20
After the digestion and extraction, the solvent is transferred in a previously weighed aluminium bowl in order to perform a cold extraction. The digestion in the vial is treated again with 5 mL n-hexane and then stirred on a magnetic stirrer for one minute. Afterwards, this solvent will be transferred in the aluminium bowl. This cold extraction is repeated three to four times. The solvent with the extracted fat is dried in the heating oven for twenty minutes at 105°C and the bowl finally weighed. Calculation of the total fat content is done by following Equation 1:
F(%)=
m2 m1
m2 - m1* 100E = Mass in g empty bowl
= Mass in g bowl with dried fat E = Mass in g weight sample
Figure 3. Chromatogram of the 37 Standard-Mix identified FAMEs.
For the derivatisation (MDM), one or two drops of the dried fat are treated in a glass vial with 10 mL water-free methanolic potassium hydroxide solution (2.5 weight-%), and a stirrer chip is added.
The alkalinec derivatisation requires the following parameters on the Discover SP-X®
Final temperature: 90°C Ramp:
Hold time: Stirrer: Power:
5 minutes 10 minutes Medium 200 Watt
After finishing the alkaline derivatisation, the vial is cooled down in a water bath. 14,.25 mL Methanol and 0,.75 mL hydrochloric acid (37 weight-%) are added, and the acidic derivatisation is generated with following parameters on the Discover SP-X®
Final temperature: 120°C Ramp:
Hold time: Stirrer: Power:
Figure 4. Chromatogram of fatty acids in a sausage sample, derivatisation according to ISO procedure and identified FAMEs.
5 minutes 6 minutes Medium 200 Watt
After finishing the derivatisation, the vial is cooled down in a water bath. 10 mL n-Hexanhexane is are added, and the vial is turned twice from bottom to top.
The generation of overpressure must be avoided. The glass vial is afterwards filled with saturated sodium chloride solution up to the top. The solvent needs to be transferred with a pipette to a 10 mL GC vial, including sodium sulphate or magnesium sulphate as drying agent (approx. 0.2 g) for possible water residue. This solution is now ready for the analysis of FAMEs using GC-FID.
GC analysis:
For identification and quantification of FAMEs, a 37 Standard-Mix (Supelco) has been used and analysed on GC-2010 Plus AF with FID detector.
The following parameters have been set on the GC: Injector:
Injection volume:
Figure 5. Chromatogram of fatty acids in a sausage sample, derivatisation according to microwave procedure and identified FAMEs.
SPL (Split 1:100) 1 µL
Injector temperature: 250°C Column type: Inner diameter: Film thickness:
Carrier gas speed:
FAME WAX 0.25 mm 0.25 µm
Detector Temperature: 250°C Mobile Phase:
Helium 35 cm / second Equation 1
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132
