Formulations for immobilized pH gradients including pH extremes

Formulations are given both for narrow (less than 2 pH units) and for wide range (up to 8 pH units) immobilized pH gradients, spanning between pH 2.5 and pH 11. The contribution from water to the buffering power (beta) at these pH extremes requires the recipes to be optimized (in terms of gradient linearity) for each desired level of beta av.     

Proteorhodopsin Photocycle Kinetics Between pH 5 and pH 9

The retinal protein proteorhodopsin is a homolog of the well‐characterized light‐driven proton pump bacteriorhodopsin. Basic mechanisms of proton transport seem to be conserved, but there are noticeable differences in the pH ranges of proton transport. Proton transport and protonation state of a carboxylic acid side chain, the primary proton acceptor, are correlated. In case of proteorhodopsin, the pK_a of the primary proton acceptor Asp‐97 (pK_a ≈ 7.5) is unexpectedly close to environmental pH (pH ≈ 8). A significant fraction of proteorhodopsin is possibly inactive at natural pH, in contrast to bacteriorhodopsin. We investigated photoinduced kinetics of proteorhodopsin between pH 5 and pH 9 by time resolved UV/vis absorption spectroscopy. Kinetics is inhomogeneous within that pH region and can be considered as a superposition of two fractions. These fractions are correlated with the Asp‐97 titration curve. Beside Asp‐97, protonation equilibria of other groups influence kinetics, but the observations do not point toward major differences of primary proton acceptor function in proteorhodopsin and bacteriorhodopsin. The pK_a of proteorhodopsin and some of its variants is suspected to be an example of molecular adaptation to the physiology of the original organisms.     

适合低PH范围测量的新型中性载体膜PH电极的研究

本文设计合成了一种在低ＰＨ范围对氢离子有很好Ｎｅｒｎｓｔ响应的新型中性载体－Ｎ，Ｎ－二辛基菸酰胺，并把它制成ＰＶＣ膜ＰＨ电极，测试了该电极的线性范围、选择性、稳定、重现性和内阻等各项性能参数，并试验了电极抗氢氟酸腐蚀的能力，该该电极用于氢氟酸的电离常数测定时获得了满意的结果。     

pH indicating resins

pH indicating resins were prepared by covalently attaching carboxylic acid derivatives of sulfthalein dyes, synthesized using a Suzuki cross-coupling, onto resin beads. The resin-bound indicators showed the expected colour changes according to pH and their behaviour was analysed using a micro UV/Vis spectrometer.     

Polyaniline pH Electrodes

Polyaniline-coated Pt, Pd, and graphite electrodes have been studied. Their potential relationship to pH follows the same principle as the pH glass electrode. They are solid state involving no redox reactions. The potential is derived from the surface charge density caused by protonation or deprotonation. They act as capacitors. The nonprotonated form of polyaniline was used for pH 6.8 to 1.2 and the protonated form of polyaniline was used for pH 6.8 to 13.0. Three regions of curves (acid, neutral, and alkaline) were obtained. The polyaniline-coated graphite electrode is recommended. It offers the advantage of sturdiness and may be used in microscale in HF solution, at high pressure. At pH 2.2, approximately 4,200 H⁺ions were adsorbed by polyaniline per nm². An insignificant number of H⁺ions were adsorbed by polyaniline at pH 6.2; this is close to its isoelectric point.     

Simulating phenol high-performance liquid chromatography retention times as the pH changes. Mobile phase pH versus buffer pH

The HPLC retention times of several substituted phenols have been measured and simulated using Advanced Chemistry Development's LC simulator, using 50% acetonitrile (ACN) as the mobile phase. For alkyl- and nitro-substituted phenols, the quality of the simulation improves when pH of the mobile phase is estimated and used in the simulation. Simply using the pH of the buffer gives simulation results that are not as close to the actual retention times. However, the opposite is the case for halogenated phenols. The pK(a) values in 50% ACN for some of these phenols have also been determined, which tend to be one unit higher than the aqueous pK(a) values reported in the literature.     

Immobilized pH gradients (IPG) simulator--an additional step in pH gradient engineering: II. Nonlinear pH gradients

While in the companion paper (Tonani, C. & Righetti, P. G., Electrophoresis 1991, 12, 1011-1021) we gave the general outline of our new computer program, immobilized pH gradients (IPG) simulator, able to simulate and optimize linear pH gradients for isoelectric focusing in immobilized pH gradients, in the present report we extend the application of such a program to: (i) convex exponential gradients, (ii) logarithmic and (iii) polynomial gradients. Such gradients are meant to give equal space to protein spots in complex protein mixtures (e.g., cell lysates, biological fluids) and follow the statistical distribution of protein pI values along the pH axis. They will prove of fundamental importance in two-dimensional maps, both because they optimize the spreading of spots in the two-dimensional plane and because of the excellent reproducibility of immobilized pH gradients. The following concave exponential recipes are given: pH 3-8, pH 3-9, pH 3-10, pH 3-11, pH 4-7, pH 4-8, pH 4-9, pH 4-10, pH 4-11, pH 5-8, pH 5-9, and pH 5-10, as well as the most extended pH 2.5-11 interval. Two interesting logarithmic gradients are described: pH 3-6 and pH 3-7 and one sigmoidal (derived with a polynomial of 5th degree): pH 3-11.     

Traceable measurements of pH

 The primary method for pH is based on the measurement of the potential difference of an electrochemical cell containing a platinum hydrogen electrode and a silver/silver chloride reference electrode, often called a Harned cell. Assumptions must be made to relate the operation of this cell to the thermodynamic definition of pH. National metrology institutes use the primary method to assign pH values to a limited number of primary standards (PS). The required comparability of pH can be ensured only if the buffers used for the calibration of pH meter-electrode assemblies are traceable to these primary pH standards. To assess the degree of equivalence, comparisons of primary measurement procedures for pH were organized in co-operation with EUROMET. Typical results will be presented. In 1998, the Consultative Committee for Amount of Substance (CCQM) decided to include the field of pH in its working programme. The first key comparison for this quantity was recently carried out on two phosphate buffer solutions. Received: 14 June 2000 / Accepted: 22 August 2000     

Associative pH sensitive system

New associative pH sensitive systems composed of a modified dextran bearing 2-carboxycyclohexyl carboxyl groups and neutral (p betaCD) or positively charged (p betaCDN(+))beta-cyclodextrin-co-epichlorhydrin copolymers have been synthesized and their properties were investigated as a function of pH by phase diagrams and viscosimetry. The affinity between the CD cavities and the hydrophobic guests (2-carboxycyclohexyl carboxyl groups) has been studied as a function of pH. The system with the neutral p betaCD shows a pH dependent behavior due to the lowest affinity of the guest at pH above 5 for the cavity. Associative phase separation is produced at low pH whereas soluble complexes occur at higher pH. The opposite trend is observed with the system containing the cationic p betaCDN(+). This behavior is due to the combination of electrostatic and inclusion complex interactions in this system. Addition of salt, by screening the electrostatic interactions, also strongly influences the response of the system.     

In-House pH Reference Materials

 In-house pH reference materials (IHRMs) traceable to the corresponding NIST pH standards have been developed. Characterization of the IHRMs was based on a comparative approach implying transmission of measurement information from the corresponding standard to the IHRM by simultaneous pH measurements in solutions of both the IHRM and the standard under the same conditions. While the measurements were performed, a statistically detectable temporal drift of the measurement system took place. This did not hinder extraction of the necessary measurement information by way of the comparative approach applied. Correspondence: The National Physical Laboratory of Israel (INPL), Givat Ram, Jerusalem 91904, Israel. e-mail: ilya_kus@netvision.net.il Received August 20, 2002; accepted November 8, 2002     

Thick-film hybrid pH sensors

The application of thick-film techniques to pH electrode construction is reported. The performance and general aspects of their operation are discussed, as a function of their construction. Particular attention is paid to the fundamental factors affecting the sensitivity of the pH-selective material (glass)after processing through standard printing conditions.     

Immobilized pH gradients (IPG) simulator--an additional step in pH gradient engineering: I. Linear pH gradients.

A new computer program, called immobilized pH gradients (IPG) simulator, is proposed for calculating and optimizing any recipe for use in isoelectric focusing in immobilized pH gradients. Unlike our previous monoprotic electrolyte gradient simulation (MGS) and polyelectrolyte gradient simulation (PGS) programs, based on minimizing CV(beta), the present program has a target function the minimization of the quadratic moment around zero of the residuals (mu 2). With this algorithm it is possible to formulate IPG recipes which have deviations from linearity well below 1% of the given pH interval (a limit set with the previous MGS and PGS programs), in fact, as small as 0.1-0.2% (in pH units). The new simulator performs 2-3 times better than the previous ones in the pH 4-10 range, and is absolutely necessary when working outside this range, at extreme pH values, where CV(beta) cannot work against the buffering power of bulk water, thus generating pH recipes with huge deviations from linearity. In the latter cases, mu 2 performs 10 times better than CV(beta). When utilizing strong titrants for extended pH intervals, the "all or none" rule has been discovered: such titrants should always be used in tandem, since omission of one of the two at either the acidic or basic extremes produces strongly distorted pH profiles. Our new, most powerful simulator also contains equations for creating nonlinear gradients, notably: concave and convex exponentials and sigmoidal (see the companion paper: Righetti, P. G. and Tonani, C., Electrophoresis 1991, 12, 1021-1027).     

Development of pH measurement system for legal traceability of pH standard solutions

The best reproducible technology of pH measurement for precise pH buffer solutions regulated by Japanese Industrial Standards (JIS) was studied. A pH meter was devised with a high resolution of +/- 0.0001 pH. An 18-bit analog-to-digital converter is used, one-bit resolution corresponding to 0.0019 mV (ca. 0.000032 pH) against an input electrode potential +/- 500 mV. Digital data were treated smoothly for some types of noise, a reproducibility of +/- 0.0002 pH being obtained with a potentiometer. A flow cell was devised to attain temperature control within +/- 0.03 degrees C and air-tight measurement prevented contamination with carbon dioxide. Also, the flow cell has a structure such that potassium chloride (KCl) inner solution effused from a ceramic junction of the reference electrode designed so as not to touch the glass membrane. A combination pH electrode (a glass electrode and a reference electrode) was assembled to minimize the dead volume of sample solution. This highly sensitive pH measuring system, consisting of a pH meter, a flow cell, a combination pH electrode, a circulating water thermostat and a peristaltic pump, was used for the certification of pH standard solutions in Japanese metrological law. The performance of this system was within +/- 0.0006 pH reproducibility and 20-30 min response time (5 min within +/- 0.0002 pH) at a sample flow rate of 3 ml min (-1).     

An organic-based pH oscillator

In a recent paper, we suggested that the acid- or base-catalyzed dehydration of a hydrated carbonyl compound provides a suitable foundation for an organic-based pH oscillator. Here we present the first experimental example of such an oscillator in a flow reactor, utilizing the base-catalyzed dehydration of methylene glycol as a source of positive feedback (OH- autocatalysis) coupled with the base-catalyzed hydrolysis of gluconolactone for negative feedback (H+ production). The large amplitude oscillations (between pH 7 and 10) are reproduced in a kinetic model of the reaction. Such experiments present new possibilities in the design of pH oscillators.     

Live cell imaging of lysosomal pH changes with pH responsive ratiometric lanthanide probes

Europium and terbium complexes of two structurally related ligands have been evaluated as optical probes to monitor changes in lysosomal pH; calibration using ionophores and fluorescent probes allows monitoring of the time dependence of lysosomal pH change, examining the green/red intensity ratio from internalised Tb-Eu complexes.     

利用手持技术探究不同酸碱环境下生物体pH变化的规律

利用手持技术数字化实验新手段做经典实验,通过pH传感器实时监测了去离子水、磷酸盐缓冲溶液、碳酸盐缓冲溶液、猪肝研磨液（新鲜和变质）和生菜研磨液（新鲜和变质）等溶液分别在酸、碱滴定过程中pH的变化,依据实验曲线和数据并结合相关生物化学知识,定量分析和比较了各物质滴定结果,从而证明在不同酸碱环境下生物体能够在一定范围内维持pH稳定并解释了其原因。     

Novel coumarin-based fluorescent pH indicators,probes and membranes covering a broad pH range

A new family of coumarin-based pH indicators was synthesized. They are sensitive to pH in either weakly acidic or weakly basic solution. The indicators possess moderate to high brightness, excellent photostability and compatibility with light-emitting diodes. The indicators were covalently immobilized on the surface of amino-modified polymer microbeads which in turn were incorporated into a hydrogel matrix to obtain novel pH-sensitive materials. When a mixture of two different microbeads is used, the membranes are capable of optical pH sensing over a very wide range comparable to the dynamic range of the glass electrode (pH 1–11). A new family of coumarin-based pH indicators is synthesized for the use in either weakly acidic or weakly basic solution. The indicators possess moderate to high brightness, excellent photostability and compatibility with light-emitting diodes. Novel pH-sensitive materials are obtained by covalent immobilization of the indicators on the surface of amino-modified polymer microbeads which in turn are incorporated into a hydrogel matrix. Sensing of pH over a very wide range also becomes possible.     

Characterisation and deployment of an immobilised pH sensor spot towards surface ocean pH measurements

The oceans are a major sink for anthropogenic atmospheric carbon dioxide, and the uptake causes changes to the marine carbonate system and has wide ranging effects on flora and fauna. It is crucial to develop analytical systems that allow us to follow the increase in oceanic pCO₂ and corresponding reduction in pH. Miniaturised sensor systems using immobilised fluorescence indicator spots are attractive for this purpose because of their simple design and low power requirements. The technology is increasingly used for oceanic dissolved oxygen measurements.     

Measuring local pH in electrochemistry

Localized pH measurements are important in various areas of electrochemistry, from corrosion to bio-electrochemistry and electrocatalysis. Different techniques are available to perform these measurements and offer numerous possibilities in terms of spatial and temporal resolution, sensitivity, and precision. In this brief review we present the recent progress made and summarize the main techniques available for localized pH measurements in electrochemistry such as scanning probe techniques (SECM, SICM, SIET), laser (confocal) fluorescence microscopy, rotating ring-disc electrode (RRDE) voltammetry, and infra-red spectroscopy, among others.