Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography

Polar betacyanin pigments together with betaxanthins from ripe cactus fruits of Hylocereus polyrhizus (Cactaceae) were fractionated by means of preparative ion-pair high-speed countercurrent chromatography (IP-HSCCC) also using the elution–extrusion (EE) approach for a complete pigment recovery. HSCCC separations were operated in the classical ‘head-to-tail’ mode with an aqueous mobile phase. Different CCC solvent systems were evaluated in respect of influence and effectiveness of fractionation capabilities to separate the occurring pigment profile of H. polyrhizus. For that reason, the additions of two different volatile ion-pair forming perfluorinated carboxylic acids (PFCA) were investigated. For a direct comparison, five samples of Hylocereus pigment extract were run on preparative scale (900 mg) in 1-butanol–acetonitrile–aqueous TFA 0.7% (5:1:6, v/v/v) and the modified systems tert.-butyl methyl ether–1-butanol–acetonitrile–aqueous PFCA (2:2:1:5, v/v/v/v) using 0.7% and 1.0% trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) in the aqueous phase, respectively. The chemical affinity to the organic stationary CCC solvent phases and in consequence the retention of these highly polar betalain pigments was significantly increased by the use of the more lipophilic fluorinated ion-pair reagent HFBA instead of TFA. The HFBA additions separated more effectively the typical cacti pigments phyllocactin and hylocerenin from betanin as well as their iso-forms. Unfortunately, similar K_D ratios and selectivity factors α around 1.0–1.1 in all tested solvent systems proved that the corresponding diastereomers, 15S-type pigments cannot be resolved from the 15R-epimers (iso-forms). Surprisingly, additions of the stronger ion-pair reagent (HFBA) resulted in a partial separation of hylocerenin from phyllocactin which were not resolved in the other solvent systems. The pigments were detected by means of HPLC-DAD and HPLC-electrospray ionization–MS using also authentic reference materials.     

Fluctuation Theory for Markov Random Walks

Two fundamental theorems by Spitzer–Erickson and Kesten–Maller on the fluctuation-type (positive divergence, negative divergence or oscillation) of a real-valued random walk \((S_{n})_{n\ge 0}\) with iid increments \(X_{1},X_{2},\ldots \) and the existence of moments of various related quantities like the first passage into \((x,\infty )\) and the last exit time from \((-\infty ,x]\) for arbitrary \(x\ge 0\) are studied in the Markov-modulated situation when the \(X_{n}\) are governed by a positive recurrent Markov chain \(M=(M_{n})_{n\ge 0}\) on a countable state space \(\mathcal {S}\); thus, for a Markov random walk \((M_{n},S_{n})_{n\ge 0}\). Our approach is based on the natural strategy to draw on the results in the iid case for the embedded ordinary random walks \((S_{\tau _{n}(i)})_{n\ge 0}\), where \(\tau _{1}(i),\tau _{2}(i),\ldots \) denote the successive return times of M to state i, and an analysis of the excursions of the walk between these epochs. However, due to these excursions, generalizations of the aforementioned theorems are surprisingly more complicated and require the introduction of various excursion measures so as to characterize the existence of moments of different quantities.     

Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. ssp. rhamnoides) by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC)—a support free all liquid–liquid chromatography technique—has been successfully used for the preparative isolation of isorhamnetin 3-O-β-d-glucoside, isorhamnetin 3-O-β-rutinoside, quercetin 3-O-β-d-glucoside, syringetin 3-O-β-d-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane–n-butanol–water, 1:1:2 v/v/v) in ‘head-to-tail’ mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-β-d-glucoside (95 mg), isorhamnetin 3-O-β-rutinoside (10 mg), quercetin 3-O-β-d-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-β-d-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC–ESI–MS–MS, 1D-NMR (¹H, ¹³C, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.     

Control of the anodic aluminum oxide barrier layer opening process by wet chemical etching

In this work, it has been shown that, through a highly controlled process, the chemical etching of the anodic aluminum oxide membrane barrier layer can be performed in such a way as to achieve nanometer-scale control of the pore opening. As the barrier layer is etched away, subtle differences revealed through AFM phase imaging in the alumina composition in the barrier layer give rise to a unique pattern of hexagonal walls surrounding each of the barrier layer domes. These nanostructures observed in both topography and phase images can be understood as differences in the oxalate anion contaminated alumina versus pure alumina. This information bears significant implication for catalysis, template synthesis, and chemical sensing applications. From the pore opening etching studies, the etching rate of the barrier layer (1.3 nm/min) is higher than that of the inner cell wall (0.93 nm/min), both of which are higher than the etching rate of pure alumina layer (0.5-0.17 nm/min). The established etching rates together with the etching temperature allow one to control the pore diameter systematically from 10 to 95 nm.