4% H2SO4 dans methanol:Toluene 90:10 (500 ml) :pour 100 ml : ajouter 9,8 ml de

toluene sec (tamis moleculaire 4A et filtre) dans 88,0 ml de methanol sec (tamis
moleculaire 3A et filter). A jouter ensuite 2.2 ml d acide sulfurique 98% (d=1,84)
(attention aux risques de projections) . Conserver le reactif dans un flacon
hermetiquement bouche, a 4C et a lobscurite. utiliser un reactif frais (moins dune
semaine)

Hexane (2000 ml): de bonne qualite analytique

NaCl 10% (1000 ml) :100g de NaCl dans 1 L deau distillee. Melanger, agiter avant
employ
NaHCO3 1% (1000 ml) : 10 g de NaHCO3 dans 1L deau distillee, melanger (ph-8)
Standard interne (facultatitf) : C21:0 ou/et C23:0 a 1 ou 0.1 mg/ml dans le
chloroforme

Reglage des pipettes et distributeurs

Reactif de transesterification: pipette graduee en verre 5 ml+ propipette
Hexane: distributeur 5 ml regle a 5 ml
NaCl 10% et NaHCO3 1% :distributeur 10 ml regle a 10 ml
Standards internes (facultatif): seringue verre de 50, 100 ou 250 l
Deroulement des operations
NB: dans le cas ou une T.E de 8 a 16 heures est choisie, prevoir de debuter
levaporation des extraits lipidiques de facon a distribuer le reactif a partir de 17
heures.
1. Evaporer au bain a sec regle a 40C maxi, d abord sous air puis sous azote ,
dans des tubes pyrex 16*160 mm, des volumes d extrait lipidique ne
depassant pas 5 mg d equivalent acides gras libres. Facultatif: ajouter la
quantite de standard intern C21:10 ou/et C23:0 a 1 mg/ml calculee par le
programme (seringue de 50,100 ou 250 l)
2. Apres obtention des culots lipidiques, mettre les tubes dans un dessicateur
(avec coupelle de silicagel bleu ou de pentoxyde de phosphore ), sous vide
(20 mm HG) pendant 15 min. verifier l absence totaled eau dans les culots
3. Distribucr dans chaque tube 5 ml de reactif de TE (pipette verre 5 ml ) Si
possible, souffler de lazote dans chaque tube . boucher hermetiquement et
vortex pendant 5 sec
4. Placer les tube dans un bloc chauffant regle a 80 C pendant 2 a 4 heures ou a
60C pendant 8 a 16 heures (ou une unit), suivant la nature de l echantillon,
verifier labsence de fuite apres 15 min . en case de fuite, examiner le col du

tube et le joint PTFE et rajouter du methanol sec. passer les tubes au vortex
pendant 10 sec . toutes les 30 minutes
5. Laisser refroidir les tubes pendant 5 min. pour chaque tube , purger le
distributeur et ajouter 5 ml d hexane (distributeur 5 ml), vortex pendant 10
sec. tube ouvert, et transfert direct dans un tube a centerifuger conique 34
mm (serie A). Ajouter a nouveau 5 ml d hexane , vortex pendant 5 sec, et
transfert dans le meme tube a centrifuge.
6. Purger le distributeur et ajouter 10 ml de NaCl 10% (distributeur 10 ml) au
contenu des tubes a centrifuge (agiter le flacon avant emploi) .boucher
fermemnt vortex pendant 55 sec . et laisser reposer jusqua ce que les deux
phases soient claires(15min. normalement).centrifuger 5 min -1000 g
(2300t/min.)a 10 c si necessaire
7. Transferer par aspiration la totalite des phases inferieures dans dautres tubes
a centrifuger coniques 34mm (serieB ). laisser les tubes serie A ouverts
8. Ajouter 2*5ml d hexane (distributeur 5 ml)au contenu des tubes a centrifuger
serie B pour recuperer le reste des EMAG presents dans la phase
inferieure. Boucher fermement vortex pendant 25 sec. et laisser
reposer jusqu a ce que les deux phases soient claires (10 min.
normalement). Centrifuge 5 min. 1000 g (2300 t/min.) a 10C si
necessaire.
9. Transferer par aspiration les phases inferieures continues dans les
tubes a centerifuger serie B dans une bouteille dont le contenu sera
jete. Prendre un minimum de phase superieure organique des tubes.
10.Transferer par aspiration les phases organiques des tubes a
centrifuge serie B aux tubes a centrifuge serie A correspondants.
11.Purger le distributeur et ajouter 10 ml de NaHCO3 1% (distributeur 10 ml) au
contenu des tubes a centrifuge serie A pour neutraliser l acidite residuelle .
Boucher, vortex pendant 5 sec .et laisser reposer jusqu a ce que les deux
phases soient claires (5 min normalement)
12.Transferer par aspiration les phases inferieures continues dans les
tubes
a centrifugeur serie A dans une bouteille dont le contenu sera jete.
Prendre um minimum de phase superieure organique des tubes.
13.Centrifuge 5 min.-1000 g (rayon de rotation 17 cm : 2300 t /min.) a
10C et aspirer leau residuelle et les residus presents au fond des
tubes dans une bouteille dont le contenu sera jete. Prendre un
minimum de phase superieure organique des tubes.
14.Transferer par aspiration les phases organiques restantes dans des
tubes 16*150 mm numerotes. Eviter d aspirer les residus restant au
fond des tubes. Boucher hermetiquement les tubes sans forcer, et
stocker au dessous de -20C

4% H2SO4 in methanol:Toluene 90: 10 (500 ml): for 100 ml: Add 9.8 ml of dry
toluene (molecular 4A and filter screens) to 88.0 ml of dry methanol (molecular 3A
and filter screens). A then add 2.2 ml sulphuric acid d 98% (d = 1, 84) (pay
attention to the risk of projections). Keep the reagent in a sealed bottle mouth, 4 c
and dark. use a fresh reagent (less dune week)

Hexane (2000 ml): good analytical quality

10% NaCl (1000 ml): 100g of NaCl in 1 L of distilled water. Mix, shake before
employees
NaHCO3 1% (1000 ml): 10 g of NaHCO3 in 1 L of water distilled, mix (ph-8)
Internal standard (facultatitf): C21:0 or / and C23:0 has 1 or 0.1 mg/ml in chloroform

Adjusting of the pipettes and dispensers

Transesterification reagent: pipette graduated glass 5 ml + propipette
Hexane: distributor 5 ml rule has 5 ml
10% and NaHCO3 1% NaCl: dispenser 10 ml rule (a) 10 ml
(Optional) internal standards: syringe glass of 50, 100 or 250 l
Conduct of operations
NB: in the case or a 8 T.E 16 hours is selected, start evaporation of lipid extracts
provide so as to distribute the reagent from 5 pm.
1 evaporate to the bath dry rule a 40 c Max, first under air and nitrogen, in tubes
pyrex 16 * 160 mm, d volumes then extracted lipid exceeding not 5 mg free
fatty acid equivalent d. Optional: Add the amount of standard intern C21:10
or / and C23:0 1 mg/ml calculated programmatically (50,100 or 250 l
syringe)
2 after obtaining lipid caps, put tubes in a desiccator (with Cup blue silica gel or
phosphorus pentoxide), vacuum (20 mm HG) for 15 min. check the absence
totaled water in caps
3 Distribucr in each 5 ml of reagent tube of TE (pipette 5 ml glass) if possible,
blow of concentration in each tube. Boucher hermetically and vortex for 5
seconds
4 place the tube in a heatblock rule 80 C for 2 to 4 hours or 60 c for 8 to 16 hours
(or a unit), depending on the nature of the sample, check absence of leakage
after 15 min. in case of leakage, examine the neck of the tube and seal PTFE
and add dry methanol. pass tubes with a vortex for 10 sec. every 30 minutes
5 cool tubes for 5 min. for each tube, flush the dispenser and add 5 ml hexane
(distributor 5 ml) d, vortex for 10 sec. tube open and direct transfer into an a
centerifuger conical tube 34 mm (series A). Add a new 5 ml d hexane, vortex
for 5 seconds, and transfer in the same tube a centrifugal.

6 purge the Distributor and add 10 ml of 10% NaCl (dispenser 10 ml) to the
contents of the tubes a centrifugal (shake the bottle before use) .boucher
fermemnt vortex for 55 seconds. and let it sit until the two phases are
expected to be claires(15min._normalement).centrifuger 5 min - 1000 g
(2300 RPM) a 10 c if necessary
7 transfer by suction all the phases below in other tubes a centrifuge conical 34
mm (serieB). leave the open series A tubes
8 add 2 * 5 ml d hexane (distributor 5 ml) to the contents of the tubes a
centrifuge series B to retrieve the rest of present in the phase EMAG
inferior. Firmly plug vortex for 25 sec. and let stand until the two
phases are clear (10 min. normally). Centrifugal 5 min. 1000 g (2300
RPM) a 10 c if necessary.
9 transfer by suction phases continuous lower in a centerifuger series B
in a bottle gift tubes' content will be thrown. Take a minimum of
phase organic tube top.
10 transfer by suction organic phases of a centrifuge tubes tubes series
B has centrifugal series matching A.
11 purge the Distributor and add 10 ml of NaHCO3 1% (10 ml dispenser) content
tubes has centrifugal series A to neutralize l residual acidity. Boucher, vortex
for 5 seconds .and let stand until has the two phases are clear (normally 5
min)
12 transfer by suction phases continuous lower into the tubes
centrifuge series A in a bottle whose contents will be thrown. Take
minimum phase um organic top of the tubes.
13 centrifugal 5 min.-1000 g (17 cm: 2300 t/min. Turning RADIUS) a 10 c
and aspirate residual water and residue present at the bottom of the
tubes in a bottle whose contents will be thrown. Take a minimum of
phase organic tube top.
14 transfer by suction remaining organic phases in 16 * 150 mm
numbered tubes. Avoid d pick up remaining residue at the bottom of
the tubes. Boucher sealed tubes without force, and store below-20 c

4% H2SO4 in methanol: 90:10 Toluene (500 ml): 100 ml: Add 9.8 ml of dry toluene
(molecular sieves 4A and filter) in 88.0 ml of dry methanol (molecular sieve 3A and
filter). A jousting then 2.2 ml of 98% sulfuric acid (d = 1.84) (attention to the risk of
splashes). Keep the reagent in a bottle mouth tightly, 4C and the dark. use a fresh
reagent (less than a week)

Hexane (2000 ml) of good analytical quality

10% NaCl (1000 ml): 100 g NaCl in 1 L of water distilled. Mix, shake before
employee
NaHCO3 1% (1000 ml): 10 g of NaHCO3 in 1L of water distilled, mix (ph-8)
Internal standard (facultatitf): C21: 0 and / or C23: 0 to 1 or 0.1 mg / ml in
chloroform

Tuning of pipettes and dispensers

Reagent transesterification: glass pipette GRADUATED 5 ml + propipette
Hexane distributor rule to 5 ml 5 ml
NaCl 10% and 1% NaHCO3: 10 ml dispenser Rule 10 ml
Internal standards (optional): glass syringe 50, 100 or 250 .mu.l
Workflow
NB: in cases where a TE 8 to 16 hours is chosen, to predict debuter evaporation lipid
extracts way to distribute the reagent from 17 hours.
1. Evaporate the bath Dry rule to 40C max, of first in air and then under nitrogen in
Pyrex tubes 16 * 160 mm, the lipid extract of volumes not exceeding 5 mg
equivalent of free fatty acids. Optional: add the amount of intern standard C21: 10
and / or C23: 0 to 1 mg / ml calculated by the program (50,100 syringe or 250 .mu.l)
2. After obtaining lipid pellets, put the tubes in a desiccator (with cup-blue silica gel
or phosphorus pentoxide) in vacuo (20 mm Hg) for 15 min. Totaled verify the
absence of water in the pellets
3. Distribucr each tube 5 ml of TE reagent (5 ml glass pipette) If possible, blowing
lazote in each tube. butcher tightly and vortex for 5 sec
4. Place the tube in a heating block rule to 80 C for 2 to 4 hours or 60C for 8 to 16
hours (or unit), depending on the nature of the sample, verify the absence of
leakage after 15 minutes. runaway box, examine the neck of the tube and PTFE and
add dry methanol joint. pass the tubes vortexed for 10 sec. every 30 minutes
5. Cool tubes for 5 min. for each tube, vent the valve and add 5 ml of hexane (5 ml
dispenser), vortex for 10 sec. open tube, and direct transfer into a tube 34 mm
conical centerifuger (series A). Add a further 5 ml of hexane, vortex for 5 seconds
and transfer to the same centrifugal tube.
6. Purge the dispenser and add 10 ml of 10% NaCl (10 ml distributor) the contents
of the centrifuge tubes (shake the bottle before use) .boucher fermemnt vortex for
55 sec. and let stand jusqua that both phases are clear (15min. normally)
.centrifuger 5 min -1 000 g (2300t / min.) 10 c if necessary
7. Aspiration Transferer the totality of lower phases in other conical centrifuge tubes
34mm (serieB). let the tubes A series open
8. Add 2 * 5 ml of hexane (5 ml dispenser) to the contents of the tubes centrifuged
Series B to retrieve the rest of FAME present in the lower phase. Boucher strong
vortex for 25 sec. and let stand until it has both phases are clear (10 min. normally).
Centrifuge 5 min. 1000 g (2300 r / min.) A 10C if necessary.

9. Transferer Aspiration continuous lower phases in the tubes has centerifuger

Series B in a bottle do not the content will be thrown. Take a minimum of upper
organic phase tubes.
10. Transferer Aspiration organic phases tubes centrifuged to Series B centrifuge
tubes corresponding A series.
11. Bleed the dispenser and add 10 ml of 1% NaHCO3 (10 ml dispenser) to the
contents of the tubes centrifuged Series A to neutralize residual acidity. Boucher,
vortex for 5 sec .and let stand up to it that the two phases are clear (normally 5
minutes)
12. Transferer Aspiration continuous lower phases in the tubes a centrifuge Series A
in a bottle whose contents will be thrown. Take um minimum of upper organic phase
tubes.
13. Centrifuge 5 min-1 000 g (17 cm turning radius. 2300 t / min) 10C and vacuum
residual water and residues present in the tube bottom in a bottle whose contents
will be thrown. Take a minimum of upper organic phase tubes.
14. Transferer Aspiration remaining organics in tubes numbered 16 * 150 mm. Avoid
suck the residue remaining in the bottom of the tubes. Boucher tightly without forci
ng the tubes and store at below -20