Patent Application: US-201415036080-A

Abstract:
a process for producing chitin nanocrystals involves contacting a chitinous material with a sufficient amount of an inorganic persulfate to produce chitin nanocrystals from the chitinous material . the process permits one - spot production of chncs from biomasses such as crustaceans , fungi , mushrooms , insects or mixtures thereof . chitin nanocrystals produced by the process comprise surface carboxylic groups and are not initially deacetylated .

Description:
a versatile method using persulfate for the preparation of cellulose nanocrystals ( cnc ) is known in the art ( leung 2011 ; leung 2012 ; lam 2013 ). however , cellulose and chitin are two different compounds . the use of persulfate to break down other polysaccharides ( e . g . chitin , xylan , dexrtran , pullulan , starch and xanthan gum ) to form nanocrystals of the polysaccharides is not a priori expected in view of the process with cellulose because each polysaccharide has unique structural and bulk physical features that may disrupt nanocrystalline formation . for example , like amino acids and unlike cellulose , chitin is negatively charged in basic conditions due to carboxyl groups and positively charged in acidic conditions due to amino groups . further , pristine chitin exhibits very high crystallinity compared to cellulose . although the main bond is 1 - 4 - β in both cellulose and chitin , the presence of n - acetyl groups of chitin can affect the hydrolysis as well as oxidation . the n - acetyl group may be oxidized or converted to other functional groups during the course of the aps treatment . to date , there are only a few reports for the production of chitin nanocrystals using acid hydrolysis ( fan 2008 ), which required the use of expensive chemicals . indeed , as is shown in the examples below , the only two polysaccharides tested that form nanocrystals from persulfate treatment are cellulose and chitin . treatment of the other polysaccharides result in nanosized spheres , not a crystalline material . ammonium persulfate ( aps ) and chitin practical - grade powder ( c7170 , ( c 8 h 13 no 5 ) n from shrimp shells ) were obtained from sigma aldrich . attenuated total reflectance ftir ( atr - ftir ) spectra were collected ( bruker tensor 27 ftir spectrophotometer ) from 4000 to 600 cm − 1 for 64 scans and 4 cm − 1 resolution using a zinc selenide ( znse ) crystal . atomic force microscopy ( afm ) micrographs of chncs were obtained from sonicated aqueous samples using a nanoscope ™ iv ( digital instruments , veeco , santa barbara , calif .) with a silicon tip operated in tapping mode . low voltage transmission electron microscopy ( lvtem ) micrographs were obtained by a delong lvem , low voltage electron microscope ( delong , montreal , qc , canada ) operating in tem mode at 5 kv . a chnc suspension ( 10 ml ) was prepared in double - distilled water at 0 . 1 wt % by sonication ( 3 min , app . 1 kj ). a 4 μl drop of the well - dispersed chnc suspension was dried on a 300 mesh , cu - 300hd grid ( pacific grid - tech ) and analyzed . wide angle x - ray scattering analysis was obtained on a bruker discover 8 diffractometer equipped with a copper ( cu kα , λ = 1 . 54184 å ) anode source , along with instrumental settings of 40 kv and 40 ma . the collected data were analyzed using winplotr ( http :// www . llb . cea . fr / fullweb / winplotr / winplotr . htm ), a graphic tool for powder diffraction to provide peak position ( 2 θ ), fwhm ( full width half maximum ), peak deconvolution , and integration intensity for calculation of the crystallinity index ( cri ). crystallite sizes were calculated using the scherrer equation d = kλ /( fwhm × cosθ ) with the scherrer constant k taken as 1 and λ = 1 . 542 å . thermogravimetric analysis ( tga ) was conducted with a netzsch sta 449f1 under nitrogen purge gas . scanning electron microscopic ( sem ) images were obtained using a hitachi sem ( s - 2600n , tokyo , japan ) coupled with an energy dispersive x - ray ( edx ) spectrometer equipped with a ln 2 - free analytical silicon drift detector ( inca x - act , oxford instruments , uk ). the sem / edx system was operated with a high vacuum mode at 20 kv , emission current of 80 μa , a working distance of 20 mm , and sample tilt of 30 °. the edx system has inca software with a database of reference spectra for elemental analysis , compositional nano - analysis and mapping . materials were immobilized on carbon tape and introduced into the chamber . to insure reproducibility , three different magnifications at three different areas were selected for each sample and data were averaged . dynamic light scattering and the - potential of the chnc materials ( 2 mg / ml ) in water were determined using a zetasizer nano - zs ( malvern instruments , malvern , uk ) in triplicate . the nmr experiments were performed on a bruker avance spectrometer operating at a 13 c frequency of 150 . 9 mhz , using proton dipolar decoupling ( tppm scheme with rf field of 80 khz ), magic angle spinning and cross - polarization . the rf field strength of 55 khz for 13 c and a ramp from 80 to 40 khz for 1 h were used for cross - polarization . the spin rate : 14 khz , contact time : 2 ms , acquisition time : 13 . 7 ms , and recycle delay : 5 s . example 1 : preparation of chitosan nanocrystals ( chncs ) and comparison to other polysaccharides chitin ( 10 g ) was added to a 1 m aps aqueous solution ( 1 l , ph 2 , conductivity about 230 μs · cm − 1 ). the suspension was heated to 60 ° c . for 12 h to give a white suspension of chncs . the suspension was centrifuged ( 12 , 000 rpm , rcf = 22 , 100 ) for 10 min . the solution was decanted , and about 200 ml of water was added to the chnc pellet , followed by 5 min of vigorous mixing and repeated centrifugation . the centrifugation / washing cycles were repeated 4 times until the solution conductivity was about 5 μs · cm − 1 ( ph about 4 ), close to that of deionized water . the product was lyophilized to yield a white powder ( 4 . 00 g ). chncs in their sodium form were prepared by adding 1 m naoh until the suspension reached ph 7 , followed by washing / centrifugation with deionized water . during the aps treatment of chitin , selective oxidation occurs at the c6 primary hydroxyl group of chitin to form carboxylic acid groups ( scheme 1 ). analogous to previous reports of cnc synthesis ( leung 2011 ; leung 2012 ), the formation of chncs occurs via cleavage of the glycosidic bonds and individualization of elementary fibrils by the formation of charged carboxylate groups on surfaces of the chitin crystallites . the resulting chncs could be subjected to a second treatment with naoh for deacetylation to form chncs with improved aqueous solubility as follows . the deacetylation of chncs was performed using a modified procedure ( zhang 2005 ). chncs ( 1 g ) were added to a 45 % naoh solution ( 4 . 5 g naoh in 10 ml of water ). the suspension was heated at 80 ° c . for 4 hours at which time the reaction mixture turned from white to brown . the brown suspension was washed with etoh , centrifuged six times at 10 , 000 rpm for 10 min , and oven dried at 60 ° c . to yield a yellow - colored product ( 700 mg ). to evaluate whether other polysaccharide - based nanoparticles could be produced using aps , chitin , dextran ( α - 1 , 6 glycosidic linkages between glucose molecules , while branches begin from α - 1 , 3 linkages ), pullulan ( three glucose units in maltotriose are connected by an α - 1 , 4 glycosidic bond , whereas consecutive maltotriose units are connected to each other by an α - 1 , 6 glycosidic bond ), starch ( amylose , α - 1 , 4 - d - glucopyranosyl and α - 1 , 6 - glucopyranosyl ), xanthan gum , and xylan ( a polymer of β - d - xylose , a pentose sugar ) were treated with 1 m aps at 60 ° c . for 16 h . the reaction products were dialyzed and evaluated with tem for their dimension as shown in fig1 . considering the backbone similarity between xanthan and cellulose , the hydrolysis of 1 , 4 - β bonds by aps was greatly dependent upon the polymer structure . it should be noted that xanthan has a particularly complicated molecular structure with the backbone β -( 1 - 4 )- d - glucose , same as cellulose . however , every alternate glucose residue has a three sugar side chain comprising two mannose residues with a glucuronic acid residue between them . the mannose residue nearest the main chain can carry a c6 acetyl group and the terminal mannose can carry a pyruvate group between c4 and c6 . no nanorods were observed from xylan , a pentose sugar with 1 - 4 - β bonds , attesting that the action of aps was only limited to chitin or cellulose , and not for other polysaccharides . indeed , the reaction of other polysaccharides with aps only yielded spherical nanoparticles whereas only the reaction of chitin with aps yielded individual rod - shape nanoparticles ( fig2 ). sem images of the chncs prepared in example 1 revealed a perceived fibrous structure prior to its dispersion in water ( fig3 ). edx spectra of various forms of chitin and their elemental analyses are presented in fig4 and table 1 , respectively . upon treatment of chitin with aps , an increase in oxygen content was consistent with the oxidation of the c6 alcohol group to the carboxylic group . deacetylation of chncs with naoh results in a decrease in the oxygen content as the acetyl groups of chitin were converted to amino groups . the dimensions and size distributions of chncs were estimated using afm . for a sample of 1069 chncs in the afm image , the average length and width were ( 149 ± 7 ) nm and ( 6 . 8 ± 0 . 1 ) nm , respectively ( fig5 ). dynamic light scattering of chncs ( 2 mg / ml solution in water ) estimated a size distribution of 112 ± 1 nm and a ζ - potential of − 42 mv . the high ζ - potential ( negative or positive above 40 mv ) conferred the stability of a colloid dispersion ( lam 2013 ), which is important for numerous potential applications such as coating , polymer fillers and drug delivery . ftir spectra of the different chitin materials are shown in fig6 with peak assignments given in table 2 . all three species exhibit distinct amide i doublet bands centered at 1620 and 1655 cm − 1 , amide ii band at 1554 cm − 1 and amide iii band at 1305 cm − 1 . for the amide i , about 50 % carbonyl groups are bonded through hydrogen bonds to the amino group inside the same chain ( c ═ o ... h — n ) as reflected by the vibration mode at 1655 cm − 1 . the remaining groups produce the same bond plus another with the — ch 2 oh group from the side chain , resulting in a decrease in the amide i band at 1620 cm − 1 . the amide ii is attributed to n - h bending vibration ( 40 - 60 %) and from the c — n stretching vibration ( 18 - 40 %). the amide iii is a very complex band resulting from a mixture of several coordinate displacements ( fig6 a ). these amide bands are consistent with the α - polymorphic crystalline structure of chitin ( jang 2004 ). the amide i peaks of chncs are more pronounced than the starting chitin , particularly the peak at 1620 cm − 1 . the inset of fig6 a also shows a distinct ν ( c ═ o ) stretching peak at 1753 cm − 1 for the coon group of chncs . the deacetylation of chncs results in several changes to the amide i bands . neutralization of the coon group in deacetylated chncs should shift the resulting coo 31 na + peak downfield leading to an absorbance increase centered at 1654 cm − 1 ( lam 2013 ). however , the loss of acetyl groups will lower both the absorbance bands at 1654 and 1621 cm − 1 as a result of decreasing amide character . the net result is a reduction in the relative ratio between the two doublet amide i peaks for deacetylated chncs in comparison to chncs . fig6 b shows the amide a band at 3262 cm − 1 for intermolecular c ═ o ... h — n hydrogen bond and the amide b band at 3103 cm − 1 for nh groups intramolecularly bonded by h . again , the vibrational bands decrease in absorbance , particularly for the amide a band of deacetylated chncs as acetyl groups are replaced by nh 2 groups , reducing the number of intermolecular c ═ o ... h — n hydrogen bonds in the system . the increase in absorbance band for the oh groups at 3437 cm − 1 could be attributed to the presence of water in addition to the hydroxyl and carboxyl group of the chitin material . it should be noted that the ftir spectra of chncs could be used to estimate the carboxylic acid and n - acetylation contents . the absorption ratio a 1740 / a 1030 between the carboxylic acid stretching band ( 1740 cm − 1 ) and the c - o stretching band of the chitin backbone ( 1030 cm − 1 ) can be used to estimate the carboxylic acid content of cellulose ( habibi 2006 ). the a 1740 / a 1030 estimated a carboxylic acid content of 0 . 058 with chncs , in agreement with the result obtained by conductometric titration as described below . conductometric titration was conducted using a known method for carboxylated cellulose nanocrystals ( leung 2011 ) to determine the carboxylic acid content or the degree of oxidation ( do ) of the chncs . chncs ( 50 mg ) were suspended in 10 ml of 0 . 01 m hcl and sonicated for 10 min to disperse the nanocrystals . the chnc suspension was then titrated against 0 . 01 m naoh and do is calculated as 203 ( ν 2 − ν 1 ) c /( w - 14 ( ν 2 − ν 1 ) c ), where c is the naoh concentration ( m ), w is the weight of chnc sample ( g ), and ν 1 and ν 2 are the volumes of naoh ( l ). the molecular weight of an n - acetylglucosamine unit with a ch 2 oh group is 203 . the molecular weight difference between an n - acetylglucosamine unit and its coon derivative is 14 . the titration was repeated three times . fig7 shows the titration plot . three segments can be observed in the conductometric titration plot . the first descending segment is the neutralization of excess hcl used in protonating the chncs . the second flat segment corresponds to the neutralization of carboxyl groups and the third ascending segment refers to the excess naoh . a do value of ( 6 . 2 ± 0 . 8 )× 10 − 2 was obtained by conductometric titration , which agreed well with the ftir estimation using the a 1740 / a 1030 ratio as mentioned earlier . the degree of deacetylation ( dd ) was determined by ftir with the results presented in table 3 . the corrected baseline ftir absorbance intensity ratio ( fan 2008 ) between the bands at 1560 and 1030 cm − 1 was obtained using the regression equation ( shigemasa 1996 ): the dd values for chitin and chncs by ftir are similar , which confirmed that oxidation of chitin by aps did not lead to deacetylation of the amide groups . an alternative method ( baxter 1992 ) has been commonly used for determining the degree of n - acetylation which takes into account the transmission bands at 3450 and 1655 cm − 1 as % n - acetylation = 115 ×( i 1650 / i 3450 ). however , the values for n - acetylation are only applicable for chitosan materials in the range of 0 - 55 %. as previously mentioned , the band at 3450 cm − 1 is attributed to oh and might include contributions from absorbed water . also , the amide i band at 1650 cm − 1 typically appears as a doublet in both α - chitin and highly acetylated materials . this leads to very subjective baseline corrections required for band intensity measurements ( shigemasa 1996 ). fig8 compares the 13 c cp - mas spectra for chncs and the chitin starting material . the c3 and c5 appear as two signal characteristics of a partially solved doublet , a typical signature of α - chitin . the doublet has been attributed to the different configurations of c3 and c5 resulting from the hydrogen bonds formed . the dd was then estimated by comparing the carbon signals of the methyl and polysaccharide backbone ( cardenas 2004 ). the dd was calculated to be 2 % for both the chitin and chnc , confirming that the aps treatment did not result in deacetylation of the amide groups . the crystal structures of chncs were studied using xrd . as shown in fig9 , chncs exhibit four diffraction peaks at 9 . 6 °, 19 . 6 °, 21 . 1 ° and 23 . 7 °, a pattern that resembles the diffraction pattern of α - chitin . after treatment with aps , the intensity of the ( 110 ) diffraction peak increased significantly , indicative of an increase of crystallinity . using the intensity method reported by lavall et al . ( lavall 2007 ), the crystallinity index ( cri ) of the chncs was estimated to be 91 . 7 %, an increase from 86 . 5 % of α - chitin . such a result was compared favorably with the literature values of 86 % for chitin nanowhiskers ( heath 2013 ). it should be noted that the oxidation of chitin led to a decrease in the thermal degradation temperatures , t d5 decrease from 298 ° c . for the chitin starting materials to 253 ° c . for chncs ( fig1 ). aqueous solubility of chncs and chitin was examined . samples of chitin , acid form chncs and neutralized chncs were dissolved in water at 1 % ( 100 mg / 10 ml ), with resulting phs of 5 . 0 , 3 . 5 and 6 . 0 , respectively . chitin was insoluble and settled to the bottom of the vial . the acid form of chncs was slightly soluble , but also settled to the bottom of the vial within 10 - 20 min . in contrast , neutralized chncs formed an opaque solution with very little sedimentation . all samples were then sonicated ( 1000 j , i . e . 10 kj / g ) on ice and left to settle for 20 min . chitin was still insoluble and sedimented . the acid form of chncs formed an opaque solution which did not sediment , whereas the neutralized chnc became more translucent ( less opaque ) compared to the non - sonicated counterpart . increasing the sonication energy to 20 kj / g had a marginal impact on the solubility . electric cell - substrate impedance sensing ( ecis ) was performed using spodoptera frugiperda sf9 insect cells and v79 chinese hamster lung fibroblast cells . these two cell lines have been used extensively for ecis testing and the culture conditions have been previously described ( male 2012 ). the ecis impedance system ( applied biophysics , troy , n . y .) comprises an 8 - well sensing chip ( 8w1 e ) with a gold electrode in each well . detailed information on ecis impedance measurement using microfabricated gold electrodes ( 250 pm ) has been reported elsewhere ( male 2012 ). cell adhesion was promoted by the addition of an extracellular matrix ( ecm ) to the gold surface as previously described . fibronectin ( 0 . 15 ml , 0 . 1 mg / ml , prepared in 0 . 85 % naci ) and concanavalin a ( con a , 0 . 40 ml , 0 . 5 mg / ml , prepared fresh daily in 50 mm pbs , ph 7 . 4 with the aid of sonication for 1 h ) were used for v79 and insect cells , respectively . after about 60 min of incubation , the wells were washed three times with 0 . 85 % nacl and then 0 . 3 ml of culture medium was placed in each well , and the impedance baseline was monitored for 1 - 2 hours at 37 ° c . in a humidified chamber of 5 % co 2 and 95 % air ( v79 cells ) or at 27 ° c . without co 2 for insect cells . the wells were then emptied before the addition of the cell - chnc solution . a chnc sample ( stock solutions in the range of 2000 ppm in culture medium ) was added ( 25 - 500 μl ) to the v79 cell suspension ( 0 . 5 ml at 2 × 10 6 cells / ml ) resulting in various concentrations ( 50 - 1000 ppm ) once the sample was made up to 1 ml with culture medium . in the case of insect cells , the chnc sample was added ( 12 . 5 - 75 μl ) to the cell suspension ( 0 . 5 ml at 6 × 10 6 cells / ml ) resulting in various concentrations ( 25 - 150 ppm ) once the sample was made up to 1 ml with culture medium having a final ethanol concentration of 2 %. for testing cytotoxic / inhibitory effects , 0 . 4 ml of each sample concentration was added to 2 or 3 wells . six concentrations of chncs including a control ( no chncs ) were tested at the same time and each experiment was performed in triplicate . after the experiment , cells were imaged using a wilovert afl 30 inverted microscope ( hund , germany ) equipped with a digital video camera ( kp - d50u , hitachi , tokyo , japan ). the impedance / resistance of each well was measured every 2 min for 20 hours at 4 khz , and the system acquires resistance , impedance and capacitance data . however , focus for study was placed on the larger changes in the resistance . the ecis 50 value derived from the time response function , f ( c , t ), was calculated as previously described ( male 2012 ). without chncs , the v79 cells descended to the well bottom within 20 min and changed their morphology from round shapes to flattened forms with much larger dimensions as observed by the video - enhanced microscope . low concentrations (& lt ; 250 ppm ) of chncs exhibited a very slight effect on the resistance signal . at higher concentrations ( 500 - 1000 ppm ), the resistance change was lower by 20 %, but the cells on the electrode surface were still well spread . this phenomenon was likely due to cells initially trying to spread flat on the electrode surface , but then becoming more spherical on the surface with time due to the influence of chncs . similarly , without chncs , the sf9 cells descended to the well bottom within 20 min and changed their morphology from round shapes to flattened forms with much larger dimensions as observed by the video - enhanced microscope . as the cells spread , the effective area available for current flow was altered , resulting in a significant increase in the resistance of the well by about 6 . 0 kω to a final resistance of 9 . 0 kω . low concentrations of chnc samples (& lt ; 50 ppm ) exhibited a marginal effect on the resistance signal with insect cells . however , as the concentration was increased ( 75 - 150 ppm ), the resistance change decreased significantly ( fig1 ). inverted fluorescent microscopy confirmed that the insect cells in the absence of chncs were intact and well spread on the con a - coated electrode surface even after washing the wells with saline three times . however , insect cells exposed to chncs (& gt ; 100 ppm ) for 24 h were spherical compared to the control cells . the chnc effect was more of an inhibitory effect at the cell - substratum binding level rather than a cytotoxic effect as dead cells were not observed . an ecis 50 value of 123 ± 16 ppm was calculated for chncs . thus , chncs are virtually non - cytotoxic . cell counting by the trypan blue exclusion assay performed at 0 , 4 . 5 , 8 , and 24 hours indicated that cell viability was still above 95 % when insect cells were exposed to 50 - 400 ppm chncs . the pattern observed correlated well with the ecis where cytotoxic effects were not observed . there was also negligible effect of the chnc sample ( 50 and 200 ppm ) on the cell growth ( doubling time from 2 . 96 x 10 6 to 4 . 88 x 10 6 cells / ml over about 24 hours as expected from the control ) over the experiment course as the cell densities were similar after 24 hours . however , at the highest concentration ( 400 ppm ) of chncs , a small effect on the cell growth was noted after 24 hours with a drop of 25 % ( 3 . 75 × 10 6 cells / ml ). thus , at concentrations below 200 ppm , chnc did not affect the viability sf9 insect cells , indicating its virtual non - cytotoxicity . the adsorption capacity of methylene blue ( mb , mw about 320 g / mol , sigma - aldrich ) on chncs is calculated as q = v ( c o - c t )/ m where v is the solution volume , c o is the initial mb concentration , c t is the mb concentration in the solution at a given time ( t ), and m is the chnc adsorbent mass ( he 2013 ). all containers used for mb solutions were of polypropylene to minimize the dye adsorption . for each initial dye concentration ( c 0 ), the amounts of mb adsorbed at a given time , q t , can be related to c t . non - linear regression analysis was then applied to estimate the equilibrium values for q e = v ( c o − c e )/ m , where c e is the equilibrium concentration of mb in solution a plot of q e vs . c e , then performed to validate the applicability of the langmuir isotherm equation , q e = q max · k l c e /( 1 + k l c 0 ) where q max is the langmuir constant related to maximum adsorption capacity and k l is the langmuir constant related to binding energy of the adsorption system . the q max value was then used for the estimation of the specific surface area ( ssa ) of chnc as ( q max / mw )× α mb × n avo where mw is the molecular weight of mb , a mb is the occupied surface area of one mb molecule ( about 1 . 3 nm 2 , assuming the mb molecule is lying flat on the adsorbent surface , 17 . 0 × 7 . 6 = about 130 å 2 ) and n avo is the avogadro number ( 6 . 023 × 10 23 mol − 1 ) ( he 2013 ). the plot of mb absorbance at 660 nm ( abs 660nm ) vs . mb concentration [ mb ] was linear up to 20 μm with a slope of 0 . 062 abs 660nm / μm [ mb ]. aqueous solutions ( 12 ml ) containing different concentrations of mb ( 100 - 1200 μm , diluted from a 4 mm stock solution in 20 mm phosphate buffer , ph 7 . 5 ), were added to samples ( 12 mg ) of chncs and rotated . small samples ( about 300 μl ) were taken every 30 s for the first 3 min and then at 4 , 5 , 10 , 20 , min . these samples were immediately centrifuged at 12 , 000 rpm and the supernatants ( after centrifugation ) were tested ( diluted 10 - 100 times depending on the concentration of mb ) for the residual concentration of mb left in solution , following any mb binding to the adsorbent , and compared to the starting concentration . the amount of mb bound ( mg ) was then calculated and the mb adsorption in mg / g of sample was determined . unless otherwise indicated , the binding experiments were performed at ambient temperature , 22 ± 1 ° c . and neutral ph . the adsorption capacity of chncs increased rapidly and reached equilibrium within 2 min ( fig1 a ) with the adsorption data well represented by the pseudo - first - order model or pseudo - second - order model ( table 4 ). as shown in fig1 b , the langmuir isotherm model ( langmuir 1918 ), q e = q max k l c e /( 1 + k l c e ), appeared to well represent the binding data for mb on chncs ( correlation coefficient of r 2 = 0 . 987 ) with a maximum mb loading capacity ( q max ) for chnc of 75 . 32 mg / g ( se : standard error = 6 . 59 ). estimated kinetic parameters of the two adsorption isotherms for mb are given in table 5 . accordingly , c e *= 1 / k l ( k l = 0 . 0023 μm − 1 ; se = 0 . 0005 ), whereby the loading is 50 % of the maximum capacity , was estimated to be 434 . 8 μm for chncs . the q max value for cnc prepared by the similar aps procedure was 101 . 2 mg / g ( he 2013 ). based on the q max estimated from the langmuir model for chncs ( table 3 ), a specific surface area ( ssa ) of 184 m 2 / g was estimated for chnc compared to the theoretical value of 410 m 2 / g . the latter was derived by considering chncs as a cylinder with a length of 149 nm , a diameter of 3 . 4 nm , and a density of 1 . 462 g / cm 3 . ssa for 1 g of chncs is estimated as n · s a where s a is the surface area of one chnc molecule or 2 π ( radius ) 2 + 2 π ( radius ) x ( length ). the number ( n ) of chnc molecules per gram is estimated as v t / v s where v s is the volume of one chnc molecule or 2 π ( radius ) 2 x ( length ) and v t is the volume for 1 g chnc = 1 / density . the production process described in example 1 was optimized to reduce the amount of ammonium persulfate ( aps ) spent and to maximize the production yield of chitin nanocrystal ( chnc ) per reaction . specifically , the amount of chitin added to the reaction was increased from 1 wt % to 15 wt % ( chitin : aps ratio from 1 : 22 . 8 to 1 : 1 . 5 ) and the resulting product was analyzed for yield , degree of oxidation ( do ), and the distribution of nanocrystals ( see table 6 ). reactions starting with 1 wt % to 5 wt % chitin resulted in similar yields and the chncs have similar degrees of oxidation ( do ). tem micrographs of these samples showed uniformly distributed nanocrystals with no significant differences in particle sizes between the samples . at 10 wt % chitin , nanocrystals were again observed with tem , but the do is significantly lower than the other samples . at 15 wt % chitin , tem showed a wide distribution of particle sizes , with the majority being in the micron range . therefore , 10 wt % chitin appears to be an optimal condition to produce chnc in high yield while minimizing the amount of aps used in the reaction . lam e , leung a c w , liu y , majid e , hrapovic s , male k b , luong j h t . 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( 2005 ) determination of the degree of deacetylation of chitin and chitosan by x - ray powder diffraction . carb . res . 340 , 1914 - 1917 . the novel features will become apparent to those of skill in the art upon examination of the description . it should be understood , however , that the scope of the claims should not be limited by the embodiments , but should be given the broadest interpretation consistent with the wording of the claims and the specification as a whole .