Chromafil xtra pvdf 45 25

Tris buffer (250.0 mM Tris, 88.5 mM citric acid, 69.38 mM glucose, 10.0 mM HEPES, 100 IU penicillin, 50 mg streptomycin, 100 mL ultrapure water; 320 mOsm and pH 7.2) was used as a base extender for production of extenders A and B, composed of 1 or 2% soybean lecithin, respectively.
To prepare soybean lecithin-based extender (SL), 1 or 2% (w/v) of soybean lecithin (P5638) were added to the base extender and kept hydrated at room temperature (25 °C) for 1 h. Afterward, the extenders were homogenized in a magnetic stirrer to form a homogeneous solution. Subsequently, the extenders were kept in an ultrasonic bath for 30 min (25 °C) for fragmentation of the micelles, following the methodology described by
Mozafari (2010) (link)
. After sonication, debris were removed. The solutions were clarified by centrifugation (2,200 × g for 30 min) and then filtered twice: first through a 3 µm pore (Cellulose Ester Filter, SSWP04700, Merck-Millipore, USA) and then through a 0.45 µm pore (Chromafil® Xtra PVDF-45/25, Macherey-Nagel GmbH & Co., Germany), as described by
De Paz et al. (2010) (link)
with modifications.

High purity acrylamide, ¹³C₃-acrylamide and 5-hydroxymethylfurfural were purchased from Sigma-Aldrich (Diesenhofen, Germany). HPLC gradient grade acetonitrile and methanol were purchased from J.T. Baker (Deventer, Holland). All other chemicals used in this study were of analytical grade. Zorbax Extend-C18 column (250 × 4.6 mm, 5 μm) was purchased from Agilent Technologies (Santa Clara, CA, USA), and 0.45 μm PVDF (polyvinyl difluoride) filters Chromafil Xtra PVDF-45/25 from Macherey-Nagel (Düren, Germany). Ultra-pure water was used throughout the experiments (MilliQ system, Millipore, Bedford, MA, USA).
Stock solutions of acrylamide (1 mg/mL), ¹³C₃-acrylamide (1 mg/mL) and HMF (1 mg/mL) were prepared by dissolving in distilled water:acetonitrile (50:50 v/v). Working solutions were prepared by diluting the stock solution with water:acetonitrile (50:50 v/v) to concentrations of 0.75, 1, 2.5, 5, 10, and 25 ng/mL for acrylamide and ¹³C₃-acrylamide; 50, 100, 250, 500, 1000 and 2500 ng/mL for HMF. Stock solutions and working standards were kept at 4 °C. Carrez I solution was prepared by dissolving 15 g of potassium hexacyanoferrate in 100 mL of water, and Carrez II solution by dissolving 30 g of zinc sulfate in 100 mL of water.