Dissolution of feather keratin in ionic liquids

Article abstract of DOI:10.1039/c2gc36556a

Keratin from various livestock industries is currently a waste material that has potential as a source of polyamide polymers that could replace fossil fuel derived materials if processing methods can be developed. In this work we have investigated methods for the dissolution and regeneration of keratin. Dissolution of keratin (from turkey feather) in ionic liquids was conducted under nitrogen at 130 °C for 10 hours. It was found that [BMIM]Cl, [AMIM]Cl and [choline][thioglycolate] could dissolve turkey feather keratin without addition of solvent or other chemicals. A significant percentage of solubility was obtained, up to 45% by weight. A water insoluble fraction was recovered by addition of water to the solution (～50%). The structure and properties of this regenerated, water insoluble fraction were investigated. Compared to the starting material, the regenerated keratin shows structural changes rather than chemical changes within the polypeptide chains. The remaining fraction, consisting of water soluble fragments, was characterised by gel electrophoresis.

Full text of DOI:10.1039/c2gc36556a

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Dissolution of keratin in ionic liquids
Azila Idris, R. Vijayaraghavan, Usman Ali Rana, Dale Fredericks, A. F. Patti and D. R. MacFarlane
Considerable amounts of keratin biopolymer can be dissolved in certain ionic liquids. In
current studies, [BMIM][Cl], [AMIM][Cl] and [choline][thioglycolate] were shown to have
good potential as solvents for keratin dissolution. Water soluble and water insoluble fractions
were obtained after regeneration of the keratin from the solution.

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ARTICLE TYPE
Dissolution of feather keratin in ionic liquids
Azila Idris,*a,b, R. Vijayaraghavan,a Usman Ali Ranaa, Dale Fredericksc, A. F. Pattia and D. R.
MacFarlanea
5
Received (in XXX, XXX) XthXXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Keratin from various livestock industries is currently a waste material that has potential as a source of
polyamide polymers that could replace fossil fuel derived materials if processing methods can be
10 developed. In this work we have investigated methods for the dissolution and regeneration of keratin.
Dissolution of keratin (from turkey feather) in ionic liquids was conducted under nitrogen at 130 °C for
10 hours. It was found that [BMIM]Cl, [AMIM]Cl and [choline][thioglycolate] could dissolve turkey
feather keratin without addition of solvent or other chemicals. A significant percentage of solubility was
obtained, up to 45 % by weight. A water insoluble fraction was recovered by addition of water to the
15 solution (~50 %). The structure and properties of this regenerated, water insoluble fraction were
investigated. Compared to the starting material, the regenerated keratin shows structural changes rather
than chemical changes within the polypeptide chains.The remaining fraction, consisting of water soluble
fragments, was characterised by gel electrophoresis.
Ionic liquids (ILs) are generally defined as salts which are
liquid below 100 °C and comprised entirely of cations and
1. Introduction
20
Keratin is a fibrous protein that can be found in feather, wool,
19
anions.18,
They offer a unique combination of properties as
human hair, finger nails, animal claws and horn.1,
These
2
50 solvents including in some cases low vapour pressure and high
thermal stability.20 The hydrophobicity/ hydrophilicity of ionic
liquids can be altered by manipulating the structures of the
cations and anions.21 In recent years, a number of ionic liquids
have been identified as solvents for the dissolution of
55 biopolymers such as cellulose, starch, wood, lignin, feather and
wool. These are generally insoluble in organic solvents but show
a reasonable solubility in certain ionic liquids.4, 22ꢀ28 Duck feather
keratin, for example, was found to be soluble in the ionic liquids
(1ꢀbutylꢀ3ꢀmethylimidazolium chloride [BMIM]Cl and 1ꢀallylꢀ3ꢀ
60 methylimidazolium chloride [AMIM]Cl).29 The same ionic
liquids have been found useful for wool dissolution by Xie et al.4
[BMIM]Cl was also found useful in the synthesis of wool
keratin/cellulose composite materials. The materials displayed a
homogeneous structure and did not show residual fiber structure.4
65 Therefore, the dissolution of biopolymers using ionic liquids
provide possibilities for the utilisation and conversion of
biopolymers into useful materials with great potential in
industrial applications.
biopolymers are plentifully available as a byꢀproduct from
poultry production and textile industries.3ꢀ5 However this kind of
protein is not easy to dissolve or extract because keratin is bound
25 by strong internal interactions that stabilise the protein structure.4,
6 In order to utilise these biopolymers and convert them to usable
materials there is a need for alternative solvents that are efficient
in dissolving keratin and preserving, at least in part, the protein
structure. The main objective of the present study therefore is to
30 investigate the use of ionic liquids (ILs) in the dissolution and
regeneration of keratin, in particular utilising the unique solvency
characteristics and high temperature properties of ILs.
Feathers are about 90 % keratin7 which contains about 7 % of
9
cysteine.8, Feather proteins are insoluble in normal organic
35 solvent due to the tight packing of the αꢀhelix and βꢀsheet in the
polypeptide chain. Additionally, a high degree of crosslinking of
the polypeptide chains is affected by cysteine residues which
participate in disulfide bridge formation.6, 8, 10 In order to extract
the keratin, cleaving the disulfide bonds can be achieved by
15
40 reduction,11ꢀ13 oxidation,12ꢀ14 sulfitolysis13,
or oxidative
In the current study we have used [BMIM]Cl, [AMIM]Cl, as
70 well as novel ionic liquids containing a thiol functional group
sulfitolysis.13, 16 Even though many reagents have a capability to
reduce the disulfide bonds, only a few, such as thiols17 and
ammonium bisulfite, have the required ability and reactivity to
preserve and maintain the protein structure. However, these
45 materials are difficult to recycle, often toxic and costly to
produce.
[choline][thioglycolate]
and
[bisꢀ(2ꢀ
ethylhexyl)ammonium][thioglycolate] as possible disulfide
bridge reducing, or exchange reagents, to dissolve and regenerate
the keratin in turkey feather. A combination of analytical
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techniques including ATRꢀFTIR, XRD, solid state NMR, TGA, 55 of neat and regenerated samples were acquired at a Larmor
DSC and gel electrophoresis has been used to investigate the
extent of keratin solubility in these ionic liquids and the
physiochemical properties of the regenerated keratin material. We
show that a significant amount of keratin (approximately 45ꢀ50
wt. %) can be dissolved in the above mentioned ionic liquids.
frequency of 300 MHz on a Bruker AVꢀ300 spectrometer. The
13C CP MAS NMR spectra of these samples were acquired using
a 4 mm rotor with Kelꢀf cap at 10 kHz spinning rate. The contact
time in the CP MAS experiments was 2.4 msec with a recycle
60 delay of 1 sec and cw decoupling. The number of scans was ~
90,000 to 100,000. In the case of 1D 1H static NMR experiments,
the pulse length was ~ 3.6 ꢁsec with a recycle delay of 5 sec and
16 to 20 scans.
5
2. Experimental
Materials preparation
The cleaned turkey feathers used in the experiments were
65 Thermogravimetric Analysis (TGA)
10 commercial materials were supplied by Shamrockcraft, purchased
from Spotlight, Clayton, Australia. Only barbs of the turkey
feathers were used in the experiments. 1ꢀbutylꢀ3ꢀ
methylimidazolium chloride ([BMIM]Cl, 98 % of purity) was
purchased from Sigma Aldrich. All other ionic liquids were
15 prepared inꢀhouse for this project as described further below. For
the preparation of these ionic liquids, allylchloride (98 %), Nꢀ
methylimidazole (99 %) , choline hydroxide (20 wt. % in H2O),
thioglycolic acid (98 %) and bisꢀ(2ꢀethylhexyl)amine (99 %)
were obtained from Sigma Aldrich. For NMR analysis,
20 deuterated chloroform (CDCl3) and deuterated methanol
(CD3OD) purchased from Merck was used. Unless otherwise
stated, all other organic solvents and reagents were used as
received from commercial suppliers.
The thermal stability of the original and regenerated materials
were investigated by TGA using a Pyris 1 in a flowing dry Argon
atmosphere between 25 to 700 °C at a heating scan rate of 10 °C
−1. The instrument was calibrated using four reference
min
70 materials, alumel, perkalloy, iron and nickel. The samples were
first dried under vacuum in an oven at a temperature of 70 °C.
These samples were then loaded in ceramic pans and equilibrated
for 15 minutes at the starting temperature of 25 °C before running
each experiment.
75
Differential Scanning Calorimetry (DSC)
Differential scanning calorimetry (DSC) was conducted on a
DSC Q100 series from TA Instruments with 5ꢀ10 mg of sample
in closed aluminium pans, at a ramp rate of 10 °C per minute. All
80 samples were cooled to ꢀ150 °C, held for 5 minutes and heated to
200 °C. Thermal scans below room temperature were calibrated
via the cyclohexane solidꢀsolid transition and melting point at ꢀ
87.0°C and 6.5 °C respectively. Thermal scans above room
temperature were calibrated using indium, tin, lead and zinc with
85 melting points at 156.6 °C, 231.9 °C and 419.5 °C respectively.
Transition temperatures are reported using the peak maximum of
the thermal transition.
25 Attenuated Total Reflectance-Fourier Transform Infrared
Spectroscopy (ATR-FTIR)
Fourier transform infrared spectra were obtained using a
Bruker IFS Equinox FTIR system coupled with a Golden Gate
single bounce diamond microꢀAttenuated total reflectance crystal
30 and
a liquid nitrogen cooled Mercury/Cadmium Telluride
detector. The FTIR was performed in the wavenumber range of
600 cmꢀ1 to 4000 cmꢀ1. The spectra were recorded with a
resolution of 4 cmꢀ1 with 50 scans. Spectra were baseline
corrected.
Electrospray Ionisation Mass Spectrometry (ESI-MS)
35
90
Electrospray ionisation mass spectra were recorded on a
Micromass Platform II API QMS Electrospray Mass
Spectrometer. Samples dissolved in methanol were subjected to a
suitable cone voltage, usually 25 V to 35 V. Measurements were
made in both the positive and negatives modes.
Powder X-ray diffraction (PXRD)
The powder Xꢀray diffraction (PXRD) patterns were obtained
at 22 ± 2 °C using Sietronics powder diffractometer. For each
XRD experiment, approximately, 1ꢀ2 g of the finely ground
40 sample was placed randomly on a locally designed flat brass
sample holder fitted with an oꢀring sealed covered Mylar sheet
providing an airtight atmosphere. CuKα1 radiation (λ = 1.540 Å)
was produced at 40 kV and 25 mA . The data were collected in
the BragꢀBrentano (θ/2θ) horizontal geometry using a 2θꢀrange
45 of 5 to 50.0° (2 θ) range with a step size of 0.02° 2θ and an
accompanying scan rate of 0.5 º / min.
95
SDS-PAGE Electrophoresis
Protein samples were diluted in 4 x NuPAGE® loading buffer
(Life Technologies) and electrophoresed using the Hoefer
miniVE vertical electrophoresis system (Amersham Biosciences)
100 in 4ꢀ12 % BisꢀTris NuPAGE® gradient gels (Life Technologies).
Proteins were stained and visualised by silver staining.
Nuclear Magnetic Resonance Spectroscopy (NMR)
Preparation of ionic liquids
1H NMR and 13C NMR spectra were recorded at 400 MHz on
50 a Bruker DPXꢀ400 spectrometer. All samples were measured as
solutions in deuterated chloroform and methanol. Chemical shifts
are reported in ppm on the δ scale, and the coupling constant is
given in Hz. Chemical shifts were calibrated on the solvent peak
1-allyl-3-methylimidazolium
chloride
[AMIM]Cl
was
prepared according to the literature30 as described in more
105 detail in the supplementary information. Structural
confirmation of the ionic liquids obtained was carried out by
spectroscopic methods including 1H NMR and mass spectroscopy
1
unless otherwise specified. Solid state 1D H static NMR spectra
2
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(electrospray ionisation). As contaminants have been shown to
Keratin Solubility
32
affect the physical properties of ILs,31,
determined by Karl Fischer titration.
water content was
The solubility experiments were conducted in glass vials,
50 which were fitted into a heating block under an inert atmosphere
of N2. To quantify the solubility, small incremental amounts of
feather material (5 wt. %) were gradually added to the ionic
liquid (1 g) and mechanically stirred until they were completely
dissolved in the ionic liquids. Initially it was observed that the
55 dissolution of turkey feather occurred rapidly. Then the
dissolution rate decreased as the viscosity of the turkey feather
solution increased. Complete dissolution was assumed to have
occurred when the feather could not be visually detected and the
ionic liquid solvent remained transparent in the glass vial. A laser
60 beam was used to more sensitively detect the presence of small
particles via light scattering. The dissolution were run at 130 °C
for 10 hours.
Apart from imidazolium salts, a series of thioglycolate ionic
liquids were also synthesised. The reaction is a simple acidꢀbase
neutralisation which involved transferring a proton from a
Bronsted acid to a Bronsted base without the use of any solvent.19
In this investigation, bisꢀ2ꢀethylhexylammonium hydroxide and
choline hydroxide were combined with one equivalent of
10 thioglycolic acid, respectively. These chemical reactions are
highly exothermic, therefore an ice bath was used to control the
temperature throughout the chemical reaction. Further details are
as follows.
5
15 [Choline][thioglycolate]
N+
HO
O
N+
Eq 1:1, 1 hour
O
+
HS
HO
OH-
OH
HS
stirred (RT), without solvent
O-
3. Results and discussion
[choline][thioglycolate]
thioglycolic acid
choline hydroxide
65 Dissolution of keratin in ionic liquids
Keratin was dissolved in two series of ionic liquids which
consisted of the imidazolium and thioglycolate systems. These
studies were undertaken in order to determine the main properties
involved in the dissolution process, for example, the effect of the
70 structure and composition of ionic liquids on their dissolving
capability. Initially, the dissolution of turkey feather was
attempted at room temperature and 65 °C. Partial dissolution was
observed at 65 °C. At 130 °C, complete dissolution was clearly
observed. This could be due to the keratin structures at some level
75 becoming unfolded at the elevated temperature,33 hence
permitting more interaction between keratin and the ionic liquids.
According to Xie et al.4, temperature has a strong effect on the
solubility and at 130 °C, better solubility of wool keratin was
noted. Therefore, based on this study, the temperature was
80 maintained at 130 °C in order to minimize any potential protein
degradation and prevent any decomposition of the ionic liquids.
The keratin that dissolved completely in ionic liquids resulted in a
viscous solution. There was no precipitation observed after the
dissolution process or after cooling to room temperature.
Scheme 1. Preparation of [choline][thioglycolate]
Choline hydroxide (15.17 ml, 0.026 moles) was initially placed in
20 a threeꢀnecked flask and equipped with reflux condenser and a
dropping funnel. An equimolar amount of thioglycolic acid (1.82
ml, 0.026 moles) was then added dropwise to the base while
stirring rapidly in an ice bath. A viscous liquid (Scheme 1) was
obtained after evaporation of the water byꢀproduct using a rotary
1
25 evaporator. (4.57 g, 91 %); H NMR (ppm, 400 MHz, DMSO)
1
δH: H NMR (ppm, 400 MHz, DMSO) δH: 4.49 (1H, broad s,
OH), 3.84 (2H, q, CH2), 3.44 (4H, m, 2 CH2), 3.12 (9H, s, 3
CH3), 1.92 (1H, s, SH); ESꢀMS: ES+m/z 104.1 [choline] +. ESꢀm/z
90.9 [thioglycolate] ꢀ. Water content (Karl Fischer): 18034 ppm
30
[Bis-(2-ethylhexyl)ammonium][thioglycolate]
85
Table S1 in the supplementary information and Figure 1 show
the limiting solubility in weight percentage of keratin in
[BMIM]Cl, [AMIM]Cl, [choline][ thioglycolate]. The limiting
solubility was found to be around 50 wt. % in all of these cases.
This was determined through the sequential addition of the
Scheme 2. Preparation of [bisꢀ(2ꢀethylhexyl)ammonium][thioglycolate]
35 Bisꢀ(2ꢀethylhexyl)amine (7.48 g, 30.9 mmoles) was loaded into a
threeꢀnecked flask and equipped with a reflux condenser under
vigorous stirring with a magnetic stirrer. An equimolar amount of
thioglycolic acid (2.72 g, 29.5 mmoles) was then added dropwise
to the flask. The flask was placed in an ice bath. Evaporation of
90 feather material until the solution became too viscous for rapid
dissolution at 130 °C. It is likely that the true solubility is even
higher than indicated here. It is interesting to note that the choride
ion seems to be as effective as the thioglycolate ion in dissolving
the keratin. In the chloride case, this high solubility is presumably
95 related to the ability of the ionic liquid chrolide ion to disrupt
hydrogen bonding in the keratin material, similar the
understanding that has emerged on the role of Clꢀ in cellulose
40 water gave [bisꢀ(2ꢀethylhexyl)ammonium][thioglycolate],
a
colourless liquid (Scheme 2). (9.8 g, 98 %); 1H NMR (ppm, 400
+
MHz, CDCl3) δH: 7.23 (2H, broad s, NH2), 3.57 (2H, s, CH2),
2.74 (4H, d, J = 6.4 Hz, 2CH2), 1.73 (2H, m, 2 CH), 1.27 (17H, m,
8CH2, SH), 0.804 (12H, m, 4CH3); ESꢀMS: ES+m/z 242.3 [bisꢀ(2ꢀ
26, 28, 34
and other biolpolymer dissolution in ionic liquids.22,
ꢀ
45 ethylhexyl)ammonium] +. ESꢀm/z 90.9 [thioglycolate] . Water
Gillespie et al.35 demonstrated that alkaline thioglycolate
100 solutions could be used to reduce the disulfide bonds in the
dissolution of wool, however there was no significant increase in
feather solubility observed in [choline][thioglycolate], compared
content (Karl Fischer): 8698 ppm
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to [BMIM]Cl and [AMIM]Cl. This suggests that disulfide
later times. Since little is known about the disulfide bridge under
cleavage is not a significant factor in the solubility limit of this
type of keratin.
these conditions, it is important to recognise that there are a
37, 38
number of cleavage reactions possible,12ꢀ15,
including
No dissolution was observed for turkey feather in [bisꢀ(2ꢀ
exchange, homolytic cleavage as well as further oxidative
5
ethylhexyl)ammonium][thioglycolate]. This is probably due to 40 processes if sufficient oxygen is present. In order to probe the
the longer chain of the cation which can therefore only penetrate
into the keratin network with difficulty. In addition, the viscosity
of [bisꢀ(2ꢀethylhexyl)ammonium][thioglycolate] is much higher
compared to [choline][ thioglycolate], thus decreasing the
reactions possibly taking place in the thioglycolate case, the
interconversion of model compounds cystine and cysteine were
studied in the thioglycolate ionic liquid. Neat cystine was added
to choline thioglycolate ionic liquids and heated to 130 °C for
10 solvating properties of the ionic liquid. This compound is also 45 about 5 hours. The resulting mixture was analysed by 13C NMR,
the only classical protic ionic liquid of the group studied and may
be prone to the proton transfer problems that have been widely
discussed in respect of protic ionic liquids. The ꢀpKa of this acidꢀ
base pair is ~7.2 which has been suggested as being sufficient for
showing 2 new peaks in the region of 170 ppm. Cysteine
dissolved in ionic liquid showed a single peak at 170 ppm. These
observations seem to indicate partial cleavage or exchange of the
cystine SꢀS bridge to form cysteine as well as the exchange
15 strong proton transfer in regard to primary amines, but 50 product, as is also suggested by the NMR data of the regenerated
insufficient for many tertiary amine based ILs.36
keratin discussed further below.
20
Figure 1.Solubility of turkey feather keratin in ionic liquids
55
Figure 3. Rate of dissolution of feather keratin in ionic liquids
25
Figure 2. Dissolved feather keratin in [AMIM]Cl
Figure 2 shows the keratinꢀ[AMIM]Cl solution after returning
to room temperature. At this point the solution was a very
viscous, clear liquid.
Regenerated keratin from ionic liquids
60
After the keratin was dissolved, it was precipitated from the
solution by addition of water at room temperature. The
regenerated keratin was separated by centrifugation for 20
minutes at 5000 rpm then dried under vacuum at 60 °C for 3
days. The regenerated keratin material was a fawnꢀcoloured hard
65 solid. Table S2 of the supplementary information and
Figure 4 show the masses obtained of the regenerated aqueousꢀ
insoluble fraction. It has been reported that feather keratin
30 Figure 3 shows the rate of dissolution of turkey feather in
[BMIM]Cl, [AMIM]Cl and [choline][thioglycolate]. Dissolution
in [choline][thioglycolate] was visually slightly more rapid in the
first ~10 minutes compared to the other two ionic liquids,
suggesting that initial cleavage of the disulfide bridges can
35 accelerate the process but that the difference becomes minimal at
4
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consists of approximately 40
% hydrophilic and 60 %
hydrophobic groups in the amino acid sequence.8 Therefore, it
was suggested that some of the dissolved keratin is insoluble in
water and the other part of the dissolved material might be
cleaved into smaller constituent polypeptide subunits which may
be water soluble. Gel electrophores is analysis was employed to
further investigate the soluble component in the water/ionic liquid
mixture produced during the regeneration step, as discussed
further below.
5
Figure 5. ATRꢀFTIR spectra of (a) raw material and regenerated keratin
40 material from (b) [BMIM]Cl, (c) [AMIM]Cl, (d) [choline][thioglycolate]
XRD Studies
Raw material and the regenerated keratin material were
examined by Xꢀray diffraction (XRD). In
Figure 6, the XRD patterns are compared. These figures show
45 crystallinity in the feather keratin fibers from the substantial 2θ
peak at about 9° (0.98 nm) which has been assigned to both αꢀ
helix and βꢀsheet structure.41, 42 The peak at about 17.8° (0.51
nm) correspond to the diffraction pattern of the αꢀhelix whereas
peak at about 19° (0.47 nm) is typical of βꢀsheet structure.41, 42
50 However, due to the overlapping signals at about 17.8° and 19°
from the αꢀhelix and βꢀsheet, both are unable to be
unambiguously assigned. It can be seen from
10
Figure 4. Recovery of water insoluble fraction of IL dissolved keratin
Figure 6, that the regenerated keratin exhibits similar diffraction
patterns, indicating regeneration of the crystallinity of the original
55 keratin. However , the peaks at about 9° and 19° are both
significantly stronger in the regenerated material, suggesting a
greater content of the βꢀsheet structure.These results are
consistent with the 13C CP MAS NMR spectra discussed further
below.
Characterisation of regenerated keratin
15 ATR studies
In order to further understand the nature of the water insoluble
keratin material regenerated from the ionic liquids, ATRꢀFTIR
measurements were used. Infrared absorption spectra of the raw
material and regenerated fraction (Figure 5) showed
20 characteristic absorption bands that are typically assigned to the
peptide bonds (ꢀCONH). These spectra provide information as to
the orientation of the constituents and the conformation of the
polypeptide chains. The Amide A vibrations ofthe peptide bond
show medium absorption bands at 3273 cmꢀ1 due to the NꢀH
25 stretching. A strong absorption band was also observed at 1627
cmꢀ1 and this was attributed to C=O (Amide I). A strong
absorption peak at 1515 cmꢀ1 was attributed to CꢀN stretching and
NꢀH bending vibrations (Amide II). A weak band was observed
at 1236 cmꢀ1 which was due to the CꢀN and CꢀO stretching, NꢀH
30 and O=CꢀN bending vibration (Amide III).39, 40 It can be seen that
the spectra are similar to the original starting material and there
are no signatures of new fuctional groups appearing in the
regenerated material. However in view of NMR results (below),
it appears that there are chemical changes occurring due to the
35 breaking of disulfide linkages and hydrogen bonds, which cannot
be observed clearly in the ATR spectra. Therefore dissolution in
these ionic liquids do not strongly affect the peptide bonds.
60
Figure 6. XRD of (a) raw material and regenerated keratin material from
(b) [BMIM]Cl, (c) [AMIM]Cl, (d) [choline][thioglycolate]
65
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5
and dynamics of the keratin molecules. Figure 7 displays the 13
C
CP MAS NMR spectra of keratin materials. All spectra in Figure
7 display a distinct asymmetric peak with maxima centred
Solid State NMR Studies
Solid state 13C NMR was conducted on the raw material and
the regenerated keratin material to investigate the local structure
between
172−174ppm.
10
Figure 7. The 13C CP MAS NMR spectra of (a) raw material, (b) regenerated keratin material from [BMIM]Cl, (c)regenerated keratin material from
[AMIM]Cl, (d) regenerated keratin material from [choline][thioglycolate]
material suggesting substantial retention of disulfide bridges in
15
This peak corresponds to the carbonyl carbons present in 35 these cases. In the thioglyoclate case a peak remains evident,
keratin. The peak at 130 ppm is ascribed to the aromatic species
present in the keratin.43 All NMR spectra in Figure 7 display this
aromatic peak, which suggests that the aromatic group containing
amino acid sequences are retained in the regenerated materials.
although less distinct. An exchange reaction in the thioglycolate
case could produce a new SꢀS bridge which would also produce
resonances near 40 ppm therefore it appears likely that some
sulfide exchange has taken place.
20 The peak at 54 ppm is related to the αꢀcarbons, while the one at
40 ppm is ascribed to the βꢀcarbons present in leucine and
cysteine residues.44 The carbon peaks at about 30 ppm to 40 ppm
are attributed to the carbon present in the proline, glutamic acid
and glutamine residues. The NMR peaks at low chemical shift
40
Since, the alkyl groups and the reduced cystine groups (ie
cysteine groups) display peaks in the same chemical shift range
around 25ꢀ29 ppm, it is hard to distinguish the reduced cysteine
uniquely in these spectra.
The two regenerated materials from [BMIM]Cl and [AMIM]Cl
25 centred at 20 ppm, 25 ppm and 31 ppm, in addition to the peak as 45 display NMR spectra that are not very different from the original
a shoulder at 17 ppm correspond to the alkyl groups of the side
chains.44 A peak at ~20 ppm is somewhat reduced in the
thioglcolate case – this region is associated with the δ−carbon of
leucine residues.44
keratin, while the [choline][thioglycolate] material shows clear
differences in the 50ꢀ65 ppm region. This is possibly due to
disruption due to the reductive cleavage of the disulfide linkages.
30
Reductive cleavage of disulfide bridges associated with
cysteine is expected to reduce the βꢀcarbon signal at 40 ppm and
produce a thiol signal around 25−29 ppm.44 The two chloride
cases show distinct peaks at 40 ppm that are similar to the raw
50
.
6
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Figure 8.The 13C CP MAS NMR spectra of (a) raw material, (b) regenerated keratin material from [BMIM]Cl, (c) regenerated keratin material from
[AMIM]Cl, (d) regenerated keratin material from [choline][thioglycolate] which were fitted with Gaussian/Lorentzian fitting functions. The error in the
fitting was ~3ꢀ5%
5
The secondary structure of keratin, which consists of αꢀhelix
Table 1. The percentage fraction of αꢀhelix and random coil + βꢀsheet of
and βꢀsheets produces a slightly different chemical shifts of the
raw material and regenerated keratin material, which were fitted using
15 carbonyl group in the NMR spectrum.43, 44, 46 Thus the estimated
percentage fraction of αꢀhelix and βꢀsheet can be estimated by the
dmfit* program 13C CP MAS NMR spectra
% Fraction
± 5
Chemical shift FWHM (Hz)
Peaks
deconvolution of the C=O peak. Figure
8 displays the
(ppm) ± 0.2
± 20
deconvoluted carbonyl NMR peaks of raw and regenerated
materials. The full width at half maximum (FWHM) of the peak
20 also provides information about the degree of motional freedom
possessed by the nuclei. The higher the value of full width at half
maximum (FWHM) shows that the nuclei posseses lower
motional freedom. Previous studies showed that such
deconvolutions usually result in two peaks, one at 175.6 ± 0.2
25 ppm, which is ascribed to the αꢀhelix, and the other at 172.7 ±0.2
ppm which is related to the βꢀsheet molecular conformations,
respectively.43, 44, 46 However, another study ascribed the NMR
peak at 172 ppm to the random coil structure as well.47 Hence, we
assume that the NMR peak ~ 172.0 ± 0.2 emerges from the nuclei
30 of both βꢀsheet and random coil structures. In the present study,
best fits for deconvoluting the carbonyl NMR peak for the raw
Raw material
33
67
α helix
random coil +β sheet
175.0
172.0
470
379
Regenerated keratin material from [BMIM]Cl
α helix
random coil + β sheet
33
67
175.6
172.7
617
437
Regenerated keratin material from [AMIM]Cl
α helix
random coil + β sheet
33
67
175.6
172.7
588
419
Regenerated keratin material from [choline][thioglycolate]
α helix
random coil + β sheet
11
89
175.6
172.7
559
494
10
*The dmfit program was used for the deconvolution of NMR into various
peaks and simulated fit.45
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material were achieved by using 175.0 ppm for αꢀhelix and 172.0
ppm for random coil + βꢀsheet. On the other hand, for all of the
regenerated materials, 175.6 ppm and 172.7 ppm respectively
gave the best fits. Table 1 shows that, in the case of the raw and
regenerated materials from [BMIM]Cl and [AMIM]Cl, the
percentage fractionof the random coil + βꢀsheet was about double
than that of the αꢀhelix and was unchanged in the regenerated
material. Conversely, the percentage fraction of random coil + βꢀ
sheet was much higher in the regenerated keratin from
5
10 [choline][thioglycolate].
A
plausible explanation of this
observation is that the dissolution of turkey feather in reducing
ionic liquids such as [choline][thioglycolate] results in much
stronger cleavage of the disulfide linkages and therefore promotes
the formation of random coil and βꢀsheet structures. The NMR
15 data in Table 1, which display lesser fraction of αꢀhelix
compared to the random coil + βꢀsheet for all materials agrees
well with their corresponding Xꢀray diffraction spectra that also
show a lesser fraction of αꢀhelix compared to random coil + βꢀ
sheet.
20
Thermal stability and phase behaviour
40
Figure 10. DSC heating scan (3rd cycle) for (a) raw material and
regenerated keratin material from (b) [BMIM]Cl, (c) [AMIM]Cl, (d)
[choline][thioglycolate]
45
The phase behaviour of the keratin materials was studied by
differential scanning calorimeter (DSC). In order to ensure that
no moisture was present in the samples, three consecutive heating
and cooling cycles were carried out; the DSC traces in Figure 10
are reported for the 3rd heating cycle. The DSC trace for the raw
50 material {Figure 10 (a)} shows an exothermic peak around ~ 230
°C, which is usually assigned to αꢀhelix disordering and
decomposition (in some cases described as a “melt”).1 In the case
of the regenerated keratin materials {Figure 10 (b), (c), (d)}, this
peak was broadened and shifted to comparatively lower
55 temperatures. These observation suggest the formation of some
greater degree of disordered/amorphous phase and loss of the αꢀ
helix in all regenerated keratin materials compared to the raw
keratin material. No obvious glass transition (Tg) was observed in
the raw keratin material {Figure 10 (a)}, however in all
60 regenerated materials, a prominent Tg was observed c.a. 170 °C ꢀ
180 °C. This tends to confirm the suggestion these materials
contain a higher degree of disordered material.
25
Figure 9. Single heating scan TGA traces of keratin samples
The thermal decomposition analyses of the raw material and
regenerated keratin material were conducted by TGA (
30 Figure 9). Thermal stability of raw material and regenerated
keratin showed that these materials were stable up to 200 °C with
the stability of regenerated keratin material being slightly higher
than the raw turkey feather. There are two stages of
decomposition that can be seen in all cases. A small weight loss
35 involved in the first stage could be due to the evaporation of
incorporated
water
near
100 °C. Between 250 °C and 400 °C, the second stage involved
with the degradation of the keratin materials.10
Characterisation of water soluble fraction
65
The development of methods to separate the water soluble
fraction from the ILꢀwater solution are underway and will be
reported in a future publication. However, it was possible to gain
some insight to the nature of the aqueous components as follows.
8
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Sodium dodecyl sulphateꢀpolyacrylamide gel electrophoresis
(SDSꢀPAGE) analysis with a standard marker was employed to
characterise and identify proteins in the water soluble fraction
segments and loss of some of the αꢀhelix structure. The
thioglycolate IL appeared to accelerate dissolution somewhat,
cause a greater degree of fragmentation in the water soluble
obtained during the regeneration of the keratin material. The 45 fraction and a greater degree of disruption of the αꢀhelix
5
protein bands were visualised by silver staining (Figure 11). The
presence of protein was confirmed and the majority of these
proteins were in the molecular weight fraction range between 10
to 40 kDa. This observation indicates that upon dissolution in
structure, however it is clear that such a reducing agent is not a
necessary component in dissolution. From the gel electrophoresis
results, it was shown that the water soluble fraction is of lower
molecular weight but still substantially polymeric. Further
these three ionic liquids, there are proteins isolated that are water 50 experimental studies of mechanical properties of a regenerated
10 soluble. As 90 % of the protein in feather samples is predicted to
be keratin,7 it is most likely that these water soluble polypeptides
are keratin related fragments. The most intense bands appeared at
10 kDa and 20 kDa in the [BMIM]Cl and [AMIM]Cl samples,
whereas in [choline][thioglycolate] only one band appeared, at
15 about ~10 kDa. The discreet bands differing by 10 kDa each are
suggestive of monomeric, dimeric and possibly trimeric forms of
the same protein sequence. This possibility is fuller supported by
the disappearance of the longer multimeric fragments in the
[choline][thioglycolate] that is thought to act as a reducing
20 agent{Figure 11(d)}. In addition, as the molecular weight of
feather keratin monomer is ~10 kDa,13, 37, 48 it is likely that these
bands are representative of keratin monomer and dimers.
keratin biomaterial are in progress to evaluate the possibility of
processing it into physical states such as fibers or films using
known techniques.
55 Acknowledgements
The financial support from the Australian Research Council and
DRM’s Federation Fellowship is gratefully acknowledged
Notes and references
60 aSchool of Chemistry, Faculty of Science, Monash University, Clayton,
VIC 3800, Australia.
kDa
bDepartment of Chemistry, Faculty of Science, University of Malaya,
50603 Kuala Lumpur, Malaysia.
cARC Special Research Centre for Green Chemistry, Monash University,
65 Clayton, VIC 3800, Australia.
E-mail: azila.idris@monash.edu.au, azila_idris@um.edu.my
50
40
30
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
70 DOI: 10.1039/b000000x/
‡ Footnotes should appear here. These might include comments relevant
to but not central to the matter under discussion, limited experimental and
spectral data, and crystallographic data.
20
15
10
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