University of Arkansas, Fayetteville
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Biomedical Engineering Undergraduate Honors
Theses
Biomedical Engineering
5-2015
Fractionation of Chitosan Using a Novel
Precipitation Method
Davis J. Ward
University of Arkansas, Fayetteville
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Ward, Davis J., "Fractionation of Chitosan Using a Novel Precipitation Method" (2015). Biomedical Engineering Undergraduate Honors
Theses. 19.
http://scholarworks.uark.edu/bmeguht/19
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Fractionation of Chitosan Using a Novel Precipitation Method
An Undergraduate Honors College Thesis
in the
Department of Biomedical Engineering
College of Engineering
University of Arkansas
Fayetteville, AR
by
Davis Ward
Table of Contents
Summary ....................................................................................................................................................... 1
Nomenclature ................................................................................................................................................ 1
Introduction ................................................................................................................................................... 2
Materials and Methods .................................................................................................................................. 4
Data and Results ........................................................................................................................................... 7
Discussion ................................................................................................................................................... 12
Conclusion .................................................................................................................................................. 14
References ................................................................................................................................................... 15
Summary
Chitosan is a derivative of chitin, a biopolymer found in the exoskeletons of crustaceans.
Chitosan is under heavy investigation in the field of drug delivery due to its wide versatility. One
such delivery mechanism is the use of hydrogels in cancer immunotherapy delivery. The
viscosity of chitosan in its use as a hydrogel depends on the length of chitosan’s N-acetyl Dglucosamine chains, which in turn determines the molecular weight of chitosan. Because there is
such a wide range of viscosity in a typical sample of chitosan, there is a need for a simple
technique to fractionate the chitosan based on its viscosity. A precipitation technique was
developed for fractionation using sodium hydroxide. There was an obvious difference in
viscosity in two of the four samples tested. Because of the low quality of the chitosan purchased,
the results for the other two samples were inconclusive.
Nomenclature
cP
Centipoise
DI
Deionized
IL-12
Interleukin-12
Mw
Molecular Weight
NaOH
Sodium Hydroxide
Na2SO4
Sodium Sulfate
1
Introduction
Chitin is the second-most abundant natural biopolymer behind cellulose (1,2). Upon
deacetylation, chitin can be converted into chitosan, a biopolymer that has been under heavy
investigation over the past few years (3). Chitosan is being researched for a variety of uses,
among which is nanoparticulate and hydrogel drug delivery systems (4). These delivery systems
can be loaded with cancer immunotherapy drugs such as human interleukin-12. The purpose of a
chitosan based hydrogel is to hold a drug like IL-12 in place at a tumor site. This property is
based on how viscous the chitosan solution is.
Figure 1. Chemical Structure of Chitosan (1,2)
Chitosan is a polymer of repeating glucosamine and N-acetylglucosamine units, similar
to the structure of cellulose (5). The length of these chitosan chains determines the molecular
weight of chitosan. The molecular weight can be characterized by the property of viscosity, and
is related to this viscosity logarithmically (6). As the molecular weight increases, viscosity also
increases. Testing viscosity is therefore an easy way to determine if there is in fact a difference in
the molecular weights of various chitosan samples. Current methods for fractionation of chitosan
are complicated, involving complex methods such as chromatography. Chromatography involves
2
running a chitosan solution through porous beads and separating the solution based on the size of
the chitosan. While this is a common method of characterizing polymers, it has an extremely low
throughput and can take a long time to collect the necessary amount of chitosan. Methods
currently in production also have a wide range of viscosities, which is inefficient and produces
varied results during experiments. It was seen that there is a need for a simple and precise way to
fractionate chitosan based on its molecular weight.
In order to test the viscosity of chitosan, it must first be solubilized. Chitosan is usually
soluble in an acidic solution due to the salt formation of the amino group (7). These tests were
done to determine if there was in fact a difference in the solubility of chitosan based on its
molecular weight. Without this solubility difference, it would be impossible to separate chitosan
by solubility based on its molecular weight.
After chitosan is in solution, it was hypothesized that the molecular weight could easily
be fractionated using a precipitation technique. In order to precipitate, the positively charged
amino group of chitosan must interact with a negatively charged base (8). This ionic interaction
will “wrap” the long chains of chitosan around the negatively charged particle, forming micro
and nanoparticles. The formation of these nanoparticles is necessary for chitosan to precipitation
out of solution. Three different methods were developed and tested. While two of these methods
proved to be ineffective, eventually a method was developed based off of the findings in Kassai
et al., which is outlined below.
3
Materials and Methods
In order to conduct this experiment, 40 mL 2% acetic acid was first prepared in a 50 mL
centrifuge tube. 400 mg chitosan1 was then added to the acetic acid to produce a solution of
chitosan that had a concentration of 10 mg/mL. The chitosan was then vortexed briefly and
placed on a rotating mixer for approximately 20 minutes at a low speed.
While the chitosan solution was mixing, 100 mL 1 molar NaOH solution was prepared.
After mixing, 4 mL 1 M NaOH was added to the chitosan solution. Figure 2 shows the
precipitated chitosan in solution.
Figure 2. Chitosan solution before centrifugation
1
Four different samples of chitosan were used, for a total of four experiments; these four samples are outline in
Results
4
The chitosan was then centrifuged at 3000 rpm for 5 minutes. This pushed all of the
precipitated chitosan towards the bottom of the centrifuge tube, as shown in Figure 3. The
supernatant was then moved to a different 50 mL centrifuge tube. The precipitate was then
washed with 5 mL DI water, vortexed briefly, and centrifuged at 3000 rpm for 5 minutes. The
water was then removed and the washing method was repeated 3 more times. The precipitation
and washing procedure was then repeated for the supernatants until no chitosan could be
precipitated.
Figure 3. Chitosan solution after centrifugation
5
After all fractions had been precipitated and washed, the fractions were brought down to 80° C for 24 hrs and lyophilized for 36 hours. All fractions were then weighed and % yield was
calculated. 40 mL 1% acetic acid solution was prepared and each fraction was placed in solution
in a 15 mL centrifuge tube at a concentration of 10 mg/mL. Viscosity tests were done on each
fraction, as well as on a standard sample taken directly from the bag.
6
Data and Results
The lowest range of viscosity tested in this experiment was 20-200 cP. While all of the
ranges were found to be fairly inaccurate, they did provide an estimate as to where the viscosities
would lie. Figure 2 shows a constant negative trend with a range of viscosities from 89.68 cP to
265.99 cP. The standard was found to be 271.75 cP. The yield for the 20-200 cP experiment was
fairly consistent between fractions and an overall yield of 65.3%.
300
Viscosity (cP)
250
200
150
100
50
0
1
2
3
4
Standard
Fraction Number
Figure 4. 20-200 cP viscosities for each fraction, as well as a standard viscosity test (271.75 cP);
viscosities from left to right are 265.99 cP, 155.56 cP, 134.81 cP, 89.68 cP
Fraction
Number
1
2
3
4
Total
Yield
(mg)
53.3
51.7
59.2
97
261.2
%
Yield
13.3%
12.9%
14.8%
24.3%
65.3%
Table 1. Yield data for 20-200 cP sample
7
The 200-600 cP test had small discrepencies between the first 3 fractions, with a range of
only 30.94 cP. The fourth fraction had the most dramatic change in viscosity at 62.59 cP, as well
as over 50% yield.
250
Viscosity (cP)
200
150
100
50
0
1
2
3
4
Standard
Fraction Number
Figure 5. 200-600 cP viscosities for each fraction, as well as a standard viscosity test (224.71
cP); viscosities from left to right are 197.73 cP, 189.41 cP, 220.35 cP, 62.59 cP
Fraction
Number
1
2
3
4
Total
Yield (mg)
% Yield
43.2
35.2
26.2
200.9
305.5
10.8%
8.8%
6.6%
50.2%
76.4%
Table 2. Yield data for 200-600 cP sample
8
The 600-1200 cP test had a much lower viscosity than the range it stated, with the highest
viscosity of only 445.34 cP. There was a negative trend as the fraction number increased, with
the lowest fraction viscosity of 85.56 cP. The second fraction had a yield of 45.4% and a total
yield of over 100%.
1200.00
Viscosity (cP)
1000.00
800.00
600.00
400.00
200.00
0.00
1
2
3
Standard
Fraction Number
Figure 6. 600-1200 cP viscosities for each fraction, as well as a standard viscosity test (1026.32
cP); viscosities from left to right are 445.34 cP, 129.13 cP, 85.56 cP
Fraction
Number
1
2
3
Total
Yield (mg)
% Yield
110.8
181.4
113
405.2
27.7%
45.4%
28.3%
101.3%
Table 3. Yield data for 600-1200 cP sample
9
The 1200-2000 cP test had the greatest number of fractions as well as the largest %
change from largest fraction to smallest fraction of 817.7 cP. Besides fraction 2 there was a
negative trend from fraction 1 to fraction 5. There was an overall positive trend in % yield from
fraction 1 to fraction 5.
1600
1400
Viscosity (cP)
1200
1000
800
600
400
200
0
1
2
3
4
5
Standard
Fraction Number
Figure 7. 1200-2000 cP viscosities for each fraction, as well as a standard viscosity test (1340.98
cP); viscosities from left to right are 869.02 cP, 225.09 cP, 321.26 cP, 230.55 cP, 51.32 cP
Fraction
Number
1
2
3
4
5
Total
Yield (mg)
% Yield
10
19.5
22.5
20.6
90.3
162.9
5.0%
9.8%
11.3%
10.3%
45.2%
81.5%
Table 4. Yield data for 1200-2000 cP sample
10
This chart shows the failed viscosity tests for two different methods that were developed.
The first three columns show three different fractions produced using sodium sulfate, which has
an upward trend but a maximum viscosity of only 24.74 cP. The fourth column is the viscosity of
the dialysis method developed, with a viscosity of only 9.42 cP.
1200.00
975.73
1000.00
Viscosity (cP)
800.00
600.00
400.00
200.00
7.84
8.83
24.74
9.42
1
2
3
Dialysis
0.00
Standard
Fraction
Figure 8. Unsuccessful tests using sodium sulfate fractionation (first three columns) and dialysis,
as well as a standard viscosity test (975.73 cP) from the 1200-2000 cP bag; viscosities from left
to right are 7.84 cP, 8.83 cP, 24.74 cP, 9.42 cP
11
Discussion
Several methods were developed and tested prior to the one outlined above. As stated in
the Introduction, sodium sulfate was used to neutralize the chitosan mixture. This in turn was
able to precipitate the chitosan, which was then separated from the supernatant. However, after
precipitation using Na2SO4 the chitosan would not go back into an acetic acid solution, which is
necessary in order to test the viscosity. This is most likely explained by how chitosan interacts
with sodium sulfate. Chitosan is made up of long chains of polymers, with positively charged
amine groups along these chains. When a negative group comes into solution with the chitosan,
it coils around this group and comes out of solution in the formation of nanoparticles and
microparticles. Because sodium sulfate is bulky in comparison to something like NaOH, the
ionic interaction energy between the nanospheres of chitosan and sodium sulfate makes it
difficult to separate from each other. These nanoparticles of chitosan wrapped around sodium
sulfate were therefore not able to go back into solution. The reason sodium hydroxide was more
effective was due to the weaker interaction between itself and chitosan. The formation of
nanoparticles was still necessary in order for chitosan to precipitate out of solution, but the small
amount of interaction between chitosan and NaOH allowed for it the two to be separated.
(9,10,11)
After using sodium hydroxide, the viscosity was less than 25 cP (Figure 6), well below
the standard sample of chitosan. Although the chitosan was able to be resolubilized into acetic
acid, the precipitated chitosan did not behave normally. It was much more difficult to
resolubilize because it would clump up, whereas the standard chitosan did not clump as easily. It
also had a much lighter color to it than normal chitosan, making it obvious that there was still
sodium hydroxide present after lyophilization. In order to combat this, dialysis was performed in
12
the next trial immediately after precipitation. This was done to separate the NaOH from the
chitosan, but no precipitate remained after 24 hours of dialysis.
The method that Kassai et al. developed was modified over the course of four
experiments using each range of chitosan. The viscosity results are shown in Figures 2-5, and the
percent yield is shown in Tables 1-4. Because each range of chitosan is solubilized slightly
differently because of their different chain lengths, a range of three to five fractions were
produced. In general, the percent yield increased as the number of fractions increased. The most
striking example of this is in Table 4. Only 10 mg of chitosan was precipitated in the first
fraction, which was only 5% of the initial amount, while the last fraction yielded 90.3 mg, a
45.2% yield. There was remarkable precipitation thresholds found in Tables 1 and 2 as well.
Figure 3 is the only test where the viscosity results align with the corresponding percent yield
results. The first three fractions taken from the 200-600 cP sample each had viscosities in the
range of 189 cP – 221 cP and a cumulative percent yield of 26.2%. This is sharply contrasted by
the viscosity of the fourth fraction, which had a viscosity of 62.59 cP and a percent yield of
50.2%, almost twice as much as the first three fractions combined. While it did not yield
decreasing viscosity results over the course of the 4 fractions, the results are still significant for
another reason. The chitosan used in this experiment was inexpensive, and therefore an
extremely wide range of viscosity was found from sample to sample. This result shows that there
was an enormous amount of disparity in the sample used. All of the first three fractions had very
similar yields, as well as similar viscosities. The last fraction had a much lower viscosity and
50% of the yield. So, rather than having a polydisperse range of chitosan, there was only two
lengths of chains in this sample.
13
Figures 2, 4, and 5 all show striking reductions in viscosities as the fraction number
increases despite the inconclusive results shown in Figure 3. The largest decrease in viscosity
happens between the first and second fractions for each of these tests, with an average reduction
of 62.2%. There are less significant reductions between the other fractions, most likely due to the
fact that each sample had a certain range of viscosity, and as more fractions were taken the closer
it was getting to the bottom of that range. The test shown in Figure 2 had the most refined
procedure, and also the best results. All four fractions had a decrease in viscosity, with an
average reduction of 29.4% per fraction.
Sources of error could be found in a few different steps of the procedure. Firstly, the
separation of precipitate from supernatant involves a simple procedure of pipetting the
supernatant out of the centrifugal flask. Some of the precipitate could also be removed while
pipetting, and some of the supernatant could still be left with the precipitate. The yield also
varied between tests. Much of the yield was lost during the multiple washing steps as the water
was separated from the precipitate. There is no definitive way of knowing if all of the chitosan
was precipitated during fractionation, which is another source of a suboptimal yield.
Conclusion
This study has shown that you can fractionate chitosan based on molecular weight with a
simple precipitation method. The implications of this are phenomenal. For example, if you need
chitosan that has a viscosity within the range of 1350-1450 cP, one can quantify the amount of
NaOH needed to remove anything larger than that in an initial fraction of the 1200-2000 range
chitosan. A second fraction can then be taken to extract chitosan that is within the precise range
necessary. Further research will be involved to quantify these precise amounts of NaOH
necessary. The process can then be scaled up to fractionate much larger amounts of chitosan.
14
References
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