Analytics

5.11.1 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The determination of Triton X-114 and cinnamic acid concentrations were performed with an Agilent 1200 series HPLC system (Agilent Technology Inc., Santa Clara, USA). The injection volume was 2 µL, the flow rate 1 ml min^–1. The mobile phase consisted of acetonitrile and water. The gradient of the two solvents was set over time as follows: 0-2 min: 60%/40%; 2 – 4.5 min: linear increase from 60%/40% to 100%/0%; 4.5 – 6 min: 100%/0%; 6 – 6.5 min: linear decrease from 100%/0% to 60%/40%; 6.5 – 10 min: 60%/40%. All measurements were performed at 55°C column temperature. The components were both detected at a wavelength of 275.8 nm, with retention times of about 2 min for cinnamic acid and about 6 min for Triton X-114. The respective calibration curves are given in appendices A14 and A15.

5.11.2 SIZE-EXCLUSION CHROMATOGRAPHY

Samples, containing a surfactant, water, and algae products were analyzed by size-exclusion chromatography (SEC) in collaboration with the “Institute for Technical and Macromolecular Chemistry,” University of Hamburg. The aim was to separate the products from the surfactant and thus qualify the extract composition.

The samples were prepared for the analysis as follows. 30 g sample was transferred into a 100 mL round-bottom flask. The sample was lyophilized. The residue was extracted three times for 5 min with chloroform, and the extract was filtered through a 0.2 µm syringe filter into a 50 mL pear-shaped flask. The solvent was completely evaporated in a rotary evaporator.

Then, the final residue was diluted in 5 g tetrahydrofuran, and one mL was measured by SEC (calibration using polystyrene).

5.11.3 DETERMINATION OF THE GALLIC ACID EQUIVALENTS

The yield of phenolic compounds after the cloud point extraction from pineapple juice was determined using the gallic acid equivalents (GAE) as a standard quantification method of total phenols content [166]. The GAE determination was conducted using the spectrophotometer Art Evolution 300 UV-VIS from Thermo

Scientific. To avoid external absorbance disturbances, two quartz SUPRASIL cuvettes from Hellma were used. Before every determination, they were filled with distilled water and used as reference cell and after, one of them was replaced with the sample.

In order to obtain the specific wavelength of the phenolic compounds among all the components in the surfactant-rich sample, the absorption spectra in a range from 190 to 400 nm was obtained separately for each compound.

The absorbance spectra were measured for the following samples: ROKAnol NL5 (0.01 wt%), gallic acid (1 mg/L), and pineapple juice (0.002 wt%). The results are presented in Figure 5.3.

Figure 5.3: Absorbance spectra of the single compounds

The spectra of the gallic acid had two maximums at 210 and 265 nm. In accordance, pineapple juice had a similar behavior to gallic acid, having a maximum absorbance peak at 265 nm. This behavior was expected due to its composition rich in phenolic compounds [76,77]. On the other hand, the surfactant ROKAnol NL5 showed only a maximum absorbance at 225 nm, and because of the low absorbance at other wavelengths, it was assumed that the surfactant did not influence in the determination of phenolic compounds.

The total phenol content of pineapple juice in the present work was measured by reading the absorbance at a wavelength of 275 nm. A calibration curve of absorbance against gallic acid concentration is shown in appendix A 16.

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5.11.4 DETERMINATION OF THE ANTIOXIDANT CAPACITY

The antioxidant capacity was used for the evaluation of the CPE from pineapple juice. The analysis was performed according to Peshev et al. [165]. The antioxidant radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) that has a maximal spectrophotometric absorbance at 517 nm was applied. The antioxidants in the sample reacted with DPPH to DPPH-H and thus decreased its the absorbance. For the analytical procedure, a standard methanolic solution containing 0.1 mmol·L^-1 DPPH was prepared. Its absorbance was measured using an UV-photometer at 517 nm after 30 minutes and was used as a reference value. Additionally, 0.25 mL of each sample were mixed with 0.75 mL standard solution and their absorbance signal was monitored every 10, 20 and 30 min as well. A baseline was obtained using pure methanol. Hence, antioxidant capacity was calculated as “quenched DPPH” in mmol DPPH/g sample according to Equation 5-12.

–—˜ ℎ˜ d ™ = (‘A− ‘<)/‘A

Equation 5-12: Calculation of quenched DPPH

A: Absorbance of the reference (R) and the sample (S)

5.11.5 DETERMINATION OF THE REDUCING SUGARS

The sugar distribution during the cloud point extraction from pineapple juice was determined via photometric analysis of total reducing sugar using DNS-reagent.

The 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540 nm [167]. The sample was diluted to match the valid absorbance range of the applied UV-photometer. Afterwards, 0.167 ml of the sample mixed with 0.333 ml 0.05 M citrate buffer and 1 ml DNS-reagent into 2 ml centrifuge tubes. The mixture is boiled in a water bath for 5 minutes and cooled in iced water immediately to stop the reaction. 0.1 ml of the boiled sample was diluted with 1.25 ml of demineralized water in a polystyrene cuvette.

Subsequently, the UV-absorbance was measured at 540 nm. A calibration for glucose was conducted before the sample analyses (appendix A 17).

5.11.6 DENSITY MEASUREMENTS

The density of surfactant solutions at a specific temperature was determined with a density meter of the type DMA 4500 M from Anton Paar GmbH. Samples of 1 mL were fed into the pre-heated measuring tube, and density values obtained when temperature equilibrium was reached.

5.11.7 VISCOSITY MEASUREMENTS

The viscosity of the samples was determined using a rheometer of the type Kinexus pro from Malvern Instruments. The geometry used for the determination was a PL65-CP-20. The sample and the measuring surface of the bottom geometry were tempered by a cryo-compact circulator of the type CF41, supplied by JULABO GmbH.

5.11.8 DETERMINATION OF THE RELATIVE PHOTOSYNTHETIC ACTIVITY

The photosynthetic activity (PA) of the green microalgae Acutodesmus obliquus was determined according to Glembin et al. [132]. The PA of the microalgae photosystem II was measured by Pulse Amplitude Measurement (PAM) with a MAXI – Imaging-PAM chlorophyll fluorimeter (Heinz Walz GmbH). Before the fluorescence took place, the samples were dark adapted to have a maximal number of active chlorophyll centers. The integrated camera captured the maximal fluorescence. The PA value was transferred to the Imaging-WIN Software. Thus, the relative photosynthetic activity (RPA) of each sample was calculated as a ratio between the sample’s PA value and the one of the reference surfactant-free microalgae culture.