Animal and vegetable fats and oils — Determination of the composition of fatty acids in the 2-position

Corps gras d'origines animale et végétale — Détermination de la composition des acides gras en position 2

Rastlinske in živalske maščobe in olja - Določanje sestave maščobnih kislin

General Information

Status
Withdrawn
Publication Date
18-Dec-1985
Withdrawal Date
18-Dec-1985
Current Stage
9599 - Withdrawal of International Standard
Completion Date
18-Dec-1997

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ISO 6800:1985 - Animal and vegetable fats and oils -- Determination of the composition of fatty acids in the 2-position
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International Standard @ 6800
~~ ~~ ~~ ~ ~
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION*MEXAYHAPOAHAR OPrAHM3AUMR no CTAHAAPTM3AUHM*ORGANlSATlON INTERNATIONALE DE NORMALISATION
Animal and vegetable fats and oils - Determination of
* the composition of fatty acids in the 2-position
Corps gras d'origines animale et végétale - Détermination de la composition des acides gras en position 2
'f i'
First edition - 1985-12-15
UDC 665.2/.3: 543.8 Ref. No. IS0 6800-1985 (E)
-
Lo
Descriptors : agricultural products, animal fats, vegetable fats, animal oils, vegetable oils, chemical analysis, determination of content, fatty
8 acids.
es
Price based on 5 pages

---------------------- Page: 1 ----------------------
Foreword
IS0 (the International Organization for Standardization) is a worldwide federation of
national standards bodies (IS0 member bodies). The work of preparing International
Standards is normally carried out through IS0 technical committees. Each member
body interested in a subject for which a technical committee has been established has
the right to be represented on that committee. International organizations, govern-
mental and non-governmental, in liaison with ISO, also take part in the work.
Draft International Standards adopted by the technical committees are circulated to
the member bodies for approval before their acceptance as International Standards by
the IS0 Council. They are approved in accordance with IS0 procedures requiring at
least 75 % approval by the member bodies voting.
International Standard IS0 6800 was prepared by Technical Committee ISO/TC 34,
Agricultural food products.
Users should note that all International Standards undergo revision from time to time
and that any reference made herein to any other International Standard implies its
latest edition, unless otherwise stated.
O International Organization for Standardization, 1985 0
Printed in Switzerland

---------------------- Page: 2 ----------------------
IS0 6800-1985 (E)
NORM E I NTE R NAT1 O NA LE
Animal and vegetable fats and oils - Determination of
the composition of fatty acids in the 2-position
4 Principle
1 Scope
This International Standard specifies a method for the deter- After neutralization, where necessary, of any free fatty acids,
purification of the test portion by column chromatography.
mination of the composition of fatty acids which are in the
2-position (B or internal position) of glyceride molecules in Partial enzymatic hydrolysis of the glycerides to yield
animal and vegetable fats and oils. 2-monoglycerides. Separation of the monoglycerides by thin
layer chromatography and determination of their fatty acid
composition by gas chromatography.
2 Field of application
Because of the nature of pancreatic lipase action, the method is
5 Reagents
applicable only to fats and oils with a melting point below
45 OC.
All reagents shall be of recognized analytical quality and the
water used shall be distilled water or water of at least equivalent
The method is not unreservedly applicable to all fats and oils,
quality.
particularly those containing substantial amounts of
-
fatty acids with 12 or fewer carbon atoms (copra oil,
palm kernel oil, butyric butter fats); 5.1 Reagents for the purification of the test
portion
0
- fatty acids with 20 and more carbon atoms and of a
high degree of unsaturation (more than four double bonds)
5.1.1 2-propano1, or ethanol 95 % (V/M.
(fish oil and marine animal oil);
-
fatty acids which have secondary groups containing
5.1.2 Hexane (if available) or light petroleum (boiling range
oxygen.
30 to 60 OC).
3 References
5.1.3 2-propanol 50 % ( V1 M or ethanol 50 % ( V/ V)
IS0 660, Animal and vegetable fats and oils - Determination
5.1.4 Sodium hydroxide solution, 0,5 mol/l.
of acid value and of acidity.
IS0 661, Animal and vegetable fats and oils - Preparation of
5.1.5 Phenolphthalein solution, 1 g per 100 ml of ethanol
test sample.
95 % (V/V).
IS0 5508, Animal and vegetable fats and oils - Analysis by
gas-liquid chromatography of methyl esters of fatty acids.
5.1.6 Activated neutral alumina, for chromatography,
Brockmann activity I, recently activated for 2 h at 260 OC and
IS0 5509, Animaland vegetable fats and oils - Preparation of
kept in a desiccator.
methyl esters of fatty acids.
5.1.7 Nitrogen.
IS0 5555, Animal and vegetable fats and oils - Sampling.
1

---------------------- Page: 3 ----------------------
IS0 6800-1985 (E)
6 Apparatus
5.2 Reagents for hydrolysis of the triglycerides
Usual laboratory equipment, and in particular
5.2.1 Diethyl ether, free from peroxides.
6.1 Apparatus for the purification of the test
5.2.2 Hydrochloric acid, 6 mol/I.
portion
5.2.3 Sodium cholate solution, 1 g/l, of enzymatic quality.
Water-bath, thermostatically controlled, and capable
6.1.1
of being maintained at 30 to 40 OC.
5.2.4 Calcium chloride (CaCI2) solution, 220 g/l.
6.1.2 Glass column, for chromatography, 13 mm internal
5.2.5 Buffer solution, 2-amino-2-( hydroxymethyl) propane-
diameter and 400 mm in length, equipped with a sintered glass
1,3-dioli), 1 mol/l, adjusted to pH 8 with 6 mol/l hydrochloric
plate and a tap.
acid (5.2.2) using a pH-meter.
6.1.3 Rotary evaporator, with 250 ml flask.
Store this solution at 0-4 OC and use within 14 days.
6.1.4 Tubing, for nitrogen bubbling.
5.2.6 Pancreatic lipase, with an activity of between 8 and
20 units/mg (see the annex).
6.1.5 Separating funnel, 500 ml capacity.
Store dry in a refrigerator. Before use, bring a portion of the
powder to ambient temperature.
6.1.6 Round-bottom flask, 100 ml capacity.
NOTE - Lipase of suitable activity is available commercially. If pre-
ferred, the lipase may be prepared and assayed in accordance with the
6.2 Apparatus for the hydrolysis
procedure described in the annex.
of the triglycerides
6.2.1 Centrifuge (if necessary).
5.3 Reagents for the isolation of the
2-monoglycerides
capacity, with ground stopper.
6.2.2 Centrifuge tube, 10 ml
5.3.1 Ethanol.
6.2.3 Vibrating electric shaker, for vigorous agitation of
the centrifuge tube.
5.3.2 Hexane (if available), or light petroleum (boiling
range 30 to 60 OC).
6.2.4 Water-bath, thermostatically controlled, and capable
of being maintained at 40 + 0,5 OC.
5.3.3 Acetone.
6.2.5 Hypodermic syringe, 1 ml capacity, with thin needle.
5.3.4 Silica powder, with binder, for thin layer
chromatography.
6.2.6 Stop-watch.
5.3.5 Developing solvent, prepared as follows :
6.3 Apparatus for the isolation of the
hexane (if available) or light petroleum : 70 ml
2-monoglycerides
diethyl ether : 30 ml
1 ml
formic acid, minimum 98 % i V/ V) :
6.3.1 Developing tank, for thin layer chromatography, with
ground glass lid, suitable for containing glass plates
5.3.6 2'. 7'-Dichlorofluorescein, indicator solution, 2 g per
200 mm x 200 mm.
litre of methanol, rendered slightly alkaline by addition of a drop
of 1 mol/l sodium hydroxide per 100 ml of the solution.
6.3.2 Spreader and rack, for preparation of the plates.
6.3.3 Glass plates, 200 mm x 200 mm.
5.4 Reagents for the analysis of the
2-monoglycerides by gas chromatography
6.3.4 Microsyringe, capable of dispensing drops of 3 to 4 pl
as a continuous uniform band.
See IS0 5508 and IS0 5509.
Alternative names : tris-(hydroxymethyl) methylamine; tris-(hydroxymethyl) aminomethane.
1)
2

---------------------- Page: 4 ----------------------
IS0 6800-1985 (E)
(5.1.4) equivalent to the free acidity of the fat or oil, with an ex-
6.3.5 Apparatus for spraying the indicator solution on to
the plates. cess of 0,5 %. Shake vigorously for 1 min, add 50 ml of water,
shake again and allow to stand. When separated, run off the
lower layer containing the soap and any intermediate layers
6.3.6 Microspatula.
(mucilages and insoluble substances). Wash the hexane or light
petroleum solution of neutralized oil with successive portions of
6.3.7 Oven, capable of being maintained at 103 k 2 OC.
25 or 30 ml of a solution of 2-propanol or ethanol (5.1.3) until
the pink colour of the phenolphthalein disappears.
6.3.8 Ultraviolet lamp, for examining chromatographic
plates, for example with a wavelength of 254 nm.
Transfer the solution into the rotary evaporator flask (6.1.3) and
remove most of the solvent by evaporating under reduced
pressure. Dry the oil at 30 to 40 OC under reduced pressure
6.3.9 Round-bottom flask, 25 ml capacity, with air con-
using a nitrogen stream (5.1.7) until the solvent is completely
denser of approximately 1 m length with ground joint.
removed.
6.3.10 Conical flask, 250 ml capacity, with ground stopper.
8.4 Purification of the test portion through
6.3.11 Conical flask, 50 ml (if necessary).
alumina
6.3.12 Filter, sintered glass, porosity P 40 (16 to 40 pm) (if
Prepare a suspension of 15 g of activated alumina (5.1.6) in
(.
necessary).
50 ml of hexane (if available) or light petroleum (5.1.2) and
pour, while shaking, into a glass column for chromatography
6.3.13 Desiccator. (6.1.2). Ensure that the alumina settles evenly and allow the
solvent level to fall to 1 to 2 mm above the upper level of the ad-
sorbent. Carefully pour into the column a solution prepared by
6.4 Apparatus for the analysis of the
dissolving a test portion of 5 g, neutralized if necessary, in
2-monoglycerides by gas chromatography
25 ml of hexane (if available) or light petroleum (5.1.2) and col-
lect all the liquid which elutes from the column in a 100 ml
See IS0 5508 and IS0 5509.
round-bottom flask (6.1.6).
Remove mo
...

SLOVENSKI STANDARD
SIST ISO 6800:1995
01-december-1995
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD'RORþDQMHVHVWDYHPDãþREQLKNLVOLQ
Animal and vegetable fats and oils -- Determination of the composition of fatty acids in
the 2-position
Corps gras d'origines animale et végétale -- Détermination de la composition des acides
gras en position 2
Ta slovenski standard je istoveten z: ISO 6800:1985
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
SIST ISO 6800:1995 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------

SIST ISO 6800:1995

---------------------- Page: 2 ----------------------

SIST ISO 6800:1995
International Standard @ 6800
~~ ~~ ~~ ~ ~
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION*MEXAYHAPOAHAR OPrAHM3AUMR no CTAHAAPTM3AUHM*ORGANlSATlON INTERNATIONALE DE NORMALISATION
Animal and vegetable fats and oils - Determination of
* the composition of fatty acids in the 2-position
Corps gras d'origines animale et végétale - Détermination de la composition des acides gras en position 2
'f i'
First edition - 1985-12-15
UDC 665.2/.3: 543.8 Ref. No. IS0 6800-1985 (E)
-
Lo
Descriptors : agricultural products, animal fats, vegetable fats, animal oils, vegetable oils, chemical analysis, determination of content, fatty
8 acids.
es
Price based on 5 pages

---------------------- Page: 3 ----------------------

SIST ISO 6800:1995
Foreword
IS0 (the International Organization for Standardization) is a worldwide federation of
national standards bodies (IS0 member bodies). The work of preparing International
Standards is normally carried out through IS0 technical committees. Each member
body interested in a subject for which a technical committee has been established has
the right to be represented on that committee. International organizations, govern-
mental and non-governmental, in liaison with ISO, also take part in the work.
Draft International Standards adopted by the technical committees are circulated to
the member bodies for approval before their acceptance as International Standards by
the IS0 Council. They are approved in accordance with IS0 procedures requiring at
least 75 % approval by the member bodies voting.
International Standard IS0 6800 was prepared by Technical Committee ISO/TC 34,
Agricultural food products.
Users should note that all International Standards undergo revision from time to time
and that any reference made herein to any other International Standard implies its
latest edition, unless otherwise stated.
O International Organization for Standardization, 1985 0
Printed in Switzerland

---------------------- Page: 4 ----------------------

SIST ISO 6800:1995
IS0 6800-1985 (E)
NORM E I NTE R NAT1 O NA LE
Animal and vegetable fats and oils - Determination of
the composition of fatty acids in the 2-position
4 Principle
1 Scope
This International Standard specifies a method for the deter- After neutralization, where necessary, of any free fatty acids,
purification of the test portion by column chromatography.
mination of the composition of fatty acids which are in the
2-position (B or internal position) of glyceride molecules in Partial enzymatic hydrolysis of the glycerides to yield
animal and vegetable fats and oils. 2-monoglycerides. Separation of the monoglycerides by thin
layer chromatography and determination of their fatty acid
composition by gas chromatography.
2 Field of application
Because of the nature of pancreatic lipase action, the method is
5 Reagents
applicable only to fats and oils with a melting point below
45 OC.
All reagents shall be of recognized analytical quality and the
water used shall be distilled water or water of at least equivalent
The method is not unreservedly applicable to all fats and oils,
quality.
particularly those containing substantial amounts of
-
fatty acids with 12 or fewer carbon atoms (copra oil,
palm kernel oil, butyric butter fats); 5.1 Reagents for the purification of the test
portion
0
- fatty acids with 20 and more carbon atoms and of a
high degree of unsaturation (more than four double bonds)
5.1.1 2-propano1, or ethanol 95 % (V/M.
(fish oil and marine animal oil);
-
fatty acids which have secondary groups containing
5.1.2 Hexane (if available) or light petroleum (boiling range
oxygen.
30 to 60 OC).
3 References
5.1.3 2-propanol 50 % ( V1 M or ethanol 50 % ( V/ V)
IS0 660, Animal and vegetable fats and oils - Determination
5.1.4 Sodium hydroxide solution, 0,5 mol/l.
of acid value and of acidity.
IS0 661, Animal and vegetable fats and oils - Preparation of
5.1.5 Phenolphthalein solution, 1 g per 100 ml of ethanol
test sample.
95 % (V/V).
IS0 5508, Animal and vegetable fats and oils - Analysis by
gas-liquid chromatography of methyl esters of fatty acids.
5.1.6 Activated neutral alumina, for chromatography,
Brockmann activity I, recently activated for 2 h at 260 OC and
IS0 5509, Animaland vegetable fats and oils - Preparation of
kept in a desiccator.
methyl esters of fatty acids.
5.1.7 Nitrogen.
IS0 5555, Animal and vegetable fats and oils - Sampling.
1

---------------------- Page: 5 ----------------------

SIST ISO 6800:1995
IS0 6800-1985 (E)
6 Apparatus
5.2 Reagents for hydrolysis of the triglycerides
Usual laboratory equipment, and in particular
5.2.1 Diethyl ether, free from peroxides.
6.1 Apparatus for the purification of the test
5.2.2 Hydrochloric acid, 6 mol/I.
portion
5.2.3 Sodium cholate solution, 1 g/l, of enzymatic quality.
Water-bath, thermostatically controlled, and capable
6.1.1
of being maintained at 30 to 40 OC.
5.2.4 Calcium chloride (CaCI2) solution, 220 g/l.
6.1.2 Glass column, for chromatography, 13 mm internal
5.2.5 Buffer solution, 2-amino-2-( hydroxymethyl) propane-
diameter and 400 mm in length, equipped with a sintered glass
1,3-dioli), 1 mol/l, adjusted to pH 8 with 6 mol/l hydrochloric
plate and a tap.
acid (5.2.2) using a pH-meter.
6.1.3 Rotary evaporator, with 250 ml flask.
Store this solution at 0-4 OC and use within 14 days.
6.1.4 Tubing, for nitrogen bubbling.
5.2.6 Pancreatic lipase, with an activity of between 8 and
20 units/mg (see the annex).
6.1.5 Separating funnel, 500 ml capacity.
Store dry in a refrigerator. Before use, bring a portion of the
powder to ambient temperature.
6.1.6 Round-bottom flask, 100 ml capacity.
NOTE - Lipase of suitable activity is available commercially. If pre-
ferred, the lipase may be prepared and assayed in accordance with the
6.2 Apparatus for the hydrolysis
procedure described in the annex.
of the triglycerides
6.2.1 Centrifuge (if necessary).
5.3 Reagents for the isolation of the
2-monoglycerides
capacity, with ground stopper.
6.2.2 Centrifuge tube, 10 ml
5.3.1 Ethanol.
6.2.3 Vibrating electric shaker, for vigorous agitation of
the centrifuge tube.
5.3.2 Hexane (if available), or light petroleum (boiling
range 30 to 60 OC).
6.2.4 Water-bath, thermostatically controlled, and capable
of being maintained at 40 + 0,5 OC.
5.3.3 Acetone.
6.2.5 Hypodermic syringe, 1 ml capacity, with thin needle.
5.3.4 Silica powder, with binder, for thin layer
chromatography.
6.2.6 Stop-watch.
5.3.5 Developing solvent, prepared as follows :
6.3 Apparatus for the isolation of the
hexane (if available) or light petroleum : 70 ml
2-monoglycerides
diethyl ether : 30 ml
1 ml
formic acid, minimum 98 % i V/ V) :
6.3.1 Developing tank, for thin layer chromatography, with
ground glass lid, suitable for containing glass plates
5.3.6 2'. 7'-Dichlorofluorescein, indicator solution, 2 g per
200 mm x 200 mm.
litre of methanol, rendered slightly alkaline by addition of a drop
of 1 mol/l sodium hydroxide per 100 ml of the solution.
6.3.2 Spreader and rack, for preparation of the plates.
6.3.3 Glass plates, 200 mm x 200 mm.
5.4 Reagents for the analysis of the
2-monoglycerides by gas chromatography
6.3.4 Microsyringe, capable of dispensing drops of 3 to 4 pl
as a continuous uniform band.
See IS0 5508 and IS0 5509.
Alternative names : tris-(hydroxymethyl) methylamine; tris-(hydroxymethyl) aminomethane.
1)
2

---------------------- Page: 6 ----------------------

SIST ISO 6800:1995
IS0 6800-1985 (E)
(5.1.4) equivalent to the free acidity of the fat or oil, with an ex-
6.3.5 Apparatus for spraying the indicator solution on to
the plates. cess of 0,5 %. Shake vigorously for 1 min, add 50 ml of water,
shake again and allow to stand. When separated, run off the
lower layer containing the soap and any intermediate layers
6.3.6 Microspatula.
(mucilages and insoluble substances). Wash the hexane or light
petroleum solution of neutralized oil with successive portions of
6.3.7 Oven, capable of being maintained at 103 k 2 OC.
25 or 30 ml of a solution of 2-propanol or ethanol (5.1.3) until
the pink colour of the phenolphthalein disappears.
6.3.8 Ultraviolet lamp, for examining chromatographic
plates, for example with a wavelength of 254 nm.
Transfer the solution into the rotary evaporator flask (6.1.3) and
remove most of the solvent by evaporating under reduced
pressure. Dry the oil at 30 to 40 OC under reduced pressure
6.3.9 Round-bottom flask, 25 ml capacity, with air con-
using a nitrogen stream (5.1.7) until the solvent is completely
denser of approximately 1 m length with ground joint.
removed.
6.3.10 Conical flask, 250 ml capacity, with ground stopper.
8.4 Purification of the test portion through
6.3.11 Conical flask, 50 ml (if necessary).
alumina
6.3.12 Filter, sintered glass, porosity P 40 (16 to 40 pm) (if
Prepare a suspension of 15 g of activated alumina (5.1.6) in
(.
necessary).
50 ml of hexane (if available) or light petroleum (5.1.2) and
pour, while shaking, into a glass column for chromatography
6.3.13 Desiccator. (6.1.2). Ensure that the alumina settles evenly and allow the
solvent level to fall to 1 to 2 mm above the upper level of the a
...

Norme internationale @ 6800
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION*MEXflYHAPOflHAfl OPrAHM3AUMR no CTAHflAPTki3AUMki*ORGANlSATlON INTERNATIONALE DE NORMALISATION
Corps gras d'origines animale et végétale -
*
Détermination de la composition des acides gras en
position 2
Animal and vegetable fats ans oils - Determination of the composition of fatty acids in the 2-position
Première édition - 1985-12-15
CDU 665.2/.3 : 543.8 Réf. no : IS0 6800-1985 (F)
Descripteurs : produit agricole, corps gras animal, corps gras végétal, huile animale. huile végétale, analyse chimique, dosage, acide gras.
Prix basé sur 5 pages

---------------------- Page: 1 ----------------------
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale
d'organismes nationaux de normalisation (comités membres de I'ISO). L'élaboration
des Normes internationales est confiée aux comités techniques de I'ISO. Chaque
comité membre intéressé par une étude a le droit de faire partie du comité technique
créé à cet effet. Les organisations internationales, gouvernementales et non gouverne-
mentales, en liaison avec I'ISO participent également aux travaux.
Les projets de Normes internationales adoptés par les comités techniques sont soumis
aux comités membres pour approbation, avant leur acceptation comme Normes inter-
nationales par le Conseil de I'ISO. Les Normes internationales sont approuvées confor-
mément aux procédures de I'ISO qui requièrent l'approbation de 75 % au moins des
comités membres votants.
La Norme internationale IS0 6800 a été élaborée par le comité technique ISO/TC 34,
Produits agricoles alimentaires.
L'attention des utilisateurs est attirée sur le fait que toutes les Normes internationales
sont de temps en temps soumises à révision et que toute référence faite à une autre
Norme internationale dans le présent document implique qu'il s'agit, sauf indication
contraire, de la dernière édition.
O Organisation internationale de normalisation, 1985 0
Imprimé en Suisse

---------------------- Page: 2 ----------------------
~~ ~~~ ~ ~
NORM E I NTE R N AT1 ON ALE IS0 6800-1985 (FI
Corps gras d'origines animale et végétale -
Détermination de la composition des acides gras en
position 2
4 Principe
I) Objet
La présente Norme internationale spécifie une méthode de Après neutralisation éventuelle des acides gras libres, purifica-
détermination dans les corps gras d'origines animale et végétale tion de la prise d'essai par chromatographie sur colonne.
de la composition de la fraction des acides gras en position 2 Hydrolyse enzymatique partielle des glycérides pour obtenir les
(position /3 ou position interne) dans les glycérides. mono-2 glycérides. Séparation des monoglycérides par chro-
matographie sur couche mince et détermination de leur compo-
sition en acides gras par chromatographie en phase gazeuse.
2 Domaine d'application
En raison des particularités d'action de la lipase pancréatique, la
5 Réactifs
méthode s'applique seulement aux corps gras ayant un point
de fusion inférieur à 45 OC.
Tous les réactifs utilisés doivent être de qualité analytique et
l'eau utilisée doit être de l'eau distillée ou de pureté équivalente.
Elle n'est pas applicable sans restrictions à tous les corps gras,
en particulier à ceux renfermant des quantités importantes
Réactifs pour la purification de la prise d'essai
5.1
- soit d'acides gras ayant 12 atomes de carbone ou moins
(huiles de coprah et de palmiste, matières grasses butyri-
ques du beurre); 5.1.1 Propanol-2 ou éthanol, à 95 % (V/V).
- soit d'acides gras ayant 20 atomes de carbone et plus et
5.1.2 Hexane ou, à défaut, éther de pétrole (intervalle de
d'insaturation élevée (plus de quatre doubles liaisons) (hui-
e distillation 30 à 60 OC).
les de poissons et d'animaux marins);
- 5.1.3 Propanol-2, à 50 % (V/V) ou éthanol, à 50 % (V/V).
soit d'acides gras possédant des fonctions oxygénées
secondaires.
5.1.4 Hydroxyde de sodium, solution à 0,5 mol/l.
3 Références
5.1.5 Phénolphtaléine, solution à 1 g pour 100 ml dans
l'éthanol à 95 % ( V/ V).
IS0 660, Corps gras d'origines animale et végétale - Détermi-
nation de I'indice d'acide et de I'acidité.
5.1.6 Alumine activée pour chromatographie, neutre, de
IS0 661, Corps gras d'origines animale et végétale - Prépara-
degré d'activité Brockmann I, récemment activée pendant 2 h
tion de l'échantillon pour essai. à 260 OC et conservée dans un dessiccateur.
IS0 5508, Corps gras d'origines animale et végétale - Analyse
5.1.7 Azote.
par chromatographie en phase gazeuse des esters méthyliques
d'acides gras.
5.2 Réactifs pour l'hydrolyse des triglycérides
IS0 5509, Corps gras d'origines animale et végétale - Prépara-
tion des esters méthyliques d'acides gras.
5.2.1 Oxyde diéthylique, exempt de peroxydes.
IS0 5555, Corps gras d'origines animale et végétale - Echantil-
tonnage. 5.2.2 Acide chlorhydrique, solution à 6 mol/l.
1

---------------------- Page: 3 ----------------------
IS0 6800-1985 (FI
5.2.3 Cholate de sodium, solution à 1 g/l, de qualité enzy- 6.1 Appareillage pour la purification de la prise
matique. d'essai
5.2.4 Chlorure de calcium, solution à 220 g/l de CaCI2 6.1.1 Bain d'eau thermostatique, réglé à une température
comprise entre 30 et 40 OC.
5.2.5 Solution tampon, de amino-2 hydroxyméthyl-2 pro-
pane diol-1,3,1) à 1 mol/l, amenée à pH 8 par de l'acide 6.1.2 Colonne en verre, pour chromatographie, de 13 mm
chlorhydrique à 6 mol/l (5.2.2) en utilisant un pH-mètre. de diamètre intérieur et 400 rnrn de longueur, équipée d'une
plaque en verre fritté et d'un robinet.
Conserver cette solution entre O et 4 OC et l'utiliser dans les
14 jours après préparation.
6.1.3 Évaporateur rotatif, avec ballon de 250 ml.
5.2.6 Lipase pancréatique, ayant une activité comprise
6.1.4 Tubulure, pour barbotage d'azote.
entre 8 et 20 unités par milligramme (voir annexe).
6.1.5 Ampoule à décanter, de 500 ml.
Elle doit être conservée déshydratée au réfrigérateur. Avant uti-
lisation pour l'analyse, une partie de la poudre doit être portée à
la température ambiante.
6.1.6 Ballon, de 100 ml de capacité.
NOTE - II existe dans le commerce des lipases ayant une activité lipa-
sique satisfaisante. Si i'on préfère, la lipase peut être préparée et con-
6.2 Appareillage pour l'hydrolyse des triglycérides
trôlée selon la technique décrite en annexe.
6.2.1 Centrifugeuse (si nécessaire).
5.3 Réactifs pour l'isolement des mono-2
glycérides
6.2.2 Tube de centrifugeuse, de 10 ml, avec bouchon rodé.
5.3.1 Éthanol.
6.2.3 Agitateur électrique vibrant, permettant une agita-
tion énergique du tube de centrifugeuse.
5.3.2 Hexane ou, à défaut, éther de pétrole (intervalle de
distillation 30 à 60 OC).
6.2.4 Bain d'eau thermostatique, réglable à 40 k 0,5 OC.
5.3.3 Acétone.
6.2.5 Seringue hypodermique, de 1 ml, munie d'une
aiguille mince.
5.3.4 Silice en poudre, avec liant, pour chromatographie en
couche mince.
6.2.6 Chronomètre.
5.3.5 Solvant de développement, préparé comme suit :
hexane ou, à défaut, éther de pétrole 70 ml
6.3 Appareillage pour l'isolement des
mono9 glycérides
oxyde diéthylique 30 ml
acide formique à 98 % (V/ V) minimum 1 ml
6.3.1 Cuve de développement, pour chromatographie en
couche mince, avec couvercle en verre rodé, susceptible de
5.3.6 Révélateur : solution méthanolique à 2 g/l de
contenir des plaques en verre de 200 mm x 200 mm.
dichloro-T, 7' fluorescéine, légèrement alcalinisée par addition
d'une goutte d'hydroxyde de sodium à 1 mol/l pour 100 ml de
6.3.2 Étaleur et socle, pour la préparation des plaques.
solution.
6.3.3 Plaques en verre, de 200 mrn x 200 mm.
5.4 Réactifs pour l'analyse des
mono9 glycérides par chromatographie
6.3.4 Microseringue, permettant de délivrer uniformément
en phase gazeuse
à 4 VI.
en une bande continue des gouttelettes de 3
Voir IS0 5508 et IS0 5509.
6.3.5 Appareil pour pulvériser le révélateur sur les
plaques
6 Appareillage
6.3.6 Microspatule.
Matériel courant de laboratoire, et notamment
Autres désignations : tris-( hydroxyméthyl) méthylamine; tris-( hydroxyméthyl) aminométhane.
1 )
2

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IS0 6800-1985 (FI
6.3.7 Étuve, réglable à 103 + 2 OC.
l’acidité libre du corps gras, plus un excès de 0,5 %. Agiter
énergiquement pendant 1 min, ajouter 50 ml d‘eau, agiter à
nouveau et laisser reposer. Après décantation, éliminer la cou-
6.3.8 Lampe U.V., pour l‘examen des plaques chromatogra-
che inférieure contenant les savons et les éventuelles couches
phiques, de par exemple 254 nm de longueur d’onde.
intermédiaires (mucilages et substances insolubles). Laver la
solution d‘hexane ou d‘éther de pétrole de l’huile neutralisée
avec des quantités successives de 25 ou 30 ml d’une solution
6.3.9 Ballon, de 25 ml, avec réfrigérant à air, d‘environ
de propanol-2 ou d’éthanol (5.1.3) jusqu‘à disparition de la
1 m de longueur avec raccord rodé.
coloration rosée de la phénolphtaléine.
Verser la solution dans le ballon de l‘évaporateur rotatif (6.1.3)
6.3.10 Fiole conique, de 250 ml à bouchon rodé.
et éliminer la plus grande partie du solvant par distillation sous
pression réduite. Sécher l’huile à 30-40 OC sous pression
6.3.11 Fiole conique, de 50 ml (éventuellement).
réduite à l‘aide d’un courant d’azote (5.1.71, jusqu’à élimination
totale du solvant.
6.3.12 Filtre, en verre fritté, de porosité P 40 (16 à 40 pm)
(éventuellement).
8.4 Purification de la prise d’essai sur alumine
a 6.3.13 Dessiccateur.
Préparer une suspension de 15 g d’alumine activée (5.1.6) dans
50 ml d‘hexane ou, à défaut, d‘éther de pétrole (5.1.21, et ver-
ser, en agitant, dans une colonne en verre pour chromatogra-
6.4 Appareillage pour l’analyse des
phie (6.1.2). Régulariser la répartition de l’alumine et laisser
mono-2 glycérides par chromatographie
s‘écouler le solvant jusqu‘à 1-2 mm au-dessus du niveau supé-
en phase gazeuse
rieur de l’adsorbant. Verser soigneusement dans la colonne une
solution préparée par dissolution de 5 g de prise d‘essai, neu-
Voir IS0 5508 et IS0 5509.
tralisée si nécessaire, dans 25 ml d’hexane ou, à défaut, d’éther
de pétrole (5.1.21, et recueillir la totalité du liquide sortant de la
colonne dans un ballon de 100 ml (6.1.6).
7 Échantillonnage
Éliminer la plus grande partie du solvant pa
...

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