The Good pH probe: non-invasive pH in-line monitoring using Good buffers and Raman spectroscopy.

In bioprocesses, the pH value is a critical process parameter that requires monitoring and control. For pH monitoring, potentiometric methods such as pH electrodes are state of the art. However, they are invasive and show measurement value drift. Spectroscopic pH monitoring is a non-invasive alternative to potentiometric methods avoiding this measurement value drift. In this study, we developed the Good pH probe, which is an approach for spectroscopic pH monitoring in bioprocesses with an effective working range between pH 6 and pH 8 that does not require the estimation of activity coefficients. The Good pH probe combines for the first time the Good buffer 3-(N-morpholino)propanesulfonic acid (MOPS) as pH indicator with Raman spectroscopy as spectroscopic technique, and Indirect Hard Modeling (IHM) for the spectral evaluation. During a detailed characterization, we proved that the Good pH probe is reversible, exhibits no temperature dependence between 15 and 40 °C, has low sensitivity to the ionic strength up to 1100 mM, and is applicable in more complex systems, in which other components significantly superimpose the spectral features of MOPS. Finally, the Good pH probe was successfully used for non-invasive pH in-line monitoring during an industrially relevant enzyme-catalyzed reaction with a root mean square error of prediction (RMSEP) of 0.04 pH levels. Thus, the Good pH probe extends the list of critical process parameters monitorable using Raman spectroscopy and IHM by the pH value.

Good's buffers as novel phase-forming components of ionic-liquid-based aqueous biphasic systems.

Aiming at the development of self-buffering and benign extraction/separation processes, this work reports a novel class of aqueous biphasic systems (ABS) composed of ionic liquids (ILs) and organic biological buffers (Good's buffers, GBs). A large array of ILs and GBs was investigated, revealing than only the more hydrophobic and fluorinated ILs are able to form ABS. For these systems, the phase diagrams, tie-lines, tie-line lengths, and critical points were determined at 25 °C. The ABS were then evaluated as alternative liquid-liquid extraction strategies for two amino acids (L-phenylalanine and L-tryptophan). The single-step extraction efficiencies for the GB-rich phase range between 22.4 and 100.0 % (complete extraction). Contrarily to the most conventional IL-salt ABS, in most of the systems investigated, the amino acids preferentially migrate for the most biocompatible and hydrophilic GB-rich phase. Remarkably, in two of the studied ABS, L-phenylalanine completely partitions to the GB-rich phase while L-tryptophan shows a preferential affinity for the opposite phase. These results show that the extraction efficiencies of similar amino acids can be tailored by the design of the chemical structures of the phase-forming components, creating thus new possibilities for the use of IL-based ABS in biotechnological separations.

Facile Determination of Aluminum Content in Industrial Brine by Investigating the Effects of Buffer Systems.

The aluminum content of concentrated (27 wt%) sodium chloride solutions could be crucial for large-scale chlor-alkali-based industries applying membrane cell electrolysis. Thus, a facile method which enables a fast and reliable protocol to determine the Al content of these solutions on ppb scale in industrial environments is fundamentally important. It was demonstrated that the increased sensitivity of colorful Al-ECR (eriochrome cyanine R) complex by the use of a cationic surfactant and specific biological buffers could effectively indicate the Al content in an extended pH interval of a concentrated saline medium under industrial conditions. The dependence of the analytical protocol on pH, temperature, time, wavelength, and the salinity of the medium was investigated. It was shown that the absorbance-based measurements of the solution should be performed at least 2-4 h after its preparation. By applying the selected two Good's buffers (HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, MOPS: 3-(N-morpholino)-propanesulfonic acid) and Tris (tris(hydroxymethyl)aminomethane), 32.8-38.1 % increase in the sensitivity was achieved for saturated NaCl solutions. Moreover, the limits of detection and quantification (LOD, LOQ) were also lowered by 19.0-29.8 %, and the salinity dependence of the calibration was also reduced.

Potential of Good's buffers to inhibit denaturation of myofibrillar protein upon freezing.

The current systematic study sought to examine the potential use of three Good's buffers (MES, MOPS and HEPES) in inhibiting myofibrillar protein (MFP) denaturation induced by acidity changes. The highest degree of acidity variation was found in the center and bottom of large bottles due to the freeze-concentration effect. Good's buffer tended to basify during freezing, and it could prevent the crystallization of sodium phosphate (Na-P) buffer. Acidification upon freezing Na-P disrupted the natural conformation of MFP and induced the formation of large proteins aggregates with tight packing. The 15 mM MES, 20 mM MOPS, and 30 mM HEPES were respectively added to neutralize the strong acidity drop induced by freezing 20 mM Na-P, and all of them significantly improved the stability of the MFP conformation (P < 0.05). This work is not only critical to meet the growing demand for protein, but also groundbreaking for broadening the applicability of Good's buffers in the food industry.

Zwitterionic betaines over HEPES as the new generation biocompatible pH buffers for cell culture.

Good's buffers have been widely applied in cell/organ culture over the past half a century as biocompatible pH stabilizers. However, the emergence of severe adverse effects, such as cellular uptake, lysosomal autophagic activation, and visible light-induced cytotoxicity, raises serious questions over its biocompatibility while underlying mechanism was unclear. Here we report that riboflavin (RF, component of cell culture medium) generates 1O2, ·OH, and O2 •- under visible light exposure during regular cell manipulation. These short half-life reactive oxygen species (ROS) react with tertiary amine groups of HEPES, producing 106.6 µM of H2O2. Orders of magnitude elevated half-life of ROS in the medium caused severe cytotoxicity and systematic disorder of normal cell functions. We have further designed and validated zwitterionic betaines as the new generation biocompatible organic pH buffers, which is able to completely avoid the adverse effects that found on HEPES and derivate Good's buffers. These findings may also open a new avenue for zwitterionic betaine based materials for biomedical applications.

Concise analysis of γ-hydroxybutyric acid in beverages and urine by capillary electrophoresis with capacitively coupled contactless conductivity detection using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid as background electrolyte.

γ-Hydroxybutyric acid (GHB), a neurotransmitter or neuromodulator in the human central nervous system, is often abused in drug-facilitated sexual assaults due to its euphoric and sedative effects. While the analysis of GHB has received continuous attention, its inherent characteristics pose challenges. In the current study, capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) was built, and Good's buffers were evaluated as the background electrolytes for CE separation and C4D detection. On this basis, a simple and efficient CE-C4D method was developed for GHB analysis. Through theoretical discussion and experimental optimization, the separation of GHB and related positional isomers α-hydroxybutyric acid (AHB) and ß-hydroxybutyric acid (BHB) was achieved within 4 min using 150 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as the running buffer. Under the optimized condition, the relative standard deviations of migration time and peak area were less than 1.1% and 4.5%, indicating good precision. The C4D signal of GHB showed a good linear relationship with GHB concentration in the range of 3-300 µM with a determination coefficient of 0.9997, and the detection limit was calculated to be 0.37 µM based on the signal-to-noise ratio of three. Furthermore, liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were comparatively studied for sample matrix purification. Combined with the optimized SPE procedure, the developed CE-C4D method has been successfully applied for the determination of exogenous GHB in spiked beverages and endogenous GHB in human urine.

Cholinium-based Good's buffers ionic liquids as remarkable stabilizers and recyclable preservation media for recombinant small RNAs.

RNA is a biopolymer of high relevance in the biopharmaceuticals field and in fundamental and applied research; however, the preservation of the RNA stability is still a remarkable challenge. Herein, we demonstrate the enhanced potential of aqueous solutions of self-buffering cholinium-based Good's buffers ionic liquids (GB-ILs), at 20 and 50 % (w/w), as alternative preservation media of recombinant small RNAs. The thermal stability of RNA is highly enhanced by GB-ILs, with an increase of 14 °C in the biopolymer melting temperature - the highest increase observed up to date with ILs. Most GB-ILs investigated improve the stability of RNA at least up to 30-days, both at 25 °C and at 4 °C, without requiring the typical samples freezing. Molecular dynamics simulations were applied to better understand the molecular-level mechanisms responsible for the observed RNA improved stability. The number of IL cations surrounding the RNA chain is similar, yet with differences found for the IL anions, which are responsible for the overall charge of the biopolymer first solvation sphere. No cytotoxicity of the studied solutions containing RNA and ILs at 20 % (w/w) was observed onto two distinct human cell lines, reinforcing their potential to act as preservation media when foreseeing biopharmaceutical applications. Finally, RNA was successfully recovered from the ILs aqueous solutions, without changes in its structural integrity, and the ILs successfully recycled and reused.

Equilibrium Studies of Dibutyltin(IV)-Zwitterionic Buffer Complexation.

Equilibrium studies in aqueous solution are reported for dibutyltin(IV) (DBT) complexes of the zwitterionic buffers "Good's buffers" Mes and Mops. Stoichiometric and formation constants of the complexes formed were determined at different temperatures and ionic strength 0.1 mol·L-1 NaNO3. The results show that the best fit of the titration curves were obtained when the complexes ML, MLH-1, MLH-2 and MLH-3 were considered beside the hydrolysis product of the dibutyltin(IV) cation. The thermodynamic parameters ΔHo, ΔSo and ΔGo calculated from the temperature dependence of the formation constant of the dibutyltin(IV) complexes with 2-(N-morpholino)ethanesulfonic acid (Mes) and 3-(N-mor-pholino)-propanesulfonic acid (Mops) were investigated. The effect of dioxane as a solvent on the formation constants of DBT-Mes and DBT-Mops complexes decrease linearly with the increase of dioxane proportion in the medium. The concentration distribution of the various complexes species was evaluated as a function of pH.