crosslinking of a gelatin solutions induced by pulsed electrical discharges in solutions

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792

Crosslinking of a Gelatin Solutions Induced byPulsed Electrical Discharges in Solutions

Isarawut Prasertsung, Siriporn Damrongsakkul,* Nagahiro Saito

This study uses discharges in solutions for the treatment of gelatin solutions in order to generatecrosslinking. The effects of plasma on the properties of gelatin solutions were investigated, thelatter including viscosity, amino acid contents, chemical analyses, and gel strengths. The resultsshow that, after short duration plasma treatments (5–10min), the viscosity of the gelatinsolutions increased, while the concentration of OH� free radicals decreased. After adding ethanolto the gelatin solutions, a greater increase in viscosity, and a greater decrease in free radicalswerefound. This suggests that ethanol providesmore free radicals that can promote the crosslinking ofthe gelatin solution during the plasma treatment, resulting in higher viscosity. The gel strengthof the gelatin was greatly enhanced by the plasma treatment of the solution. The resultsregarding the free amino acid contents showed that the crosslinking degree of plasma-treatedgelatinwas higher than that of the untreated gelatin. FTIRmeasurements show that after plasmatreatment, the IR bands at 1668 and 1558 cm�1, corresponding to the amide I and II groups of
gelatin, shifted to higher wavenumbers, i.e. 1 672 and1564 cm�1, respectively. This suggests that cross-linking has occurred between gelatin molecules. Theresults show that discharges in solutions may be ableto induce crosslinking reactions in the gelatin.Electrical discharges in solutions can be chemical-free, alternative crosslinking methods.
I. PrasertsungChemical Engineering Program, Department of IndustrialEngineering, Faculty of Engineering, Naresuan University,Phitsanulok 65000, ThailandI. PrasertsungResearch Unit on Functionalized Material for Chemical,Biochemical and Biomedical Technology, Faculty of Engineering,Naresuan University, Phitsanulok 65000, ThailandS. DamrongsakkulDepartment of Chemical Engineering, Faculty of Engineering,Chulalongkorn University, Bangkok 10330, ThailandE-mail: [email protected]. DamrongsakkulPlasma Technology and Nuclear Fusion Research Unit,Chulalongkorn University, Bankok 10330, ThailandN. SaitoDepartment of Molecular Design and Engineering, GraduateSchool of Engineering, Nagoya University, Furo-cho Chikusa-ku,Nagoya 464-8603, JapanN. SaitoEcoTopia Science Institute, Nagoya University, Furo-cho Chikusa-ku, Nagoya 464-8603, Japan

Plasma Process. Polym. 2013, 10, 792–797� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com
DOI: 10.1002/ppap.201200148
1. Introduction

Gelatin is a protein derived from the partial hydrolysis of

native collagen, themost abundant structuralprotein found

intheskin,tendon,cartilageandanimalbones.Duetoseveral

advantages, suchas its biological origin, non-immunogenic-

ity, biodegradability, biocompatibility, and commercial

availability at relatively low cost, gelatin is widely used in

the pharmaceutical, food, and medical industries. Gelatin

hasbeenusedasabasematerial, substitutinga largeportion

of collagen to produce scaffolds without affecting the

biological properties.[1] However, it is necessary to crosslink

gelatin-based scaffolds to decrease their solubility, and

enhance their mechanical properties prior to biomedical

applications. Crosslinking of gelatin is typically performed

by various methods, such as chemical treatments, de-

hydrothermal treatment, ultraviolet irradiation, or electron

beam irradiation. The crosslinking degrees of gelatin films

Figure 1. Schematic diagram of discharge in solution system.

Crosslinking of a Gelatin Solutions Induced by Pulsed Electrical Discharges

vary depending on the methods employed. Among the

crosslinking methods, chemical treatments are the most

widelyusedduetotheirhighefficiency inthestabilizationof

soluble materials. However, the disadvantage of chemical

crosslinking is the possible chemical residuals, whichmight

cause toxicity and irritation.[2]

Plasmatreatmentsarealternativemethodsthathavebeen

employed to crosslink polymericmaterials. This treatments

are able to subtract hydrogen, and to form free radicals at or

near the surface, which then interact to form crosslinks.[3]

Gerenser et al.[4] reported that polyethylene could be

crosslinked by argon plasma treatments. The crosslinking

depth depended on the treatment time, and plasma power,

with the maximum being found to be �160mm.[4] In our

previous study, pulsed inductively coupled plasmas (PICP)

were applied to induce the crosslinking of gelatin films.[5]

The crosslinking degree was, however, very low compared

to the that achieved by conventional de-hydrothermal

technique. This could potentially be due to the fact that

the plasma was not able to treat the bulk of the gelatin

film, resulting in a low degree of overall crosslinking.

Among the plasma treatment methods, electrical dis-

charges in solutions are liquid-phase plasma methods,

which were first proposed by Saito et al.[6] They have been

widely utilized in nanomaterial synthesis, surface mod-

ifications, water treatment, sterilization, and decomposi-

tion of toxic compounds.[7] These systems are able to

produce highly active species such as hydroxyl radicals

(OH�), hydroperoxyl radicals (HO2�), free electrons (e-),

superoxide anions (O2�), and atomic oxygen anions

(O�).[8] Since the molecular density of the liquid phase is

much higher than that of the gas phase, it would be

reasonable to expect a higher reaction rates and chemical

reaction variability.[9] Moreover, it is very easy to control a

homogenous solution in contact with plasma.

It was therefore our aim to apply electrical discharges to

solutions in order to crosslinking gelatin. The effects of

plasmas on the crosslinking of gelatin were explored. The

plasma-treated gelatin solutions were then characterized,

monitoring the viscosity, gel strength, free amino contents,

and chemical characteristics.

Table 1. The experimental set of gelatin samples.

Samples Addition of ethanol [vol%]

0%e 0

5%e 5

10%e 10

20%e 20

40%e 40

2. Experimental Section

2.1. Discharge in Solutions Setup

The setup of the discharge in solutions,modified fromour previous

study,[10] is shown in Figure 1. The discharge in solutions was

operated at atmospheric pressure. The pulsed electric discharge

was generated at the needle electrodes in the glass reactor.

The tungsten needle electrodes (1mm in diameter) were fixed

with Teflon and covered by an insulator (mullite) tube. The

distance between the electrodes was set at 0.2mm. A high

frequency bipolar pulsed DC power supply was connected to the

needle electrodes.

Plasma Process. Polym. 2013, 10, 792–797� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.2. Plasma Treatments of Gelatin Solutions

TypeA gelatin solutions (Nitta Gelatin, Japan) in the concentration

range of 1.25–2.5wt/vol% were prepared. To apply the plasma to

the gelatin solution, the frequency, voltage, and pulse width were

fixed at 15KHz, 1.6 kV, and 2ms, respectively. The temperature of

the gelatin solutionduring the plasma treatmentwas controlled at

22� 2 8C. In order to maintain a homogeneous aqueous solution,

the solution was continuously stirred. Ethanol was added to the

gelatin solution in order to enhance the concentration of free

radicals, such as OH�, H�, and O�, generated by the solution plasma

system.[11] The set of gelatin samples with various ethanol

concentrations is summarized in Table 1. The period of plasma

treatment was varied from 0 (untreated) to 30min.

2.3. Characterization of Plasma-Treated and

Untreated Gelatin Solutions

2.3.1. Viscosity Measurements

The viscosities of plasma-treated and untreated gelatin solutions

were determined at 20 8C using a viscometer (Vibro SV-100, Japan).

793www.plasma-polymers.org

I. Prasertsung, S. Damrongsakkul, N. Saito

794

2.3.2. Determination of H�, OH� and O� Species in Plasma-

Treated Gelatin Solutions

To characterize the species in plasma-treated gelatin solutions,

optical emission spectroscopy was used to monitor the light

emitted fromtheplasma in thewavelengthrangeof200–1000nm.

The emissionwasdetected throughaquartz glasswindow,withan

optical fiber placed 1mm in front of the glass chamber. Data were

acquired using the Avantes software.

2.3.3. Determination of Gel Strength

The plasma-treated and untreated gelatin solutions were kept in

the refrigerator at 5� 1 8C for 16–18h prior to the determination of

gel strength using a modified version of the method described

by Wainewright[12] which uses a texture analyzer (Rheoner II,

YAMADEN, Tokyo). The penetration test of the gel was carried out

using a flat-bottomed plunger (0.5 inch in diameter) at a crosshead

speed of 1mms�1. The maximum force when the plunger

proceeded into the gel to a depth of 4mm was determined.

2.3.4. Determination of the Free Amino Content

The plasma-treated and untreated gelatin solutions were cast

into Teflon molds and air-dried overnight. The free amino acid

contents of the plasma-treated and untreated gelatin films were

determined using the TNBS assay.[13] The concept of thismethod is

to react the free amino groups of the gelatin, which indicate

uncrosslinked groups, with 2,4,6-trinitrobenzene sulfonic acid

(TNBS). Briefly, gelatin films were treated with 0.5% TNBS solution

and4%sodiumhydrogencarbonate (NaHCO3,pH8.5) andheatedat

40 8C for 2h. The uncrosslinked primary amino groups of gelatin

react with TNBS and form a soluble complex. This solution was

further treatedwith6 NHCl at 60 8C. The absorbance of the solutionwasmeasured using spetrophotometry at 415nm. The free amino

Figure 2. The %increase in viscosity of the plasma-treated gelatin soluof treatment time and ethanol concentration (concentration of gelatinvol%).


content of gelatinwas thendetermined froma standard curve ofb-

alanine.

2.3.5. Chemical Analyses

The chemical analyses of plasma-treated and untreated gelatin

films (thickness of 100� 20mm) were performed using FTIR

spectroscopy (Digilab, FTS 7000 Series).

2.3.6. Statistical Analyses

Statistical analyseswere performed by the paired t-test, using SPSSfor Windows (version 13.0 Statistical Package for Social Sciences

(SPSS), USA). A P-value<0.05 was considered to be significantly

different.

3. Results and Discussion

3.1. The Effects of Ethanol Concentration and

Treatment Time on the Viscosity and OH_Radical

Content of Gelatin Solutions

The effects of the ethanol concentration and plasma

treatment time on the viscosity of plasma-treated gelatin

solution are shown in Figure 2. The results show that after

5min of plasma treatment, the viscosity of the gelatin

solutionwassignificantly increased.After10minofplasma

treatment, the viscosity of gelatin seemed to reach a

plateau. The viscosity of plasma-treated gelatin solution in

ethanol was higher than that in pure distilled water. The

viscosity of gelatin solution was increased with increasing

ethanol concentrations from 5 to 20 vol%. Further increase

in the ethanol concentration from 20 to 40 vol% tended to

tion as a functionsolution 1.25wt/

slightly lower the viscosity of the gelatin

solution.

The emission spectrum of plasma-

treated gelatin solution, determined at

a treatment timeof1min, asa functionof

ethanol concentration was compared to

that of pure water, as presented in

Figure 3. The spectra of the gelatin

solutions showed strong peaks at the

wavelengths of 309.0, 486.0, 656.5, and

777.3 nm, corresponding to OH� radicals,Hb, Ha, andO

� radicals, respectively.[14] It

was noted that the intensity of these

peaks increased with increasing the

ethanol concentration.

Figure 4 shows the intensity of the

peak assigned to OH� radicals (wave-

length of 309.0 nm) as a function of the

ethanol concentration and treatment

time. The results indicate that with very

short treatment times (1–2min), the

gelatin solutions containing higher

DOI: 10.1002/ppap.201200148

Figure 3. OES spectrum of pure water and plasma-treated gelatin solutions (1.25wt/vol%) containing various concentrations of ethanol after being treated for 1min.


ethanol concentrationshadhigher amounts ofOH� radicals.The amount of this type of radical gradually decreased

when increasing the treatment time up to 10min. After

plasma treatments for 10min, the amount of OH� radicalsseemed to stabilize. This result corresponds to the viscosity

of plasma-treated gelatin solution, i.e., the viscosity of the

gelatin solution did not change after 10min of plasma

treatment.

3.2. The Effects of the Solution Concentration on the

Viscosity of the Gelatin Solution

Theeffects of the solutionconcentrationson theviscosityof

plasma-treated gelatin solution are shown in Figure 5. It

Figure 4. The effects of the ethanol content and the treatmenttime on the OES intensity at the wavelength of 309.0 nm (OH�

radical peak) determined from the plasma-treated gelatinsolution (1.25wt/vol%).

Figure 5. The effein viscosity of gel

Figure 6. The gegelatin solutions2.5wt/vol% andsignificant differsample.


could be seen that, after 5min of plasma

treatment, the viscosities of the gelatin

solutions at all concentrations increased

by 80–89%. The viscosities of gelatin

solutionsat the concentrations of 1.8and

2.5wt/vol% were still increased when

increasing the treatment time to 10min.

After that, they slightly decreased. On

the other hand, the viscosity of the

1.25wt/vol% solution remained un-

changed after increasing the treatment

time beyond 5min.

3.3. The Properties of the Plasma-

Treated Gelatin

3.3.1. Gelatin Gel Strength

Figure 6 shows the plasma effects on the

gel strength of gelatin after the sol-gel

cts of the gelatin concentration on the %increaseatin solution (ethanol content of 20 vol%).

l strength of plasma-treated and untreated(gelatin concentration and ethanol content of20 vol%, respectively). The asterisk marksence at p<0.05 relative to the untreated


Figure 7. The free amino content of the plasma-treated anduntreated gelatin solutions (gelatin concentration and ethanolcontent of 2.5wt/vol% and 20 vol%, respectively). The asteriskmarks significant difference at p<0.05 relative to untreatedsample.

I. Prasertsung, S. Damrongsakkul, N. Saito

796

transition. The results indicate that after 5min of plasma

treatment, the gel strength was significantly increased by

96%, compared to that of the untreated sample. However,

plasma treatments longer than 5min did not further

enhance the gel strength of the gelatin.

3.3.2. Free Gelatin Amino Acid Content

The free amino acid content of gelatin samples with and

without plasma treatment is shown in Figure 7. It could be

seen that the free amino acid content of the plasma-treated

gelatin samples was significantly lower than that of the

untreated one. However, an increase in treatment time

from 5 to 30min had no effect on the free amino acid

content of the gelatin.

3.3.3. FTIR Spectra of the Gelatin Films

Figure 8 shows the FTIR spectra of the

plasma-treated and untreated gelatin

samples. The characteristic peaks of

gelatin films were found at 3 348,

1 668, and 1 558 cm�1, corresponding to

OH stretching, COO-asymmetric stretch-

ing, and amide II stretching in gelatin,

respectively.[15] After plasma treatment,

the transmittance peaks at 1 668 and

1 558 cm�1 were shifted to 1 672 and

1 564 cm�1, respectively, while the one

at 3 348 cm�1 remained unchanged.

Figure 8. FTIR spectra of the untreated and plasma-treated gelatin films at differenttreatment time (gelatin concentration and ethanol content of 2.5wt/vol% and 20 vol%,respectively).

4. Discussion

Discharges insolutionswereproduced to

treat gelatin. During plasma treatment


of the gelatin solution, free radicals, such as hydroxyl, were

generated by the reaction of oxygen radicals and water:[16]

O� þH2O ! 2OH� ð1Þ

Tomoda et al.[17] previously reported that hydroxyl

radicals could induce the crosslinking process of gelatin

molecules. They proposed the crosslinking mechanism of

gelatin solution as follows:

RH� þ OH� ! R� þH2O ð2Þ

R� þ R� ! R� Rðcrosslinked moleculeÞ ð3Þ

where R represents gelatin molecules.

When crosslinking occurs, it subsequently results in an

increased viscosity of the gelatin solution, as shown in

Figure 2. From the proposed crosslinking mechanism of

gelatin, it is obvious that the OH� radicals could greatly

affect the crosslinking process. In order to observe the

effectsofOH_radicalsontheviscosityofgelatinsolution, the

amount of free radicals present during plasma treatment

was enhanced by the ethanol addition. The results showed

that the viscosity of gelatin solution increased with

increasing the ethanol concentration. This could be

attributed to an increase in the radical content, resulting

in more crosslinking. This corresponds to the results

regarding the effects of the ethanol concentration on the

amount of OH_radicals, as shown in Figure 3. An increase in

the ethanol concentration could provide a greater amount

of hydroxyl radicals generated during plasma treatment,

which enhance the crosslinking process, and finally resulte

in increased viscosity of the gelatin solution. Nonetheless,

DOI: 10.1002/ppap.201200148


an increase in the ethanol concentration from20 to 40 vol%

didnot further increase theviscosity of the gelatin solution.

Examining the effect of treatment time on the viscosity of

the gelatin solution, the viscosity was increased after short

plasma treatments (around 5–10min) and remained

constant when longer plasma treatments (>10min) were

used. This result corresponds to the amount of OH_radicals,

as seen in Figure 4. The amount of free radicals decreased

and seemed to become constant when plasmawas applied

for >10min. As mentioned earlier, the free radicals

generated by plasma treatment can be used to generate

the crosslinking process of gelatin; therefore, the decrease

in the free radical content during plasma treatment was

due to the progression of the crosslinking reaction. When

increasing the concentration of the gelatin solution, the

percentage increase in the viscosity after plasma treatment

was higher, as shown in Figure 5. This confirmed that an

increase in the gelatin concentration provided more

crosslinked molecules, thus promoting the crosslinking

reaction. An increase in the gel strength of plasma-treated

gelatin samples, shown in Figure 6, confirmed the presence

of gelatin crosslinks. The result corresponds to the report by

Rajeev et al.[18] who found that the gel strength of UV-

treated gelatin solution was significantly increased com-

paredtotheuntreatedsample.Theassessmentof theamino

acid content of plasma-treated and untreated gelatin in

Figure 7 shows that the amino acid content of the gelatin

was significantly decreased as a result of the plasma

treatment. It iswell-knownthat the freeaminoacidcontent

of gelatin is used to form the crosslinked molecules.[19] The

decrease in the free amino acid content revealed that the

discharge in solution could induce the crosslinking process

of gelatin. The FTIR results of the plasma-treated and

untreated gelatin films in Figure 8 indicate that, after

treatmentwithplasma for5 to30min, the transmittanceat

1 668 and 1 558 cm�1, corresponding to the amide I and II

groups of gelatin, were shifted to a higherwavenumbers of

1 672 and 1 564 cm�1, respectively. The amide I and II

absorption bands are useful peaks for IR analysis of the

secondary structure of proteins like gelatin.[18] This shift

might be possibly be due to the crosslinking that had

occurredbetweengelatinmolecules.[18,20]However, further

investigations are required to characterize the possible

quantitative changes in functional groups.

5. Conclusion

In this study, an electrical discharge was used to treat

gelatin solutions. Plasma treatment can induce gelatin

crosslinking, as indicated by the increased viscosity,


decreased free amino acid contents, and increased gel

strength. The addition of ethanol (< 20%) to the gelatin

solutionswas showntoenhancecrosslinking, as it provided

more free radicals required for the progression of the

crosslinking reactions. The results presented in this work

suggest that electrical discharges could be effective

methods to crosslink gelatin solutions instead of chemical

methods that might cause toxicity.

Acknowledgments: Financial support from The Royal GoldenJubilee PhD Program, Thailand Research Fund, ChulalongkornUniversity through the Ratchadaphiseksomphot EndowmentFund, and the Chulalongkorn University Centenary AcademicDevelopment Project are gratefully acknowledged.

Received: October 30, 2012; Revised: May 2, 2013; Accepted: May20, 2013; DOI: 10.1002/ppap.201200148

Keywords: crosslinking of gelatin; electrical discharges insolutions; FTIR measurements; hydroxyl radicals; viscosity ofgelatin

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crosslinking of a gelatin solutions induced by pulsed electrical discharges in solutions

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