Crosslinking of Gelatin with Formaldehyde; a 13C NMR Study

Klaus A lbert, Bernadette Peters, Ernst Bayer*

Institute for Organic Chem istry, A u f der M orgenstelle 18, D-7400 Tübingen
Ulrich Treiber, and Michael Zwilling
Du Pont de Nem ours (D eutschland) G m bH . Dornhofstraße 10, D-6078 Neu-Isenburg
Z. Naturforsch. 41b, 3 5 1 -3 5 8 (1986); received N ovem ber 22, 1985
A q ueous Formalin Solution C om positions, G elatin Cross-Linking. Gelatin Hardening,
Static and Continuous Flow , 13C NM R
The different species formed when gaseous formaldehyde is dissolved in aqueous medium are
investigated by a l3C N M R flow-through technique and the spectra of commercial aqueous form ­
aldehyde solutions containing m ethanol com pletely interpreted.
I3C H ,0 was used to study the crosslinking reaction with gelatin which leads to m ethylene and
oxym ethylene bridges betw een gelatin-am ino-groups. On ageing, hardened gelatin shows an
increase in the number o f m ethylene bridges.

Introduction N M R flow-through experim ent

Formaldehyde is widely used for crosslinking pro­
teins: e.g. in the leather and photographic industry,
in the biological and medical sciences. This crosslink-
ing-reaction however isn’t yet fully understood
[1 -5 ]-
The different com pounds present in aqueous form ­
aldehyde solutions are of interest for these reactions,
and the structures of the species have been studied
by 13C NM R (e.g. [6 , 7], This paper describes the
measurement of form aldehyde solutions with in situ
formation of the oligom eric hydrates by using a con­
tinuous flow through technique.
E xperim ental
Preparation of gelatin solutions and hardening.
600 mg gelatin (inert gelatin from R ousselot,
France) were allowed to swell 10 min in 5 ml distilled
water, then dissolved at 38 °C with further 3.4 ml
more distilled water and 1 ml D 20 . The resulting
solution had a concentration of 0.2744 mM/1. One
drop sodiumazide solution (65 mg/ml) was added.
Prior to m easurem ent, 4 ml of this solution were
transfered to a 10 mm NM R tube and degassed in an
ultrasonic bath. To harden the gelatin, 7.23 u\ 30%
formaldehyde solution (N o 1 from Ruehl Chem ie,
stabilized with 0.5% m ethanol and N o 2 from Emes-
co Chemikalien without m ethanol) or 1 1 /<1 20%
I3CH20 (Merck, Sharp & D ohm e, Canada) were ad­
ded to the above m entioned gelatin preparation.
* Reprint requests to Prof. Dr. Ernst Bayer.
Verlag der Zeitschrift für Naturforschung, D -7400 Tübingen
0 3 4 0 -5 0 8 7 /8 6 /0 3 0 0 -0 3 5 1 /$ 01.00/0
Paraformaldehyde was heated to 170 °C [8] and
the generated gaseous formaldehyde transferred
with a N 2 stream into 100 ml phosphate buffer solu­
tion (pH 7.2) containing 5% (v/v) D 20 . A t the same
time the solution was circulated by a tubing pump
(1 ml/min) through a ,3C NM R flow through cell,
which is described elsewhere [9, 10]. A fter an initial
period of 5 min, 13C m easurements were conducted
in time intervals of three minutes. After one hour the
solution contained 5% form aldehyde, as quantita­
tively determinated with dim edone [ 11].
N M R measurements
Spectra of 13CH:0 hardened gelatin and the flow
through spectra were measured on Bruker WM 400
(100.6 M Hz) NM R spectrometer. U p to 40 K scans
with a pulse-repetition-rate (prr) of 0.7 sec were ad­
ded in the case of the hardening experim ents. The
flow-through spectra were accumulated with 512
scans and prr = 0.344 sec. All other measurements
were conducted with a Bruker NM R spectrometer
W H 90 (22.63 MHz) by accumulating up to 100 K
scan at prr = 1 . 8 sec. R eference was the signal of
external DSS. For field-frequency-stabilization a co­
axially centered tube containing D 20 was used in the
measurements of formaldehyde solutions.
R esu lts and D iscu ssion
The 13C spectrum of commercial form aldehyde so­
lution (1), shows a variety of 15 resonances (Fig. 1).
A first discrimination is possible by determination of
the multiplicity by spin-echo measurements and by
the chemical shifts. The signals between 50 and
 352 K. Albert et al. ■Crosslinking o f G elatin with Form aldehyde

f )
60 ppm can be assigned to - O C H ?-groups and those
between 80 and 100 ppm to —O C H 2-groups in differ­
ent chemical environm ents. Since further assign­
ments cannot be m ade, a determination of the differ­
ent species by reference specira is needed. A
L’C NM R flow through experim ent as described
above shows the rapid formation of a species with
peak at 84.7 ppm (Fig. 2). Because of the quick reac­
tion between form aldehyde and water this signal can
be assigned to the m onom er formaldehyde hydrate
C H 2(O H )2 (A ).
During the time interval 9 - 1 2 min a second signal
occurs at 88.3 ppm which must be assigned to the
dimer reaction product (B) of two hydrate
molecules:
2 CH 2(O H )2 h o - c h 2- o - c h 2- o h + h 2o
A fter further nine m inutes (tim e period
21 —24 min) two more signals at 88.8 ppm and
91.4 ppm with the intensity relation 2:1 can be ob ­
<— ppm
served, indicating the presence of the trimer (C).
Fig. 1. 13C NM R spectrum (22.63 M Hz) o f the commercial The signal at 91.4 ppm is in Fig. 2a hardly to see;
formalin solution 1 (30% form aldehyde, 0.5% methanol in
water). Fig. 2 b shows therefore the time interval 3 3 —36 min

t Imin 1

15-1 8

1 2 -1 5

9-12

J _________ 6 - 9

3-5

0-3 18-21

I' - r—r
95 90 85 95 90 85
r-' 1 I 1 —r- ’
-ppm 95 90 85
■ppm
-ppm
Fig. 2. a) l3C NM R flow-through spectra (100.6 M Hz); formation of form aldehyde hydrate (A ), the dim eric (B ) and
trimeric (C) species upon transferring C H 20 to aqueous medium (pH 7.2);
b) L1C NM R flow-through spectrum (tim e interval: 33 —36 min) with higher am plitude.
 K. Albert et al. ■Crosslinking of Gelatin with Form aldehyde
I'M

t— i— |— f— i— i— i— |— i— i— i— *— |— *— *— i * r T 1 1 1 r
100 75 50 25 o 1— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i p

— ppm 10 0 ■*--ppm 75 50 25 0
Fig. 3. 13C NM R spectra (22.63 M Hz) o f different formalin solutions; a) formalin solution 2 (30% solution); b) formalin solution 2 upon addition of
methanol; c) formalin solution 1 (30% solution with 0.5% methanol); d) formalin solution 1 upon addition o f ethanol.
354
with higher amplitude. This intensity relation indi­
cates the assignment of the resonance at 88.8 ppm to
the two terminal CH;OH-groups of (C ), whereas the
peak at 91.4 ppm results from the central
—C H 2—O — unit.
By this experim ent, the shift ranges in the
13C NM R spectra of pure formaldehyde solutions can
be defined: The monomer (A ) gives a signal at
84.7 ppm, terminal CH2O H — groups peak between
88 ppm and 90 ppm and —CH 2—O — units inside of
the oligom ers show resonances at 91—92 ppm. O bvi­
ously a prolongation of the oligom eric chain results
in a slight shift to lower fields of NM R signals, e.g.
the change from (B) to (C) results in such a shift of
the terminal CH2OH signal.
With these results, the NM R spectrum of the com ­
mercial m ethanol-free formaldehyde solution N o 2
can be fully understood (Fig. 3 a). The signals at
89.0 ppm, 91.9 ppm and 92.3 ppm which occur in
addition to the above m entioned resonances of (A ),
(B) and (C) are in the two ranges of —CH2OH and
—C H 2—O — shifted to lower fields indicating the pre­
sence of a tetrameric (D ) and pentameric (E) form.
This conclusion can be drawn by an incremental
analysis as given in [6 , 7],
On adding an equivalent volum e of m ethanol
((K ), 51.6 ppm) to the formaldehyde solution 2
three additional resonances at 57.2 ppm, 92.3 ppm
and 95.8 ppm occur; two of them can be assigned to
the m onom ethylhem iacetal of formaldehyde ((F);
Fig. 3b): 57.2 ppm representing —OC H3 and
92.3 ppm for the terminal —CH2OH group of this
com pound respectively.
The signal at 95.8 ppm shows the presence of the
m onom ethylhem iacetal of dimeric form aldehydehy-
drate (G ). This resonance, which is shifted to lower
field, relates to the —O C H 2-group of (G) adjacent to
the acetal bond. The —OC H3 and —CH2OH signals
of this com pound are not detectable in the noise.
These assignments are in agreement with the incre­
mental analysis given in [6].
Signals o f the dimethylacetal of formaldehyde are
not observed; the reference spectrum of the pure
com pound shows resonances at 55.8 and 95.2 ppm.
The addition of ethanol ((L ), 19.7 ppm (C H 3) and
59.8 ppm (C H 2O H )) to the formaldehyde solution
N o 1 (Fig. 3 c) results in the formation of the
ethylhem iacetal ((M ), 16.9 ppm (C H 3), 65.5 ppm
(C H 20 ) and 90.7 ppm (terminal CH2OH); Fig. 3d ).
Ethanol predominantly reacts with form aldehyde-
K. A lbert et al. ■Crosslinking o f G elatin with Formaldehyde
hydrate (A ). This conclusion can be drawn from the
ratio of the integrals.
With this reference data set, the spectrum of form­
aldehyde solution N o 1 in Fig. 1 can be fully as­
signed; the shift-areas (from high to low field) are:
a) free m ethanol (K; 51.6 ppm);
b) —O C H 3-groups of hemiacetals with different
chainlength (F, G, H; 5 7 .2 —58.1 ppm);
c) form aldehydehydrate (A ; 84.7 ppm);
d) terminal CH2OH groups of formaldehyde oligo­
mers and o f their hem iacetales respectively (B, C,
D , E , and G, H; 8 8 .3 —89.0 ppm);
e) inner C H 20 groups of hemiacetals and oligomers
in ß-, y-, d-, etc. position to —O C H 3 and the ter­
minal C H 2OH group o f the m onom ethylhem i­
acetal (C, D , E and G , H and F; 9 1 .4 —92.3 ppm).
The terminal CH2OH group of F is shifted to
lower field in comparison to the other CH2OH
groups, because of its a-position to —OC H 3;
f) inner C H 20 groups of hemiacetals of the oligo­
mers in a-position to —OC H3 (G , H, J;
9 5 .8 —96.5 ppm).
A survey o f the detectable species in the aqueous
form aldehyde-m ethanol system is given in Table I.
The given assignment is confirmed by [6 , 7], where
the observed shift phenom ena are summarized in a
system o f increm ents, which correspond to the date
in this work within ± 2 ppm.
Trioxan, a possible cyclic trimeric form of form­
aldehyde with a 13C N M R resonance at 96.2 ppm
(from the reference spectrum of the pure compound)
is neither formed in the flowthrough experiment nor
found in the formalin solution 2. We thus tentatively
conclude the absence of this species in the methanol
containing formalin solution 1.
Hardening of Gelatin
The spectra of the educts and products of the
crosslinking reaction of gelatin with formaldehyde
are shown in Fig. 4; the strong resonance at
84.6 ± 0 .2 ppm from (A ) is present in the spectrum
of the hardened gelatin in comparison to the unhar­
dened form. The signals of oligomers and hem i­
acetals are not detectable, they obviously were con­
sumed by the hardening reaction. One additional sig­
nal at 51.4 ppm is observed in the spectrum of the
hardened gelatin.
To obtain deeper insight into the mechanism of the
crosslinking reaction a hardening experiment was
K. Albert e ta l. • Crosslinking o f G elatin with Form aldehyde 355

Table I. Species in aqueous form aldehyde-m ethanol solutions. The ppm values of 3C NM R resonances are given in
brackets.

[CH2o] CH 3OH
Formaldehyde; not detectable in aqueous solutions M ethanol (K; 51.6 ppm)
(H 20 ) H em iacetals
4
Hydrate (A ) h o - c h 2- o h h o - c h 2o - c h ,
(84.7 ppm) (F; 92.3* ppm, 57.2 ppm)
Dimeric (B) H O - C H 2- O C H 2- O H H O - CH 20 - C H :0 - CH ,
(88.3 ppm) (G ; 88.6* ppm, 95.8 ppm, 57.9 ppm)
Trimeric (C) h o - c h 2o - c h 2o - c h 2- o h h o - c h 2o - c h 2o - c h 2o - c h ,
(88.8 ppm, 91.4 ppm) (H ; 88.6* ppm, 91.9* ppm, 96.3 ppm, 58.1* ppm)
Tetrameric (D ) h o - c h 2o - c h 2o - c h 2o - c h 2- o h - H O - c h 2o - c h 2o - c h 2o - c h 2o - c h 3
(89.0* ppm, 91.9 ppm) (I; 88.6* ppm, 91.9* ppm, 96.5 ppm, 58.1 ppm)
Pentameric (E) h o - c h 2o - c h 2o - c h 2o - c h 2o - c h 2- OH
(89.0* ppm, 92.3* ppm)

Overlap with other resonances.

conducted with 13CH20 (Fig. 5). The 13C NM R spec­

FORMALIN (30%)
trum of this formaldehyde solution is shown in
Fig. 5 b. Obviously a small quantity of the form­
aldehyde in this solution has undergone a dispropor­
tion to methanol (K) and formic acid (168.6 ppm ), so
that beside to the m onom er hydrate (A ) and the di­
meric (B ) their hemiacetals F and G are formed.
In Fig. 5 a and 5d the spectra of unhardened gela­
tin and of the hardened product are depicted and
HARDENED GELATIN
b) Fig. 5 c shows the difference spectrum of both pro­
ducts with 12 signals. The peak at 27.0 ppm obvi­
ously is an artefact arising from incorrect substrac-
tion o f two resonances o f high intensity. One signal
in the range of the carbonyl functions can’t yet be
classified. A t 84.7 ppm the high intensity resonance
of unreacted formaldehyde hydrate is present.
The remaining nine signals represent the reaction
products between gelatin and the formaldehyde solu­
tion. A t 74.1 ppm and at 67.7 ppm the N-m ethylol
resonance of lysine-CH2OH (N) and arginine-
C H 2O H (O ) are observed. These lines coincide with
the assignments in [5] for polylysine and polyarginine
m ethylol with a difference of + 2 .5 and + 2 .8 respec­
tively. Shift differences in the same order o f mag­
nitude (ca. + 3 ) are present between the ppm values of
Fig. 4. 13C NM R spectra (322.63 M Hz) o f
the form aldehyde spectrum in [5] and these reported
a) unhardened gelatin;
b) formaldehyde-hardened gelatin; here. Such methylol compounds react under elim ina­
c) formalin solution 1 used for hardening. tion o f water and form = N —CH2—O —CH2—N = -
356 K. Albert et al. ■Crosslinking o f G elatin with Formaldehyde

d) Hardened Gelatin

Difference Spectrum h-h ([ a N_ 0 P T Q R

IW
B A
b) 13C -Formalin
G F

a) Unha rdene d Gelatin

Fig. 5. 13C NM R spectra (1ÜÜ.6 M Hz) of a) unhardened gelatin; b) l3C formalin solution (20% ); c) difference spectrum of
hardened and unhardened gelatin; d) hardened gelatin.

Table II. R esonances and formulae of the

(N ) H O - C H ,- N H - C H ,- C H ,- C T T - C H ,- C H - C O -
proposed reaction products o f formaldehyde
I with gelatin.
NH-
74.1 ppm; lysinem ethylol
(O ) H O -C H ,-N = C - N H - C H ,- C H .- C H ,- C H - C O -
I I
2 nh n h -
67.7 ppm; argininemethylol
(S) ^n - ch 2- o - ch :- n ^
72.0 ppm; oxym ethylene bridge
(P) Arg. = N —CH ; —N H -L y s.
62.0 ppm; arginine-lysine-m ethylene bridge
(T) Arg. = N - C H 2- N = Arg.
56.0 ppm; arginine-arginine-m ethylene bridge
(H . H ') ^ N - C H 2- 0 - ( C H 2- 0 ) „ - C H 2- N /
96.1 ppm. 99.5 ppm; crosslinking under participation of
oligom eric formaldehyde species
(O ) -C -N H -C H ,-N /
II “ X
O
51.6 ppm; am ide-N H 2/am ine-N H 2-crosslinking
(R ) —C —N H —C H ,—NH —C —
II “ II
o o
45.2 ppm; am ide-N H 2/am ide-N H 2-crosslinking
K. Albert et al. • Crosslinking o f G elatin with Form aldehyde 357

bridges (S) [2]. An analysis with increments from [6] difference only in an increase in the intensity of the
proves that the signal at 72.0 ppm results from such a crosslinking resonance at 51.6 ppm (Q ) on aging
species (e.g. 67.7 ppm (ArgC H 2O H )+ 9 .4 p p m (ß -C )— (Fig. 6). This phenom enon can be explained by the
5.1 ppm (y-N) + 0.3 ppm ((3-C) = 72.3 ppm). expulsion of nascent formaldehyde from oxym ethyl­
From such oxym ethylene bridges crosslinking ele­ ene bridges, which is then available for further
ments = N —CH2—N = (P, T) with a m ethylene bridge hardening reactions. A decrease of the excessive
are formed by elimination of one m olecule form­ m onohydrate (A ) is not observed.
aldehyde. The broad resonances at 62.0 ppm and at
56.0 ppm show the reduced flexibility of this rigid
crosslinking unit. These assignments are consistent
with the data of CH20-crosslinked amino acid m odel
substances in [5]: 54.9 ppm for the reaction product of
polyarginine and 59.3 ppm for the bond between poly­
arginine and polylysine. According to [5] no lysine-
lysine crosslinking is observed.
The low field signals at 96.1 ppm (H ) and 99.5 ppm
(FT) might indicate a reaction of formaldehyde
oligomers with gelatin-NH2 to form longer oxymethyl-
enebridges, e.g. = N —C H 20 —CH20 —CH2—N = .
With increments from [6] and starting from the ppm
value of the central C-atom in trimeric formaldehyde
hydrate (C; 91.4 ppm) a resonance at 94.2 ppm for the
central C-atom in the bridge is expected. The reso­
nances H and H' also indicate the presence of N-
hemiacetal compounds = N —(C H 20 ) „ —CH2—O C H 3. 53 52 51 50 49

Increment calculations [6] with n = 2 result in a ppm ■«- p p m

value of 97.2 ppm for the C-atom near to the —O C H 3
group.
In addition to the discussed reaction products,
crosslinking between either gel.am ide-N H 2 and
gel.am ine-N H 2 or two gel.am ide-N H 2 is possible.
The crosslinking model substances [5] give reso­
nances at 50.5 ppm (am ide-am ine), 41 and 45 ppm
(amide-am ide) respectively. This fits to the remain­
ing two signals in the difference spectrum 5 c:
51.6 ppm (Q) and 45.2 ppm (R ). The amino com ­
pound of these methylene bridges might be lysine or
arginine, and as am ide-com ponent, glutamin and as­
paragine could be involved.
Aging of Hardened Gelatin
A comparison of the spectra of freshly hardened
gelatin and of the 11 months old sample shows a
Fig. 6. I3C NM R spectra (22.63 M Hz) o f hardened gelatin
(with formalin solution 1) in the range o f 4 8 —54 ppm;
a) im m ediately after the hardening reaction; b) after 11
monihs.
Summary
The different formaldehyde species in aqueous so ­
lution show different reactivity with gelatin. Excess
of form aldehyde hydrate remains nearly unchanged,
whereas the oligomers and methylhemiacetals react
with gelatin. The crosslinking reaction takes place on
amino acids and amides in a first step under form a­
tion o f m ethylols with subsequent condensation to a
crosslinked product containing oxym ethylene and
m ethylene bridges.
358
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