Soybean total oil content and profile measurements
In use from 2008-11-01
Soybean has been the major oil seed used for biodiesel resource in US. Soybean has high content of oil and protein. This protocol is used to measure total oil content and oil compositions in soybean seed. Total oil content is measured by Accelerated Solution Extractor (ASE). After in-situ transesterification, oil composition is measured via Gas Chromatography (GC) with flame ionization detector (FID). The major five fatty acids that are produced in soybeans are palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and alpha – linolenic acid (C18:3).
Oil content measurement
The total oil content measurement followed the method of Matthäus, B. and Brühl, L. (2001)
1. Grind the soybean seed by using Foss water-cooled Knifetec 1095 sample mill (Tecator AB, Höganäs, Sweden) for 10 s.
2. Check the moisture content
3. Perform three replicates
4. Make sure that the tops and bottom of the cells are clean
5. Screw the bottom of the cell on to the cylinder
6. Place the Glass fiber filter (Cat. No. 600004-2129-DB, Environmental express) in to the bottom of the cell using the tamping rod
7. Add 1.0 g of powdered sample in to the labeled stainless-steel cell for total oil extraction by Accelerated solvent Extractor ASE 200 (Dionex, Idstein, Germany)
8. The dead volume of the extractor cell is filled with Ottawa sand (Fisher Scientific, Rochester, NY)
9. Screw the top of the cell on to the center cylinder
10. Check the weights of the cell, and record them. Make sure not to tilt the cells
11. Place them on the top carousel of ASE
12. Assemble the labeled collection vials, along with the collection vials for rinsing (R1, R2, R3, R4) in the bottom carousel
13. Set the following condition on the ASE system:
Preheat for 6 min, heat for 6 min, oven temperature at 105°C, static time for 10 min, flush volume 70%, purge time for 60s, 2 static cycles, and extraction pressure at 1000 psi. After process, extraction solvent hexane will be evaporated by purging oxygen free compressed nitrogen (AGA Gas) above the surface. The residue hexane will be removed in oven at 100°C for about 70 min.
14. Turn on the power to the extractor
15. Open the valve on the ASE nitrogen cylinder and also on the regulator
16. Select the method and START
17. After extraction, open the top screw and place the cells in the oven set at 50°C for over night
18. Check the dry weights
The total oil content is calculated by the equation as follow (ref AOCS):
C = 100 x Qw / (W x (1- moisture))
Ow (g) is the total oil extracted from ground sample, W (g) is the weight of ground sample, and moisture% is the moisture percentage of the ground sample measured by A & D Moisture analyze
Fatty acid composition
1. Label the aluminum crushing trays (100 holes) with labeling tape
2. Fill each well of the tray with 2 seeds for each sample
3. Stack the trays as they are filled, always keeping the top tray covered with a glass plate
4. Crush the seed in the aluminum crushing tray using the hydraulic press, wipe the pegs of the crushing plate with hexane and a paper towel
5. Turn the wheel on the front of the press clockwise until it is tight. Using the lever on the right hand side of the press to crush the seed to the pressure of 40,000 lbs
6. Add 400uL n-hexane to the crushed seeds in each well to cover the seeds
7. Cover the tray with a glass plate so that the hexane does not evaporate
8. Let the tray sit for a minimum of two hours
9. Load the plastic trays with GC vials. The vials are labeled and loaded in the trays with the sample positions as in the crushing tray
10. Transfer 100ul from each well in to the respective vial
11. Add 500μl of 1 N sodium methoxide solution in to each GC vial
12. Shake the vials in the plastic tray by sliding the tray back and forth or on the vortex mixer for 30 minutes or until the oil droplets are gone
13. After 30 minutes, add 150uL distilled water to each vial. The liquid in the vial should turn cloudy.
14. Add 1250μL of hexane in to each vial
15. Let them sit still till the suspension separates in to two phases. The top layer contains the fatty acid methyl esters dissolved in hexane, and the bottom layer contains the sodium methoxide, water and glycerol.
16. The samples are ready for analysis on the gas chromatograph or store at 4C until you are ready to start the analysis
Matthäus, B. and Brühl, L. 2001. Comparison of different methods for the determination of the oil content in oilseeds. Journal of the American Oil Chemists’ Society 78: 95-102.
Authors:: Juan Gao, Dechun Wang, Pavani Tumbalam