2.2. Coffee Samples Ultrafiltration

To separate the high molecular weight (HMW) material from the low molecular weight (LMW) material, all samples were individually submitted to a fractionation procedure by ultrafiltration (Figure 1). The ultrafiltration occurred on an ultrafiltration module—Labscale TFF System (Millipore), operating at room temperature.

Schematic representation of the ultrafiltration process with photos of the successive Permeates and Retentate for espresso coffee and instant coffee brews. * Permeates 1 and 2 represent the most abundant fractions of Low Molecular Weight (LMW) compounds. ** Retentate represents the fraction of High Molecular Weight (HMW) compounds.

Using a pellicon XL ultrafiltration module ultracel membrane with cut-off 100 kDa, and working among 10 and 20 psi transmembrane pressures, the initial 500 mL of solution in the reservoir was concentrated to 10% of the initial volume (50 mL, Retentate 1). The permeated solution was recovered, concentrated in a rotary evaporator at 40 °C, frozen and freeze-dried in a freeze drier (−50 °C, 0.1 mbar) (SCANVAC CoolSafe 9 L, Labogene, Allerød, Denmark) (Permeate 1). The ultrafiltration process was repeated by adding distilled water to Retentate 1 reaching a volume of 500 mL in the reservoir. The ultrafiltration process re-started until the solution in the reservoir was down to 50 mL (Retentate 2). The permeated solution (Permeate 2) was recovered as described for Permeate 1. The ultrafiltration process was repeated for several cycles until the conductivity of the last permeate was below 75 µS·cm⁻¹. A total of 6 and 5 permeate fractions were recovered, respectively, from expresso coffee 1 and instant coffee 1 samples. For analytical purposes, an aliquot of 4 mL was collected from each Retentate, representing each cycle of the ultrafiltration sequential process. All fractions obtained were freeze-dried (−50 °C, 0.1 mbar) (SCANVAC CoolSafe 9 L, Labogene, Allerød, Denmark) and stored under an anhydrous atmosphere.

To disclose possible adsorption phenomena during the ultrafiltration process, an experiment was performed under diluted conditions, dissolving 0.50 g of caffeine and 1.0 g of the final Retentate in 500 mL of water.