Presence Of Buffer Research Articles

Membrane optode for uranium(VI) ions preconcentration and quantification based on a synergistic combination of 4-(2-thiazolylazo)-resorcinol with 8-hydroxyquinaldine

A membrane optode was developed utilizing the 8-hydroxyquinaldine (HQ) facilitated preconcentration of UO 2 2+ ions and subsequent colored complex formation of UO 2 2+ with 4-(2-thiazolylazo)-resorcinol (TAR) in optode matrix. The composition of the membrane optode was optimized by scanning several extractants immobilized in different plasticized polymer matrices. It was observed that the chelating agent HQ along with an indicator TAR immobilized in the tri-(2-ethylhexyl)phosphate (TEHP) plasticized cellulose triacetate matrix (CTA) was best suited as an optode for the UO 2 2+ ions in aqueous samples. On sorption of UO 2 2+ in the optode matrix, TAR changes color of the optode from yellow to magenta having a maximum absorbance ( λ max) at 546 nm. The uptake of UO 2 2+ ions in the optode was found to be pH dependent and was maximum (>90%) at pH above 3. The acetate buffer (0.1 mol L −1 sodium acetate + 0.1 mol L −1 acetic acid) was found to be necessary for the stable response. The optimum equilibration time for the optode (2 cm × 1 cm) was found to be 30 min in 10 mL aqueous sample containing acetate buffer (pH 4.75). The equilibration time was found to increase with increase in aqueous sample volume. The optode response was found to be linear in the UO 2 2+ ions concentration range of 0.01–0.11 μmol L −1 in tap water as well as aqueous solutions containing 0.1 mol L −1 NaCl or NaNO 3. The tolerance to the presence of several cations and anions in the determination of UO 2 2+ ion was studied. It was observed that the optode in the presence of buffer can tolerate presence of large amounts of interfering cations (Ce 4+, V 4+, Eu 3+, Al 3+, Fe 3+, Ni 2+, Cd 2+, Co 2+, Pb 2+, Hg 2+, Cu 2+ and Th 4+ ions) without hindering the sorption of UO 2 2+ ions in the optode matrix. The present work indicated that 50 ppb UO 2 2+ ions in 100 mL sample can easily be quantified using this optode. The optode was found to be fully reversible, can readily be regenerated by equilibrating it with 0.1 mol L −1 HNO 3 and reusable up to three cycles. The applicability of the developed optode in real samples was studied by determining uranium in the ground water samples spiked with a known quantity of UO 2 2+ ions.

In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the ubiquitin-mediated destruction of both misfolded and normal ER-resident proteins. ERAD substrates must be moved from the ER to the cytoplasm for ubiquitination and proteasomal destruction by a process called retrotranslocation. Many aspects of retrotranslocation are poorly understood, including its generality, the cellular components required, the energetics, and the mechanism of transfer through the ER membrane. To address these questions, we have developed an in vitro assay, using the 8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We have now directly demonstrated in vitro retrotranslocation of full-length, ubiquitinated Hmg2p-GFP to the aqueous phase. Hrd1p was rate-limiting for Hmg2p-GFP retrotranslocation, which required ATP, the AAA-ATPase Cdc48p, and its receptor Ubx2p. In addition, the adaptors Dsk2p and Rad23p, normally implicated in later parts of the pathway, were required. Hmg2p-GFP retrotranslocation did not depend on any of the proposed ER channel candidates. To examine the role of the Hrd1p transmembrane domain as a retrotranslocon, we devised a self-ubiquitinating polytopic substrate (Hmg1-Hrd1p) that undergoes ERAD in the absence of Hrd1p. In vitro retrotranslocation of full-length Hmg1-Hrd1p occurred in the absence of the Hrd1p transmembrane domain, indicating that it did not serve a required channel function. These studies directly demonstrate polytopic membrane protein retrotranslocation during ERAD and delineate avenues for mechanistic understanding of this general process.

Co-solvent solubilization of some poorly-soluble antidiabetic drugs

Co-solvent solubilization approach has been used to enhance the solubility of seven antidiabetic drugs: gliclazide, glyburide, glipizide, glimepiride, repaglinide, pioglitazone, and roziglitazone. Solubility in water, phosphate buffer (pH 7.4), six co-solvent solutions prepared in water as well as phosphate buffer (pH 7.4) and pH-solubility profile of various drugs have been determined at 25°C. Aqueous solubility of various drugs was found to be less than 0.04 mg/mL. Solubility of gliclazide, glipizide and repaglinide increased by 3-6 times by using phosphate buffer (pH 7.4) as solvent. Solubility enhancement by pH modification was not sufficient. Significant enhancement in solubility could be achieved by the use of co-solvents. The combined effect of co-solvent and buffer was synergistic and enormous increase in solubility of sulfonylureas and repaglinide could be achieved. In the case of glitazones, however, co-solvent alone caused significant enhancement; the presence of buffer had negative effect on the solubilization potential of the co-solvents. Up to 763, 316, 153, 524, 297, 792 and 513 times increase in solubility could be achieved in the case of gliclazide, glyburide, glimepiride, glipizide, repaglinide, pioglitazone and rosiglitazone, respectively.

Efficient Approach to Determine the pKa of the Proton Release Complex in the Photocycle of Retinal Proteins

This work utilizes a photoelectrochemical approach to study the pH dependence of proton release and uptake in the photocycles of two retinal proteins, bacteriorhodopsin (BR) and archaerhodopsin 4 (AR4). By detecting photoinduced potentials that originate from the proton concentration changes (DeltaH(+)) generated by proteins near the indium tin oxide (ITO) electrode, we show that the kinetics of release and uptake can be followed in a broad pH range, and the pK(a) of the proton release complex (PRC) can be easily determined under different conditions. Nonoriented protein films were deposited on the electrode, and photovoltage in an electrochemical cell was detected after illumination with a green flash. The kinetics of proton release and uptake could be measured as light-induced decreases and increases of the photopotential. A kinetic analysis was performed, and a formula describing proton fluxes of wild-type BR and AR4 and D96N mutant of BR was derived. Three componentsfast proton release, slow proton release, and proton uptakewere found in the wild-type retinal proteins; two components, fast and slow proton releases, were found in the D96N mutant. The pH dependence of the fraction of fast release over the whole release was used to determine the pK(a) for proton release in the photocycles of these retinal proteins. Measurements were also performed in conventional buffer solutions and crown ether. The presence of buffer in 10-50 mM concentration did not abolish the light-induced signals, indicating that the electrode response is much less sensitive to buffers than pH-sensitive dyes in a suspension due to a higher protein/buffer ratio near the electrode. This feature enables us to study effects of chemicals with high buffer capacity, and significant effects of buffers and crown ether on proton pumping behaviors of retinal proteins were revealed. In comparison with the classic pH-sensitive dye approach, the photoelectrochemical approach is convenient and efficient for measurements of transient proton concentration changes (DeltaH(+)) generated by a proton pump and thus might be utilized as a powerful tool for the investigation of light-driven proton pumping mechanisms in a wide pH range.

Kinetic study of nitrosation of methylformamide

AbstractNitrosation of methylformamide (MFA) has been studied kinetically. The results obtained show that the reaction rate is first order with respect to both the MFA and proton concentration. The absence of catalysis by nucleophilic anions and the observed general base catalysis is interpreted in terms of formation of a protonated nitrosamide intermediate, being the proton transfer the slow step of the process. The primary solvent isotope effect observed corroborates this mechanism. From this study, we were able to obtain the values of the rate constants for the nitrosation process. The catalytic constants in the presence of buffers were also obtained and the analysis of the Brönsted slopes suggests a process with a transition state more similar to the reactants than the products. The absence of electron donating groups in MFA leads to a very low value of the bimolecular rate constant of the nitrosation reaction. Copyright © 2009 John Wiley & Sons, Ltd.

Microsensor technology for measuring H + flux in buffered media

Nearly all experiments designed to quantify phenomena in biological systems are conducted in the presence of buffers. Most analytical techniques measure free proton activity, and do not account for the activity of protons associated with buffer molecules, potentially leading to an underestimation of biophysical proton transport. In applications where total proton flux activity at the level of biological membranes is of interest, a dynamic biophysical approach is required. This paper presents a simplified method and data filtering algorithm for measuring electrophysiological, transmembrane H + flux activities in buffered media based on the self-referencing microsensor technique. A simplified mathematical model was developed that accounts for chemical H + buffering in a background matrix of buffer molecules. The model and buffer correction algorithm were validated for various biochemically relevant buffers over the 6.1–10.7 pH range. The buffer correction algorithm was used to filter the output of a self-referencing ion-selective H + sensor for the measurement of proton flux. Special emphasis has been placed on the use of pH sensitive microelectrodes when operating as self-referencing flux sensors, but the analytical method is also applicable to the use of static electrodes and pH sensitive dyes.

Rate-Dependent Carbon and Nitrogen Kinetic Isotope Fractionation in Hydrolysis of Isoproturon

Stable isotope fractionation permits quantifying contaminant degradation in the field when the transformation reaction is associated with a consistent isotope enrichment factor epsilon. When interpreted in conjunction with dual isotope plots, isotope fractionation is also particularly useful for elucidating reaction mechanisms. To assess the consistency of epsilon and dual isotope slopes in a two-step reaction, we investigated the abiotic hydrolysis of the herbicide isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) using a fragmentation method that allows measuring isotope ratios in different parts of the molecule. Carbon and nitrogen position-specific isotope fractionation, as well as slopes in dual isotope plots, varied linearly with rate constants k(obs) depending on the presence of buffers that mediate the initial zwitterion formation. The correlation can be explained by two consecutive reaction steps (zwitterion formation followed by dimethylamine elimination) each of which has a different kinetic isotope effect and may be rate-limiting. Intrinsic isotope effects for both steps, extracted from our kinetic data using a novel theoretical treatment, agree well with values computed from density functional calculations. Our study therefore demonstrates that more variable isotope fractionation may be observed in simple chemical reactions than commonly thought, but that consistent epsilon or dual isotope slopes may nonetheless be encountered in certain molecular fragments.

441 POSTER The G-quadruplex ligand RHPS4 potentiates the antitumor activity of camptothecins in preclinical models of solid tumors

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K+ and Na+ ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15 mer deoxynucleotide, d(G2T2G2TGTG2T2G2), labeled with 5′-fluorescein and 3′-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K+ ions bind with higher affinity than the Na+ ions. The enthalpy changes, ΔH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.

A new peptide-based method for the design and synthesis of nanoparticle superstructures: construction of highly ordered gold nanoparticle double helices.

Left-handed gold nanoparticle double helices were prepared using a new method that allows simultaneous synthesis and assembly of discrete nanoparticles. This method involves coupling the processes of peptide self-assembly of and peptide-based biomineralization of nanoparticles. In this study, AYSSGAPPMPPF (PEPAu), an oligopeptide with an affinity for gold surfaces, was modified with an aliphatic tail to generate C12-PEPAu. In the presence of buffers and gold salts, amphiphilic C12-PEPAu was used to both control the formation of monodisperse gold nanoparticles and simultaneously direct their assembly into left-handed gold nanoparticle double helices. The gold nanoparticle double helices are highly regular, spatially complex, and they exemplify the utility of this methodology for rationally controlling the topology of nanoparticle superstructures and the stereochemical organization of discrete nanoparticles within these structures.

Degradation of magnesium and its alloys: Dependence on the composition of the synthetic biological media

Magnesium and its alloys are highly degradable metals that are potentially useful as biomaterials, especially in orthopaedic and cardiovascular applications. However, the in vivo corrosion has proved to be too high. Because of the complexity of in vivo conditions, a careful study of the corrosion of magnesium in synthetic solutions that simulate the in vivo environment is necessary as a first approach to predict the actual in vivo situation. The aim of this work was to evaluate the influence of the electrolyte composition on the corrosion behavior of magnesium and two Mg-alloys in synthetic biological media. Pure magnesium and its alloys (AZ31 and LAE442) were employed in the experiments. Electrochemical potentiodynamic polarization curves were recorded in sodium chloride and PBS electrolytes with different chloride ion and albumin concentration. Optical and SEM observations complemented by EDX analysis were made. The results showed that magnesium corrosion is localized in chloride- and albumin-containing buffer solutions. They also showed that the chloride concentration and the presence of buffer and protein strongly affect the electrochemical behavior of magnesium and magnesium alloys.

Kinetic study of nitrosation of guanidines

AbstractThe kinetics of the nitrosation reaction of three guanidines, dicyandiamide, N,N′‐dimethyl‐N″‐cyanoguanidine and guanidine have been studied. The nitrosation rate is first order with respect to both the guanidine and acid concentration. The absence of catalysis by nucleophilic anions, the observed general acid–base catalysis and the observed deuterium isotope effect lead us to propose mechanism for the nitrosation of guanidines similar to that which operates in the case of the amides and ureas, in which a slow proton transfer is the rate determining step. From this mechanism we were able to obtain the values of the rate constants for the nitrosation and denitrosation processes. The catalytic constants in presence of buffers were also obtained and the analysis of the Brönsted slopes suggests a process with a transition state more similar to reactants than products. Copyright © 2008 John Wiley & Sons, Ltd.

Calcium waves in systems with immobile buffers as a limit of waves for systems with nonzero diffusion

We study the existence and properties of calcium waves in the presence of buffers. The model represents a reaction–diffusion system of equations with some diffusion coefficients equal to zero. They correspond to immobile buffers. The proof of the existence of travelling waves is carried out by passing to zero in the diffusion coefficients.

Binding of Caspase-3 Prodomain to Heat Shock Protein 27 Regulates Monocyte Apoptosis by Inhibiting Caspase-3 Proteolytic Activation

Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.

Hu Proteins Regulate Polyadenylation by Blocking Sites Containing U-rich Sequences

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.

Effect of Formulation Factors on <i>In Vitro</i> Permeation of Diclofenac from Experimental and Marketed Aqueous Eye Drops through Excised Goat Cornea

The effect of formulation factors on permeation of diclofenac from some experimental and marketed aqueous eye drops through excised goat cornea was evaluated. Raising the pH of formulation from 6.0 to 8.0 or diclofenac concentration from 0.05 to 0.15% (w/v) or adjusting tonicity with mannitol or addition of viscolizing agent decreased apparent permeability coefficient (Papp). Formulation (pH 7.4) containing sodium metabisulfite or EDTA or combination of methyl and propyl paraben showed significantly (p<0.05) higher Papp whereas benzalkonium chloride (BAC) had no effect and sorbic acid (SA) had reduced permeation. Surprisingly marketed drops containing BAC or SA, showed significantly (p<0.05) higher Papp and decreased in the order of Difen>Voveran>NSAID>Dicol>Diclolab. Lower pH (7.1-7.3) and surface tension of drops indicating presence of surfactant, could mediate increased permeation and presence of buffer could cause irritation on in vivo instillation. The marketed formulations showed corneal hydration >83% suggesting corneal damaging potential.

Biodegradable Polymeric Vectors for Gene Delivery to Human Endothelial Cells

Endothelial cells are an important cell type to both cardiovascular disease and cancer, as they play critical roles in vascular function and angiogenesis. However, effective and safe gene delivery to primary endothelial cells in the presence of serum proteins is known to be particularly challenging. A library of biodegradable poly(beta-amino esters) was synthesized for use as potential vectors. Promising vectors were optimized for high efficacy and low cytotoxicity to human umbilical vein endothelial cells (HUVECs) in serum. Vector parameters including polymer type, polymer weight, and DNA loading were varied, and biophysical properties including particle size, zeta potential, and particle stability over time were studied. While many of the poly(beta-amino ester) vectors have similar biophysical properties in the presence of buffer, their biophysical properties changed differentially in the presence of serum proteins, and the properties of these serum-interacting particles correlated to transfection efficacy. Leading poly(beta-amino ester) vectors were found to transfect HUVECs in the presence of serum significantly higher (47 +/- 9% positive, n = 10) than the best commercially available transfection reagents including jetPEI (p < 0.001) and Lipofectamine 2000 (p < 0.01). These results demonstrate the potential of a new class of biomaterials, poly(beta-amino esters), for effective human endothelial cell gene therapy.

ANALYTICAL SOLUTIONS FOR NONLINEAR CABLE EQUATIONS WITH CALCIUM DYNAMICS I: DERIVATIONS

The interaction between membrane potential and internal calcium concentration plays many important roles in regulating synaptic integration and neuronal firing. In order to gain a better theoretical understanding between the voltage-calcium interaction, a nonlinear cable equation with calcium dynamics is solved analytically. This general reaction-diffusion system represents a model of a cylindrical dendritic segment in which calcium diffuses internally in the presence of buffers, pumps and exchangers, and where ion channels are sparsely distributed over the membrane,in the form of hotspots, acting as point current sources along the dendritic membrane. In order to proceed, the reaction-diffusion system is recast into a system of coupled nonlinear integral equations, with which a perturbative expansion in dimensionless voltage and calcium concentration are used to find analytical solutions to this general system. The resulting solutions can be used to investigate, the interaction between the membrane potential and underlying calcium dynamics in a natural (non-discretized) setting.

Osmolyte‐induced protein folding free energy changes

Upon addition of protecting osmolyte to an aqueous solution of an intrinsically unstructured protein, spectral observables are often seen to change in a sigmoid fashion as a function of increasing osmolyte concentration. Commonly, such data are analyzed using the linear extrapolation model (LEM), a method that defines a scale from 0%-100% folded species at each osmolyte concentration by means of extending pre- and post-folding baselines into the transition region. Defining the 0%-100% folding scale correctly for each osmolyte is an important part of the analysis, leading to evaluation of the fraction of folded protein existing in the absence of osmolytes. In this study, we used reduced and carboxyamidated RNase T1 (RCAM-T1) as an intrinsically unstructured protein, and determined the thermodynamic stability of RCAM-T1 induced by naturally occurring osmolytes. Because the folded fraction of the protein population determined by experiments of thermal and urea-induced denaturation is nonzero in the absence of osmolytes at 15 degrees C, the commonly used LEM can lead to false values of DeltaG[stackD-->N0] for protein folding due to the arbitrary assumption that the protein is 100% unfolded in the presence of buffer alone. To correct this problem, titration of the protein solution with urea and extrapolating back to zero urea concentration gives the spectral value for 100% denatured protein. With fluorescence as the observable we redefine F/F0 to F/F0extrap = 1.0 and require that the denatured-state baseline have this value as its intercept. By so doing, the 0%-100% scale-corrected DeltaG[D-->N0] values of RCAM-T1 folding in the presence of various osmolytes are then found to be identical, with small error, demonstrating that DeltaG[D-->N0] is independent of the osmolytes used. Such a finding is an important step in validating this quantity derived from the LEM as having the properties expected of an authentic thermodynamic parameter. The rank order of osmolyte efficacies in stabilizing RCAM-T1 is sarcosine > sucrose > sorbitol > proline > betaine > glycerol.

Ganglionic Adrenergic Action Modulates Ovarian Steroids and Nitric Oxide in Prepubertal Rat

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The purpose of this work was to analyse the ganglionic adrenergic influence on the ovarian release of steroids and NO and the possible steroids/NO relationship. The experiments were carried out in the ex vivo coeliac ganglion-superior ovarian nerve (SON)-ovary system of prepubertal rats. The coeliac ganglion-SON-ovary system was incubated in Krebs Ringer-bicarbonate buffer in presence of adrenergic agents in the ganglionic compartment. The accumulation of progesterone, androstenedione, oestradiol and NO in the ovarian incubation liquid was measured. Norepinephrine in coeliac ganglion inhibited the liberation of progesterone and increased androstenedione, oestradiol and NO in ovary. The addition of alpha and beta adrenergic antagonists also showed different responses in the liberation of the substances mentioned before, which, from a physiological point of view, reveals the presence of adrenergic receptors in coeliac ganglion. In relation to propranolol, it does not revert the effect of noradrenaline on the liberation of progesterone, which leads us to think that it might also have a "per se" effect on the ganglion, responsible for the ovarian response observed for progesterone. Finally, we can conclude that the ganglionic adrenergic action via SON participates on the regulation of the prepubertal ovary in one of two ways: either increasing the NO, a gaseous neurotransmitter with cytostatic characteristics, to favour the immature follicles to remain dormant or increasing the liberation of androstenedione and oestradiol, the steroids necessary for the beginning of the near first estral cycle.

Pseudo‐Isotachophoresis Stacking: I. Investigating the Early Steps

Pseudo ITP (p‐ITP) is a simple method for concentration directly on the capillary, especially for samples containing high levels of salts or proteins. By moving the sample closer to the window after injection, we investigated the early steps of stacking without the influence of the separation step (the separation buffer). About one third of the capillary was filled with the sample in the presence of salts and acetonitrile. Salts such as chromate ions moved fast as leading ions, not as a sharp peak but as a wide wave; one side of it having a high ultra‐violet absorbency while the other side does not. The width of the wave is related to the salt concentration. Behind the salts, the anionic analytes moved rapidly forming sharp peak(s). As the concentration of the chromate ions are increased better stacking occurred. In the absence of salts or acetonitrile the wave did not form and the stacking was greatly diminished. Unexpectedly, the stacking was also accompanied by some separation, which was affected by the presence of buffers in the sample. This simple approach is useful for investigating other types of stacking.