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Alternative B: Accelerated 74-Hour Extraction at 25–60
°
C
C. Apparatus
(a) Covered water bath capable of maintaining a temperature of up to 60°C for extended
periods. Ensure the mean temperature in the system is 50.0, 55.0, or 60.0 ± 1.0°C by
monitoring incoming and exit temperatures to the manifold at comparable locations. Before
Extraction Nos. 2, 3, and 4 begin, it is necessary to preheat the bath several degrees (
see
X, Y,
and Z in the
Extraction
section below) above the desired temperature to account for initial
heat exchange and temperature equilibration with manifold and columns. Within 10 min, the
bath should be
stabilized at temperature X, Y, or Z.
(b) Reversible peristaltic pump capable of delivering 4.0 (±0.1) mL/min continuously for 54 h.
Pump heads capable of using 16 to 40 tubes are used for an eight to 20 column apparatus,
respectively (Lsmatc ® No. 78006–00 by Cole-Parmer, Vernon Hills, IL).
(c) Extraction apparatus consisting of two parts (illustrated in Figures
2015.15C–F
). Example
equipment as a parts list can be found in Appendix A of ref. 2 in the online
J. AOAC Int
.
(1) Vertical jacketed chromatography columns enclosing inner column of 2.5 × 30 cm (e.g., Ace
Glass, Vineland, NJ; No. 5821–24, filter removed with Teflon ® adapter No. 5838–51). PTFE rods
(6 mm × 15 cm) should be used to avoid channeling of air or caking. All fittings needed to attach
column, pump tubes, and transfer tubing (e.g., 2.4 mm id tubing). Standardize the length of
tubing for each column (typically about 0.75 meter) and secure fittings.
(2) Constant temperature water circulation manifold and pump system capable of maintaining
adequate (minimum 4 L/min) flow and stable temperature for each column. Insulation is typically
required to maintain a stable temperature. Two inline, symmetrically placed thermometers (Figure
2015.15E
) are used tomonitor temperature to input and outflow of manifolds. Attach roll clamps
and flow monitors to column manifold tubing to ensure balanced flows and uniform
temperatures.
(d) Solvent/extract reservoirs [500 mL volumetric flasks (e.g., Kimball Chase Life Science, Vinland,
NJ; No. 28100–500)] with three-hole stoppers and properly placed rigid tubing attached transfer
tubing and to pump (
see
Figure
2015.15E
). Make sure return tube remains approximately 2
cm from the bottom of the flask to prevent pickup of any precipitates.
(e) Detection equipment capable of analyzing liquids at moderate to high (100–10 000 mg/L)
nutrient levels. Analysis falling below the LOD or LOQ should be noted.
(f) 250 mL graduated cylinders (8–20).
D. Reagents and Reference Materials
Candidates for 2016 Method of the Year
211