose-tris ratio of 0.713
g trehalose/ g tris, the corresponding value was 5.43%
for the 11.41 g trehalose/ g tris concentration with
p<0.05 between the sets. Similar results demonstrating
a lower DBMC for higher sugar content solutions were
observed for all buffer- sugar combinations in this
study.
3.2. Effect of Sugar on the Tg of Baked Samples
3.2.1. Effect of Trehalose Concentration on Baked
Samples Tg
Figure 2 shows the plot of Tg as a function of trehalose
concentration. The trend shows that both trehalose- buffer
solutions Tg asymptotically approach approximately
105˚C upon increasing trehalose concentration. The tre-
halose-tris samples increased their Tg too from a low
starting point due to tris’ low Tg value. Figure 2 also
shows that for trehalose-citrate samples fell towards the
105˚C value due to the elevated Tg of citrate. Trehalose
tris buffer at 0.713g trehalose/g tris concentration showed
0
20
0 24 6 8101214
Time (weeks)
40
60
80
100
120
140
DBMC
(a)
0
0246810121
Time (weeks)
5
10
15
20
4
DBMC
(b)
Figure 1. (a) Shows the drying kinetics of trehalose tris solu-
tions in our 7% humidity environment. The blue diamonds
(♦) represent the drying kinetics of 0.713g trehalose/g tris,
the pink square (■) represents 2.85g trehalose/g tris, and
the yellow triangle (▲) represents 11.41g trehalose/g tris.
The second graph is a zoomed view showing the equilibra-
tion of samples over time.
an average Tg of approximately 30˚C. As trehalose con
tent was then increased to 11.41g trehalose/ g tris the av-
erage glass transition increased to around 106˚ C. When
using the citrate buffer the lowest trehalose concentration
of 1.485 g trehalose/g citrate produced an average glass
transition temperature of approximately 125˚C. When our
trehalose mass ratio was increased to 23.76g trehalose/g
citrate an average Tg of approximately 102˚C was ob-
served. During the heating and cooling of samples no
crystallization was present in any of the thermographs
irrespective of the salt/sugar mixture content. The trendli-
nes in the figure, which were fitted using the Gordon
Taylor model, show that the values for Tg assimilate
themselves with the majority mass fraction component of
the solution. Samples with low sugar concentrations as-
similated Tg’s with the buffer involved (tris Tg ≈28.6°C
citrate Tg ≈130°C) and move towards that of trehalose
(Tg≈115°C).

598 J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605
SciRes Copyright © 2009 JBiSE
0
20
0510 1520 25
g trehalose / g buffer
40
60
80
100
120
140
160
Tg (˚C)
Figure 2. Shows glass transition as a function of treha-
lose concentration. The square blocks (■) represent the
trehalose with citrate buffer while the diamond symbol
(♦) show trehalose combined with tris buffer. The figure
also depicts the Gordon Taylor depicted by the black
dashed lines.
3.2.2. Effect of Sucrose Content on Baked Samples Tg
Figure 3 suggests the Tg of sucrose glasses as a function
of sucrose concentration. Sucrose glasses demonstrated a
similar asymptotic behavior with increasing sucrose
content. Sucrose-tris samples showed an increase in their
glass transition as sucrose concentration increased be-
cause pure sucrose has a larger Tg than tris. Sucrose-
citrate samples showed a decrease in Tg as sucrose con-
centration was increased as the Tg of sucrose is below
that of what we found for citrate. At mass ratio 0.713g
sucrose/g tris, the average Tg was 45˚C. When sucrose
concentration was increased 11.41 g sucrose/g tris the
samples produced and average Tg of 48˚ C. The 1.485 g
sucrose/g citrate concentration samples average glass
transition temperature was approximately 103˚ C. As our
mass ratio of sucrose increased all the way to a concen-
tration of 23.76 g sucrose/g citrate the glass transition
fell to approximately 46˚C. The trendlines fitted to the
experiment data show that the samples follow the
Gordon Taylor model of the two component models.
Similar to the trehalose results, Tg assimilates itself with
the majority fraction of the samples. The data sets ex-
hibit a trend of approaching a Tg slightly below that of
pure sucrose (Tg≈60˚C) as the concentration of sucrose
increases.
3.3. Role of Moisture in Modulating
Thermophysical Behavior of
Sugar Based Buffers
First, water activity curves were generated from sample
weight measurements taken after equilibration under
different relative humidity environments used in the
calculation of DBMC. These curves allowed us to de-
termine effect of both sugars and buffers to retain mois-
ture at equilibrium, and determine the Tg of the solution.
In the corresponding sections we show sample plots of
0
0510 15 20 25
g sucrose / g buffer
20
40
60
80
100
120
140
Tg (°C)
Figure 3. Shows glass transition as a function of sucrose
concentration. The diamond symbol (♦) represents the su-
crose with tris buffer while the square blocks (■) show su-
crose combined with citrate buffer. Gordon Taylor model-
ing is depicted by the black dashed lines.
DBMC vs. aw and Tg vs. DBMC for a high and low
sugar-buffer combination. Finally, we show the 3-D sur-
face contour of Tg as a function of moisture and sugar
content by using the Gordon Taylor equation.
3.3.1. Effect of Moisture on Trehalose Tris Solutions
Figure 4 shows that over the range of relative humidity
environments trehalose tris samples tended to equilibrate
to specific moisture contents and then hold this in the
range. The lower concentration of 0.713g trehalose / g
tris show an initial jump in residual moisture content
followed by a leveling in the 0.07 to 0.21 aw where
points are statistically the same (p>0.05). We then show
a significant increase in moisture from aw of 0.21 to 0.53
(p<0.05). The 11.41 g. trehalose/g. tris concentration
solution shows a similar trend however after the initial
jump in moisture the next successive four points are sta-
tistically the same (p>0.05). The figure also shows that
residual moisture content was affected by trehalose con-
centration in the solution. Under similar equilibration
environment, solutions containing higher concentrations
0
5
10
15
20
25
30
35
00.1 0.20.30.40.50.6
Aw
DBMC
Figure 4. Shows trehalose tris solutions DBMC as a function
of water activity. The diamond symbol (♦) represents 0.713g
trehalose/g tris, while the square blocks (■) show 11.41 g
trehalose/g tris.

J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605 599
SciRes Copyright © 2009
of trehalose equilibrated to lower end moisture contents
then lower trehalose concentration solutions.
although low concentration of trehalose, such as the
0.713g trehalose/g tris level, has a much lower Tg than the
11.41g trehalose/g tris level, the lower concentration is
far less affected by the addition of moisture into the ter-
nary solution.
Figure 5(a) shows trehalose-tris solutions exhibit a
linear trend of decrease in Tg with increasing residual
moisture. It also shows that samples with higher treha-
lose concentration undergoes a more drastic decrease in
glass transition upon gaining moisture as compared to
lower trehalose concentrations. The slopes illustrate that
The Gordon Taylor modeling in Figure 5(a) shows
that our 0.713g trehalose/g tris concentration prediction
misses three of the experimental points; however fall
directly upon the other three data points in the set. The
three missing points seem to be outliers of the general
linear trend of the decreasing glass transition as moisture
increases. The largest sugar concentration of 11.41 is
modeled quite effectively. The Gordon Taylor modeling
was also used to determine values for k for all trehalose-
tris concentrations to create the surface plot shown in
Figure 5(b). Figure 5(b) shows that the rate at which Tg
decreases is a function of sugar concentration. The result
also indicates the possibility for in intermediate maxi-
mum in larger moisture levels.
-60
-40
-20
0
20
40
60
80
100
120
140
051015 2025 30
DBMC (%)
Tg (°C)
3.3.2. Effect of Moisture on Trehalose Citrate Solutions
Figure 6 shows equilibrated moisture content in treha-
lose-citrate solutions as a function of water activity (aw).
The lowest trehalose concentration of 1.485 g treha-
lose/g citrate seems to continue on a gradual increase as
(a)
moisture content as aw increases. We see significant
difference between the 0.015 and 0.21 aw environments
(p<0.05) followed by a continued increase in the next
successive points (p<0.05).The largest concentration of
23.76g trehalose/g citrate shows gradual gain before
reaching a plateau around 12% DBMC. Figure 6 also
shows that especially at the largest aw environment
there is a large difference in the moisture capacities for
the samples (p<0.05) where the samples containing
larger trehalose concentrations equilibrate to lower
DBMC’s.
Figure 7(a) shows the predictable decrease in Tg of
trehalose-citrate solutions as residual moisture increases.
The lowest concentration of 1.485 g trehalose/g citrate
shows a average Tg of 122°C at approximately 6%
DBMC and it is reduced to 7°C at the largest DBMC of
31%. The 23.76g trehalose/g citrate yielded an average
Tg of 98°C at a DBMC of 25% and 20°C at 12% DBMC.
The plot also shows that the trehalose - citrate solutions
show decreasing linear slopes of -5 indicating that the
decrease in glass transition seems to be more affected by
the moisture content. The figure also shows larger con-
centration of trehalose had slightly lower glass transition
over the range of moisture content. The possibility of
salts precipitating out when these solutions were equili-
brated in the larger RH environments is a possibility for
the large variation in the 23.76 concentration data at ap-
proximately 12% DBMC.
(b)
Figure 5. (a) Shows glass transition of trehalose tris solutions
as a function of dry basis moisture content. The diamond
symbol (♦) represents 0.713 g trehalose/g tris and the triangle
(■) represents 11.41 g trehalose/g tris. The figure also shows
Gordon Taylor modeling of glass transition of trehalose tris
solutions as a function of dry basis moisture content. The blue
dashed line represents Gordon Taylor modeling of the 0.713g
trehalose/g tris while the black dotted line shows Gordon
Taylor modeling of 11.41g trehalose/g tris solutions; (b)
shows a 3D surface plot of the Gordon Taylor models of our
ternary trehalose tris moisture solution using our calculated k
values. This surface plot includes all intermediary solution
values for k plotted which were not shown in Figure 5(a).
The Gordon Taylor model was then used to create the
surface plot shown in Figure 7(b). The plot was created
from calculated k values for intermediary solutions and
JBiSE

600 J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605
SciRes Copyright © 2009 JBiSE
-5
0
5
10
15
20
25
30
35
00.1 0.2 0.3 0.4 0.5 0.6
Aw
DBMC
Figure 6. Shows trehalose citrate solutions DBMC as a function of water activity. The diamond
symbol (♦) represents 1.485g trehalose/g citrate while the square blocks (■) show 23.76g treha-
lose/g citrate.
-40
-20
0
20
40
60
80
100
120
140
160
Tg (°C)
010203040
DBMC (%)
(a)
(b)
Figure 7. (a) Shows glass transition of trehalose citrate solu-
tions as a function of dry basis moisture content. The diamond
symbol (♦) represents 1.485g trehalose/g citrate and the square
blocks (■) represents 23.76g. trehalose/g. citrate. The figure
also shows Gordon Taylor modeling of glass transition of tre-
halose tris solutions as a function of dry basis moisture content.
The blue dashed line represents Gordon Taylor modeling of the
1.485g trehalose/g citrate while the black dotted line shows
Gordon Taylor modeling of 23.76g trehalose/g citrate solutions;
(b) shows a 3D surface plot of the Gordon Taylor models of
our ternary trehalose citrate moisture solution using our calcu-
lated k values. This surface plot includes all intermediary solu-
tion values for k plotted which were not shown in Figure 7(a).
then the plotting of the Gordon Taylor equations in a 3D
plot. We see that the profile of the surface is fairly flat
due to the similarity Tgs of trehalose and citrate.
3.3.3. Effect of Moisture on Sucrose Tris Solutions
Figure 8 shows the sucrose-tris solutions equilibrated to
different DBMC’s as a function of aw. Both concentra
tion samples show a gain in moisture at 0.015 aw envi-
ronment. From this point forward we see that both
sam-ples show a general trend of decrease in equilibrated
DBMC (p<0.05 for both concentrations) before showing
an increase at our highest aw. Figure 8 also shows that
the larger sugar concentration solutions equilibrate to
lower end moisture contents across the entire aw range.
While this trend is more prevalent in the 0.21 and 0.53 aw
environments; however even in the lower RH environ-
ments samples with lower sugar concentrations still
equilibrated to larger average end moisture contents
while being just barely significantly different at the 0.21
aw environment (p≈0.05).
Figure 9(a) shows the sucrose-tris samples’ rapid de-
creases in Tg with even low levels of moisture. Our low-
est sucrose concentration 0.713g sucrose/g tris yielded
average Tg’s of approximately 20°C in the vicinity of
10% DBMC and -4°C at a DBMC of 21%. Larger con-
centration of sucrose samples yielded slightly lower Tg’s
than the smaller sucrose concentration solution. The
11.41 g sucrose/g tris solutions experimental values fall
above and below our predicted values in the larger

J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605 601
SciRes Copyright © 2009 JBiSE
-30
-20
-10
0
10
20
30
40
50
05101520 25
DBMC
0
5
10
15
20
25
00.1 0.2 0.3 0.4 0.5 0.6
Aw
DBM C
Tg (°C)
Figure 8. Shows sucrose tris solutions DBMC as a
function of water activity. The diamond symbol (♦)
represents 0.713g sucrose/g tris while the circles (●)
show 11.41g sucrose / g tris.
DBMC’s but effectively show the trend. The Gordon
Taylor models were then used to create the surface plot
shown in Figure 9(b). The surface plot shows that the Tg
for the range of sucrose tris concentrations varies far
more drastically as a function of moisture content as
opposed to sucrose concentration. At low moisture con-
tents we see that Tg is almost invariant between the con-
centrations of sucrose.
3.3.4. Moistures Effect on Sucrose Citrate Solutions
Figure 10 shows sucrose-ctrate samples equilibrated
DBMC at the different relative humidity environments.
We see that initially both the 1.485g sucrose/g citrate
and the 23.76g sucrose/ g citrate gain significantly dif-
ferent moisture in the 1.5% RH environment (p<0.05).
The next two aw levels both solutions show a plateau
(p>0.05 for both 23.76g sucrose/g citrate and 1.485g
sucrose/g citrate). From this point the lower concentra-
tion shows the expected gain of moisture as a function of
increasing relative humidity (p>0.05 between successive
points), however the 23.76 g sucrose/ g citrate solution
shows a decrease in it moisture contents (p>0.05 be-
tween successive points).
In Figure 11(a) we see that sucrose-citrate solutions
decrease Tg as their residual moisture content increases.
The 1.485 g sucrose/ g citrate solution yielded an aver-
age Tg of 64°C at 5% DBMC and 46°C at 18% DBMC.
The 23.76 g sucrose/ g citrate yielded an average Tg of
37° at 5% DBMC and 8°C at 16% DBMC. Figure 11(a)
also shows that Tg is affected by sucrose content. Lower
concentrations of sucrose yielded higher glass Tg’s when
equilibrated to the same level as their higher concentra-
tion counterparts. The Gordon Taylor model slightly
overestimates the glass transition of solutions. Baked
solutions at this concentration had fairly large error bars
showing large variation in Tg. This could cause an over-
estimation of Tg since we used this value for Tg 2 in the
Gordon Taylor equation. Should Tg 1 be lower it would-
flatten the entire curve and possibly hit all experimental
points as well providing a better fit for our baked predict-
(
%
)
(a)
(b)
Figure 9. (a) Shows glass transition of sucrose tris solutions
as a function of dry basis moisture content. The diamond
symbol (♦) represents 0.713 g sucrose/g tris and the square
blocks (■) represent 11.41g sucrose/g tris. We can see here
that as DBMC increases the trend is that our glass transition
decreases in respect to a DBMC of 0%. The trend seems to
be fairly flat over the general area of DBMC’s we have
mapped. The figure also shows Gordon Taylor modeling of
glass transition of trehalose tris solutions as a function of dry
basis moisture content. The blue dashed line represents
Gordon Taylor modeling of the 0.713g sucrose/g tris while
the black dotted line shows Gordon Taylor modeling of 11.41
g sucrose / g tris solutions; (b) shows a 3D surface plot of the
Gordon Taylor models of our ternary sucrose tris moisture
solution using our calculated k values. This surface plot in-
cludes all intermediary solution values for k plotted which
were not shown in Figure 9(a).
tion as well.
The Gordon Taylor models were then used to create
the surface plot shown in Figure 11(b) from calculated k
values for all solutions. The surface plot shows that Tg
decreases at a similar rate for increasing moisture con-
tent regardless of sucrose concentration. The surface is

602 J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605
SciRes Copyright © 2009 JBiSE
similar to a plane in which all solutions undergo the
same rate of decreasing Tg with residual moisture with
the difference in levels being a function of baked solu-
tions Tg.
0
5
10
15
20
25
00.1 0.2 0.30.4 0.5 0.6
Aw
DBMC
4. DISCUSSION
The current thermophysical study of the ternary solu-
tions was driven by a larger goal of obtaining optimal
excipient conditions for desiccation preservation of
mammalian cells. Majority of studies showing the sugar
stabilization effects are derived from the food preserva-
tion or pharmaceutical literature [28,29,30,31,32]. Di-
saccharides, particularly trehalose and sucrose, have
shown to be important in the preservation of cells and
biologics [2,3,4,5,6,7,9,10,11]. These sugars exhibit lar-
ger glass transition temperatures and possess excellent
water replacement abilities. In combination with these
sugars, tris and citrate salts were chosen for being indus-
try standards for bovine sperm preservation [18,19,20,
33]. The complicated ternary solutions in this study were
chosen based upon previous research showing their abil-
ity to sustain cellular life. The thermophysical properties
of preservation medium were important in understanding
which would be applicable as a desiccation medium.
Solutions which undergo glass transition at less than
ambient are clearly not appropriate since stability of me-
dium is essential.
4.1. Baked Samples
The completely dried samples behaved exactly as the
Gordon Taylor (Fox equation) two-component model
predicted [12]. The variation in Tg of a given sugar-
buffer mixture was a function of the glass transition of
each component and its weight fraction. Similar trend
for other mixtures have been observed in varying de-
grees by several studies [6,12].
The Tg of trehalose-tris and Trehalose-citrate mixtures
converged to a limit of 105°C as trehalose concentration
increased. The limit was approached both from above
and below as the tris and citrate buffer were found to
have a Tg at approximately 28.6°C and 133°C respec-
tively. Though we would expect Tg to reach that of the
pure sugar, extrapolation out to the pure limit may not be
accurate for complex materials containing salts as de-
scribed by Mazzobre et al [34]. Since we approach this
limit from very different starting points, we assume that
the lowered limit for Tg is caused by a common sub-
stance found in both biological buffers, in our case the
citric acid monohydrate. This commonality between the
buffers could be the most logical reason for both solu-
tions to approach a common Tg roughly 10°C below that
of pure trehalose. The deviation from the expected limit
of the pure substance has been shown in previous studies
in the literature. Jeong-Ah Seo and coworkers demon-
strated that when different monosaccharides are com-
bined with disaccharides, glass transition deviated from
Figure 10. Shows sucrose citrate solutions DBMC as a
function of water activity. The diamond symbol (♦) repre-
sents 1.485g sucrose/g citrate while the circles (●) show
23.76g sucrose/g citrate.
-40
-20
0
20
40
60
80
100
120
140
0510 15 20
DBMC (%)
Tg (°C)
(a)
(b)
Figure 11. (a) Shows glass transition of sucrose citrate so-
lutions as a function of dry basis moisture content. The
diamond symbol (♦) represents 1.485g sucrose / g citrate
and the square blocks (■) represent 23.76g sucrose/g citrate;
(b) shows a 3D surface plot of the Gordon Taylor models of
our ternary sucrose citrate moisture solution using our cal-
culated k values. This surface plot includes all intermediary
solution values for k plotted which were not shown in Fig-
ure 11(a).

J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605 603
SciRes Copyright © 2009 JBiSE
the expected value both on the high and low side of the
Gordon Taylor prediction as a result of size and shape of
molecules involved [35].
Similar to the trehalose results, the Tg of sucrose-
buffer solutions also converged to a common limit at
47°C as the sucrose content increased. Again the limit of
the solutions approached both from above and below due
to the Tg’s of our buffers involved and still they converge
upon a common value which falls approximately 10°C
below that of pure sucrose (Tg ≈60°C). For similar rea-
son as with trehalose, we again make the argument that
since we approach a common limit found below the Tg of
the sugar, the structure must be altered by a common
component of the buffers, the citric acid monohydrate.
Trehalose based mixtures consistently showed larger
Tgs than their sucrose equivalents. The glass transition
temperature of trehalose is approximately two times lar-
ger than that of sucrose making it far superior in terms of
the thermophysical property of glass transition. In com-
paring tris to citrate from a thermophysical standpoint,
citrate clearly dominates with a roughly five times larger
Tg than that of tris. However the Tg of the pure buffer is
very weak and it is in combination with sugars that the
glass transition becomes stronger and more prevalent.
Based on Freeze dried results, Kets and coworkers have
shown that the citrate was able to increase the glass tran-
sition of sucrose [6]. These numbers are slightly larger
but comparable to our results. Even though the Tg of tris
buffer could not be correlated to any literature value, our
experimental results were quite clear and consistent.
Hence, from a purely thermophysical standpoint, solu-
tions containing larger fractions of citrate salt produce
consistently larger Tg values than their tris counterparts.
4.2. Role of Moisture
4.2.1. Water Activity
Water activity curves are extremely important in this
ternary study in order to predict a solution’s ability to
retain or release moisture under different relative humid-
ity environments. Moisture content is also greatly re-
sponsible for a solution’s glass transition temperature. Its
role as a plasticizer has been shown in many similar
studies of wide varieties of solution composition [2,12,
36,37].
The general trend observed from our water activity
study is that larger sugar concentrations equilibrate to
lower end moisture contents. At the largest aw values, we
consistently see that the lower sugar concentration solu-
tions have a significantly larger DBMC, which is due to
the more hygroscopic nature of salts compared to sugars.
Trehalose solution isotherms vary depending on sugar
concentration. Solutions containing lower concentrations
of trehalose equilibrated to larger end moisture contents.
Solutions containing high concentrations of trehalose
seem to plateau at constant moisture content after an
initial increase in moisture content. The leveling shows
the formation of stable trehalose dihydrate which results
in the resilience of high trehalose concentration solutions
to gain moisture [34]. On the other hand, sucrose water
sorption isotherms show that the sucrose solutions con-
tain large amounts of moisture at lower water activity.
However the moisture content decreases when exposed
to larger aw environments. This is due to the fact that
anhydrous sucrose crystallizes above this water activity
[34]. Sucrose-tris solutions show more of a leveling than
a decrease, possibly due to the fact that there is a lower
sucrose concentration in relation to the buffer for our tris
solutions. Mazzobre and coworkers also showed a very
similar trend in there isotherms for sucrose-potassium
chloride solutions. Their trend shows that as aw increased
moisture content decreased until levels which fall above
our range of study. A comparison of our sucrose iso-
therms to trehalose isotherms show that sucrose tends to
equilibrate to lower DBMC’s in the upper aw range
whereas the reverse is true for lower aw range.
On the other hand, it is difficult to dr aw distinctions
between tris and citrate salts in terms of water activity.
Depending on the specific water activity examined it
seems as if each sugar buffer solution show something
slightly different.
4.2.2. Glass Transition
In comparing trehalose to sucrose one very important
trend arises about there rate at which glass transition
decreases as a function of moisture. Crowe and cowork-
ers showed in their Stabilization of Dry Mammalian
Cells study that at upon the gain of moisture sucrose and
trehalose begin to assimilate Tg’s. They show that at ap-
proximately 10% DBMC the Tg of trehalose and sucrose
seem to be approximately 10°C different compared to
dry states where trehalose has a Tg roughly two times
larger than sucrose. This shows that the rate of change at
which sucrose’s glass transition decreases as a function
of moisture content is less than that of trehalose, a trend
which our data replicates. This was determined by fitting
data points with a linear regression and comparing the
slopes of the 3D surface plots. The surface plots show
that in order to reach a similar Tg at 10% DBMC the tre-
halose graph shows a sharper decrease in Tg as a function
of moisture content. When we compare solutions con-
taining the largest sugar concentrations between sucrose
and trehalose utilizing the same buffer the trehalose so-
lutions slope is approximately twice as large as the su-
crose samples. This may have far reaching implication in
trying to stabilize mammalian cells near room tempera-
ture.
A notable difference between trehalose-tris and tre-
halose-citrate solutions lies on the rate at which Tg de-
creases as a function of moisture content. Comparing
the 3-D contour plots, while sucrose samples exhibit
similar rates for both the tris and citrate buffers treha-
lose does not. While the trehalose-citrate samples show

604 J. Reis et al. / J. Biomedical Science and Engineering 2 (2009) 594-605
SciRes Copyright © 2009 JBiSE
a rapid decrease in their glass transition upon the arri-
val of moisture, the trehalose tris-samples are far less
affected.
When comparing tris and citrate buffers samples, the
major difference is that the rate at which Tg decreases as
a function of moisture content varies between the buffers.
Comparing the 3D plots 7b and 11b, both sugars in
combination with the citrate buffer show fairly constant
rates of decreasing glass transition within that particular
sugar buffer solutions range of sugar concentration. Tris
samples on the other hand show a more varied rate
which seems to increase as a function of sugar concen-
tration. While sucrose-tris samples seem to present a
very slight increase in the rate at which Tg decreases with
increasing moisture, trehalose-tris samples show large
variation in their slope of their 3D surface contour. The
lowest concentration of trehalose exhibits a slope of ap-
proximately -1 in as a function of moisture content while
our largest trehalose concentration produces a signifi-
cantly larger slope of approximately -10 almost identical
rates for Tg of trehalose water solutions. This is a clear
difference between the tris and citrate buffer.
4.2.3. Modeling of Glass Transition
The use of the Gordon Taylor was chosen for its accu-
racy and overall simplicity. Other far more complex
equations such as Millers equation and the Miller-Fox
equation include specific component parameters such as
excess thermal expansion coefficient and excess volume
coefficients. Our buffer solution in itself is a complicated
solution making these coefficients difficult to determine.
Shah and Schall compared the Fox, Miller, and Miller
Fox’s equations ability to predict Tg [12]. When we look
at the percent differences of the data which they compare
to the experimental data we see that even for the least
precise Fox equation the percent differences all fall be-
low 9%. Without any given standard deviations of their
experimental data it is difficult to even further comment
on deviations between experimental and model data.
When we examined the average percent difference for
Shah and Schall work, we see that the Miller Fox equa-
tion is the most accurate with an average 1.86% percent
difference while the least accurate Fox equation falls in
at an average 2.98%. The additional 1% average accu-
racy hardly seems to warrant the usage of the far more
complex equation.
5. OVERALL CONCLUSIONS
Increasing sugar concentration allows solutions to
equilibrate at lower moisture contents.
Trehalose solutions yielded larger values for Tg at
lower moisture contents than their sucrose counterparts.
Citrate solutions yielded larger glass transitions than
their tris counterparts.
The rate of change of Tg with moisture content, d Tg /d
(moisture content), had very different behavior depend-
ing on which sugar was present as the excepient. While
sucrose content did not change that behavior, the pres-
ence of trehalose had a strong influence-an increasing
trehalose concentration caused solutions to increase d Tg
/d (moisture content) when compared to lower trehalose
solutions.
Based on the current results, a combination of treha-
lose and citrate would be the preferred composition for
storage applications.
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