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FTIR analysis of natural and synthetic collagen

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DOI: 10.1080/05704928.2018.1426595

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FTIR analysis of natural and synthetic collagen

Tehseen Riaz, Rabia Zeeshan, Faiza Zarif, Kanwal Ilyas, Nawshad

Muhammad, Sher Zaman Safi, Abdur Rahim, Syed A. A. Rizvi & Ihtesham Ur
Rehman

To cite this article: Tehseen Riaz, Rabia Zeeshan, Faiza Zarif, Kanwal Ilyas, Nawshad
Muhammad, Sher Zaman Safi, Abdur Rahim, Syed A. A. Rizvi & Ihtesham Ur Rehman (2018) FTIR
analysis of natural and synthetic collagen, Applied Spectroscopy Reviews, 53:9, 703-746, DOI:
10.1080/05704928.2018.1426595

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APPLIED SPECTROSCOPY REVIEWS
2018, VOL. 53, NO. 9, 703–746
https://doi.org/10.1080/05704928.2018.1426595

FTIR analysis of natural and synthetic collagen

Tehseen Riaza, Rabia Zeeshana, Faiza Zarifa, Kanwal Ilyasa, Nawshad Muhammada,
Sher Zaman Safia, Abdur Rahima, Syed A. A. Rizvib, and Ihtesham Ur Rehmanc
a
Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS Institute of Information
Technology, Lahore, Pakistan; bDepartment of Pharmaceutical Sciences, College of Pharmacy, Nova
Southeastern University, Fort Lauderdale, Florida, USA; cDepartment of Materials Science & Engineering, Kroto
Research Institute, University of Sheffield, Sheffield, UK

ABSTRACT KEYWORDS
Collagen is the most abundant protein in humans and animals, FTIR; collagen; molecular
comprising of one third of the total proteins that accounts for three structure; characterization;
quarters of the dry weight skin in humans. Collagen containing a range biomedical materials
of proteins has been reported for tissue engineering applications, but,
only a small number of studies related to chemical structure evaluation
of collagen are found in the literature. Collagen can be obtained from
both the natural and synthetic sources and offers a wide range of
biomedical applications due to its excellent biocompatibility and low
immunogenicity. Hence, it is important to identify chemical structural
properties of collagen and Fourier transform infrared (FTIR) appears to
be a technique of choice to study their chemical structure. This review
aims to highlight the use of FTIR to study collagen-based biomaterials,
using it for characterization of collagen extracted from various sources.
Characterization of collagen-based materials used in wound healing,
skin substitutes, derma fillers, and aging of skin, collagen containing
drug delivery agents, collagen-based materials used in tissue
engineering, bone regeneration, and osteogenic differentiation is
discussed in detail. FTIR analysis of collagen-containing materials used
for dental applications, cleft-palate, and in alveolar-ridge preservation
has also been highlighted.

Introduction
Collagen is one of the extracellular matrix (ECM) fibrous proteins present in multicellular
animals and most abundantly in mammals and is the major component of their bone
and skin (1). The collagen molecule consists of three polypeptide chains organized in a triple
helical conformation (2). A regulatory role of collagen in tissue development has made it the
most studied biomolecule. To date, 29 distinct types of collagen have been found and their
structural characterization showed typical triple helical structure of all types. Being the most
abundant protein on the earth, collagen can be extracted from various sources, including

CONTACT Ihtesham Ur Rehman i.u.rehman@sheffield.ac.uk Department of Materials Science &

Engineering, Kroto Research Institute, University of Sheffield, Broad Lane, Sheffield, UK;
Nawshad Muhammad [email protected] Interdisciplinary Research Centre in Biomedical
Materials (IRCBM), COMSATS Institute of Information Technology, Lahore, Pakistan.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/laps.
© 2018 Taylor & Francis Group, LLC
704 T. RIAZ ET AL.

living animals such as alligators (3) and kangaroos (4). For the tissue engineering applica-
tions, common sources of collagen involved porcine skin, bovine tendons, and skin. Another
considerable source of collagen are marine life forms, which include fish skins (5, 6), jellyfish
(7), and sea sponges (8, 9). Squid species is largely explored for food purposes (10). The pro-
duction of squid skin collagen represents a way for valorization of by-products. Recombi-
nant human collagen has been found potentially less immunogenic than the other sources
and expected to have future collagen scaffolds application (11).
Collagen (either extracted or from acellular matrix) has a wide range of biomedical applica-
tions due to its superior biocompatibility and low immunogenicity. Collagen-based biomate-
rials and scaffolds have been used to study the cells proliferation, differentiation as well as
phenotype expression. Collagen-based biomaterials are reported (12, 13) for bone and carti-
lage tissue regeneration and promising nerve guide (14, 15). Recent findings show that colla-
gen-based biomaterials are leading materials for cardiovascular regenerative medicine (16,
17). Moreover collagen-based wound dressings have been used for burn coverage applications
(18, 19). Collagen-based scaffolds have also shown applications in delivering drugs, cells, pro-
teins, and nucleic acids on long-term basis (20–22). Collagen gel is reported as a potential
material for future clinical studies on gene therapy delivered by collagen matrix (23).
In the characterization of collagen-based biomaterials, several in situ and non-invasive
analysis techniques have been exploited. These techniques help to get detailed insights into
the physicochemical properties of the materials. These techniques include Infrared (IR)
spectroscopy (24), Raman spectroscopy (25), dielectric spectroscopy (26), and Differential
scanning calorimetry (27).
Among all these techniques, vibrational spectroscopic techniques, both Raman and IR, have
significant potential in the field of biomedical analysis due to quick, nondestructive, affordable,
and reliable results. Moreover, only few micrograms of the material are required with minimum
sample preparation. These techniques also provide molecular-level information, functional
groups investigations and bonding types conformations. Spectral bands in vibrational spectra
are molecule specific and provide direct information about the biochemical composition.

1. FTIR analysis of collagen extracted from various sources

1.1. FTIR of collagen extracted from skin
Tamilmozhi et al. (28) isolated acid-solubilized collagen (ASC) and pepsin-solubilized colla-
gen (PSC) from the skin of sail fish (Istiophorus platypterus) and characterized extensively
by Fourier transform infrared (FTIR). The spectra of both ASC and PSC showed amide A
band (associated with N–H stretching) positions at 3423 and 3337 cm¡1, respectively. The
amide B band of ASC and PSC was observed at 2928 and 2924 cm¡1, respectively. Polypep-
tide backbone C–O stretching vibration was found in the range of 1600–1700 cm¡1. The for-
mation of hydrogen bond between N–H stretch and C–O (Gly) of the fourth residue was
confirmed by the absorption peaks at 1654 and 1646 cm¡1 for ASC and PSC, respectively.
N–H in-plane bend and the C–N stretching vibrations were noted at 1549 cm¡1 for PSC
and 1560cm¡1 for ASC. Amide II and amide III were found at the same wavenumber
1240 cm¡1, further confirming the hydrogen bonding for the both ASC and PSC. Therefore,
the IR spectra of both ASC and PSC from the skin of sail fish differed slightly and suggested
that pepsin hydrolysis mostly did not affect the triple helical structure of collagen.
 APPLIED SPECTROSCOPY REVIEWS 705

FTIR study of collagen extracted from outer skin of squid (Doryteuthis singhalensis) was
reported by Veeruraj et al. (29). It was found that the spectra of both ASC and PSC were like
those of collagens from other fish species reported in the literature. Amide A bands were
found at wavenumber of 3307 and 3428 cm¡1 for ASC and PSC, respectively, which repre-
sents the hydrogen bonding of N–H group with a carbonyl group of the peptide chain.
Asymmetrical stretching of CH2 was shown by amide B band positions of ASC and PSC at
wavenumbers of 2928 and 2927 cm¡1, respectively. The amide I band in the frequency range
from 1600 to 1700 cm¡1 was observed showing the peptide secondary structure and hydro-
gen bonding between N–H stretch (X position) and CDO (Gly). The amide II band of ASC
and PSC at 1541 and 1544 cm¡1, respectively and amide III band at 1236 and 1239 cm¡1,
respectively represent N–H bending vibrations coupled with C–N stretching vibration and
C–H stretching. Furthermore, FTIR confirmed the triple helix, high extent of intermolecular
structure, and similar secondary structure of the proteins of both ASC and PSC.
Sionkowska et al. (30) reported the isolation of collagen from fish, Brama australis. The
IR spectra taken from thin films of collagen showed amide A, amide B, amide I, amide II,
and amide III, the typical bands for collagen type I. The N–H stretching vibration of amide
A peak from B. australis was observed at 3318 cm¡1 and amide B peak was found at
3079 cm¡1. The changes of protein secondary structure of B. australis collagen was con-
firmed by amide I bond appeared at 1655 cm¡1 (stretching vibration of CDO in polypeptide
backbone of protein). The amide II band was found at 1548 cm¡1.
Zhang et al. (31) studied the physical and chemical properties of collagen (acid-soluble
and pepsin-soluble) extracted from the skins of Nile tilapia (Oreochromis niloticus) and
channel catfish (Ictalurus punctatus). FTIR spectra showed the intact triple helical structure
of collagens with the appearance of typical amide II band of collagen at 1538–1548 cm¡1.
Amide A bands appeared in the range 3292–3315 cm¡1 proved that N–H group of peptides
is involved in hydrogen bonding. Amide I band was observed at 1656 cm¡1 while amide III
band was found in range 1232–1238 cm¡1. Overall FTIR represents the characteristic amino
acids and intact collagen molecule.
Chen et al. (32) analyzed the FTIR spectra of ASC, which was extracted from scales and
skin of tilapia. These FTIR spectra matched well with those of collagen extracted from the
other fish species reported in literature. The main peaks included amide A and B, as well as
amide I, II, and III. The amide A bands of tilapia scales collagen and skin collagen were
observed at 3318 and 3321 cm¡1, respectively, associated with N–H stretching vibrations
and confirmed the hydrogen bonding. The amide B (asymmetrical stretch of CH2) bands
were found at 2925 and 2924 cm¡1 for scales and skin collagen, respectively. The amide I, II,
and III bands of skin collagen and scale collagen showed that collagen from scales had stron-
ger hydrogen bonds. A strong CDH stretching was found to be occurred at 1453 cm¡1 for
both collagens. Moreover, a triple helical structure was indicated by the absorption ratios of
0.93 and 1.1 for skin and scales collagen, respectively. Overall FTIR showed that both
extracted collagens were of type I collagen and maintained their triple helical structure.
In a study by Veeruraj et al. (33), ASC and PSC was extracted from the skin marine eel
fish (Evenchelys macrura). The FTIR spectra suggested that pepsin hydrolysis did not affect
the triple helical structure of collagen. In the spectra, N–H stretching bands at 3421 and
3395 cm¡1 confirmed the existence of hydrogen bond respectively for ASC and PSC. The
asymmetrical stretch of CH2 (Amide B band) of ASC and PSC was observed at 3079 and
3079 cm¡1, respectively. Spectral peaks in the region of 1649 and 1653 cm¡1 confirmed the
706 T. RIAZ ET AL.

formation of peptide secondary structure (amide I band). An amide III band of both colla-
gens at 1454 cm¡1 revealed that both collagens did not denature during extraction and
exhibited triple helical structure. The amide II band (N–H bending vibration coupled with
C–N stretching vibration) in the range 1541–1542 cm¡1 confirmed the secondary structure
of both ASC and PSC collagens.
Safandowska et al. (34) reported the extraction of collagen from fish skin of silver carp
(Hypophthalmichthys molitrix) that could have medical applications. In FTIR investigation,
the absorption peaks found in extracted collagen are assigned to symmetric stretching of car-
boxylate salts (1403 cm¡1, amide I (1629 cm¡1), amide II (1548 cm¡1), amide III
(1451 cm¡1), and ester bond (1081 cm¡1)).These are close to the collagens from other fish
sources (35). The collected collagens were treated with dehydration treatment and cross-
linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydrox-
ysulfosuccinimide. After the treatment, the amide I, II, and III were slightly shifted to higher
wavenumbers indicating the formation of amide bonds between carboxyl groups and avail-
able amino groups.
Liu et al. (36) conducted study on extraction and characterization of an advanced collagen
aggregate (Ag-col) from porcine acellular dermal matrix (pADM). The FTIR spectra of Ag-
col revealed the typical characteristic peaks of amide A, B, I, II, and III, which are associated
with the special triple helical conformation of intact fibrillar collagen (37).
Zhang et al. (38) extracted collagen from skin of Rana chensinensis skin using an acid
enzymatic extraction method. In FTIR of the pure extracted collagen, a strong peak observed
at 3334 cm¡1 attributed to the stretching vibration of the N–H bond of amide A. Amide B
(C–N stretching vibration) band appeared at 3087 cm¡1. Amide I, amide II, and amide III
vibrations of the collagen appeared at 1658, 1537, and 1261 cm¡1, respectively.
An FTIR study on both ASC and PSC extracted from the skin and bone of Spanish mack-
erel (Scomberomorous niphonius) (39) confirmed that these collagens were mainly composed
of type I collagen possessing slight molecular structure differences. FTIR also showed that
PSCs had lower content of high-molecular weight crosslinks as compared to ASCs.
Lee et al. (40) compared the content and concentrations of collagen in skin samples from
sea-rainbow trout (S-RT) and freshwater-rainbow trout (F-RT). The amide A band of ASC
from S-RT skin appeared at 3328 cm¡1 and from F-RT skin appeared at 3332 cm¡1 confirm-
ing the involvement of hydrogen bonding. The amide B bands for ASCs from S-RT and F-
RT skins were observed at 2889 and 2898 cm¡1, respectively. The secondary structure of a
protein in two collagens was confirmed by amide I band, and appeared at 1674 and
1696 cm¡1, respectively for S-RT and F-RT skins. The amide II band of ASCs from S-RT
and F-RT skins appeared at 1546 and 1554 cm¡1, respectively. The triple helical structure
from amide band III was confirmed. The amide III band of collagens from ASCs in S-RT
and F-RT skins appeared at 1210 and 1214 cm¡1. FTIR spectra also showed that collagen
from S-RT and F-RT skins had similar secondary structures. FTIR also suggested that differ-
ent rainbow trout habitats (seawater and freshwater) do not affect the amino acid composi-
tion and molecular weight properties of ASCs from S-RT and F-RT skins.
Extraction of collagens from the body wall of tropical sea cucumber (CSC) and PSC of Sti-
chopus monotuberculatus (PSC-Sm) is described in a study (41). The FTIR spectra of both
collagens (PSC-Sm and CSC) included the main peaks of amide A, amide B, amide I, amide
II, and amide III with the slight difference in frequency positions. It was observed that amide
II band in CSC appeared at higher wavenumber than PSC-Sm amide band. This showed that
 APPLIED SPECTROSCOPY REVIEWS 707

PSC-Sm had more hydrogen bonds between adjacent to a chains. PSC-Sm showed good
activity of collagen and triple helical structure.
Ramanathan et al. (42) carried out extraction of ASC from the skin of a fish, Arothron
stellatus for tissue engineering purposes. FTIR spectra showed the characteristic peaks of
amide A, amide I, II, and III at 3328, 1658, 1556, and 1243 cm¡1, respectively. The triple
helical structure of the collagen was confirmed by absorption intensity (of approximately
equal to 1) between amide III and II.
Rizk et al. (43) reported on collagen extraction from buffalo skin for useful medical appli-
cations. The main FTIR peaks included amide A (3299 cm¡1), amide B (2919 cm¡1), amide
I (1628 cm¡1), amide II (1540 cm¡1), and amide III (1234 cm¡1), as shown in Figure 1. The
peaks were matched well with those of other animal’s collagen (44). Overall, the FTIR con-
firmed the existence of helical structure of buffalo skin collagen.
In another study (45), ASC from the fish skin of Salmo salar, African catfish, Clarias gar-
iepinus, and Baltic cod, Gadus morhua was extracted. An asymmetrical stretch of alkyl
groups was found at 2920, 2924, and 2925 cm¡1 for Salmon, African catfish, and Baltic cod,
respectively. The amide bands for African catfish, Salmon, and Baltic cod collagens spectra
were found at 1633, 1637, and 1649 cm¡1, respectively representing the different secondary
structures of these collagens. The amide II band position appeared in the range 1539–
1543 cm¡1 for three collagens. The IR spectra of all collagens indicate that their overall
chemical compositions are similar; however, a slight difference was observed in secondary
structure.
Krishnamoorthi et al. (46) reported the type I collagen extracted from outer skin of Sepia
pharaonis. The ASC and PSC were characterized by FTIR. The FTIR spectrum of both ASC
and PSC (Figure 2) reported the major peaks comparable with that of the standard collagen.
Elango et al. (47) carried out study on extracted shark catfish (Pangasius pangasius) skin
collagen (Type I). Shark catfish skin collagen films were prepared with four crosslinking
agents: Hexamethylene diisocyanate (HMDC), sorbitol, glycerol, glutaraldehyde (GTA), k-
carageenan (kCGN), and transglutaminase (TG). FTIR study on these films showed that col-
lagen films made with kCGN, Sorbitol, TG, and HMDC exhibited similar vibrations. The
films prepared with the biological crosslinking agents (kCGN &TG) had similar peaks for
amide A and amide B.

Figure 1. FTIR spectrum of acid-soluble buffalo skin collagen (43) (reproduced with permission of
publisher).
708 T. RIAZ ET AL.

Figure 2. FTIR spectrum of standard collagen (A), ASC (B), and PSC (46) (reproduced with permission of
publisher).

In another study by Wu et al. (44), PSC was extracted from the skin of black carp (Mylo-
pharyngdon piceus). The main absorption peaks were 3300 (amide A), 2924 (amide B), 1633
(amide I), 1548 cm (amide II), and 1238 cm¡1 (amide III), consistent with the typical colla-
gen and collagen from the skin of Rachycentron canadum (48) confirming the triple helical
structure and formation of hydrogen bonding.
ASCs were extracted from skins and scales of silver carp, grass carp, black carp, and bighead
carp by Liu et al. (49). FTIR study characterized them as type I collagen and confirmed triple heli-
cal structures. The spectra in the amide I region of ASC from the four carp species were character-
ized. The highest absorption was observed at 1656 cm¡1, showing a high content of b-turn
structures in all ASC. However, a-helix (1647 cm¡1) was not observed for all ASC.
In another study by Chi et al. (50), ASC from the skin of hammer head shark (Sphyrna
lewini) (ASC-H) was characterized and compared with those of calf skin collagen (CSC).
The configuration of type I collagen (with triple helical structure) was confirmed by amide I,
amide II, and amide III bands and the absorption ratio of 1.0 between amide III and
1452 cm¡1 bands.
 APPLIED SPECTROSCOPY REVIEWS 709

Wang et al. (51) studied the collagen isolated from Amur sturgeon. Collagen was
extracted using acetic acid (ASC), sodium chloride (SSC), and pepsin. In FTIR of collagens,
triple helical structure of three collagens was confirmed from the absorption ratio of amide
III band (1240 cm¡1) and 1452 cm¡1 band. The appearance of amide A band for PSC at
lower frequency as compared to ASC and SSC indicated that more N–H group of PSC
involved in hydrogen bonding. The amide I and amide II bands indicated the higher degree
of molecular order in ASC.
Nagai et al. (52) extracted collagen from emu (Dromaius novaehollandiae) skins having
the potential applications in pharmaceutical and biomedical fields. These FTIR spectra indi-
cate the existence of helical structure of the collagen extracted from the emu skins and spec-
tral patterns were found to be same as other species collagens. The percentage of secondary
structural components (a-helix (9 %), b-sheet (35 %), b-turn (18 %), and others (random
coil structure (20 %))) of emu skin collagen were calculated as major findings of this study.
An ASC has been extracted from grass carp skin by Liu et al. (53). The FTIR spectra of
ASC (extracted using eight combined conditions) showed an absorption ratio in the range
1.03–1.05 between the amide III and the 1454 cm¡1 band which confirms that the intact tri-
ple helical structure of collagen is well maintained.
Kittiphattanabawon et al. (54) isolated PSC from the splendid squid (SC) skin and type I
collagen from calf skin (CC). SC and CC collagens exhibited FTIR spectra with a slight dif-
ference due to variations in amino acid compositions and sequence. SC and CC showed
amide A bands at 3290 and 3296 cm¡1. Amide B positions were found at 2921 and
2933 cm¡1 for SC and CC, respectively. Amplitudes of amide A and amide B bands in SC
and CC showed different secondary structures. C–OH stretching vibrations of the carbohy-
drate moieties for SC and CC exhibited absorptions at 1031, 1060, and 1081 cm¡1. FTIR
results suggested that extracted collagens might contain carbohydrates attached to hydroxy-
lysine residues of the polypeptide chain by O-glycosidic bonds.
Rui et al. (55) carried out a study on collagen extracted from Antarctic and Sub-Antarctic
squid for tissue engineering applications. FTIR spectra of ASC and PSC were taken, isolated
from the skin of I. argentines, skin of Kondakovia longimana, and from the muscle of K.
longimana. Collagen was identified by FTIR characteristic spectral bands: amide I
(1659 cm¡1), amide II (1555 cm¡1), and amide III (1240 cm¡1). A strong signal at
3281 cm¡1 was observed corresponding to the stretching of O–H bonds. ASC obtained from
both the muscle and skin of K. longimana exhibited characteristic bands similar to type I col-
lagen, while PSC showed poorly defined bands with impurities. Skin of I. argentinus collagen
showed the well-defined and expected type I collagen with high degree of purity.

1.2. FTIR of collagen extracted from fish scales

Kozlowska et al. (56) reported on extraction and characterization of ASC and PSC from fish
scales of northern pike. In both of the samples, collagen bands appear at amide A
(3312 cm¡1), amide B (3076 cm¡1), amide I (1653 cm¡1), and amide II (1548 cm¡1). The
amide III band observed in the range (1235–1237 cm¡1) in both collagens was similar to the
other collagen species (57).
Chen et al. (58) reported on pepsin-soluble type I collagen extracted from red drum fish
scales. The FTIR spectrum of PSC represented the absorption bands reported for the colla-
gen, amides A, I, II, and III at 3338, 1658, 1548, and 1240 cm¡1, respectively. The amide A
710 T. RIAZ ET AL.

band of PSC observed at 3328 cm¡1, showed that N–H groups in PSC were involved in
hydrogen bonds, by which collagen triple helical structure is held together. The IR ratio of
»1.0 between 1240 (amide III) and 1454 cm¡1 bands confirmed the triple helical structure
of PSC. The amide I band at 1658 cm¡1, associated to the stretching vibrations of carbonyl
groups (CDO bond) confirmed the peptide secondary structure. The assignment for the fish
scale collagen band for the region between 1200 and 1350 cm¡1 were CH2 deformation at
1338 cm¡1, C–N stretching and N–H deformation at 1240 cm¡1.
Barzideh et al. (59) reported the FTIR of PSC from the ribbon jellyfish (Chrysaora sp.,
morphotype 1) umbrella (JPSC). Main FTIR spectral peaks were amide A (3314 cm¡1),
amide B (2924 cm¡1), amide I (1653 cm¡1), amide II (1551 cm¡1), and amide III
(1239 cm¡1) consistent with the collagen FTIR peaks in literature. The absorption intensity
ratio between amide III (1239 cm¡1) and 1454 cm¡1 was found to be 0.85 for JPSC, which
indicated that the triple helical structure of collagen has not been severely disrupted.
Mori et al. (60) carried out a study on collagen extracted from the scales of pacific saury
fish (Cololabis saira). The FTIR spectra are similar to the reported for collagens. Spectral
peaks of amide A (N–H stretching) were found within the region of 3315–3330 cm¡1 and
amide II (N–H bending, C–N stretching) peaks were found at 1550–1554 cm¡1. The bands
in region 1200–1400 cm¡1 showed the amide III (N–H in-plane bending). C–N stretching,
CH2 vibration of the glycine backbone and that of the proline side chain) at 1238–
1240 cm¡1, have also been described previously in the literature. Moreover, a spectral band
in the region of 2958–2980 cm¡1 representing the CH2-asymmetric vibration mode and that
of the CH3-asymmetric vibration mode has also been observed. Extracted collagen exhibited
a shift of amide I peaks from 1660–1658 cm¡1 to 1652–1656 cm¡1 after heat denaturation.
The content of triple helical structure was found to decrease after denaturation. The
1235 cm¡1/1454 cm¡1 ratio, amide III/amide I ratio, and the amide III/1454 cm¡1 ratio
decreases after denaturation. Overall, the FTIR data indicate that all three collagen exhibited
unfolding of the triple helices due to heat denaturation.
Zhang et al. (61) reported on extraction of ASC and PSC from the jellyfish (Cyanea noza-
kii Kishinouye). Both collagens showed the identical FTIR results. The amide A band (N–H
stretching) positions of ASC and PSC were found at 3429 and 3431 cm¡1 respectively, con-
firming the hydrogen bonding. The amide B band (the asymmetrical stretch of CH2) of the
ASCs and PSCs was observed at 2933 and at 2934.50 cm¡1, respectively. The amide I band
(secondary structure of protein) of the Cyanea nozakii collagens was observed at around
1639 cm¡1. The amide II bands of ASCs and PSCs were detected at 1578 cm¡1. The absorp-
tion between the 1236 and 1452 cm¡1 suggests the helical arrangement of the two collagen
samples. Overall, FTIR investigations showed that the spectra obtained for collagens from
jellyfish were different from spectra of collagens from other fish species because of peak
shifting to lower or higher frequencies.
Huang et al. (62) used a novel extrusion–hydro-extraction process to extract collagen
from tilapia (Oreochromis sp.). FTIR spectra of this collagen possessed five major adsorption
bands in the amide band region, including amide A (3304–3315 cm¡1), amide B (2922–
2940 cm¡1), amide I (1644–1653 cm¡1), amide II (1541–1548 cm¡1), and amide III (1237–
1239 cm¡1).
In another study (63), ASC and PSC from seabass (Lates calcarifer) scale were character-
ized by FTIR. The amide A bands of ASC and PSC from seabass scales were found at 3285
and 3311 cm¡1, respectively, which showed the increased free amino groups by pepsin as a
 APPLIED SPECTROSCOPY REVIEWS 711

result of hydrolysis of telopeptide region. The amide B bands of both collagens were found at
3075 and 3086 cm¡1. Amide I band occurs at 1657 and 1650 cm¡1 for ASC and PSC from
seabass scales. Both collagens exhibited the amide II band at 1553 and 1548 cm¡1. Bands of
both amide I and amide II of PSC shifted to lower wavenumber, compared with those of
ASC, suggesting higher proportion of hydrogen bond in PSC. Amide III bands were found
at wavenumber of 1456 and 1459 cm¡1 for ASC and PSC, respectively. Overall results of
FTIR suggested that hydrogen bonds were involved in both ASC and PSC. Moreover, triple
helical structure of both collagens has been confirmed by the intensity ratios between the
peak heights of amide III and of 1450 cm¡1.

1.3. FTIR of collagen extracted from other sources

Santos et al. (64) reported on type I collagen extraction and purification from bovine pericar-
dium, which has potential bioengineering applications. In the FTIR spectrum of extracted
collagen, main functional groups of collagen were observed. The observed spectral peaks
were amide I (1680–1620 cm¡1), amide II (1580–1480 cm¡1), and amide III (1300–
1200 cm¡1). The strong hydroxyl band in the region of 3200–3600 cm¡1 was found to be
overlapped with the amide A band (3360–3320 cm¡1). The peak at 1360–1340 cm¡1 was
observed representing wagging vibration of the proline side chains present in the type I col-
lagen found in biological tissues.
Jeevithan et al. (65) isolated the type II PSCs from skeletal and head bone of silvertip
shark, which can be suitable for biomedical applications. FTIR analysis exposed that the sec-
ondary structure of skeletal and head PSC are similar. The amide A peak of skeletal and
head PSC were found at 3331 and 3414 cm¡1, respectively. The amide B band appeared at
2957 and 2932 cm¡1 for head and skeletal collagen, respectively. The amide I peaks were
observed at 1655 and 1659cm¡1 for head and skeletal collagens, respectively. The amide II
bands were found at 1550and 1547 cm¡1 for skeletal and head PSC, respectively, which are
attributed to C–N stretching vibration (18–40%) and N–H in-plane bending (40–60%).
Muyonga et al. (66) also observed the amide II peak at 1540–1558 cm¡1 for Nile perch skin
collagen.
Kaewdang et al. (67) extracted the ASC and PSC from the swim bladders of yellowfin tuna
(Thunnus albacares). FTIR of ASC and PSC showed similar characteristic spectral peaks col-
lagen (amides A, B, I, II, III). The amide A spectral peaks were observed at 3275 and
3303 cm¡1 for ASC and PSC, respectively. Amide B bands were observed at 2919 and
2920 cm¡1, with a higher amplitude in PSC which could be related to higher wavenumber in
amide A. Amide I spectral peak of ASC and PSC was observed at 1643 and 1648 cm¡1,
respectively. The absorption ratio of 1 between amide III and 1454 cm¡1 band of both shows
collagens did not denature during the extraction. This ratio also confirmed the triple helical
structure of collagen. The amide II of both collagens appeared at 1544–1549 cm¡1, showing
that both ASC and PSC had similar secondary structure.
In another study by Tylingo et al. (68), collagen was extracted from jellyfish mesoglea.
FTIR spectra of collagen were taken with different degrees of crosslinking. The main charac-
teristic bands (amide A, I, II and III) were observed for collagen. The amide A band was
found at 3300 cm¡1, amide I band was observed at 1650 cm¡1, amide II band at 1530 cm¡1,
and amide III band was found at 1240 cm¡1. They reported that in the FTIR spectra of colla-
gen, amide III bands of crosslinked collagens were much weaker than that of untreated ones,
712 T. RIAZ ET AL.

which showed that crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

(EDC) influenced the structure of collagen. Moreover, the amide B band at 2930 cm¡1 was
very weak in crosslinked collagens. The intensity of band at 2362 cm¡1 was found to increase
with the increase in degree of crosslinking.
Kim et al. (69) reported on collagen extraction from duck’s feet for tissue engineering
applications. FTIR spectra showed that duck feet’s collagen (DFC) exhibited broader amide I
and II bands (1628, 1550 cm¡1, respectively) as compared to porcine collagen (PC). The dif-
ference in IR spectra between PC and DFC was attributed to structural differences between
porcine skin and duck’s feet and may also be affected by extraction process of collagen.
Liang et al. (70) reported on FTIR of collagens from the cartilage of Amursturgeon (Aci-
penser schrenckii). FTIR confirmed the triple helical structure of collagens from the absorp-
tion ratio of 1.0 between 1240 cm¡1 (amide III) and 1454 cm¡1 bands. The amide A bands
of ASC and PSC indicated more N–H group of ASC was involved in hydrogen bonding. The
amide I and amide II band of PSC and ASC indicated that PSC had more molecular order
and thus more intermolecular crosslinks in PSC.
Barros et al. (71) obtained the FTIR spectra of collagen extracted from marine sponges. The
FTIR spectra of collagen contained the following bands: amide A band at 3425 cm¡1 associ-
ated with the N–H stretching and confirmed hydrogen bonding. Amide B in the range 3277–
3294 cm¡1 represented asymmetrical stretch of CH3. Amide I, associated with stretching
vibrations of the carbonyl groups (CDO bond), was observed in the frequency range 1640–
1655 cm¡1 for different extracts. The amide II band appeared at 1522 cm¡1 for all extracts.
The presence of the amide III (C¡H stretching) in the range 1240–1250 cm¡1 suggested the
helical structure. A secondary structure was observed in collagen with CO2 acidic water.

2. FTIR analysis of collagen-based materials used in wound healing and skin

substitutes
Wound healing property of collagen-chitosan films has been investigated. ASC was isolated
from Sepia kobiensis mantle. Characteristic frequencies at 431, 2924, 2854, 1645 and 1238
cm¡1 corresponded to amide regional bands of A, B, I, II, and III, respectively. Frequencies
between 1600 and 1700 cm¡1 correspond to the polypeptide’s carbonyl groups (CDO bond)
stretching vibrations and constitute as a marker for the secondary structure of the peptide.
Spectral peak at 1645 cm¡1 was assigned to amide I band, which relates to the CDO stretch-
ing absorption band. This amide I band governs the peptide’s secondary structure. (72)
The fabrication of a fish collagen/alginate (FCA) sponge scaffolds have been investigated
due to their potential as functional skin substitutes for wound healing. The scaffolds were
functionalized by chitooligosaccharides (COSs) with the help of crosslinking agent, 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The FTIR analysis was done to
shed light on the structural modifications pre and post crosslinking. In case of Fish collagen,
the spectra exhibited five characteristic bands (absorption). Absorption band at approxi-
mately 3299 cm¡1 corresponds to amide A which is associated with vibrations of N–H (s)
group. Absorption band for amide B at around 3074 cm¡1 corresponds to C–H stretching,
whereas amide I band at 1647 cm¡1 corresponds to the bending vibrations of N–H group.
Similarly, amide II vibrations around 1534 cm¡1 associated with the stretching of C–N
group. Furthermore, for amide III, frequencies at approximately 1234 cm¡1 (combination/
union of two peaks) are related to in between deformation (N–H) and stretching (C–N).
 APPLIED SPECTROSCOPY REVIEWS 713

Table 1. Different spectral peak positions in FTIR spectra of pure materials and fabricated scaffolds (76)
(reproduced with permission of publisher).
Absorption peaks (cm¡1)

Types of fabrication Amide A Amide B Amide I Amide II Amide III C–O–C

FC 3299 3074 1647 1534 1234 —

SA — — — — — 1035
FCA-NCL 3285 3072 1602 1556 1238 1032
FCA-CL 3299 3076 1648 1550 1235 1035
FCA/COS1 3295 3077 1636 1544 1238 1033
FCA/COS2 3295 3075 1636 1548 1240 1030
FCA/COS3 3294 3076 1637 1548 1240 1030
FCA/COS4 3299 3089 1636 1546 1246 1035

NCL D non-crosslinked, CL D crosslinked by EDC.

These spectral bands confirmed the extraction of collagen (73–75). The homogenous blend-
ing of FC and SA is indicated by the merger of IR peaks corresponding to non-crosslinking
of FCA. Spectral positions for the raw material versus fabricated scaffolds are tabulated in
Table 1. No apparent modifications in the structure of alginate or the secondary structure of
collagen were observed by addition of crosslinking agent EDC. This was indicated by rela-
tively unaltered stretching peaks(s) of FCA scaffolds (crosslinked). The absorption bands
also suggested that the peaks remained significantly unaltered; however, a downshift in spec-
tral bands have been reported; specifically, in the regions corresponding to amide A and O–
H bands. The downshift in the region approximately between 3650 and 3300 cm¡1 is attrib-
uted to the loss of water bonded to scaffolds (FC–FC and FC–SA) crosslinked with EDC
(76). They reported that the spectral peaks remained constant with respect to wavenumber,
whereas in parallel, there was a decrease in spectral bands attributed to amide A and O–H
region. This change is due to the removal of water from the scaffolds with increasing molec-
ular weights of COSs. Collectively, it can be inferred that all the scaffolds had uniform cross-
linking of COOH and NH2, where the addition of increasing molecular weight of COSs
further enhanced the crosslinking (76).
Collagen peptides have also been reported to be used for the modification of carboxymethyl
cellulose (CMC), where collagen influences the antioxidant properties of the material following
a reduction in reactive oxygen species in vitro (77). The materials were also compatible with
fibroblast cells, hence signifying the utility of these materials in addressing wound healing pro-
cesses. FTIR spectra of CMC and CMCC (carboxymethyl cellulose collagen) was analyzed
(Figure 3). Characteristic peak at 2922 cm¡1 correspond to C–H anti-symmetrical stretching,
while peaks at 1422, and 1624 cm¡1 appear due to the carboxylate groups stretching vibrations
(symmetric and asymmetric). Due to presence of intermolecular and intramolecular hydrogen
bonds and –OH groups, a visible band is seen at 3434 cm¡1. In the FTIR spectrum of
CMCC, characteristic spectral peaks at 1651 and 1548 cm¡1 were assigned to amide I and
amide II, respectively, which showed spectral shift when compared to CMC. Degradation of
CMCC due to the presence of H2O2 did not disrepute the presence of the amide groups,
which is evident from the presence of characteristic spectral peaks for the amide group at
1654 and 1545 cm¡1 (77).
3D scaffold fabrication as cumin loaded nano-graphene oxide (NGO), functionalized by
type I collagen has been explored for potential wound healing agent, an emerging prospect in
tissue engineering (78). FTIR spectra were analyzed to depict collagen’s and NGO’s interaction
714 T. RIAZ ET AL.

Figure 3. FTIR spectra of the carboxymethyl cellulose (CMC), the carboxymethyl cellulose derivative
(CMCC, DS D 0.29, Mw D 1.78 £ 104) and the degradation of CMCC (DCMCC, DS D 0.35, Mw D 8.2 £ 104)
(77). (Reproduced with permission of publisher).

via –CONH2 group. The spectrum of NGO collagen is supported by previous studies (79, 80).
Investigation of collagen I indicates that the absorption bands at 1626, 1539, and 1390 cm¡1
correspond to amide I stretching vibrations (CDO), amide II (bending of N–H group), and
amide III’s stretching vibrations (-CONH2 group), respectively. Stretching vibrations due to
–NH2 group is represented by the peak at 3282 cm¡1, whereas peak at 3078 cm¡1 is attributed
to the Fermi resonance overtone of the band at 1539 cm¡1, and stretching vibrations of C–H
corresponds to 2927 cm¡1. C–N amine stretching vibrations correspond to peak 1236 cm¡1
(80). The spectrum of NGO indicate the presence of –COOH and –OH groups characterized
by the presence of peaks at 384, 1700, 1640, and 1035 cm¡1 (79). No new peaks were evident
when comparing CFNGO spectrum with Collagen and NGO’s spectrum. However, presence
of intense characteristic peaks of collagen and NGO signified the interaction via –CONH2
group between NGO’s –COOH group and collagen’s free NH2 group.
Nanofabrication of P(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] fiber
matrices with incorporated collagen has been investigated as potential wound dressing
(81). FTIR analysis was carried out to identify components of the matrices (Table 2). Col-
lagen peptides were characterized by the presence of absorption bands of amide I and
amide II at 1634 and 1526 cm¡1, respectively, followed by a broadband at 3300 cm¡1 cor-
responding to N–H stretching vibrations (82). In case of polymer P(3HB-co-4HB), ester
carbonyl groups contribute to the band at 1720 cm¡ 1 as reported earlier (83, 84). The
amide I, amide II, and ester carbonyl group were seen in the P(3HB-co-4HB)/collagen pep-
tides construct. Absorption band at 3340 and 3190 cm¡ 1 correspond to primary amide
group (82). It is possible that in case of P(3HB-co-4HB)/collagen peptides construct, these
bands are masked by bands corresponding to bands of P(3HB-co-4HB) copolymers as
given in Table 2. The FTIR spectra characterized by the presence of functional groups and
assigned peaks is presented in the table below the spectra.
 APPLIED SPECTROSCOPY REVIEWS 715

Table 2. The functional groups and mode of vibration of nanofibrous P(3HB-co-4HB)/collagen peptides
(81) (reproduced with permission of publisher).
Construct Wavelength range (cm¡1) Assignments

P (3HB-co-4HB) 1740–1680 CDO carbonyl group stretching

Collagen peptides 3500 NH2 bonding
1600–1500 NH2 bending, CDODN stretching (amides I and II)
1660–1535 N–H stretching vibration
P (3HB-co-4HB)/Collagen peptides 3518 H bonded O–H groups, NH2 bonding
1740–1680 CDO carbonyl group stretching
1600–1500 NH2 bending, CDO, CDN stretching (amides I and II)
1660–1535 N–H stretching vibration

Grafting of guar gum scaffold films with ethylene diamine and collagen (fish scale)
followed by crosslinking with ceftazidime has shown promise in the field of wound
healing owing to its superior biocompatibility (85). FTIR spectra were analyzed for
native guar gum (GG), carboxymethyl guar gum (CMGG), aminated carboxymethyl
guar gum, collagen, and aminated CMGG-Ceftazidime-Collagen (ACCC) films. Due to
O–H and C–H stretching vibrations, absorption bands at 3279 and 2918 cm¡1 (respec-
tively) are seen for GG. Furthermore, in case of GG, the absorption band at 1140 cm¡1
is attributed to the alcoholic C–O conjugation and band at 1007 cm¡1 is due to the
glycosidic linkage (pyranose ring) in its backbone. Upon modification to CMGG,
absorption at 1603 and 1454 cm¡1 is characterized as –COO¡ stretching vibration
(symmetric and asymmetric), which confirm the presence of COOH group in CMGG.
Bending vibrations of CH2–O–CH2 are assigned to peaks at 1072 and 1028 cm¡1 (86).
The spectrum of aminated CMGG confirms EDA conjugation with CMGG along with
the presence of amino functional groups in CMGG, which is represented by the peaks
at 3278, 1580, and 1412 cm¡1. In case of collagen, absorption bands at 1626 cm¡1 is
due to stretching vibrations of C–O for amide I, 1539 cm¡1 due to N–H bending vibra-
tions (amide II), and 1390 cm¡1 indicative of stretching vibrations (amide III) of
–CONH2linkages in collagen. Peak at 3282 cm¡1 and at 1539 cm¡1 correspond to
stretching vibration of NH2. Fermi resonance overtone band led to a peak at 3078
cm¡1, whereas band at 2927 cm¡1 corresponded to C–H stretching vibration. Stretch-
ing vibrations due to C–N of amines was indicated by peak at 1236 cm¡1 (80). The
ACCC spectrum affirmed the presence of both collagen and CMGG in the film, which
was represented by characteristic peaks at 1641, 1528, and 1011 cm¡1.
The potential of duck’s feet collagen/silk hybrid scaffold in dermal substitution has been
reported recently (87). FTIR spectra of porcine collagen, duck feet collagen, silk scaffolds,
and duck feet collagen–silk scaffolds were compared. In case of porcine collagen, the amide
I and II bands were indicated by the presence of characteristic peaks at 1634 and
1550 cm¡1, respectively. Whereas broader bands of amide I and II at 628 and 1550 cm¡1,
respectively, were seen in the case of duck feet collagen. This variation could be attributed
to the structural difference between the two collagens. Absorption peaks at 1638 and
1516 cm¡1 were seen in the case of silk. Absorption bands of amide I at 1639 cm¡1 and
amide II at 1535 cm¡1 were seen in case of duck feet collagen–silk scaffolds (87).
Fernandes et al. (88) reported the fabrication of collagen-chitosan porous scaffolds as
potential dermal substitutes. FTIR spectra exhibited characteristic peaks at 1650, 1560, and
1235 cm¡1 corresponding to amide I, amide II, and amide III, respectively (Figure 4).
716 T. RIAZ ET AL.

Figure 4. FTIR spectra for chitosan and collagen-chitosan samples (88) (reproduced with permission of
publisher).

The absorption peaks are attributed to the stretching and bending of specific functional
groups for amide I, amide II, and amide III as reported earlier (89).The spectrum of the colla-
gen-chitosan scaffold indicated presence of characteristic bands as of raw materials collagen and
chitosan. In case of collagen-chitosan scaffolds, the interaction has been reported to be facilitated
by the hydrogen bonding between the –OH, –NH2, and –CDO groups of collagen with –OH
and –NH2 groups of chitosan (90). The interaction is further promoted by the acidic media
which promotes protonation of amino groups of chitosan which stimulates electrostatic interac-
tions between chitosan’s NH3C groups and collagen’s –COO¡ groups (corresponding to aspartic
and glutamic acid). Changes in the relative amount of collagen and chitosan also influence the
absorption bands intensities. Owing to lower collagen quantity, the amide I band decreases to
correspond to a small shoulder to amide II, which is similarly to the pure chitosan spectrum;
whereas, amide II band’s intensity increases following an increase in the chitosan quantity. How-
ever, increasing chitosan results in a decrease in amide III band intensity as shown previously
(89). Collagen’s triple helical structure plays a significant role in its biological and mechanical
functioning. Helix integrity is determined by the absorbance ratio at 1235 and at 1450 cm¡1. Pre-
vious studies have indicated that the absorbance ratio is close to 1 for the intact helix (91). For
the collagen-chitosan scaffold, the ratio was evaluated to be 1.06, reiterating that inclusion of chi-
tosan did not hamper the triple helix of collagen necessary for its function (88, 91).
Electrospun scaffolds composed of PLGA/Collagen composite were prepared as skin substi-
tutes with therapeutic prospect for skin related injury and burn treatment (92). Characterization
by FTIR identified absorption bands in the region 1650–1660 cm¡1 corresponding to amide I,
where the peptide CDO groups contribute to stretching vibrations. Region between 1540 and
1555 cm¡1 correspond to the C–N stretching vibrations and N–H bending of amide II. Absorp-
tion band at 1235 cm¡1 corresponding to amide III due to the stretching and binding vibrations
of C–O and N–H, respectively. Region between 3300 and 3400 cm¡1 signifies the presence of a
middle amide A which is due to the N–H stretching (93, 94). In case of PLGA, the region of
1540–1660 cm¡1 lacks intense peaks. Interestingly, the region above 3200 cm¡1 indicates pres-
ence of a weaker peak, which in case of collagen corresponds closely to the amide A region.
 APPLIED SPECTROSCOPY REVIEWS 717

Figure 5. FTIR spectroscopy for collagen, ES0, and EScoat samples (93). (reproduced with permission of
publisher).

Collagen grafting on PLGA scaffolds has been explored for their promise as skin substitutes
(93). Modification by collagen has resulted in enhancing the bioactivity of the scaffold’s sur-
face, and hence reiterating its potential in skin bioengineering. Figure 5 depicts FTIR spectra
of PLGA, collagen, and the collagen-reinforced scaffolds. Spectrum for collagen shows charac-
teristic bands for amide I in the spectral region of 1650–1660 cm¡1 due to peptide’s CDO’s
stretching vibration. Spectral region between 1540 and 1555 cm¡1 indicated stretching (C–N)
and bending (N–H) vibrations, which corresponded to amide II. Spectral region between 3300
and 3400 cm¡1 corresponds to a broad and middle amide A due to N–H stretching (94–96).
Analysis of PLGA spectrum in the region between 1540 and 1660 cm¡1 region lacked any
characteristic intense peak. In case of collagen, the region spanning amide A indicates pres-
ence of a weaker peak.
Bacterial cellulose in combination with collagen as hydrogels has been reported to be a pro-
spective candidate for skin regeneration. Bovine tendons were used to extract the type I colla-
gen. The FTIR spectra analyzed for characterization of the individual components as well as
the composite hydrogel. The extracted collagen’s triplex helix was intact as estimated by the
relative absorbance intensity values of the 1235 and 1450 cm¡1 bands, which corresponded to
»1. Bands for collagen and bacterial cellulose (BC) indicated typical band patterns whereas
bands at 1450 and 1235 cm¡1 were also seen for collagen. The spectrum for the hydrogel (col-
lagen-BC) indicated variation in band intensity as compared to the pure collagen, possibly
contributed by the interaction of collagen with BC. This interaction was also evident from the
band shift, i.e., a 11 cm¡1 (blue shift) of 1439 cm¡1 and a 20 cm¡1 (red shift) of band
1260 cm¡1.Whereas, variation in band intensities of collagen-BC hydrogel as compared to
pure collagen could be attributed to presence of other components in the hydrogels (97).

3. FTIR of collagen containing dermal fillers

A comprehensive literature was carried out on the FTIR spectroscopy of collagen-based
material for dermal reconstruction and skin aging. Injectable dermal fillers have recently
718 T. RIAZ ET AL.

attracted the attention (e.g., surgical procedures, filler formulation, crosslinking) for aesthetic
use as well as cosmetic improvement of the skin (98, 99). The FTIR spectra of collagen
source isolated from human umbilical cord and crosslinked hyaluronic acid composite filler
was reported by Z-Hun Kim et al. (100) as a dermal filler for tissue reconstruction. The spec-
trum showed characteristic peaks of amide bending (1636, 1553, and 1237 cm¡1) in collagen.
The HA spectra exhibited a peak at 1300 cm¡1 that did not appear in the uncrosslinked HA
which may be attributed to the carboxyl groups and hydroxyl groups of HA converted into
ether chains crosslinked with the butanediol diglycidyl ether (BDDE) (100).
Xiaoxuan et al. (101) utilized FTIR spectroscopy to investigate the formation of novel
hydrogels (HCD Hydrogels) using Human Like collagen (HLC) and chitosan using Dialde-
hyde starch (DAS) for making it potentially promising for tissue cavity fillers, skin patch
scaffolds, and wrinkle treatments. The FTIR spectral results showed the characteristic peaks
of amide I (v(CDO)) at 1664 cm¡1 and amide II (corresponding to the amide I (n(CDO))
and amide II of (d(N–H) and v(C–N)) of RCONHR0 at 1630 cm¡1 that corresponds to the
characteristic peaks of HLC. After the formation of the HCD-1.0 hydrogel, while comparing
curve b with d, weakening of peaks were observed. Some characteristic peaks of DAS disap-
peared in curve d after the formation of the HCD-1.0 hydrogel. A new peak was observed
associated with (v(CDN)) at 1646 cm¡1. Similarly, four new peaks associated with the vibra-
tional coupling from the two C–O–C groups between 1150 and 1025 cm¡1 further con-
firmed the formation of covalent bonds by acetalization and Schiff base reactions.
Novel crosslinked human Acellular Dermal Matrix (ADM) demonstrated its suitability
adequate for use as a biologic tissue substitute. Crosslinked collagen materials with Dermal
matrix has been found helpful in reconstructive surgery. The FTIR for e-beam irradiated
ADM (0, 10, 15, 25, and 35 kGy) and unirradiated ADM (controls) have been documented
in attenuated total reflection mode using a single diamond crystal window
Collagen crosslinking can be investigated by comparing the area ratio of these two sub-
bands (1690:1660) (103, 104). Increased absorption ratio of 1660:1690 characteristic peaks
was observed after irradiating at 25 kGy and 35 kGy. Area ratio of the amide I sub-bands
1660 and 1690 cm¡1 and peak shifts in the amide I and II band after irradiation at 35 kGy
were also observed as can be seen in Figure 6. The results showed effective crosslinking by e-
beam irradiation at doses exceeding 25 kGy (102).
Hydrolysates non-tanned collagen waste can be advantageously used as hydration sub-
stances without further modification in cosmetic preparations (105). The collagen hydroly-
sate based wet white leather wastes pre-tanned with oxazolidine (hydrolisate A), titanium-
aluminium complex (hydrolysate B), and oxazolidine-resorcinol (hydrolysate C) was studied
by Madalina et al. (106). Combining resorcinol and oxazolidine together increase the shrink-
age temperature upto 100 C as tanning agents (107).
During chronological aging, modifications in dermal structural proteins cause morpho-
logical alterations due to type I collagen rearrangement and reorientation with aging that
have not been investigated until now (108). In this study, polarized-FTIR imaging was used
to evaluate the molecular modifications of dermal collagen alterations associated with chro-
nological skin aging performed on eighty skin samples from 41 to 80-year-old women
donors operated for breast cancer resection. Breast skin is considered as a good model for
studying chronological aging as it is relatively less exposed to sunlight. Rat tail tendon, aged
2 months, was used as a reference sample to investigate skin tissue sections to measure the
effect of polarization on the type I collagen IR signal as shown in Figure 7.
 APPLIED SPECTROSCOPY REVIEWS 719

Figure 6. FTIR spectrometry analysis of acellular dermal matrix (ADM) before and after e-beam irradiation
(102) (reproduced with permission of publisher).

The FTIR spectra showed the characteristic IR band vibrations particularly at the level of
the amide I (1658 cm¡1 ) and amide II (1550 cm¡1) of type I collagen and at 1451, 1399, 1339,
1282, 1236, and 1203 cm¡1 assigned to CH2 and CH3 wagging and deformation and stretching
vibrations of C–N bond (109). Consequently, the area of the amide I band (CDO) and amide
II band (C-N) parallel or perpendicular to the axis of collagen fibers indicates the orientation
of collagen peptide links. The C-N and C-C skeletal stretch vibrations at 1240 cm¡1 observed
to be sensitive to the polarization (110).
Prior to analyzing based on the results obtained on the rat tail tendon, skin specimens
were selected using a similar approach. The maximum of the amide A band was located at
3310 and 3295 cm¡1 for the epidermis and the stratum corneum. The spectrum of stratum

Figure 7. IR spectra of rat tail tendon in the 900–1800 cm¡1 range in (a) non-polarized mode (b), with IR
radiation polarized perpendicular (c) to the x-axis of the motorized plate (108). (reproduced with permis-
sion of publisher).
720 T. RIAZ ET AL.

corneum presented higher lipid content according to the more intense signal observed at the
C–H stretching vibration level at 2950 cm¡1.

4. FTIR analysis of skin aging

Solar radiation has a profound effect on premature skin aging and skin cancer (111,
112). Effects of solar radiation on collagen (obtained from tail tendons of young albi-
nos’ rats) and collagen/synthetic polymer blends in the form of thin films and solutions
has been studied using FTIR spectroscopy. Positions of amide A band shifted to lower
wavenumbers after solar irradiation due to the scission of hydrogen bonds to maintain
the helical structure of collagen but position of amide I and II bands remained on the
same position as given in Table 3.
It was observed that integral absorbance of collagen amide bands (A, I, and II) decreased
after UV irradiation. For collagen/ PVA, integral absorbance for amide A band remain
unchanged after UV irradiation and integral absorbance of amide bands (A and II) are simi-
lar. For collagen/PVP blends, the changes of integral absorbance of amide bands (A and II)
was found to be like those for pure collagen. Thus, FTIR spectroscopy was helpful to prove
that solar irradiation causes the changes in conformation of the collagen molecule (113).
Photo-aging and photo-degradation are the deleterious effect of chronic exposure to sun
light of materials made of natural polymers (111, 112). IR spectra of collagen film showed
bands at 1647, 1547, and 1240 cm¡1 for the amide I, II, and III bands, respectively. Similarly,
amide A band of collagen (associated with the NH– stretching frequency and O–H group) is
usually found at 3315–3330 cm¡1, amide B at 3080 cm¡1. It was further investigated that
amide A and the amide I bands shifted to lower wavenumbers after solar radiation, amide II
and III positions remain less altered. FTIR spectra of collagen/chitosan blends has been dis-
played exhibiting the amide bands B, I, and II positions almost at the same wavenumbers
before and after solar irradiation.
There was a difference observed for the amide A band position. Shifting of amide A band
is smaller for collagen/chitosan film than for collagen film due to the scission of hydrogen
bonds. For collagen/chitosan blends, the changes of integral absorbance of amide bands A
and I are similar to those for pure collagen (114).

Table 3. Characteristic bands of collagen and collagen-based biomaterials after solar radiation (113)
(reproduced with permission of publisher).
Characteristic bands (cm¡1)

Sample Amide A Amide B DCH2 Amide I Amide II Time of irradiation (days)

Collagen 3315 3079 2936 1638 1547 1

3311 3071 2935 1634 1546 2
3309 3078 2940 1635 1547 3
3301 3064 2950 1634 1547 4
Collagen/PVP blend 3329 3087 2935 1644 1550 1
3322 3088 2953 1644 1550 2
3323 3086 2953 1645 1551 3
3321 3091 2955 1641 1549 4
Collagen/PVA blend 3316 3079 2937 1642 1548 1
3321 3067 2937 1642 1550 2
3318 3080 2938 1648 1549 3
3310 3088 2928 1641 1548 4
 APPLIED SPECTROSCOPY REVIEWS 721

Aging process involves the modifications of ECM proteins which contribute to various
pathological phenotypes. Different collagen 3D constructs extracted from tail tendons of
rats (newborns, young, and old adults) were analyzed with the IR imaging system (Spotlight
300, Perkin Elmer) to check the influence of aging in collagen 3D matrix model.
Up-shifting of the Amide I peak from 1656 cm¡1 for the newborn collagen to 1632 cm¡1
for the young-adult and old-adult collagen, show consequent alterations for collagen confor-
mation and 3D network. Similarly, increase in intensity ratio peak at 1032 cm¡1 (C–OH
bonds) indicates that advanced glycation end products (AGEs) are correlated with the fluo-
rescence-AGEs accumulation (115).
Exposure to ultraviolet light results in lower fibril organization level causing premature
skin aging and skin cancer. Shifting of amide A peak to lower wavenumber (3424–
3411 cm¡1) after irradiating collagen for 2 h takes place due to thermal break-up of hydro-
gen bonds and N–H stretch coupled with H-bond from H2O to stabilize the helix structure.
Amide I band at 1645 cm¡1 showed vibration of amide carbonyls along the polypeptide
backbone attributed to random coil transformation of collagen (116).
Slight shifting of Amide II to lower wavenumber (1562–1560 cm¡1 ) further suggested
that absorbance is not proportional to the increase of the coil conformation (117). The
results further showed that photo degradation of collagen occurs in parallel “fingerprint”
region (1200–1350 cm¡1 ) assigned to specific tripeptides (Gly-Pro-Hyp)n of collagen (118).
Exposure to UV irradiation is largely dependent on the degree of hydration of collagen.
Xing et al. (119) used FTIR spectroscopy to investigate the structural changes in collagen
caused by UV irradiation. The spectrum showed shifting of characteristic NH-stretching
vibration frequency of collagen at 3330 cm¡1 to a lower wavenumber at 3318 cm¡1, indicat-
ing thermal break-up of hydrogen bonding along the collagen backbone. Similarly, it was
observed that native collagen showed the amide I band components at 1692, 1660, and
1615 cm¡1 and UV irradiated collagen amide I band at 1697 and 1652 cm¡1. Shifting of
band centered at 1615 cm¡1 to a lower frequency indicates a structure change. It was further
noticed that shifting of the band at 1660 cm¡1 to a lower frequency (1652 cm¡1) takes place
after UV irradiation. Increased peak area at 1652 cm¡1 indicated a helix-coil transformation
of collagen. The peak area was enhanced for UV-irradiated collagen at 1697 cm¡1 further
suggesting decomposition of aggregated collagen fibrils. The findings further revealed that
amide II bands in native collagen IR spectra at 1528 and 1574 cm¡1 shifted to 1526 and
1573 cm¡1 after treatment with UV corresponds to the disruption of the collagen and three
distinct components in UV irradiated collagen IR spectra at 1526, 1573, and 1588 cm¡1.
Metreveli et al. (120) in their study by adding ascorbic acid as antioxidant, found that
interaction between ascorbic acid and collagen molecule has been found helpful to increase
the photo-stability of collagen molecule. Degree of the intensities of bands (amide A, amide
B, amide I, and amide II) decreased with the increased UV radiation dosage. Noticeably,
width of amide A band narrows sharply after treatment with UV radiation. Furthermore,
the intensities of amide bands also decreased in the presence of ascorbic acid (depending on
the concentration of antioxidant) under UV radiations much more slightly than for pure
collagen.
Natural and synthetic polymers undergo photo-aging and photo-degradation that may
lead to loss of material structure and function when exposed to electromagnetic radiation
(121). Amide A bands at 3330–3325 cm¡1, amide B (3080 cm¡1), amide I (1650 cm¡1), and
amide II (1550 cm¡1) bands relates to the peptide linkages of collagen as shown in Figure 8.
722 T. RIAZ ET AL.

Figure 8. FTIR spectra of components of collagen (122) (reproduced with permission of publisher).

Different bands observed at (3330–3325 cm¡1) due to amide A, amide B (3080 cm¡1), amide
I (1650 cm¡1), and amide II (1550 cm¡1) bands relates to the peptide linkages of collagen as
shown in Figure 8. The modification of the position of band of amide A components sug-
gests the change in structural conformation of collagen. No change was observed for amide
B band after UV treatment. The amide I band corresponds to the vibration of amide carbon-
yls along the polypeptide backbone while the amide groups of the triple helical state are
linked with a peak at 1549 cm¡1 for amide II bands (122).

5. FTIR analysis of collagen containing drug delivery agents

Collagen and polycaprolactone (PCL) composite-based biomaterials have also been explored
for their potential as drug delivery agents. In this present study, collagen-PCL composites
were loaded with insulin for potential release analysis. The eventual drug entrapment effi-
ciency and patterns of release by diffusion are related to alterations in the collagen’s micro-
structure which are optimized during the fabrication process. The ability to fabricate
biomaterial based composites with specific pore size and developing mechanistic insight into
patterns of controlled drug release is presently a challenge that is being explored widely
(123–125).
Evidence of the secondary structure of collagen was supported by the FTIR spec-
trum. Characteristic peaks corresponding to the amide region I, II, and III were seen at
frequencies of 1660, 1550, and 1242 cm¡1, respectively. The frequencies related to
stretching vibration of C–O groups (amide I), stretching vibration of N–H and C–H
groups (amide II) and stretching vibrations of C–N and N–H in-plane bending (amide
III) were also observed. Furthermore, comprehensive analysis of the secondary structure
of collagen has been done in related studies (126–128). Frequency at 1452 cm¡1 corre-
sponds to stretching vibration of cyclic proline’s C–N group. Proline’s side chain and
glycine backbone’s wagging vibrations (CH2 groups) correspond to the band character-
ized in the region spanning 1200 and 1400 cm¡1. The CH2 groups in proline’s side
 APPLIED SPECTROSCOPY REVIEWS 723

chain contributing to wagging vibrations correspond to band position at 1334 cm¡1.

Amide bond positioning is governed by the variability in both the strength of the
hydrogen bonding and protein’s transition dipole coupling. In case of PCL, frequencies
at 1728 and 1180 cm¡1 relate to characteristic peaks relating to carbonyl group and n’s
(C–O–C) (129). Specific orientation toward the surface plane is evident by the absorp-
tion band at 1180 cm¡1 (130). The spectra of CPCL-1 and CPCL-2 indicated absence
of shoulders at 1334 and 1149 cm¡1, which were present in case of the parent protein.
This is attributed to the remodeling of the wagging vibration (CH2 group) owing to the
glycine backbone and proline side chain (131).
The role of chitosan nanoparticles functionalized by collagen peptides as drug carriers for
the treatment of cancer has been addressed in this study (132). The collagen peptides (CP)
contribute to the stability of the chitosan nanoparticles (CN). These NPs were characterized
by FTIR spectroscopic spectra. Chitosan nanoparticles were characterized by the presence of
specific peaks at 3258, 2927, and 2832 cm¡1, whereas peak at 1541 cm¡1 contributed by the
bending vibrations of NH2 group. Peak at 1395 cm¡1 corresponds to the primary alcoholic
group’s (¡OH) vibrations (89, 133). Stretching vibrations indicated by bands at 3380 and
1620 cm¡1 corresponded to NH2 and CDO groups, respectively. CPCN NPs spectrum indi-
cated the presence of characteristic C–N peaks as well as distinct intense peaks spanning of
800–1500 cm¡1. In case of CP, peaks at 1632, 1554 and 1230 cm¡1 represented amide I, II,
and III bands of collagen, respectively as reported earlier (134). Presence of supplementary
peaks at 1205 and 1260 cm¡1 along amide III’s band at position 1230 cm¡1 are characterized
by the following components. The first relates to amide linkages stretching vibrations (C–N)
and plane bending (N–H). The second is attributed by glycine backbone and proline side
chain’s wagging vibrations (CH2 group) (134). Peaks at 1541 and 1395 cm¡1 show a marked
reduction in sharpness with increasing concentrations of CP in the nanoparticles. In parallel,
formation of a new amide I band is indicated by the broad peak at 1632 cm¡1. Incorporation
of CP resulted in the formation of the new amide I bond which was not clearly visible due to
overlapping of amide I bond attributed by collagen. It was concluded that the hydrogen
bonding between the end groups of CP (-COOH and –NH2) and C–N (-OH and –NH2)
contributed to the shift of amide I (original) bond to a higher wavenumber. The peak attrib-
uted to these end groups showed a reduction in their intensities which is resulted by the
simultaneous increase in the utility of these groups in hydrogen bond formation. This hydro-
gen bonding between C–N and CP is attributed by –COOH and –NH2 end groups of CP,
whereas the other end group –OH of hydroxyproline contributes to hydrogen bonding
between the chains. Comparable investigations were made in recently reported studies,
where it is postulated that the –OH and –NH2 end groups of C–N groups form hydrogen
bonds with nanocrystalline cellulose (135, 136).
Barbaresso et al. (137) studied the delivery systems using anti-inflammatory drug and
analgesic in dentistry systems that contained collagen as support and niflumic acid as a
drug. Type I collagen and niflumic acid gels were crosslinked with different concentrations
of glutaraldehyde and then freeze-dried to obtain collagen matrices (spongious form). The
IR spectra represent the main wavenumbers obtained for samples of collagen, niflumic acid,
and collagen with niflumic acid and crosslinking agent. In the polymeric structure, collagen
peaks can be easily recognized with bands for amide I, amide II, and amide III at 1630, 1549,
and 1339 cm¡1 respectively, accompanied by specific bands for associated amides at 3294
and 3081 cm¡1 (137).
724 T. RIAZ ET AL.

6. FTIR analysis of collagen materials used in tissue engineering

Composite membranes of poly(hydroxyalkanoate) (PHA) and maleic anhydride-grafted
PHA (PHA-g-MA) with collagen have been reported to be a promising tool in biomedical
applications. Composites had improved mechanical properties, showed compatibility with
human foreskin fibroblasts cells, and facilitated collagen production. Comparison of FTIR
spectra of PHA, PHA-g-MA, PHA/Col (10 wt.%), and PHA-g-MA/Col (10 wt.%) was done
(Figure 9). Both PHA and PHA-g-MA polymers demonstrated transitions at 3300–3700
cm¡1, 1700–1760 cm¡1, and 500–1500 cm¡1 (characteristic of PHA), whereas shoulders at
1785 and 1852 cm¡1 were seen in PHA-g-MA, (characteristic feature of MA group). In case
of PHA/Col (10 wt.%), peak at 3200–3500 cm¡1 (characteristic of the N–H stretching vibra-
tion) intensifies due to the presence of ¡NH group of collagen. Characteristic peaks at fre-
quencies 3286(amide, N–H stretching), 2924(amide, C–H stretching), 1640(amide I, CDO
stretching), 1550(amide II, N–H bending), and 1230 cm¡ 1 (amide III, N–H bending) corre-
spond to amide group in collagen. Spectra for PHA/Col (10 wt.%) indicated that due to the
presence of PHA’s hydrogen bonding, peak frequencies at amide I shifted to 1633 cm¡ 1 as
compared to 1641 cm¡ 1 for pure collagen. Comparing the FTIR peaks of PHA/Col
(10 wt.%) and PHA-g-MA/Col (10 wt.%) composite revealed presence of an additional peak
at 1726 cm¡ 1 due to the vibrational ester linkage (138).
Hybrid scaffolds of collagen–poly (dialdehyde) (PDAGG) guar gum modified with
growth factors have been reported for potential application in tissue engineering. The FTIR
spectra was obtained for comparison between collagen and collagen–poly (dialdehyde) guar
gum hybrid scaffolds. It was seen that characteristic peaks for amide I, amide II, and amide
III groups were observed at frequencies 642, 1554, and 1240 cm¡1, respectively. An intense

Figure 9. FTIR spectra of (A) poly(hydroxyalkanoate) (PHA), (B) maleic anhydride-grafted PHA (PHA-g-MA),
(C) PHA/collagen composite (PHA/Col (10 wt.%)), (D) PHA-g-MA/Col (10 wt.%), and (E) Col. (138) (repro-
duced with permission of publisher).
 APPLIED SPECTROSCOPY REVIEWS 725

stretching absorption at 1620 cm¡1 due to the formation of imine bond is evident in case of
hybrid scaffold. This could be contributed by the crosslinking between PD-AGG (aldehyde
groups) and collagen’s amine groups. This imine bond supports the formation of a homogenous
hybrid scaffold. The crosslinking also might have contributed to the loss of characteristic peaks
for PDAGG. Similarly, due to the amide I group in collagen, the peak at 1642 cm¡1 became less
sharp. Increase in PDAGG content of the hybrid scaffold resulted in an increase in intensity and
sharpness at 1620 cm¡1. This is followed by a similar intense pattern at 1028 and 770 cm¡1, indi-
cating presence of anhydroglucose rings in the hybrid scaffolds (139)
Collagen’s application in tissue engineering is being widely investigated. A study explored the
extraction and application of ovine collagen for biomaterial based therapy. The FTIR spectra of
the extracted collagen and control collagen type I from rat tail were compared. The OTC spec-
trum presented characteristic peaks for amide I, amide II, and amide III at frequencies 1632,
1548 and 1237 cm¡1, respectively. The absorption peak at 3302 cm¡1 corresponded to –NH
stretching, whereas peak at 2923 cm¡1 indicated asymmetrical stretching of CH2 (140).
Collagen modified Poly lactic acid (PLA) composite were synthesized and evaluated for
their potential as therapeutic agents in the field of biomedicine, where addition of collagen
resulted in improved degradability of PLA (141). In case of PLA, weak peak at 3510 cm¡1
corresponds to stretching vibrations of hydroxyl and carboxyl group’s O–H (Figure 10).
Stretching vibrations of the carboxyl groups (CDO) correspond to sharp peak at 1759 cm¡1.
Spectrum for collagen-PLA (b) indicates the presence of O–H, NH2, and N–H correspond-
ing to amide group as indicated by the peak at 3329 cm¡1, which is clearly absent from the
spectrum of PLA. A wider peak at 1759 cm¡1 corresponding to CDO is seen as compared to
PLA (a). Collagen reinforcement on to PLA is confirmed by the presence of peaks at 1671
and 1526 cm¡1 corresponding to stretching and bending vibrations of amide groups (CDO)
and (N–H), respectively.
Report of the synthesis of a collagen fascicle system (CSF) has shown promise in tendon
repair and regeneration. The FTIR spectra in this case were used to identify collagen dena-
turation where the CSF was crosslinked with either NHS/EDC or NHS/EDC C EDGE. The
extent of denaturation was evaluated by taking two reference peaks, i.e., 1235 cm¡1 corre-
sponding to an amide III band, whereas it is also variable to the content of collagen’s triple
helix and at 1450 cm¡1, which relates to changes in –CH2 and –CH3 groups and it remains
consistent with the degree of denaturation (142). The triple helical content was proportional
to the peak height ratio A(1235 cm¡1):A(1450 cm¡1). The peak height ratio was 1.13 § 0.01
for the acid swollen collagen, whereas it was 0.95 § 0.01 for the unprocessed gelatin. Cross-
linked SCFs indicated peak height ratios between values of 1.02 § 0.01 when NHS/EDC was
incorporated, whereas the peak height ratio was 1.02 § 0.01 with NHS/EDC C EGDE. These
peak ratios reflect that the collagen structure has undergone partial denaturation.
Fabrication of biomimetic ECM by electrospinning collagen and chitosan for potential
smooth muscle regeneration has been presented in this study. Characteristic peaks in specific
regions owing to the presence of amide I, amide II, and amide III was evident. Previous stud-
ies have elaborately discussed the FTIR spectra of chitosan and collagen electrospun fibers
(143). Influence of crosslinking between collagen and chitosan fibers and native materials
can be compared by the FTIR spectra. The table further illustrates the amide absorption
bands of the electrospun fibers with variable compositions of chitosan. No compelling shift
in the amide absorption band was noted. However, a band shift from 1640 to 1680 cm¡1 for
amide I band was observed in case of electrospun nanofibers (20%) chitosan content. A
726 T. RIAZ ET AL.

Figure 10. FTIR spectra (a) and (b) collagen-PLA (141) (reproduced with permission of publisher).

decrease in amide III band corresponds to pure collagen. Hence, it can be inferred that fol-
lowing increase in stability, no compelling change incurred in the crosslinked collagen and
chitosan fibers when compared to the raw components (144).
A novel method for fabrication of dialdehyde bacterial cellulose (DBC)/collagen peptide
(Col-p) nanocomposite, where the binding of collagen on the BC is enhanced, has shown
great potential in tissue regeneration (145). FTIR spectra of all the components and the com-
posite supported the success of the fabrication (Figure 11). Characteristic absorption bands
for DBC were observed in the regions 1745 and 889 cm¡1. 889 cm¡1 corresponding to a dif-
fusion band is attributed to the hydrated structure (145), whereas vibrational frequencies at
1745 cm¡1 correspond to presence of carbonyl groups which support the oxidation of BC
(146). Analysis of the DBC/Col-p composite spectrum identified 1545 cm¡1 as novel regions
for amide II bands. Characteristic bands in the region of 2700–3000 cm¡1 were attributed to
stretching of NHC3, NHC2, or NHC groups (147) which support the presence of collagen
peptide in the fabricated composite membrane. Bands around 1649 cm¡1 indicate alteration
in the peak intensities corresponding to –CDN– band (147). Comparing DBC with compos-
ite membrane DBC/Col-p, peak corresponding to stretching of CDO bond, i.e., at
1745 cm¡1, disappeared in the composite membrane spectrum. It can be inferred that the
missing peaks and corresponding intense peak at approximately 1649 cm¡1 validate the fab-
rication of composite nanofibers with collagen peptide incorporated successfully (148).
Collagen nano-gold composites have been studied and shown to contribute significantly
to vascular regeneration. These composites have shown to provide the necessary ECM sig-
nals that facilitate migration and differentiation of Mesenchymal stem cells (MSC) (149). In
yet another study by the same group, collagen-gold coated catheter showed strong potential
in vascular regeneration. Analysis of the FTIR spectrum validated the presence of collagen
in the composites of collagenCAu. This was shown by the amide I and amide II peaks at
around 1655 and 1540 cm¡1, respectively. Furthermore, the absorption peak at 3340 cm¡1
corresponds to N-H stretching vibrations (149). The immobilization of gold in the collagen
is evident from the shift in absorption peak from 1540 cm¡1 as seen in pure collagen to
1535 cm¡1 for collagenCAu (Figure 12) (150).
 APPLIED SPECTROSCOPY REVIEWS 727

Figure 11. Left: FTIR spectroscopy of BC, DBC, and DBC/Col-p composite membranes; right: FTIR spectra
around 1800–1500 cm¡1 (BC: bacterial cellulose, DBC: dialdehyde bacterial cellulose, Col-p: collagen pep-
tide) (145) (reproduced with permission of publisher).

Collagen is a vital component of the ECM; hence it is widely explored for its potential in
regenerative medicine. This study focuses on the fabrication and characterization of colla-
gen-based heparin scaffolds for promoting stem cell differentiation into hepatocytes. The
study provided evidence supporting these scaffolds as potential therapeutic agents in the
liver regenerative medicine (151). FTIR spectral analysis was carried out for non-crosslinked
collagen, heparin, and crosslinked collagen with or without heparin. The spectrum of non-
crosslinked collagen demonstrated an absorption band around 3300 cm¡1 which is due to
the amine-free and carboxyl groups. Absorption peak at 1430 cm¡1 corresponded to stretch-
ing vibrations (symmetric) due to the COO– groups. It was also visible that after crosslink-
ing, due to hydrogen binding, the absorption corresponding to amine and carboxyl groups
widened. Similarly, a minor increase in peak intensity around 1650 cm¡1 was seen after
crosslinking. This was due to the CDO stretching vibrations corresponding to amide I. These
variations provide conclusive evidence of crosslinking. The intensities of carboxyl and amine
bonds are lower for the heparin collagen and heparin spectra when compared with the spec-
trum of collagen. In case of heparinized collagen, the stretching vibrations due to N–H and
O–H are seen near 3400 cm¡1. The increase of bending vibrations due to N–H around
1540 cm¡1 is identified as the amide II band as seen in the spectrum.
Tendon tissues endure high physiological loads and hence are associated with mus-
culoskeletal injuries. This present study addresses the fabrication, characterization, and
compatibility of chitosan-collagen/poly (l-lactic acid) (PLLA)/alginate scaffold for
regeneration of flexor tendons (152). Chitosan was characterized by the presence of
specific absorption peaks at 1404, 1560, 2878, and 3352 cm¡1 (153). The spectrum for
728 T. RIAZ ET AL.

Figure 12. FTIR spectra of collagen and collagen with different concentrations of nanogold (Au). (150)
(reproduced with permission of publisher).

collagen indicates the presence of characteristic peaks at 1235, 1560. and 1650 cm¡1 as
previously reported (110). Spectrum for chitosan collagen demonstrates the presence of
characteristic peaks for both chitosan and collagen, reiterating their presence. Spectrum
for PLLA indicates the presence of characteristic peaks at 83, 1182, 1450, and
1751 cm¡1 (154). Spectrum for alginate showed characteristic peaks at 1024, 1751, and
3358 cm¡1. Spectrum for the composite scaffold confirms the presence of all the com-
ponents depicted by their characteristic peaks.

6.1. FTIR analysis of collagen-containing biomaterials used in regeneration of bone

Wenpo et al. (155) synthesized the Collagen-Hydroxyapatite/kappa-Carrageenan (COL-
HAP/KCAR) composite that is similar to those of natural bone. They extracted the collagen
from rabbit skin and then used with the collagen in combination with Ca(NO3)2 and
(NH4)2HPO to obtain COL-HAP composite in situ. Kappa-Carrageenan, a kind of plant-
polysaccharides, was also employed to improve the properties of the composite (155).
The FTIR spectra show the typical bands of collagen in the COL-HAP, COL-KCAR, and
COL-HAP-KCAR composites. N–H stretch coupled with hydrogen bond appeared at 3400–
3440 cm¡1 for amide A. C-H asymmetrical stretch appeared at 2920–2930 cm¡1 for amide
B. C-O stretch/hydrogen bond coupled with COO– appeared at 1600–1700 cm¡1 for amide
I. N–H bend coupled with C–N stretch was found at 1540–1580 cm¡1 for amide II, and N–
H bending coupled with C–N stretching was found at at 1220–1300 cm¡1 for amide III
band. These amide bands were known to be related to the degree of molecular order and to
be involved with the triple helical structure of collagen, especially the amide I band; it could
be a sensitive marker of secondary structure. Normally, the amide I band is strong, the amide
II band is weak, and the amide III band is moderate (39, 156). In collagen, the characteristic
band (1087 cm¡1) may be the vibration coupling of specific amino acid side chains or pep-
tide groups. For example, Tyr’s (COH) at 1169–1260 cm¡1, Asp and Glu’s v(CO) at 1160–
 APPLIED SPECTROSCOPY REVIEWS 729

1253 cm¡1, His’ v(CN) and d(CH) at 1104, 1090, 1106, and 1094 cm¡1, Thr’s n(CO) at
1075–1150 cm¡1 (NC) at 1092 cm¡1, Ser’s v(CO) or v(CC) at 983 cm¡1, v(NC), d(CH), and
v(CC) at 1064 cm¡1, gt CH2 at 1063–1295 cm¡1, v(CC) and d (CH) at 1012–1016 cm¡1
(157).
With the addition of carrageenan, and the synthesis of hydroxyapatite (HA), the frequen-
cies of the band assigned to the amide I region remained constant at 1640–1700 cm¡1. This
clearly indicates, whether in collagen, COL-HAP or COL-HAP/KCAR, the secondary struc-
ture of collagen remains unchanged, but it shifts from 1640 to 1634 cm¡1 and from 1634 to
1630 cm¡1. The band II and the band III are not detectable in the blends of COL-HAP, or
COL-HAP/KCAR, but shifts from 1543 to 1580 cm¡1, and from 1237 to 1240 cm¡1 for
COL-KCAR, respectively. Generally, the lower wavenumber of bands corresponds to higher
hydrogen bonding potential (39).
After that, Long et al. (158) fabricated a 3D porous scaffold based on collagen fiber and
bioactive glass for bone tissue engineering. An ideal scaffold for bone tissue engineering
should have interconnected porous structure, good biocompatibility, and mechanical prop-
erties well-matched with natural bones. Collagen is the key component in the ECM of natu-
ral bones, and plays a key role in bone regeneration.
The FTIR spectra they studied showed the results of the Collagen fiber scaffold and the
Collagen/BG scaffold in the range of 4000–600 cm¡1 as shown in Figure 13. The spectra
show a broadband from 3100 to 3600 cm¡1 in two samples, assigned to O–H stretching
vibration in the collagen due to the strong hydrogen bond of intramolecular and intermolec-
ular type. The peak at 2907 cm¡1 is ascribed to the stretching vibrations of CH groups. In
the spectrum of collagen, the peak at 1093 cm¡1 is attributed to stretching vibrations of C–
O. In the Collagen/BG scaffold, the peak at 1086 cm¡1 is related to Si-O-Si asymmetric
stretching vibrations which shows the presence of bioactive glass (158).
Chen et al. (159) prepared the Collagen/hydroxyapatite nanocomposite scaffolds by in
situ precipitation and freeze-drying approach. It was revealed that the inorganic phase in the
nanocomposite was carbonate-substituted HA with low crystallinity (159). As presented, the
FTIR spectra of the collagen showed absorbance at 1639, 1551, and 1238 cm¡1, which were
ascribed, respectively, to amide I, amide II, and amide III of collagen (Figure 14). From the
FTIR spectra of the Col/HA nanocomposite, the bands at 1092, 1035, 961, 603, and
566 cm¡1 corresponded to different vibration modes of phosphate group in HA, while the
bands at 3570 and 632 cm¡1 represented hydroxyl group as stretching and bending vibra-
tion. Bands assigned to carbonate group at 1482, 1452, 1424, and 874 cm¡1 were also
observed. This agreed with the fact that HA crystals prepared using the precipitation method
contained carbonate ions (160). In comparison with pure collagen, the peaks for the amide I
of the Col/HA composite show no significant shift. However, the amide II and amide III
adsorption peaks are not easily detected. A weak shoulder appeared in the composite which
confirm that Collagen was involved in the intermolecular crosslinking reaction. Also, a
strong chemical interaction occurred between the inorganic and organic components in
Col/HA nanocomposite.
The peak at 3080 cm¡1 belongs to the stretching vibration of amide hydrogen bonding
(NH), but it weakened in the Col/HA nanocomposite system. This is due to the fact that
amino groups of Collagen react with glutaraldehyde via Schiff’s base linkage (159).
Cunniffe et al. (161) also studied the composites of collagen. They developed and
characterized the collagen nano-hydroxyapatite (nHA) composite scaffold for bone tissue
730 T. RIAZ ET AL.

Figure 13. FTIR spectra for (a) CO fiber scaffold and (b) CO/BG scaffold (158) (reproduced with permission
of publisher).

engineering. Bone regeneration requires scaffolds that possess suitable mechanical and bio-
logical properties. This study sought to develop a novel collagen-nHA biocomposite scaffold
via two new methods. Firstly, a stable nHA suspension was produced and added to a colla-
gen slurry (suspension method), and secondly, porous collagen scaffolds were immersed in
nHA suspension after freeze-drying (immersion method). The FTIR spectra showed the
presence of nHA in the collagen-nHA composite scaffolds made by both methods (S-100
and I-High). Characteristic peaks for HA are in the region of 500–600 cm¡1, the asymmetric
bending and the stretching band of the (PO4)3- group is found at1063 cm¡1. In addition,
characteristic peaks for collagen are seen at 2800–2950 cm¡1 (CDO group), 3420 cm¡1
(C–H stretching), and 1652 cm¡1 (N–H stretching) (161).
Begam et al. (162) studied the different composites of HA with different polymers and
investigated these composites in the field of bone tissue engineering. The present study

Figure 14. FTIR spectra of (a) collagen; (b) the Col/HA nanocomposite (159) (reproduced with permission
of publisher).
 APPLIED SPECTROSCOPY REVIEWS 731

reports the, in vivo performance of zinc doped HAp and HAp/collagen composite (HAC)
using bone morphogenetic protein-2. It was done for a span of two months on New Zealand
rabbit model (162). The narrow sharp peak at 3570 cm¡1 reveals O–H group. The asymmet-
ric v4 stretching vibration of PO4 groups is observed at 568 and 599 cm¡1. The bands at
1423 and 1454 cm¡1 are observed for the carbonate groups. In the FTIR spectra for collagen,
the broad peak at 3300 reveals the amide A band. CO stretching of amide I was found at
1636 cm¡1. The amide III peak was observed at 1349 cm¡1. C–N stretching of amide I and
II was observed at 1560 cm¡1. A typical protein band was seen at 675 cm¡1. From HAp/col-
lagen composite it was observed that the composite consists of peaks of HAp as well as peaks
of collagen. The presence of collagen was confirmed by peaks of amide A at 3350 cm¡1,
amide I at 1639 cm¡1, and amide II at 1558 cm¡1. Wide peak at 3416 represents the
hydroxyl group for HA. POH stretching was observed at 872 cm¡1. Bands 1023 cm¡1,
570 cm¡1, and 606 cm¡1 showed the presence of phosphate groups for HA (162).

6.2. FTIR analysis of collagen composites for guided bone regeneration

Sun et al. (163) proposed that highly flexible hydroxyapatite/collagen (HAP/Col) composite
membranes are deemed to be significant for guided bone regeneration application owing to
their similar chemical composition to that of natural bone, excellent bioactivity, and good
osteoconductivity. However, the mechanical strength of the HAP/Col composite membranes
is usually weak, which leads to difficult surgical operations and low mechanical stability dur-
ing the bone healing process. Herein, the highly flexible ultra-long hydroxyapatite nano-
wires/collagen (UHANWs/Col) composite biopaper sheets with UHANWs weight fractions
ranging from 0% to 100% are facilely synthesized (163).
The FTIR spectra of the UHANWs/Col composite biopaper with different weight fractions of
UHANWs were investigated as shown in Figure 15. The broad absorption peak of the samples
at around 3420 cm¡1 is attributed to the adsorbed water (164). From the FTIR spectrum of the
pure UHANWs inorganic paper, one can see that the absorption peaks around 1098, 1031, 964,
605, and 563 cm¡1 are attributed to the characteristic absorption peaks of PO43¡ groups in HA,
and the absorption band at around 3572 cm¡1 derives from the stretching mode of hydroxyl
(-OH) in UHANWs, overlapping with the broadband of adsorbed water. The FTIR spectrum of
the pure collagen membrane shows strong absorption bands at around 1645, 1550,
and1240 cm¡1, which belong to the amide I (CDO), amide II (N–H), and amide III (C–N) in
collagen. Moreover, the absorption peak of pyrrolidine ring centered at 1449 cm¡1 is also
observed. The above results demonstrate that the integrity of the collagen structure is well pre-
served in the UHANWs/Col composite biopaper (165). The FTIR spectra of the UHANWs/Col
composite biopaper with different UHANWs weight fractions (25, 50, 70, and 90 wt.%) show
both characteristic absorption peaks of collagen and HA. With increasing UHANWs weight
fraction in the UHANWs/Col composite biopaper, the relative intensities of the characteristic
peaks of HA are significantly increased and those of collagen are decreased (165).
Marelli et al. (166) worked on the 3D mineralization of dense nanofibrillar of collagen-
bioglass hybrid scaffolds. Scaffolds for bone tissue engineering must meet a number of
requirements such as biocompatibility, osteoconductivity, osteoinductivity, biodegradability,
and appropriate biomechanical properties. A combination of type I collagen and 45S5 Bio-
glass may meet these requirements, however, little has been demonstrated on the effect of
Bioglass on the potential of the collagen nanofibril16lar 3D mineralization and its influence
732 T. RIAZ ET AL.

Figure 15. FTIR spectra of UHANWs/Col composite biopaper (163) (reproduced with permission of
publisher).

on the structural and mechanical properties of the scaffolds. In this work, rapidly fabricated
dense collagen-Bioglass hybrid scaffolds were assessed for their potential for immediate
implantation. Hybrid scaffolds were conditioned, in vitro, in simulated body fluid (SBF) for
up to 14 days and assessed in terms of changes in structural, chemical, and mechanical prop-
erties. While ATR-FTIR microscopy revealed the presence of hydroxyl-carbonated apatite
on the surface and within the two hybrid scaffolds at days 7 and 14. FTIR confirmed that the
triple helical structure and typical banding pattern of fibrillar collagen was maintained as a
function of time in SBF (166).

6.3. FTIR analysis of collagen composites for osteogenic differentiation

Bone defect is a major clinical problem observed in a variety of conditions, such as trauma or
osteopathia, and treatments such as autologous or xenogeneic bone grafts have severe short-
comings (167). Huang et al. (168) investigated the effects of hydroxyapatite/collagen com-
posite on the osteogenic differentiation of rat bone marrow. The HA/Col composite was
developed by bioinspired mineralization. The FTIR spectra was taken for pure HA and
HA/Col composite. Several characteristic bands showed the presence of HA. Besides the typ-
ical peaks of phosphate bands in HA, the HA/Col composite also showed the characteristic
peaks at 1655 and 1550 cm¡1, corresponding to the amide I and II in Collagen component,
respectively. The CO32- bands appeared at 1410–1460 and 876 cm¡1 in the spectra of both
HA and HA/Col composite, which indicated the substitution of PO with CO32¡groups. The
broadband peak in the range of 3000–4000 cm¡1 represent the –OH functional group of
absorbed water in the collagen/HA composites (167).

7. FTIR analysis of collagen-based materials for dental applications

Akkouch et al. (169) design a new natural/synthetic bioactive bone scaffold for potential use
in bone replacement application. They developed a tri-component osteogenic composite
 APPLIED SPECTROSCOPY REVIEWS 733

scaffold made of collagen (Col), HA, and poly(L-lactide-co-e-caprolactone) (PLCL). This

Col/HA/PLCL composite scaffold was combined with human osteoblast-like cells obtained
by differentiation of dental pulp stem cells (DPSCs) to engineer bone tissue in vitro. The
chemical structure of the bone nodules was assessed by FTIR analysis in the region of 400–
1800 cm¡1. The bands at 1650, 1550, and 1270 cm¡1 were attributed respectively to the
amide I (CDO stretching of proteins), amide II (C–N stretching and N–H bending of pro-
teins), and amide III regions of the Coll. The bands at 1425 cm¡1 were assigned to the
v3(CO32¡) mode of the carbonate in the HA. The bands at 1070 and 1040 cm¡1 were
assigned to asymmetric absorptions of the phosphate groups v3(PO)43¡ in the HA, and the
band at 1130 cm¡1 corresponded to the absorption of v3(HPO43¡), which was the non-stoi-
chiometric hydroxyapatite (newly formed HA). Thus, our results show that the osteoblast-
like cells displayed the ability to produce new mineralized bone matrix when cultured on the
Coll/HA/PLCL 3D scaffold (169).
Fu et al. (170) studied the difference between stem cells and their extracellular microenvi-
ronment. The study is of critical importance in the stem cell-based therapeutics in regenera-
tive medicine. Mineralized collagen is the main component of bone ECM, but the effect of
interfacial properties of mineralized collagen on subsequent cellular behaviors is unclear.
This study examined the role of surface chemistry of nanoscale mineralized collagen on
human periodontal ligament stem cell (hPDLSC) fate decisions. The intrafibrillarly mineral-
ized collagen (IMC), fabricated by a biomimetic bottom-up approach, showed a bone-like
hierarchy with nano-hydroxyapatites (nHAs) periodically embedded within fibrils (170).
All FTIR spectra displayed typical peaks of collagen: the peak between 1585 and
1720 cm¡1 (amide I). Absorbance band for Amide II (1500–1585 cm¡1 was attributed to the
CDO stretching vibration), arising from angular deformation of NDH bond, was observed
in the IMC and COL. Compared with the IMC and COL, the intensity of amide I and amide
II bands in the extrafibrillarly mineralized collagen (EMC) scaffold decreased quite markedly
to the extent that the amide II peak nearly disappeared. Typical vibration bands of phosphate
(900–1200 cm¡1) indicating mineral particles, were identified in both the IMC and EMC
scaffolds. Furthermore, the vibration band (855–890 cm¡1) of carbonate apatite was also
observed in the IMC, resulting in a similar mineral phase to natural bone. The broad peak
between 3000 and 3400 cm¡1 (O–H band) identified in the IMC and COL was related to the
absorption of water in the scaffolds (170).
The recently developed micro-FTIR, which offers a unique insight at the molecular scale
to monitor the structural changes of bio-samples (171), was applied to determine the micro-
structural details on chemical composition and spatial distribution of calcification of the
scaffolds. The IR spectrum of the IMC showed the presence of phosphate, carbonate (from
carbonate substitution for hydroxyl and phosphate groups in HA), amide I and II bands,
which is quite similar to that of natural bone (172). Compared with the COL, the intensity
of amide I and II bands in the IMC only decreased a little, whereas that in the EMC reduced
substantially. This may be because a large quantity of HAs deposited around the collagen
fibril surface and covered up the CDO stretch (amide I) and N–H in-plane bend (amide II).
Previous study has demonstrated that calcium phosphate crystals could block the 1D vibra-
tion CDO stretch and the 2D vibration N–H in-plane bend during collagen mineralization
(173). Besides, the peak intensity of O–H band in the EMC was much lower than that in the
IMC and COL, indicating that the calcium ions may chelate with hydroxyl group at the sur-
face of collagen fibrils and then grow into micro-scale particles.
734 T. RIAZ ET AL.

Qian et al. (174) constructed a biomimetic structure and incorporated bioactive mol-
ecules into it to achieve the favorable cell response. To explore the effect of electrospin-
ning nanofibrous architecture and collagen I incorporated modification on tuning
osteoblast response, a resorbable membrane composed of poly (lactic-co-glycolic acid)/
poly (caprolactone) (PLGA/PCL; 7:3 w/w) was developed via electrospinning. COL I
was blended into PLGA/PCL solution to prepare composite electrospun membrane.
Notably, relatively better cell response was delivered by the bioactive electrospinning
based membrane which was fabricated by modification of 3,4 dihydroxy-pheny-lalanine
and COL I (174).
In FTIR spectra, there were two characteristic absorption bands at 1756.4 and 1725.3 cm¡1
attributed to the free ester groups in the pristine PLGA and conjugated ester groups in PCL,
respectively. The absorption band of PLGA/PCL was similar to the electrospun PLGA/PCL
except the strength. When the collagen was added, the typical bands of collagen were not found
due to lower concentrations of collagen (175). However, the peak strength of spectra of the free
ester groups in the pristine PLGA and conjugated ester groups in PCL changed. It indicated that
the surface composition had changed. After the introduction of pDA, the corresponding conspic-
uous peaks of pDA–PLGA/PCL ES appeared at 3000–3400 cm¡1, which were attributed to the
N–H stretching and the O–H stretching, respectively. The peak strength of methyl and methy-
lene groups was decreased, respectively. For the FTIR spectra of electrospun COL I–pDA–
PLGA/PCL, a band attributed to the CDO stretching vibration at 1653.2 cm¡1 and a band to N–
H bending at 1557.9 cm¡1 for amide were seen. This result suggested that the collagen was
grafted on the pDA–PLGA/PCL ES surface (176).

8. FTIR analysis of collagen composites for cleft palate

Cleft palate is a congenital malformation that generates a maxillofacial bone defect around
the mouth area. Sangkert et al. (177) synthesized the scaffolds for bone tissue engineering in
cleft palate to reduce this issue. Silk fibroin was selected as the scaffolding material because
of its good biocompatibility, high stability, and non-toxicity. Silk fibroin scaffolds were pre-
pared by freeze-drying before immerging in a solution of collagen, decellularized pulp, and
collagen/decellularized pulp (177).
As previously mentioned for the collagen, the FTIR spectrum showed the important peak of
–OH groups at 3500 cm¡1 (122). In this research, the –OH groups of collagen appeared at a
wavenumber of 3303 cm¡1 that shifted to a lower wavenumber (Figure 16). This indicated that
the –OH groups interacted with the other groups. This interaction came from the self-assembly
of the collagen molecules, which showed that they could organize themselves into a higher order
structure. The wavenumber of the –OH groups in the coated silk fibroin scaffolds shifted to a
higher wavenumber. This result indicated that the –OH groups could vibrate freely in the decel-
lularized pulp. Collagen/decellularized pulp showed –OH groups at 3464 cm¡1 which is possibly
the merged peak of collagen and decellularized pulp. Furthermore, it demonstrated that the
–OH groups in the collagen/decellularized pulp could vibrate freely (177).

9. FTIR analysis of collagen-based materials used in alveolar ridge preservation

Recently Wang, et al. (178) prepared the composites of collagen type I and in this study, they
prepared a novel composite of type I collagen and HA with b-tricaleium phosphate (TCP)
 APPLIED SPECTROSCOPY REVIEWS 735

Figure 16. FTIR spectra (A) collagen, (B) decellularized pulp, and (C) collagen/decellularized (177) (repro-
duced with permission of publisher).

scaffold (CHTS) by incorporating type I collagen and bovine calcined bone granules, pre-
pared as a mixture of 50% HA and 50% TCP, by freeze drying.
The FTIR spectra of these scaffolds showed typical peaks at 1030 and 602/566 cm¡1,
indicative of the n3 and n4 vibrations of PO43– of HA (Figure 17). The peaks at 1547 and
1600 cm¡1 indicated the presence of COO¡ groups in collagen. Compared with the relatively
low peaks of the Bio-Oss Collagen, the collagen component in the CHTS did not change sig-
nificantly after the composite process.

Figure 17. FTIR patterns of the CHTS and Bio-Oss Collagen (178) (reproduced with permission of
publisher).
736 T. RIAZ ET AL.

Conclusion
Collagen is the major component of bone and skin of mammals. It has been extracted from
various sources for development of biomaterials. The FTIR technique is a powerful method
for a rapid and thorough chemical structural characterization of collagen properties from a
range of sources. Identifying precisely and accurately chemical structural properties of colla-
gen from different sources is a challenge. Collagen-based biomaterials (from natural and
synthetic sources) can be easily characterized and distinguished using non-destructive FTIR
spectroscopic technique, which provides excellent molecular-level information, types of
functional groups, bonding types, and molecular conformations.

Acknowledgements
This work has been supported by IRCBM, COMSATS Institute of Information Technology Lahore
and Higher Education Commission of Pakistan under NRPU program for project No 4146.

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