Simplicity uv apparatus

All chemicals and standards had a purity grade higher than 97%. Sotolon was purchased from SAFC (St. Louis, MO, USA). Absolute ethanol was from Sigma-Aldrich (Steinheim, Germany); formic acid, tartaric acid, and UPLC grade methanol were from Panreac (Barcelona, Spain). Ethyl acetate was from Fisher Scientific (Leicestershire, UK). The type 1 ultrapure water was obtained with a Simplicity® UV apparatus from Millipore (Milford, MA, USA).
A synthetic fortified wine was prepared and consisted in a solution containing 6 g/L of tartaric acid in a 180 mL/L ethanol/water solution, pH adjusted to 3.5 with a 1 M sodium hydroxide (Panreac) solution. Standard stock (400 mg/L) and working (200 mg/L) solutions of sotolon were rigorously prepared in ethanol and water, respectively. Seven calibration points were prepared in synthetic wine and in a fortified wine, within the validation range 1–2000 μg/L.
All eluents were filtered through a hydrophilic polypropylene 0.2 μm pore size membrane filter (Pall Corporation, Ann Arbor), before being used. Wine extracts were filtered using Chromafil PTFE 0.2 µm syringe filters (Macherey-Nagel, Düren, Germany), before being analysed.
A sample set of 44 fortified wines with different ages (3–115 years old), sweetness degrees (including dry (5), medium-dry (6), medium-sweet (10), and sweet wines (23)), and ethanol contents (18 to 20%) were analysed.

Ammonium acetate (NH₄OAc, ≥99.99%), ammonium hydroxide (ACS reagent), and acetic acid (ACS reagent) were purchased from Sigma-Aldrich (Steinheim, Germany). The electrolyte solution was prepared by dissolving 40 mM of Ammonium acetate with water of ultrahigh quality (UHQ) delivered by a Simplicity UV apparatus (18.2 MΩ × cm at 25 °C, Millipore, Billerica, MA, USA). The solution was adjusted to different pH levels ranging from 4.0 to 9.0 (with 1.0 step increase) with ammonium hydroxide or acetic acid. Lastly, the solution was filtered through a surfactant-free cellulose acetate membrane with 0.20 µm pore size syringe filters (Sartorius, Göttingen, Germany).

ammonium acetate (NH₄OAc, ≥99.99%) and ammonium hydroxide (ACS reagent) were both purchased from Sigma‐Aldrich (Steinheim, Germany). The nES GEMMA electrolyte solution was prepared by dissolving 40mM of ammonium acetate with water of ultra‐high quality (UHQ) delivered by a Simplicity UV apparatus (18.2 MΩ × cm at 25°C, Millipore, Billerica, MA, USA). The solution was adjusted to pH 7.0 with ammonium hydroxide and filtered through a surfactant‐free cellulose acetate membrane with 0.20‐μm pore size syringe filters (Sartorius, Göttingen, Germany).

Ammonium
acetate (NH₄OAc, ≥99.99%) and ammonium hydroxide
(ACS reagent) were both purchased from Sigma-Aldrich (Steinheim, Germany).
The GEMMA electrolyte solution was prepared by dissolving 40 mM ammonium
acetate with water of ultrahigh quality (UHQ) delivered by a Simplicity
UV apparatus (18.2 MΩ × cm at 25 °C, Millipore, Billerica,
MA, USA). The solution was adjusted to pH 7.0 with ammonium hydroxide
and filtered through a surfactant-free cellulose acetate membrane
with 0.20 μm pore size syringe filters (Sartorius, Göttingen,
Germany).
AF4 carrier buffer (PBS) was prepared by dissolving
sodium chloride (≥99.5%), monopotassium phosphate (≥99.0%),
potassium chloride (≥99.5%, all from Sigma-Aldrich), and disodium
phosphate (≥99.5%, Merck, Darmstadt, Germany) in UHQ water.
The elution buffer additionally included 0.02% (w/v) sodium azide
(Merck) as an antimicrobial agent. The pH was adjusted to 7.4 with
ammonium hydroxide and filtered through a 0.1 μm pore size polyethersulfone
membrane filter (VacuCap, Pall, NY, USA).
Sample preparation
for AFM measurements required UHQ water and
nitrogen gas (≥99.999%, Messer Austria GmbH, Gumpoldskirchen
Austria) for rinsing and drying.