Millex hv hydrophilic pvdf

In the present study, chemicals with analytical purity were used. Organic acid standards (citric acid, tartaric acid, oxalic acid, malic acid, succinic acid and fumaric acid), sugar standards (glucose, fructose, and sucrose), and vitamin C standards (l-ascorbic acid) were obtained from Sigma–Aldrich (St. Louis, MO, USA). The other chemicals were obtained from Merck (Darmstadt, Germany).
For organic acid extraction, the method by Bevilacqua and Califano [18 (link)] was modified. About 200 g of samples was fragmented and 10 g from each sample was delivered to centrifuge tubes. The 10 ml of 0.009 N H₂SO₄ was added to the samples and the samples were homogenized with Heidolph Silent Crusher M, Germany. Then, the samples were mixed for an hour with a shaker (Heidolph Unimax 1010, Germany) and centrifuged at 14.000 × rpm for 15 min. The supernatants were passed through coarse filter paper, then twice through a 0.45 mm membrane filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, USA), and last in a SEP-PAK C18 cartridge. The concentration of organic acids was determined by HPLC using an Aminex column (HPX-87H, 300 mm × 7.8 mm, Bio-Rad) fitted on an Agilent 1100 series HPLC G 1322 A, Germany) [18 (link)]. Organic acids were detected at 214 and 280 nm wavelengths. The mobile phase, 0.009 N H₂SO₄ was passed through a 0.45 μm filter membrane.

The samples were kept in
a deep freezer (−80 °C) until the analysis. In the study,
citric acid, tartaric acid, malic acid, succinic acid, and fumaric
acid contents of organic acids were determined in a service tree fruit.
The method given by Bevilacqua and Califano^{26 (link)} was modified and used for the extraction of organic acids. 50 g
of the service tree samples were taken and transferred to centrifuge
tubes. 20 mL of 0.009 N H₂SO₄ was added to these
samples and homogenized (Heidolph Silent Crusher M, Germany). Then,
it was mixed for 1 h on a shaker (Heidolph Unimax 1010, Germany) and
centrifuged at 15,000 rpm for 15 min. The aqueous fraction separated
in the centrifuge was first passed through a coarse filter paper,
then twice through a 0.45 μm membrane filter (Millipore Millex-HV
Hydrophilic PVDF, Millipore, USA), and finally through the SEP-PAK
C18 cartridge. Samples were analyzed in an HPLC instrument (Agilent
HPLC 1100 series G 1322 A, Germany). An Aminex HPX-87 H, 300 mm ×
7.8 mm column (Bio-Rad Laboratories, Richmond, CA, USA) was used in
the HPLC system, and the device was controlled by a computer with
an Agilent package program. The DAD detector (Agilent, USA) in the
system is tuned to 214 and 280 nm wavelengths. In the study, 0.009
N H₂SO₄ passed through a 0.45 μm membrane
filter was used as the mobile phase.

Organic acids identified by the technique reported by Bevilacqua and Califano [56 (link)]. Organic acid standards used in research; oxalic acid, tartaric acid, malic acid, citric acid and fumaric acid standards were prepared at the levels of 100, 200, 300, 400, 500, 600, 700 and 800 ppm and formed curves. Each sample (50 g) mixed with 80 mL of 0.009 N H₂SO₄ (Heidolph Silent Crusher M, Berlin, Germany), then homogenized for 1 h with a shaker (Heidolph Unimax 1010, Berlin, Germany). The mixture centrifuged for 15 min at 15,000 rpm, and supernatants filtrated twice with 0.45 µm membrane filter following filtration with coarse filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, Burlington, MA, USA) and run through a SEP-PAK C18 cartridge. Organic acid readings performed with HPLC using Aminex column (HPX—87 H, 300 mm × 7.8 mm, Bio-Rad Laboratories, Richmond, CA, USA) at 214 and 280 nm wavelengths, on Agilent package program (Agilent, Santa Clara, CA, USA).