cme320_lab03

STONY BROOK UNIVERSITY
COLLEGE OF ENGINEERING AND APPLIED SCIENCES
Chemical and Molecular Engineering Program
Chemical Engineering Laboratory II:
CME 320
Hydrogels - Rheology
By
Marcin Kielkiewicz
Team Members: Jennifer Imbrogno & Kathryn Margaret Caducio
TA: Clement Marmorat
Submitted to:
Prof. Miriam Rafailovich and Dr. Pinkas-Sarafova
Submitted: April 15, 2015

Marcin Kielkiewicz 108225444 CME 320 Hydrogels April 15, 2015
Abstract
The properties of hydrogels, specifically pure gelatin and mTG cross-linked gelatin were compared to
determine the effect cross-linking has on rheological properties, thermal stability at body temperature,
solvent absorbance rate, and drug release. Two hydrogels, one with 10% weight by volume of gelatin in
deionized water was prepared with the other as 10% weight by volume mTG cross-linked hydrogel (with a
3:1 ration of mTG: gelatin). mTG catalyzes the formation of covalent bonds across gelatins molecular
structure, which are normally held together in gelatin by hydrogen bonds. This cross-linking strengthens
the hydrogel matrix as proven by the tests performed. Dynamic shear rheometry over a range of shear from
1-8000 Pa showed that the elastic modulus dominates over the range of shear for both gels, however it is
stronger in hardgel; this is attributed to cross-linking across gelatin macromolecules. The hardgel sample
was unexpectedly destroyed at a lower shear than pure gelatin, which can be attributed to a greater water
content between cross-linked gelatin molecules than within the molecular matrix as is the case for pure
gelatin. It was observed that dehydrated gelatin has a higher rate of water absorption than cross-linked
gelatin due to a decrease in pore size caused by cross-linking. Gels saturated with blue dye were also tested
for the release of dye at 37 °C in both dilute ethanol and deionized water. The hardgel remained in gel form
at elevated temperature, and more dye was released in deionized water than in dilute ethanol. From this we
can conclude that ethanol does not readily interchange with the solvent present within the hydrogel. Pure
gelatin samples “melted” at this temperature, and released all their dye in less than one hour. From these
results we canconclude that cross-linked gelatin has the potential to actasa biodegradable, extended release
drug delivery system.
Introduction
A hydrogel is a viscoelastic material
composed of a network of polymer chains that are
hydrophilic and highly absorbent; a typical
hydrogel can be composed of over 90% water by
weight. This property puts hydrogels in a unique
class of materials that have both sold solid
(elastic) and liquid (inelastic) properties.
Hydrogels are often homogenous materials
produced by either biological or synthetic
processes. Due to their substantial water content
hydrogels possess a degree of flexibility very
similar to vertebrate tissue. From a biomedical
perspective, they show promise in a number of
areas including drug delivery and regenerative
medicine. Hydrogels are already widely used as
three-dimensional scaffolds for cell and tissue
culture environments, as they are excellent
mimics of the in vivo state.1,2
One of the most common hydrogels of
biological origin is hydrated gelatin. Gelatin is
derived from collagen which is a highly abundant
structural protein found in various fibrous
tissues in vertebrates, including tendons,
ligaments, skin, blood vessels, corneas,cartilage,
etc. The collagen in each tissue is somewhat
unique in order to serve a specific biological
purpose in every species, and each type of
collagen varies across species as well. Therefore
it is impossible to assign a single structure to
collagen. However, all types of collagen are
composed of a triple helix, which generally
consists of two identical chains (α1) and an
additional chain that differs slightly in its
chemical composition (α2). The three primary
amino acids that compose all types of collagen
are glycine, proline, hydroxyproline, and alanine
which in sum account for over 50% of the total
amino acid content in the molecules. The
remainder is a combination of over a dozen amino
acids in varying concentrations.3-5

On an industrial scale, collagen is
harvested from animal tissue and hydrolyzed
under either acidic or basic conditions. This is
done in order to purify and decontaminate the raw
collagen. In this process collagen is broken down
into a simpler molecules of gelatin which share
all the properties of collagen, the only difference
being molecular weight.6,7
Because gelatin
is inexpensive and biodegradable its use for drug
delivery systems and tissue engineering has long
been sought after. A major challenge in utilizing
gelatin for this purpose has been its
decomposition at 35 °C,8
which is below human
body temperature, which lies at approximately
36.8° C ± 0.4 °C.9
The cause of decomposition is
the unwinding of the triple helix structure and
subsequent “melting”, which transforms the
hydrated gelatin from a hydrogel into a solution.
This renders gelatin a non-biocompatible
material when it is intended to remain in its gel
form for extended periods of time within the
human body. In order
to overcome this problem, we tested the effect of
cross-linking the triple helix structure of gelatin
with the microbial transglutaminase enzyme
(mTG). This enzyme catalyzes the formation
of isopeptide bonds between free amine groups
and the acyl group at the terminus of side chains
of protein or peptide bound glutamine. Bonds
formed by transglutaminase exhibit high
resistance to breakdown into smaller peptides and
amino acids.10
We predicted that by cross-linking
the triple helix structure the strands would be
unable to disassociate at body temperature and
remain stable at 37 °C for longer periods of time
compared to gelatin. Samples of pure
gelation and mTG cross-linked gelatin were
created and then compared through the following
tests. Their elastic modulus and viscous modulus
was measured over a range of shear using a
rheometer, Other samples were placed in an
incubator at 37 °C to observe whether they would
“melt”. The absorbent properties of both
dehydrated gels were measured, and the release
of blue dye (which simulated a drug) was
measured in both in pure water and dilute ethanol
to simulate the effects of drug release in an
inebriated patient versus a sober one. These tests
served as preliminary measurements to see
whethercross-linking affectsthe properties of the
hydrogel and whether it could serve as a
biomaterial for use within the human body.
Method and Materials
Our first hydrogels were created using
the following procedure: a 10% weight by
volume (w/v) sample of pure gelatin hydrogel
was prepared by adding 3.925 g of dehydrated
gelatin to 39.2 mL of deionized water at room
temperature in a plastic test tube. The mixture
wasthen heatedin an incubator at37 °Cto initiate
gel formation. This procedure was performed
because gelatin is relatively insoluble in cold
water but hydrates readily in warm water.11
By
cooling the solution below 37° C the triple helix
structure “recrystallized” and the hydrogel was
formed. A 10% w/v hard gel composed of a 3:1
mixture of gelatin: mTG was prepared via the
same procedure.
Two similarly sized samples of pure
gelatin and hard gel were subjected to a dynamic
shear rheometry test which measured the elastic
modulus and viscous modulus of each sample. A
dynamic shear rheometer is a device that is used
to measure the effects of shear on the hydrogels.
In the device a sample is placed between two
plates, and a minimum pressure is exerted onto
the sample to hold is stationary between the
plates. While one plate is held stationary the
second plate oscillates angularly relative to the
opposite plate. A predetermined range of torque
values are applied to the sample to determine its
rheological properties.12
Rheological properties
are often measured at a precise temperature

therefore external temperature control was
exercised. In our experimental setup a Bohlin
Gemini HR rheometer connected to an ETO
electronics heating unit was used. The
temperature was held constant at 25 °C for both
samples. A range of shearfrom 1 to 8000 Pa were
applied until the sample reached its critical shear
and wasdestroyed. Critical shearis defined asthe
shear required to cause atom planes to slip past
one another; in other words this is the point where
the viscous modulus exceeds the elastic modulus,
hence the sample becomes more liquid-like than
solid and is “liquidated” causing it to slip away
from between the plates.
The next test measured the rates of
absorbance of pure gelatin compared to hard gel
(3:1 mixture of gelatin: mTG). 10% w/v pure
gelatin hydrogel was placed in an incubator at 37
°C until the hydrogel “melted”. Using a
micropipette 3.000 mL of the warm gelatin
solution were added to a petri dish. This was
repeated two more times to give a total of three
samples of pure gelatin. Conversely, three
samples of hard gel were prepared by adding
2.250 mL of warm gelatin solution to each petri
dish. 0.750 mL of mTG was subsequently added
to each petri dish. Care was taken to avoid the
introduction of air bubbles into each petri dish.
Any bubbles that were substantial in size were
removed with the micropipette. The samples
were left to gel over several hours and then
dehydrated in air at room temperature for one
week. After one week the mass of each petri dish
was measured [iBalance 211TM
] and recorded.
Excess water was then added to each petri dish
and the gels were allowed to swell at room
temperature. After 67 minutes excess water was
drained and the mass of each petri dish was
recorded once more. The average mass of each
sample set was calculated using Eq. 1. The
population standard deviation of each sample set
was calculated suing Eq. 2.
[Equation 1] x̄ = N-1∑ 𝑥𝑁
𝑖=1 i
[Equation 2] σ = √∑ (𝑥𝑁
𝑖=1 i - x̄ ) ÷ N
From the information obtained from the
above test,the volume fraction of the solvent (ɸs,t)
within the hydrogel at a specific time t was
determined using Eq.3. This parameter is useful
for calculating the Gibbs free energy of mixing
according to the Flory-Huggins theory. In this
theory each polymer segment is described by a
position in a lattice which is as large as the
solvent molecules. Flory and Huggins gave an
expression for the enthalpy of mixing, which
would be zero for an ideal solution but is given
by Eq. 4. By using the Boltzmann relation for an
increase in entropy due to mixing in combination
with the probability function and Sterling
approximation an expression for the molar
entropy change of mixing is given by Eq. 5.14
From these two equations the Gibbs free energy
is easily calculated. This calculation was not
performed, however it has been included to show
the reader the significance of the volume fraction
parameter. The relative uncertainty in the volume
fraction is given by Eq. 6. Note that the
uncertainty in the density of water is negligible
and is omitted.
[Equation 3] ɸs,t =
𝑉𝑠
𝑉𝑝 + 𝑉𝑠
=
1
ρs
𝑄𝑚
𝜌𝑝
+
1
𝜌𝑠
Qm =
𝑚𝑎𝑠𝑠 (ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑑 𝑝𝑜𝑙𝑦𝑚𝑒𝑟)
𝑚𝑎𝑠𝑠 (𝑑𝑒ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑑 𝑝𝑜𝑙𝑦𝑚𝑒𝑟)
ρs = density (solvent)
ρP = density (polymer)
[Equation 4] ΔHm = zn1r1 ɸpΔw12
[Equation 5] ΔSm = -R∑ 𝑥𝑁
𝑖=1 iln[xi]
[Equation 6] δ% ɸs,t = ɸs,t*(δ%Qm + δ%ρP)
In a final test the theoretical release of a
drug from gelatin and hardgel was observed in
both deionized water and an ethanol/water

solution. Pure gelation and hard gel were both
saturated with water containing a blue dye for an
extended period of time prior to initiating this

solution. Pure gelatin and hard gel were both
saturated with water containg blue dye prior to
initiating this experiment. The blue dye was
intended to simulate a theoretical drug. The
ethanol solution was meant to emulate the
bloodstream conditions of a highly inebriated
patient while the pure water emulated those of a
sober patient. Four equal volume samples of
gelatin and hard gel were prepared (two each),
and one of eachgelwasplaced in either a testtube
with deionized water or one with a 35% ethanol
by volume solution. 3.000 mL of deionized
water was added to two test tubes, while 1.500
mL of 70% ethanol in water was added to two
other test tubes and diluted with an additional
1.500 mL. All solutions were made using a
micropipette. The four test tubes were sealed in
an incubator at 37 °C and wrapped with
aluminum foil (to prevent light from interacting
with the samples) and kept there for 47 minutes.
The extent of release of blue eye was estimated
by eye and the samples were visually compared
relative to one another.
.
Results and Discussion
The results of the rheometry test for both the pure
gelatin hydrogel and the hardgel are shown in Fig.
1 and Fig. 2, respectively. From the data obtained
it is clear that the elastic modulus (G’) is
dominant and linear in both samples over a range
of shear from 1-1000 Pa. G’ is greater for the
hardgel than for pure gelatin in this range. This
result can be explained by the cross-linking in the
hardgel, which in theory could occur within the
triple helix structure of gelatin as well as across
individual triple helix strands. The cross-linking
across strands increases the internal organization
within the polymer matrix and in turn increases
the ability of the hydrogel to resist shear stress.
Beyond 1000 Pa of shear stress the viscous
modulus in both samples began increasing
exponentially while the elastic modulus begins
decreasing. The rate of change in both samples
differed significantly, with a decomposition of
the hard gel occurring at a critical shear of
approximately 5500 Pa. The critical shear for
pure gelatin was extrapolated to a value of
roughly 13500 Pa, more than twice as large as
that of the hardgel. This result was unexpected,
but it can also be explained by the cross-linking
within the hardgel. The cross-linking in the
hardgel reduces the pore sizes within the polymer
matrix, hence there could be more water residing
outside of the polymer structure than within it
when compared to pure gelatin, which has larger
pores. Since more water rests outside of the
polymer matrix individual planes of gel can begin
to slide past one another at a lower shear,because
the planes are largely composed of water and are
therefore more “liquid-like” in nature than of
those found in gelatin.

Figure 2. Results obtained for the
rheometry test for the hardgel.
For the absorption test, the mass of the
dehydrated hardgel and gelatin, their hydrated
counterparts, and related calculations are shown
in Table 1. The standard mass of a 60 mm * 15
mm petri dish was 2.268 g, which was used to
calculate the mass of hydrogel in each dish. The
uncertainty of 0.001 g in the electronic scale was
omitted in favor of calculating a standard
deviation and using one standard deviation as the
error in the mean mass of gelatin in each petri
dish. It wasfound that dehydration of both gelatin
and hardgel samples resulted in a loss of nearly
equal volumes of water; the average mass of pure
gelatin in eachpetridish was0.666 g ± 11.7% and
for the hardgel it was 0.617 ± 7.9%. The mass of
hardgel wasexpectedto be smaller because 0.750
mL less of gelatin solution was used to make it
when compared to pure gelatin. Although the
initial masses were similar, the rates of
absorbance differed significantly between the
two gels. Pure gelatin was able to absorb two
equivalents of its mass in water weight to yield a
hydrogel with a mass of 1.768 g ± 6.9%. The
hardgel absorbed only one equivalent of its
weight in water with a final mass of 1.294 g ±
4.2%. The different rates of absorbance can be
explained by cross-linking/lack of cross-linking;
the hardgel has smaller pores and does not absorb
water as quickly as gelatin which has larger pores
and swells at a faster rate.
Using Eq. 3, Eq. 6, and the data from
Table 1, the value of δ% ɸgelatin,67 min was found to
be 0.782 ± 22.3% while the value of δ% ɸhardgel,67
min was0.739 ± 15.8%. Since anunknown amount
of moisture was present in the air, the samples
could not have been completely devoid of water.
Figure 1. Results
obtained for the
rheometry test of
pure gelatin hydrogel.

Assuming the samples retained 8% to 13% of
their initial water concentration, which is a
common value for air dried samples, the density
of gelatin at that level of water content is (1.35 ±
0.05) g/cm3
.13
The density of water at 25 °C is
known with high precision to be 0.997 g/cm3
therefore the uncertainty in it is negligible.14
In a final test the release of blue dye from
gelatin and hardgel was observed in both
deionized water and an ethanol/water solution.
After 47 min in the incubator, both gelatin
samples dissolved in ethanol and pure water,
releasing all of their blue dye. The hardgel
samples did not dissolve in either solution,
however it appeared that the sample placed in
water released more dye than the one in ethanol.
This seems to indicate that ethanol cannot readily
passthrough the pores within the polymer matrix,
and as the pores get smaller the rate of solvent
interchange decreases. This test also proved that
mTG cross-linked gelatin is stable at 37 °C in
both water and ethanol.
TABLE 1
Sample # Pure Gelatin
(dehydrated) w/
Petri Dish
Pure Gelatin
(hydrated) w/
Petri Dish
Hardgel
(dehydrated) w/
Petri dish
Hardgel
(hydrated) w/
Petri dish
1 2.831 g 3.867 g 2.950 g 3.638 g
2 3.021 g 4.141 g 2.875 g 3.527 g
3 2.950 g 4.099 g 2.831 g 3.521 g
Pure Gelatin
(dehydrated)
w/out Petri Dish
Pure Gelatin
(hydrated)
w/outPetri Dish
Hardgel
(dehydrated)
w/out Petri dish
Hardgel
(hydrated)
w/out Petri dish
1 0.563 g 1.599 g 0.682 g 1.370 g
2 0.753 g 1.873 g 0.607 g 1.259 g
3 0.682 g 1.831 g 0.563 g 1.253 g
Pure Gelatin
(dehydrated)
Average Mass
Pure Gelatin
(hydrated)
Average Mass
Hardgel
(dehydrated)
Average Mass
Hardgel
(hydrated)
Average Mass
0.666 g 1.768 g 0.617 g 1.294 g
Pure Gelatin
(dehydrated)
Std. Deviation
Pure Gelatin
(hydrated)
Std. Deviation
Hardgel
(dehydrated)
Std. Deviation
Hardgel
(hydrated) Std.
Deviation

0.078 g 0.120 g 0.049 g 0.054 g
Conclusion
The properties of mTG cross-linked gelatin and
pure gelatin were compared using several tests.
mTG cross-linked gelatin had a higher elastic
modulus than pure gelatin, but the cross-linked
sample could not withstand shear as well as
gelatin. The absorbance rate test showed that
cross-linking decreases the pore size within the
polymer matrix hence limiting the rate of solvent
absorption. Cross-linked gelatin was only able to
absorb
2
3
of the waterabsorbedby pure gelatin. As
a drug delivery system, cross-linked gelatin
showed promise because it didn’t decompose at
body temperature yet managed to release blue
dye in both wateranddilute ethanol, albeit slower
in the latter. Pure gelatin, on the other hand,
decomposed quickly at body temperature
releasing all the dye in less than one hour,
limiting its potential effectiveness as an extended
release drug delivery method.
References
1) Elisseeff, Jennifer. "Hydrogels: Structure Starts to
Gel." Nature Materials. 7.4 (2008): 271-73.
2) J.M. Van Bemmel. “Der Hydrogel und das
kristallinische Hydrat des Kupferoxydes.” Z. Anorg.
Chem. 5 (1894) S. 466.
3) Lullo, G. A. Di. "Mapping the Ligand-binding Sites
and Disease-associated Mutations on the Most
Abundant Protein in the Human, Type I Collagen.
“Journal of Biological Chemistry. 277.6 (2001):
4223-231.
4) Sikorski, Zdzisław E. Chemical & Functional
Properties of Food Proteins. Lancaster, PA:
Technomic, 2001.
5) Szpak, Paul. "Fish Bone Chemistry and
Ultrastructure: Implications for Taphonomy and
Stable Isotope Analysis." Journal of Archaeological
Science. 38.12 (2011): 3358-372.
6) "Gelatin." Food Chemistry 45300. PDF. Purdue
University.
7) Ward, A. G., and A. Courts. The Science and
Technology of Gelatin. London: Academic, 1977.
8) Cole, CGB (2000), "Gelatin", in Francis,
FJ, Encyclopedia of Food Science and Technology,
2nd edition, John Wiley & Sons, pp. 1183–1188.
9) Longo, Dan L. Harrison's Principles of Internal
Medicine. New York, N.Y.: McGraw-Hill, 2012.
10) Clarke, D.d., M.j. Mycek, A. Neidle, and H.
Waelsch. "The Incorporation of Amines into
Protein." Archives of Biochemistry and Biophysics. 79
(1959): 338-54.
11) "Solubility." PB Gelatins. Tessenderlo Group.
Web. 13 Apr. 2015.
13) Members, GMIA. "Gelatin Handbook." Gelatin
Manufacturers Institute of America. Web. 15 Apr.
2015.
14) "Water Density Calculator." Web. 15 Apr. 2015.

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