Advances in Biological Chemistry, 2011, 1, 24-28 ABC
doi:10.4236/abc.2011.12004 Published Online August 2011 (http://www.SciRP.org/journal/abc/).
Published Online August 2011 in SciRes. http://www.scirp.org/journal/ABC
Reliability of fourier transform infrared spectroscopy in the
characterization of human skin
Maria O. Longas1*, Kenya Cheairs1, Michelle M. Puchalski1, Jung I. Park2
1Department of Chemistry and Physics, Purdue University Calumet, Hammond, USA;
2St. Margaret Mercy Health Care Center, Hammond, USA.
Received 24 June 2011; revised 3 July 2011; accepted 5 August 2011.
ABSTRACT
Fourier transform infrared (FT-IR) spectroscopy, an
organic molecule characterizing tool, is used here to
differentiate young (36  2.87 years) and aged (78 
1.25 years) skins, based on glycosaminoglycan (GAG)
and protein functional groups. Female breast mas-
tectomy-skin, FT-IR spectroscopy revealed intensity
differences that were quantified on GAG and protein
standard curves, and assigned to the corresponding
functional groups. Band intensity reductions at 78-
years include: 34.37% (w/w) 1259 - 1223 cm–1, sul-
fate (SO42–)/sulfonate (SO3–) S=O/phosphate (PO42־)
P=O stretch; 32.00% (w/w) (1383-1262 cm-1, GAG-
methyl C-H/C-C-H); and 35.60% (w/w) 1738 - 1646
cm–1, C=O stretch: N-acetylated GAG’ s, Amide I , and
others. Intensity increments at 78-years are 63.32%
(w/w) (1636 - 1523 cm–1, Phe/Trp/ Tyr -C=C, Amide II);
27.02% (w/w) [1511 - 1457 cm–1, protein (CH2)/
(CH3) stretch]; and 41.90% (w/w) (1218 - 1139 cm–1,
Phe/Trp/Tyr C-H/C-N/C-C6H5 vibrations). The data
speak to the power of FT-IR spectroscopy as a non-
invasive tool to diagnose tissue disorders such as skin,
liver, kidney or any other type that would require a
noninvasive tool like FT-IR, to prevent further dam-
age during the diagnosis. These results also demon-
strate an age-mediated decrease of skin-GAG content,
and GAG-N-acetylation, in addition to protein com-
position concentration increments.
Keywords: Infrared Spectroscopy; Glycosaminoglycans;
Skin; Age
1. INTRODUCTION
Infrared (IR) spectroscopy is a useful technique to iden-
tify organic, functional groups [1]. Monosaccharides, ho-
mopolysaccharides, glycosaminoglycans (GAG’s) and
amino acids have been characterized, using IR spectros-
copy [2-4]. This technique has also been employed suc-
cessfully in the analysis of age-mediated GAG altera-
tions [5], elucidation of protein secondary structure [6],
and protein conformational changes during reaction [7,8].
Its value in determining relative hydration states of hya-
luronic acid (hyaluronan, HA) and chondroitin sulfate
have been demonstrated [9]. Different types of cells may
be characterized using FT-IR spectroscopy [10], and
various pathologic tissues may be classified [11].
Specifically, FT-IR has been used to investigate the
following cases: Examination of the stratum corneum
barrier function in vivo [12]; detection of conformational
changes during differential apoptosis of HL60 cells [13];
and monitoring their apoptosis progress [14]. Evidence
of proton wires created by proton radicals of the O-H···O
bond group, with at least four other O-H···O groups on
either side of this chain, can act as an electrical wire to
separate a positive charge [15]. Finally, the addition of a
13C at a specific position of the amino acid tyrosine in a
protein, changes the FT-IR vibration position of the C-C
aromatic tyrosine ring to a different frequency; this fa-
cilitates detection of structural alteration when the pro-
tein folds, unfolds or forms aggregates [16]. The uses of
FI-IR continue to accumulate. This paper demonstrates
the reliability of FT-IR spectroscopy in the characteriza-
tion of whole human skin as a function of age.
2. MATERIALS AND METHODS
Glycosaminoglycan standards were purified from calf
ligamentum nuchae, following procedures described pre-
viously [17,18]. Water was filtered, passed through or-
ganic removal and mixed-bed ion-exchange cartridges
(Millipore Corp., Bedford, MA), and distilled. Purified
water was collected in glass, stored under nitrogen until
used and tested for free amino acids, sugars, and organic
compounds commonly found in water (chlorinated hy-
drocarbons and polyaromatic molecules). For this pur-
pose, 2L of purified water was concentrated to 200 l. A

M. O. Longas et al. / Advances in Biological Chemistry, 2011, 1, 24-28 25
50 l aliquot was subjected to reaction with phenyli-
sothiocyanate to get phenylthiohydantoin (PTH) amino
acids, which were characterized and quantified using
high performance liquid chromatography (HPLC) [19];
another 50 l aliquot was subjected to gas
chro-matography/mass spectrometry (GC/MS), and ana-
lyzed for chlorinated hydrocarbons and polyaromatic
molecules; and a third 50 l portion was subjected to
silylation as described by Pierce (P.O. Box 117, Rockford,
IL), and tested for silylated sugars, utilizing GC/MS. The
results show that, under the conditions used, purified
water was devoid of detectable amino acids, sugars or
the aromatic compounds indicated above. All other re-
agents were of the best quality commercially available.
Disposable, breast skin of Caucasian women (excised
during mastectomy and certified as normal by the pa-
thologist in service) was obtained at area hospitals within
two hr from surgery and stored at –70˚C until used. The
patients who donated the skin went to the hospital at
their own will, and signed consent forms authorizing the
hospital to dispose of the tissues removed from their
bodies, following approved procedures, or to use them
for teaching or scientific research.
2.1. Fourier Transform Infrared Spectroscopy
Frozen skin was allowed to warm up to room tempera-
ture and scanned on the epidermal side using a Perkin
Elmer Spectrum 1000 FT-IR spectrophotometer equipped
with Spectrum software and LiTaO3 detector. Just prior
to use, the instrument was calibrated with compounds of
known IR bands; the energy was adjusted to the same
level before each run, and known standards were scanned
periodically to ascertain band position. Spectra from
standard goat/anti-rabbit IgG were obtained in 250 scans
in triplicate at a resolution of 4.0 cm–1, and at sampling
intervals of 1.0 cm–1 at room temperature, using solid KBr
blanks. The Amide I band of this protein at 1650 - 1645
cm–1 was quantified and used as sensitivity calibration; its
area did not deviate from ±2.0% from the assigned value.
Whole, Caucasian, female breast skin of varying ages
was then scanned under the conditions indicated above.
The 250 FT-IR scans were selected after preliminary
experiments demonstrated that spectra collected, utiliz-
ing these conditions, had a higher degree of reproduci-
bility when compared to spectra collected in just a few
scans. Namely, band areas were more prominent and
symmetric; triplicates of spectra collected in 250 scans
yielded more reproducible data than 8 or 10 spectra col-
lected in a few scans; and the signal to noise ratio was
significantly reduced.
2.2. Standard Curve Generation
Highly purified dermatan sulfate (DS) of calf liga-
mentum nuchae was dissolved in water at a concentra-
tion of 1 mg/ml. Samples containing 2 to 20 µg of DS,
50 mg of FT-IR grade KBr and water to a total volume
of 150 µl were lyophilized to dryness. The control con-
tained KBr (50 mg) and water (150 µl). The dry mate-
rial was triturated thoroughly, and subjected to FT-IR
spectroscopy as described above. DS concentrations
between two and seven µg produced linear absorbance
curves. Goat anti-rabbit IgG, D-GlcA, D-GalNAc,
D-GlcNAc, and L-Phe were also employed to generate
standard curves, under the same conditions, and thus
confirm the DS bands.
2.3. Chemometrics
Fourier-transform-IR spectra collected in % transmittance
were converted to absorbance and normalized, utilizing
Spectrum, the software provided by Perkin Elmer, Inc.
for the IR spectrophotometer employed. Baseline correc-
tion, spectra smoothing, optimization, and area band in-
tegration were carried out using the same software. This
is a good program that comes with that FT-IR. It pro-
vides the basis to get the area required, and the means to
convert bands into their respective areas bands. Band
areas on the skin spectra were then identified, using pro-
tein (goat anti-rabbit IgG) and DS FT-IR standard curves,
using the corresponding functional groups. Band identity
was also corroborated using the free sugars, and amino
acids listed in the following paragraph. The averages of
triplicate spectra from three different skins of every age
group are reported here.
3. RESULTS AND DISCUSSION
Typical FT-IR spectral patterns of whole, Caucasian fe-
male, breast skin at 36  2.87 and 78  1.25 years appear
in Figure 1. Except for band intensities, these spectra
were the same for all the skins analyzed, and displayed
bands characteristic of GAG’s, proteins, DNA and
phospholipids (among others). The bands were quanti-
fied on standard curves of DS functional groups, such as
those shown in Figures 2 and 3. Although a standard
curve was developed for every DS functional group de-
tected, only two of them are shown (Figures 2 and 3).
The data were corroborated using standard curves of
goat anti-rabbit IgG, free D-GlcA, D-GalNAc,
D-GlcNAc and L-Phe (not shown).
The FT-IR spectroscopy band in the 1738 - 1646 cm–1
region originates from: the C=O stretch of monosubsti-
tuted amides like those found in N-acetylated GAG’s [3],
protein backbone (Protein Amide I) [6], ceramides [20],
phospholipid esters [21] and the −COOH asymmetric
vibrations [1]. Figure 2 shows the standard curve of the
DS band in the 1738 - 1646 cm–1 region that was used to

M. O. Longas et al. / Advances in Biological Chemistry, 2011, 1, 24-28 26
Figure 1. Typical FT-IR Spectra of whole Caucasian female
breast skin. A, at 36 ± 2.87 years. B, at 78 ± 1.25 years. Spectra
were collected in 250 scans in triplicate, utilizing skin from three
different subjects of every age-group. Shaded, band areas were
quantified on standard curves of DS functional groups. See the
text for additional information.
Figure 2. Standard curve of the FT-IR absorbance band area in
the 1738 - 1646 cm–1 region of the DS spectra. Refer to the text
for additional information.
Figure 3. Standard curve of the FT-IR absorbance band area in
the 1383 - 1262 cm–1 region of the DS spectra. See the text for
more details.
quantify the corresponding FT-IR bands on the spectra
of human skin (Figure 1). The area of this band de-
creased by 35.60% (w/w) in the older group (Ta bl e 1 ).
Such band is made out of contributions from molecules
known to increase with age like proteins [22], and from
reactive carbonyl compounds that are believed to cause
“the carbonyl stress” [23], minus reductions from mole-
cules that decreased, such as GAG-N-acetyl groups [5,
18] ceramides [20], and phospholipids [21]. The 35.60%
(w/w) decrease of band intensity at 1738 - 1646 cm–1 in
the older group, indicates that there were more losses
than gains in the number of molecules containing func-
tional groups that absorb IR light in this region.
Because of the various positive and negative contribu-
tions to band intensity in the 1738 - 1646 cm–1 region
(C=O stretch) described above, the degree of
age-mediated GAG-N-deacetylation was calculated util-
izing the FT-IR spectroscopy band in the 1383 - 1262
cm–1 domain that is more specific for N-acetyl moieties
(Figure 3). This band is known to originate from C-H
and C-C-H vibrational modes of methyl groups in
monosubstituted amides (as in GAG’s) coupled to car-
bohydrate C-C-H, O-C-H and C-O-H vibrations3. Its
intensity decreased by 32.00% (w/w) in the skin spectra
of the 78  1.25- year-olds (Table 1). These results sug-
gest an age-mediated N-deacetylation of GAG’s, and
confirm previous data obtained utilizing purified mole-
cules [5,18].
The 34.37% (w/w) reduction of band intensity at 1259
- 1223 cm–1 (Ta ble 1) in the older group suggests a loss
of DNA as the skin ages, since this band may arise from
the P=O stretch of phosphates (PO4
2–) as in nucleic acids
that are known to decrease with aging, in particular mi-
tochondrial DNA [24,25]. Stretching vibrations of the S
= O bond of sulfates (SO4
2–) and sulfonates (SO3–) (as in
GAG’s) also appear in the latter region, but previous
data obtained using purified molecules indicate that
GAG sulfate composition is not significantly affected
during aging [5,18].
The areas calculated for every group was estimated on
the basis of standard curves generated from groups
originating from goat anti-rabbit IgG and from the cor-
responding DS FT-IR bands. These data show that in
whole Caucasian female breast skin, GAG concentration
and degree of GAG-N-acetylation decreased with aging,
while protein content increased. Although the actual
numbers obtained in this study are not the same as the
ones reported previously [5,18,26], these data demon-
strate an age-related decrease in GAG concentration and
GAG-N-acetylation, and a significant increment in pro-
tein content. These results speak to the power of FT-IR
spectroscopy as a noninvasive tool to diagnose tissue
disorders such as skin, liver, kidney or any other type

M. O. Longas et al. / Advances in Biological Chemistry, 2011, 1, 24-28 27
Table 1. Quantitation of skin FT-IR spectroscopy absorbance bands.
Age
(Years) Functional Group Wavenumber (cm–1)
Band Area
(g)
Change
(%)
36  2.87
78 ± 1.25
aS=O and P=O stretch vibes
aS=O and P=O
1259 - 1223
1259 - 1223
0.11  0.02
0.07 ± 0.02
34.37↓
36  2.87
78  1.25
bmethyl C-H/C-C-H & C-C-H,
0-C-H & C-O-H of CHO’s
1383 - 1262
1383 - 1262
1.63  0.15
1.11  0.09
32.00
36  2.87
78  1.25
cC=O + Asym −COOH stretch
Protein Amide I
1738 - 1646
1738 - 1646
5.49  0.77
3.53  0.47 35.60
36  2.87
78  1.25
Phe, Trp, Tyr C=C-vibes
Protein Amide II
1636 - 1523
1636 - 1523
1.80  0.32
0.66  0.11
63.32
36  2.87
78  1.25
Protein -(CH2), (CH3)-vibes
1511 - 1457
1511 - 1457
0.455  0.02
0.624  0.09 27.02
36  2.87
78  1.25
C-H, C-N; Phe, Trp, Tyr
C-C6H5-vibes
1218 - 1139
1218 - 1139
0.820  0.14
1.410  0.11
41.90
aS=O stretch of sulfates (SO42–) and sulfonates (SO3–) as in GAG’s, and P=O stretch of PO42– as in DNA. bCH3 of monosubstituted amides, cacetamido group of
GAG’s, and phospholipid esters. Absorbance band areas of triplicate spectra from three different skins of every age-group were used to determine the corre-
sponding concentrations, using DS standard curves; the averages are shown. See the test for more information. , decrease; , increase.
that would require a noninvasive tool like FT-IR, to pre-
vent further damage during the diagnosis.
4. ACKNOWLEDGEMENTS
This work was supported by Purdue University Calumet Scholarly
Research Release Award and by the NSF NS-FILIP DUE 9650816
grant to M. O. Longas.
REFERENCES
[1] Solomons, T.W. and Fryhle, C.B. (2004) Oganic Chemis-
try. 8th Edition, John Wiley & Sons Inc., New York, 79-
93.
[2] Orr, S.F.D. (1954) Infra-red spectroscopic studies of
some polysaccharides. Biochimica et Biophysica Acta, 14,
173-181. doi:10.1016/0006-3002(54)90156-0
[3] Cael, J.J., Isaac, D., Blackwell, H.J., Koenig, J.L., Atkins,
E.D.T., Sheehan, J.K. (1976) Polarized infrared spectra of
crystalline glycosaminoglycans. Carbohydrate Research,
50, 169-179. doi:10.1016/S0008-6215(00)83848-3
[4] Venyaminov, Y. and Kalnin, N.N. (1990) Quantitative IR
spectrophotometry of peptide compounds in water (H2O)
solutions. I. Spectral parameters of amino acid residue
absorption bands. Biopolymers, 30, 1243-1257.
doi:10.1002/bip.360301309
[5] Longas, M.O., Russell, C.S. and He, X-Y. (1986) Bio-
chimica et Biophysica Acta, 884, 265-269.
[6] Venyaminov, Y. and Kalnin, N.N. (1990) Quantitative IR
spectrophotometry of peptide compounds in water (H2O)
solutions. II. Amide absorption bands of polypeptides
and fibrous proteins in α-, β-, and random coil
con-formations. Biopolymers, 30, 1259-1271.
doi:10.1002/bip.360301310
[7] Kötting, C. and Gerwert, K. (2005) Proteins in Action
Monitored by Time-Resolved FTIR Spectroscopy. Chem
Phys Chem, 6, 881-888. doi:10.1002/cphc.200400504
[8] Pinakoulaki, E., Koutsoupakis, C., Stavrakis, S., Agge-
laki, M., Gambaro, G., Papadopoulos, V. and Daskalakis,
C., Varotsis, (2005) Structural dynamics of heme-copper
oxidases and nitric oxide reductases: Time-resolved step-
scan Fourier transform infrared and time-resolved reso-
nance Raman studies. Journal of Raman Spectroscopy, 36,
337-349. doi:10.1002/jrs.1313
[9] Servaty, R., Schiller, J., Binmdier, H. and Arnold, K.
(2001) Hydration of polymeric components of cartilage
—an infrared spectroscopic study on hyaluronic acid and
chondroitin sulfate. International Journal of Biological
Macromolecules, 28, 121-127.
doi:10.1016/S0141-8130(00)00161-6
[10] Gaigneaux, A., Ruysschaert, J.M. and Goormaghtigh, E.
(2002) Infrared spectroscopy as a tool for discrimination
between sensitive and multiresistant K562 cells.
Euro-pean Journal of Biochemistry, 269, 1968-1973.
doi:10.1046/j.1432-1033.2002.02841.x
[11] Crupi, V., Venuti, V. and Majolino, D. (2004) Spec-
troscopy, 19, 22-30 & 42.
[12] Bommannan, D., Potts, R.O. and Guy, R.H. The Society
for Investigative Dermatology, Inc., 1990, 95, 403-408.
[13] Zhou, J., Wang, Z., Sun, S., Liu, M. and Zhang, H. (2001)
A rapid method for detecting conformational changes
during differentiation and apoptosis of HL60 cells by
Fourier-transform infrared spectroscopy. Biotechnology
and Applied Biochemistry, 33, 127-132.
doi:10.1042/BA20000074
[14] Gasparri F. and Muzio M. (2003) Monitoring of

M. O. Longas et al. / Advances in Biological Chemistry, 2011, 1, 24-28 28
apoptosis of HL60 cells by Fourier-transform infrared
spectroscopy. Biochemical Journal, 369, 239-248.
doi:10.1042/BJ20021021
[15] Stoyanov, E.S., Stoyankova, I.V. and Reed C.A. (2008)
IR Spectroscopic Properties of H(MeOH)n
+ Clusters in
the Liquid Phase: Evidence for a Proton Wire. European
Journal of Chemistry, 14, 3596-3604.
doi:10.1002/chem.200701746
[16] Beyermannn, M., Tremmel, S., Oschkinat, H., Bienert, M.
and Hainz, F. (2005) spectroscopyNOW.com
[17] Meyer, K. (1958) Fed Proc. 17, 1075-1077.
[18] Longas, M.O., Russell, C.S., He, X-Y. (1987) Evidence
for structural changes in dermatan sulfate and hyaluronic
acid with aging. Carbohydrate Research, 159, 127-136.
doi:10.1016/S0008-6215(00)90010-7
[19] Tsunasawa, S., Kondo, J. and Sakiyama, F.J. (1985)
Biochemistry, 97, 701-704.
[20] Coderch, L., López, O., Maza, A. and Parra, J.L. (2003)
Ceramides and Skin, 4,107-129.
[21] Schroeder, F., Goetz, I. and Roberts, E. (1984)
Age-related alterations in cultured human fibroblast
membrane structure and function. Mechanisms of Ageing
and Development, 25, 365-389.
doi:10.1016/0047-6374(84)90010-1
[22] Gniadecka, M.,Nielsen, O.F., Christensen, D.H. and Wulf,
H. (1998) Structure of water, proteins, and lipids in intact
human skin, hair, and nail. Journal of Investigative Der-
matology, 110, 393-398.
doi:10.1046/j.1523-1747.1998.00146.x
[23] Schleicher, E.D., Bierhaus, A., Häring, H-U., Nawroth,
P.P. and Lehmann, R. (2001) In: D’Angelo, A., Favaro, S.
and Gambaro, G. Eds., Chemistry and Pathology of Ad-
vanced Glycation End Products, Advanced Glycation
End Products in Nephrology, Contrib. Nephrol., Basel,
139, 1-9.
[24] Proksch, E., Feingold, K.R., Man, M.Q. and Elias, P.M.
(1991) Journal of Clinical Investigation, 87, 1668-1673.
doi:10.1172/JCI115183
[25] Alexeyev, M.F., LeDoux, S.P. and Wilson, G.L. (2004)
Barrier function regulates epidermal DNA synthesis.
Clinical Science, 107, 355-364.
doi:10.1042/CS20040148
[26] Rocquet, C. and Bonté, F. (2002) Acta Derma-
tovenerologica Alpina, Pannonica Et Adriatica, 11, 1-59.
Abbreviations
DS: dermatan sulphate;
DNA: deoxyribonucleic acid;
FT-IR: Fourier transform infrared;
GC/MS: gas chromatography/mass spectrometry;
GAG: glycosaminoglycan;
HA: hyaluronic acid, hyaluronan