Journal of the Society of Leather Technologists and Chemists, Vol. 90 p.

29

MODIFICATION OF CHROME-TANNED LEATHER WASTE

HYDROLYSATE WITH EPICHLORHYDRIN
R. KARNAS, M. MLDEK and K. KOLOMAZNK
F. LANGMAIER, P. MOKREJS,
Fakulty of Technology, Tomas Bata University, Nm. TGM 275, CZ 276 72, Zln, Czech Republic.

Summary
Wider use of chrome-tanned leather waste hydrolysate as a secondary industrial raw material is impeded by the
unclear economic effect of most proposed practical applications. More attention should deservedly go to
hydrolysate utilised as a biodegradable packing material (packing for agricultural chemicals including herbicides,
insecticides, pesticides, fertilizers etc.) The appropriate cross-link density enables us to control the water
solubility of hydrolysate, its biodegradation and the rate at which active substances are released from such
packing. The properties may significantly influence the economic effect of such an application. Increasing the
cross-link density of hydrolysate by reacting it with dialdehydes easily leads to the formation of thermoirreversible gels which are difficult to process. The main attention, therefore, is aimed at cross-linking hydrolysate
with epichlorhydrin.
In an aqueous environment, epichlorhydrin reacts with the primary amino groups of hydrolysate as a
monofunctional agent through its chlorine atom. At temperatures around 60C an equilibrium is attained in about
60 min. characterised by approx. 80% of the primary amino groups present having reacted. Reaction of the
oxirane ring of epichlorhydrin proceeds at considerably higher temperatures (~200C). Cross-linking of
hydrolysate with epichlorhydrin is thus a two-stage reaction, which may be regarded as an advantage for some
applications.

Introduction

exactly the reason why the hydrolysates obtained are

incapable of competing in price with current types of
agricultural fertilisers. In addition, their employment as a
component in livestock feed comes up against low nutritive
value of collagen proteins (polypeptides) and against the
suspicion that feed mixes prepared from them may play no
minor role in spreading the BSE syndrome.

Development of leather manufacture, chiefly in Europe,

is limited to a considerable extent by the large volume of
protein waste produced, which is estimated, by particular
authors, at 40-60% of the mass of starting raw material.
The problem has admittedly received attention since the
end of the 1930s but, solid protein waste, however, has
been mostly disposed of until now to landfill despite the
material in question being a potential secondary industrial
raw material.
When considering processing solid leather waste, the
starting operation is partial hydrolysis making possible
subsequent chemical modification of the hydrolysate in an
aqueous medium, as well as quite simple separation and recleaning of polypeptides formed from substances
potentially contaminating the collagen waste from various
stages of leather manufacture. The preferred procedure is
partial enzymatic hydrolysis, which is the least demanding
on energy, capable of being applied to leather waste both
tanned, greased or otherwise treated as well as non-tanned
collagen waste including that from the manufacture of
meat product casings and also collagen waste from
abbatoirs. Hydrolysates of non-tanned collagen waste can
be used to advantage without further modification as
humectants in cosmetic preparations for skin and hair
care.1-4 As far as concerns the processing of solid tanned
waste from leather production, enzymatic hydrolysis is
interesting for its potential of relatively easily separating
Cr3+ tanning compounds which may be ecologically
controversial.
Hydrolysates, obtained as quite dilute aqueous
solutions, can be stored only with difficulty. Evaporating
excess quantities of water (after separation of tanning
chrome salts or other substances contaminating
hydrolysate) is quite an expensive matter and complicates
to a certain extent the application of hydrolysates in
industrial practice.
In the widely discussed application of protein
hydrolysates as fertilisers in agricultural technology, this is

Glues
The mean molecular mass of hydrolysates obtained
through enzymatic procedures usually ranges within limits
of 15-50kDa.5 These are capable, in a similar fashion to
gelatine or glues, of producing thermo-reversible gels
although, of course, at considerably higher concentrations
of dry matter. The rigidity of such gels (similarly to glues)
depends on their mean molecular mass (weighted mean
Mw) and dry matter concentration. At dry matter
concentrations of 20-35 % (w/w), gels of enzymatic
hydrolysates usually exhibit rigidities of 150-350g -1
(Bloom).6 Their quality, assessed in a standard manner, is
thus not too high and makes it impossible to consider their
application as adhesives for functional bonds with the
exception, maybe, for sticking labels (e.g., on glass bottles
and such) that should be removable with water. The
economic advantage of such applications, when compared
with lower quality glues or even starch adhesives, is hardly
appreciable.
In aqueous solutions, collagen hydrolysates behave like
substances with positive sorption, but do not display
stronger surface activity if not further processed. This is the
basis for contemplating their use in the form of additives
for retarding concrete setting in building. Nevertheless, the
necessity to concentrate hydrolysate solutions (for storage)
is a factor limiting the economy of these applications even
in such considerations.
The environmental necessity to process leather waste
and the benefits of processing it through enzymatic
hydrolysis are not, then, sufficiently supported by the
above mentioned applications as having any economic
29

advantage. Achieving an adequate economic effect is more

likely to result from utilising specific hydrolysate
properties, possibly emphasised through appropriate
chemical modification of their functional groups, than by a
high demand for non-modified hydrolysates.

activate nucleophile groups of a protein matrix (mainly

-NH 2 or -OH groups) with formation of epoxide
compounds which subsequently react with remaining
nucleophile groups of polypeptides. Reactivity of the
functional groups of peptides considered goes down in the
sequence -SH > -NH2 > -OH > -COOH.
Mention may be even sporadically found of
epichlorhydrin reacting with guanidine or imidazole
groups.15 It is assumed that cross-linking of proteins with
epichlorhydrin has the character of a two-stage process that
may bring certain advantages. These problems are dealt
with in this work.

Surfactants
One of the more realistic possibilities consists in using
leather waste hydrolysates for manufacturing surfactants of
Lamepon type - through acylation of primary amino
groups of hydrolysates with acyl chlorides of fatty acids
(anionic surfactant types), or through their quaternation,
which is best with benzyl chloride (various types of
amphoteric surfactants).7 The relatively high mean
molecular mass of enzymatic hydrolysates results in
reduced solubility of surfactant derivatives made from them
in water, which leads to a lower production yield.
Acceptable yields can be attained if the mean molecular
mass of enzymatic hydrolysates is reduced to below
approx. 1.0kDa. That is more readily achieved through acid
hydrolysis (e.g. in HCl) than through alkaline hydrolysis or
through prolonged enzymatic hydrolysis.8 Subsequent
acylation or quaternation has to be performed in an alkaline
medium (pH>10.5 ),which somewhat complicates this
industrial application.

Experimental procedures
Starting materials:
Collagen hydrolysate (H) in powder form was prepared
by enzymatic hydrolysis of chrome-tanned leather waste by
a procedure according to reference 16 and drying in
pulverising drier. Its characteristics are shown in Table I.
TABLE I
Characteristics of the collagen hydrolysate used in the work

Dry substance %
92.99
Amide nitrogen in dry substance %
14.85
Ash in dry substance %
4.94
Cr content in dry substance ppm
28.15
Ca content in dry substance ppm
2746.62
Mg content in dry substance ppm
4798.00
0.216
Primary amino groups in dry substance mmol-NH2.g-1
17.75
Average molecular weight (numerical mean MN), kDa

Adhesives
On closer study of the properties of hydrolysate, it was
found that its addition to aminoplast type adhesives, at
approx. 5% (w/w) level, may considerably limit emissions
of formaldehyde gas from their cured adhesive films. The
effect is more pronounced if addition of hydrolysate is
accompanied with adapted, optimised conditions for curing
adhesive bonds.9-11 Results evaluating formaldehyde
emissions from cured films by the official bottle
method 12-13 confirmed that added hydrolysate may reduce
formaldehyde emissions from free adhesive films by as
much as 70%.

Epichlorhydrin (EPICH), 99% from Sigma-Aldrich,Cz,

Sigma-Aldrich Cat. 2005-2006, E 1055-500ml, CAS No.
106-89-8, boiling point 115-117C Sodium hydroxide,
analytical quality, from Lachema Co., CZ

Working procedure

Packaging materials
A topic much discussed lately, concerns problems of
biodegradable polymers based on renewable raw material
sources, due to their employment in packing technology.
Besides renewability of raw material sources, the property
they are appreciated for is degradation rate, on termination
of package life. That, (degradation rate) when compared
with hitherto most widespread synthetic polymers based on
non-renewable crude oil, is several times greater and poses
virtually no environmental hazards. Among biodegradable
materials, proteins of both animal and vegetable origin
occupy a prominent position. It was proved that treating
hydrolysates of chrome-tanned leather waste with
glutaraldehyde allowed the production of biodegradable
materials14 applicable to packing of chemicals in farming
technology (herbicides, insecticides, fertilisers, etc.). In
addition, the degree of hydrolysate cross-linking with
glutaraldehyde allows efficient control of the rate at which
active substances are released from such packing into
surroundings, which is a very interesting effect from the
practical point of view.
Similar results may be probably also attained by reacting
hydrolysates with other cross-linking agents. One of them,
easily and economically available, is epichlorhydrin, which
was more applied in the past to grafting cellulose materials
(e.g., for immobilising various enzymes, etc). It is known
that under favourable conditions epichlorhydrin can

The stock solution of hydrolysate of chrome-tanned

leather waste was prepared by dissolving powdered
hydrolysate in 0.01N NaOH to make a solution of 5.0%
(w/w) concentration, whose pH level was maintained at
11.0. To 50ml stock solution in a reaction flask under
reflux, heated over a water bath to 60C, epichlorhydrin
was added with good stirring over 15 min, in a quantity
corresponding to mass fractions from 0.05 to 0.376. The
reaction was performed at this temperature for 60 min
maintaining good stirring.
After cooling the reaction mixture to room temperature,
an aliquot proportion of the mixture was employed for
spectrophotometric determination (by reaction with
ninhydrin) of primary amino group content.17 The results
obtained are graphically represented in Fig. 1, and confirm
that under these conditions a certain equilibrium state is
established in the reaction mixture.
In the first series of experiments, an excess of
epichlorhydrin was provided over the primary amino
groups of hydrolysate (see data in Table II). No gel
formation occurred, and as that is normally a side-effect of
hydrolysate cross-linking, we conclude that, epichlorhydrin
under the given conditions does not behave like a
bifunctional, cross-linking, agent.
Lower reactivity of the oxirane cycle of epichlorhydrin
(as compared with the chlorine atom) to -SH, -NH2 and

30

absorption in the 901-917cm-1 range, possibly in the less

specific 840-854 cm-1 range.19 More specific absorption in
the range 901-917cm-1 was employed in FTIR
measurements, and the values thus determined of starting
and/or final contents of the oxirane ring of liquid reaction
mixtures are included in Table II, together with contents of
unreacted and reacted amino groups.
Relatively lower reactivity of oxirane groups when
cross-linking collagen hydrolysates may be compensated
(similarly to epoxide resins) by an elevated reaction
temperature. Based on analogous cross-linking of epoxide
resins, we may assume that such a reaction will take place
at a temperature above 120-150C. Therefore, this stage of
the reaction was studied with solid films prepared from
liquid reaction mixtures through vacuum drying at a
pressure of 1.0mm Hg and at 60C. The actual crosslinking reaction was observed with these films employing
differential scanning calorimetry (DSC) over the
temperature interval 25-250 or 300C (DSC 2010, TA
Instruments, Del., U.S.A.). Exothermal DSC peaks relating
to a condensation reaction of oxirane ring with primary
amino groups of hydrolysate were detected in the
temperature range 190-230C. A typical record of such
measurements is shown for illustration in Figure 3.

Figure 1. Reacted primary amino groups of hydrosylate (-NH2) relative

to mass fraction of epichlorhydrin in initial reaction mixture.

Heat flow (W/g)

even -OH groups is a problem often discussed.18 For the

sake of completeness in the experiments we performed, the
time dependence of unreacted chlorine atoms and oxirane
rings of epichlorhydrin (EPICH) was investigated
employing an FTIR technique using a commercial IR
spectrophotometer Genesis FTIR (ATI Matsson, U.S.A.).
The Cl-C bond is usually assigned absorption peaks in
the frequency range of 686-648cm-1 or 731-723cm-1, which
are quite serrated. Their exact position depends on the
structure of remaining carbon-chain, and from the
viewpoint of analytical determination, they are not
regarded as being too reliable. Nevertheless, the time
course of relative absorbance in both the regions mentioned
(648-686cm-1 and 723-731cm-1) is shown in Fig. 2 and
demonstrates in convincing manner the decrease in Clgroups of the epichlorhydrin in reaction mixture.

Temperature (C)

Figure 3. Typical DSC results for dried film of reaction mixture of

hydrosylate with epichlorhydrin for the thermal interval of 25C-300C
at dT/dt = 5C/min.

Relative absorbance

EPICH weight fraction in reaction mixture XEPICH = 0.234

EPICH weight fraction in reaction mixture XEPICH = 0.355
EPICH weight fraction in reaction mixture XEPICH = 0.376

In the experiments of series I, as follows from data of

Table II, considerable proportions of primary amino groups
of hydrolysate react with epichlorhydrin in the liquid
phase. Following condensation of the oxirane ringin the
solid phase may be then unfavourably affected by the ratio
of epichlorhydrin to primary amino groups in the dried film
of reaction mixture. Thus, we introduced, in addition,
experimental series II (performed in liquid phase through
same procedure) immediately before preparing the solid
film, hydrolysate content in reaction mixture was increased
by 30% ( related to starting content of H in reaction
mixture), which corresponds to increasing the content of
primary amino groups in the dried film of reaction mixture
by 0.167mmol. Experiments of series II are also included
in Table II.
The presence of free (unreacted) EPICH in films used
for DSC measurements is unlikely due to the sample
preparation (EPICH: b.p.760 117.9C, b.p.400 98C, b.p.200
62.0C and b.p.100 62.0C).20 The area of exothermal
DSC peaks detected may be thus regarded as proportionate

Reaction time, H
Figure 2. Time dependence of IR absorption peaks in region 648-686cm-1
and 713-731cm-1 characteristic of -Cl group in liquid reaction mixture
when modifying the hydrosylate with epichlorhydrin in alkaline
environment.

To the contrary, determination of the content of oxirane

rings in various reaction mixtures is often based on IR

31

TABLE II
Characteristics of chrome-tanned leather waste hydrolysate modified with epichlorhydrin
XEpich

Primary amino groups

Unreacted
mmol

Epichlorhydrin
Starting

Reacted
mmol

Ratio of EPO:NH2 groups

Reacted

mmol

Mmol

Starting
mmol

Reacted
mmol

Series I.
0.053

0.2293

0.3097

57.40

0.5841

0.3033

51.93

1.08

0.98

0.094

0.1428

0.3952

73.32

1.0710

0.3394

31.69

1.99

0.86

0.074
0.131
0.171
0.203

Series II.

0.1916
0.1034
0.0746
0.0846

0.3474
0.4269
0.4644
0.4544

64.45
79.20
86.16
84.30

0.8276
1.5578
2.1420
2.6289

0.3236
0.3643
0.3863
0.4015

39.10
23.38
18.03
15.32

1.53
2.89
3.97
4.88

0.93
0.85
0.83
0.88

0.053

0.3564

0.1820

33.77

0.5841

0.3042

52.08

1.08

1.67

0.234

0.2889

0.2502

53.50

3.1644

0.4155

13.13

5.87

1.66

0.094
0.171
0.286
0.335
0.376

0.3545
0.2608
0.1379
0.1051
0.1041

0.1845
0.2782
0.4011
0.4339
0.4339

34.23
51.61
74.41
80.50
80.50

1.0710
2.1420
4.1380
5.2091
6.2310

to the enthalpy of the cross-linking reaction of oxirane ring

of EPICH, with the remaining -NH2 groups of H for,
according to finds by various authors,21,22 its value lies in a
quite narrow interval of 102.5 2.5kJ/mol. Results of DSC
measurements are summarised for ready survey in Table
III.

0.3399
0.3864
0.4234
0.4419
0.4569

31.74
18.04
10.23
8.48
7.33

1.99
3.97
7.68

29.66
11.56

1.84
1.39
1.06

1.02
1.05

Agents applicable to modifying hydrolysate of chrometanned leather waste are substances of bifunctional
character, capable of entering into cross-linking reactions
with its functional groups. Choice is limited by a
requirement to perform the cross-linking reaction in an
aqueous or, at most, an aqueous-alcoholic environment
(solubility of hydrolysate). In the not very wide
concentration range of both reactants, cross-linking
reactions usually lead to formation of initially thermoreversible, subsequently thermo-irreversible gels swelling
in water.
The ability of swelling in water gradually deteriorates
with increasing cross-link density, and the gel acquires the
character of thermosetting (reactoplastic), poorly soluble,
non-fusible material whose processing by standard
procedures is somewhat more complicated. At the same
time, its biodegradability also deteriorates.
An alternative solution is cross-linking of hydrolysate
with bifunctional cross-linking agents possessing markedly
different reactivities of their functional groups as, for
example, epichlorhydrin. In an an aqueous-alkaline
environment such a reaction does not lead to gel formation
(phenomenon accompanying cross-linking of collagen
hydrolysates) despite the fact that a decrease in the primary
amino groups of the hydrolysate demonstrates clearly
enough the reaction of both substances. A state of
equilibrium is established in the reaction mixture,
characterised by 70-85% of initially present primary amino
groups of hydrolysate having reacted (see Figure 1).
The detected decrease in -Cl groups of epichlorhydrin in
the reaction mixture corresponds to a decrease in primary
amino groups, while the found decrease in oxirane rings of
epichlorhydrin merely ranges within limits of experimental
error. In an aqueous-alkaline environment, epichlorhydrin
then binds to hydrolysate at one point and actual
hydrolysate cross-linking does not occur in this stage of the
reaction. The mutual ratio of reacted EPICH and -NH2

Discussion

Synthetic polymers, nowadays so widespread in

packaging technology, are a source of certain problems
because of their limited recycling possibilities. A
considerable proportion of packages, on finishing their life,
are disposed of by incineration or to landfill. Disposal of
polymeric packages by incineration has to be performed at
high temperatures to prevent formation of degradation
products possessing unfavourable (often carcinogenic)
properties. This has a negative effect on costs of their
disposal. The long degradation time of synthetic polymers
in landfills, currently ranging up to hundreds of years,
exerts pressure for permanent expansion of landfills
necessitating subsequent recultivation. Biodegradable
polymers, for which the time required for microbial
degradation in landfill falls in a range of weeks or, not more
than months, thus represent a very attractive alternative in
this field. In addition, these polymers come from renewable
raw material sources (mostly animal or vegetable proteins),
and this may also be a very significant circumstance for the
future.
Hydrolysates of chrome-tanned leather waste, without
certain chemical modification, are not directly exploitable
in this field, above all due to their ready water solubility.
However, increasing their cross-link density (essentially
analogous to tanning) enables us to arrive at materials well
suited for application in packing technology, and,
moreover, regulation of cross-link density allows to achieve
time-controlled release of active substances from these
packings.
32

XEPICH

TABLE III
Characteristics of DSC exothermal peaks of dry films of reaction mixtures from TABLE 1

Ratio
EPICH:NH2
in film

Experiments of series I.
1.323
0.053
1.689
0.074
0.094
2.377
0.131
3.523
0.171
5.178
0.203
4.746
Experiments of series II
0.053
0.588
0.094
0.659
0.171
0.916
0.234
0.924
0.286
1.416
0.355
1.661
0.376
1.742

ThermaL coordinates and enthalpy of

Exothermal DSC peak

Tinit. oC

Tmax oC

Tfinal oC

Enthalpy
Q (J/g)

mmol/g of
cross-links
in film

203.0
195.4
190.7
190.5
190.8
193.8

215.0
209.2
201.5
202.9
203.1
204.6

233.8
233.0
232.3
232.9
232.0
230.0

0.31
1.06
0.82
1.11
1.84
1.92

0.0033
0.010
0.008
0.011
0.018
0.019

203.1
201.5
185.0
181.5
187.2
183.8
181.5

212.0
211.2
202.4
200.4
205.6
201.5
201.50

237.2
237.1
236.0
232.5
227.8
230.0
230.8

19.20
16.78
14.38
13.95
8.40
5.60
4.80

0.188
0.164
0.141
0.136
0.082
0.055
0.047

Note:
*)
Mass fraction of epichlorhydrin (EPICH) in liquid reaction mixture with hydrolysate
**) Calculated from mean value of enthalpy of condensation reaction of epoxy groups with
amino groups 102.15 kJ/mol see / 20,21 /

groups of H (see data in Table II) ranges within the limits

1:0.83-1.84, so that amino groups of hydrolysate obviously
bond to epichlorhydrin as a virtual monomer unit. Rather
similar conclusions were arrived at by authors studying the
immobilisation of some biologically active proteins.18
Utilisation of epichlorhydrin in this reaction largely
depends on its mass fraction in the initial reaction mixture.
With a low mass fraction, the percentage proportion of
reacted epichlorhydrin fluctuates around 50%, this
proportion gradually decreases with increasing mass
fraction of EPICH in the starting reaction mixture until,
with a mass fraction of epichlorhydrin exceeding approx.
0.29, the percentage of hydrolysate-bonded epichlorhydrin
is less than 10 % (related to its initial quantity in reaction
mixture). Quite a large quantity of unreacted EPICH thus
remains in the reaction mixture and has to be removed prior
to its further processing. These facts are shown by data in
Table II.
According to results of DSC measurements, the reaction
of the oxirane ring takes place (again very probably with
-NH2 groups of H) only at temperatures of 180-200C (see
Table III). Cross-linking of H accordingly requires
application of the two-stage technique well-known in the
field of epoxide or other types of thermosetting polymers.
The area of the exothermal peak on DSC curves, which
corresponds to the reaction of oxirane ring with functional
groups of hydrolysate, characterises its energetic content
and is proportionate to the enthalpy of the cross-linking
reaction (unreacted EPICH was removed by drying the
reaction mixture). According to findings by some
authors,21,22 molar enthalpy of the reaction of oxirane ring
with amino groups corresponds to practically constant level
102.15 2.5kJ/mol, so that the energetic content of the
DSC peak enables us to estimate the density of cross-links
formed in films under observation. Such data, together with
thermal coordinates of corresponding exothermal DSC
peaks and contents of oxirane rings of a given film, are
summarised in Table III.

The data presented make it obvious that cross-link

density is affected by the ratio of EPICH to primary amino
groups in the film. When amino groups dominate (approx.
up to 2.1-2.2 in the ratio of EPICH : NH2- groups), the
content of cross-links in film decreases with an increase in
this ratio, while with further increase of the ratio to over 2.2
produces a slight increase in cross-link density of films.
Such a dependency is shown in Figure 4.
This find is interesting in view of potentially regulating
the cross-link density in the end-product, which affects not
only processing properties but also solubility in an aqueous

Figure 4. Influence of ratio of epichlorhydrin (oxirane groups) to

primary amino groups in epichlorhydrin cross-linked
hydrosylate films.

environment and hence, as a consequence, also its

biodegradation rate. This circumstance is particularly
interesting from a practical viewpoint as it offers potential
for time-controlled releasing of active substances from
33

such packages,23 and that may be utilised in a number of

modern applications for chemicals in farming technology,
whether those in question are fertilisers, herbicides,
pesticides, insecticides, etc., where the non-foodstuff
character of initial protein material (hydrolysate of chrometanned leather waste with very low content of Cr
compounds - see data in Table I) has no essential
significance for packing such materials, while the
economic aspects are undoubtedly more interesting.
Modification of hydrolysates with epichlorhydrin to
produce biodegradable packing materials is to be carried
out as a two-step process: in alkaline solution (pH11.0)
epichlorhydrin is bonded to hydrolysate -NH2 groups with
its chlorine group representing the reaction centre. At this
stage the hydrolysate solution (preferably with 10-15% of
dry matter content) may be used directly. The reaction
product remains water-soluble and the obvious casting
method may be used for film preparation. Curing of the
films proceeds in the second stage by heating the films to
210C for 5 minutes.
The biodegradation rate of the films depends on crosslink density. The experiments in this direction are not yet
fully finished, but preliminary results lead to conclusion,
that with cross-links density of 0.1-0.15mmol/g the
biodegradation time does not exceed 1-2 months. As the
experiments were carried out at laboratory scale only, the
economy of the whole process remain to be investigated.

4.
5.
6.
7.
8.
9.
10.
11.

12.

13.
14.

Acknowledgement

15.
16.

The work presented was executed within the framework

of grant VZ MSM7088352102. The authors wish to
extend their thanks to the Ministry of Education of the
Czech Republic for financial support to this work.

17.
18.
19.

(Received July 2005, revised August 2005)

20.
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