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FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Preparation of apricot saturated juice. 300 g of frozen apricot were homogenized together with 150 mL
of water UP (ultra pure) and 200 g of (NH4)2SO4. The mixture was centrifuged at 10,000xg rpm during
30 min to 4 ºC, and the saturated juice (supernatant) was recovered.
Liquid-liquid extraction method (L-L).
In a cold bath, where blended 50 mL of saturated juice, with 30 mL of CH2Cl2 and 48 µg of 4-nonanol
as internal standard (IS). The mixture was agitated during 30 minutes on a gaseous nitrogen saturated
atmosphere. Later, the mixture was centrifuged at 10,000xg at 4ºC during 15 min (this extraction was
done twice). Watery phase was removed and the organic phase (CH2Cl2 + volatile compounds) was
object of microdistillation process.
Concentration using reflux (microdistillation). The microdistillation process is the final step, commonly
used in the extraction techniques with solvents here mentioned. The organic phase (CH2Cl2 + volatile
components) coming from any of the methods (L-L, SDE and C18) was concentrated, the following
way: the organic phase was dehydrated with Na2SO4 and filtrated through glass fiber, collecting the
filtrate in a flask of conical bottom of 250 mL. The sample filtrated was distilled in a Vigreux column,
heating the flask of conical bottom in a bath at 45ºC, to concentrate the volume of the sample
approximately to 0.5 mL. The extract concentrated was stored -20 ºC in a 2 mL vial, until the moment
of its analysis in GC-FID.
Clarification of the saturated juice.
210 mL of the saturated juice was defrosted in a bath of water at room temperature (15 to 20 ºC) and
treated as Boulanger (1999), liquefied using a mixture of cellulose (5 g/L), pectinase (2 g/L) PVP (0.2
g/L) at 25 ºC for 90 min, and centrifuged (30 min, 10 000xg) at 4 ºC. The clear supernatant (saturated
juice clarified) was used in the reverse phase chromatography.
Reverse phase chromatography (C18).
The C18 column (Varian®, Walnut, CA, USA) was activated passing through 25 mL of CH3OH and
later 25 mL of water UP. 50 mL of the saturated juice clarified were mixed with 48µg of 4-nonanol as
IS and filtrated through the column C18, at flow rate of 1.5mL/min, the free aroma compounds (free
fraction) were adsorbed in the solid phase of the column. Later, the column was washed with 30 mL of
water UP, to elute the polar components, the free fraction was eluted of the column with 30 mL of
CH2Cl2. Finally this fraction was conduced to the microdistillation process.
Simultaneous distillation extraction (SDE).
Preparation of the sample.
100 g of frozen Apricot were mixed with 200 mL of phosphate buffer (pH 8), 48 µg of 4-nonanol as IS
and 0.2 mL of antifoaming, during 4 min. The final pH was on a 7± 0.2 range.
Parameters in the SDE.
The flask with the sample it was assembled to the Likens-Nickerson apparatus and warmed at 100
to120 ºC range, until boiling. Simultaneously in the other section of the apparatus a small flask was
assembled, with 30 mL of CH2Cl2, this was heated to 45 ºC. The heating of the sample stayed 2 hours.
The solvent was recovered and stored at -20 ºC until their use in the micro distillation process.
Solid Phase Microextraction (SPME).
A puree was prepared with 50 g of frozen Apricot and 50 mL of water UP, in a coldbath. 5 g of this
puree was placed in a 20 mL vial and 5 mL of a saturated solution of NaCl were added. The vial was
sealed tightly and incubated at 40 ºC during one hour, later, the needle of the syringe SPME was
inserted and the fiber (Carboxen/PDMS de 65 µm, Supelco, Bellefonte, PA) was exposed in the head
space inside the vial during 20 minutes. After, the fiber SPME was retracted carefully; the needle of
the vial was taken out and immediately injected in a GC-FID exposing the fiber during 4 min in the
injector. The quantification of the concentration of volatile compounds in SPME it is determined by
means of a standard curve of 4-nonanol.
GC-FID conditions: A Varian 3300 (Walnut Creek, CA, USA) chromatograph equipped with
split/splitless injector and flame ionization detector (FID) was used for all GC analysis. A fused silica
capillary column (J&W Scientific, Folsom, CA, USA) was employed (30m x 0.25 mm i.d., film tickness,
0.25 µm). The temperature program was: increased of 40 to 200 ºC (at 3 ºC/min), then from 200 to
250 ºC (at 5 ºC/min) and maintained for 5 min. The injector temperature was maintained at 250 ºC and
the detector temperature was 300 ºC.