It has been demonstrated that freezing-induced acidity changes have an impact on the structural integrity, degree of aggregation, and chemical stability of frozen food and pharmaceutical products. The stability of the compounds in solutions is maintained by the presence of buffers. However, many buffers are unsuitable for applications involving freezing as this process substantially alters the acidity. In this study, we determine the effect of initial pH, concentration, and cooling rate on the freezing-induced change in acidity of phosphate buffered saline (PBS) in the frozen state via UV-VIS spectroscopy. Furthermore, we examine the impact of individual salts present in PBS and discuss the mechanisms affecting the resulting acidity that we approximate via Hammett acidity function (H2-).
- MeSH
- Phosphates * chemistry MeSH
- Hydrogen-Ion Concentration MeSH
- Buffers MeSH
- Saline Solution * chemistry MeSH
- Freezing MeSH
- Publication type
- Journal Article MeSH
The concept of "pH memory" has been established in the literature for the correlation between the pH of a pre-lyophilization solution and the ionization state of freeze-dried powder (lyophile). In this paper, the concept of "pH memory" is explored for the system of an aqueous solution, a frozen solution, and a lyophile. Sodium and potassium phosphate buffers in the pH range of 5-9 were frozen and lyophilized with sulfonephthalein indicators as acidity probes, and their Hammett acidity functions were compared to the initial pH of the aqueous solution. The results show that the acidities of the lyophiles are somewhat changed compared to the initial pHs, but the acidities in the frozen state differ more substantially. The Hammett acidity functions of the frozen buffers were found to be markedly dissimilar from the initial pH, especially in the sodium phosphate frozen at 233K, where an increase in the initial pH led to a decrease in the Hammett acidity function of the frozen state at a certain pH range. The large acidification observed after freezing the sodium phosphate buffer was not detected in the lyophiles after the sample had been dried; the phenomenon is explained considering the formed crystals analyzed by X-ray powder diffraction. The results suggest that monitoring the final acidity of a lyophile is not sufficient to predict all the acidity changes throughout the whole lyophilization process. The importance of well-controlled freezing and lyophilization conditions follows from the results of the research.
- MeSH
- Phosphates chemistry MeSH
- Hydrogen-Ion Concentration * MeSH
- Freeze Drying MeSH
- Buffers * MeSH
- Freezing MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Solutions of three Good's buffers (HEPES, MOPS, and MES), both pure and mixed with sodium phosphate buffers (Na-P), are investigated in terms of the freezing-induced acidity changes in their operational pH ranges. The Good's buffers have the tendency to basify upon freezing and, more intensively, at lower pHs. The acidity varies most prominently in MES, where the change may reach the value of two. Importantly, the Good's buffers are shown to mitigate the strong acidification in the Na-P buffer. Diverse concentrations of the Good's buffers are added to cancel out the strong, freezing-induced acidity drop in 50 mM Na-P that markedly contributes to the solution's acidity; the relevant values are 3 mM HEPES, 10 mM MOPS, and 80 mM MES. These buffer blends are therefore proposed to be applied in maintaining approximately the acidity of solutions even after the freezing process and, as such, should limit the stresses for frozen chemicals and biochemicals.
- MeSH
- Phosphates * MeSH
- Ions MeSH
- Hydrogen-Ion Concentration MeSH
- Buffers MeSH
- Freezing MeSH
- Publication type
- Journal Article MeSH
Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.
- MeSH
- Phosphates * chemistry MeSH
- Histidine * chemistry MeSH
- Hydrogen-Ion Concentration MeSH
- Citric Acid chemistry MeSH
- Freeze Drying * methods MeSH
- Magnetic Resonance Spectroscopy * methods MeSH
- Buffers MeSH
- Spectrophotometry, Ultraviolet methods MeSH
- Trehalose * chemistry MeSH
- Publication type
- Journal Article MeSH
Humanin (HN), Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-IIe-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, recently discovered in the human brain, is an important neuroprotective peptide. Some derivatives of HN show even higher biological activity, for example [G-14]-HN, where Ser at position 14 is replaced with Gly. As structurally related HN peptide derivatives have similar chemical properties, their separation by CE is difficult. In this work, the electrophoretic behaviour of HN derivatives including [G-14]-HN, a tryptophan HN derivative [W-14]-HN, several other HN derivatives and HN fragments was studied. While phosphate buffer was used as the general BGE, the effects of the buffer concentration and various additives were examined, including sulphate, heptane sulphonate, 2-morpholinoethanesulphonic acid N-[tris(hydroxymethyl)methyl]-2-aminoethane sulphonic acid (TES), sulphated-beta-CD and beta-CD. Separation efficiency of 200,000 theoretical plates was achieved in a BGE of 80 mM phosphate at pH 2.5 where seven out of nine major peaks were partially separated. By investigating the influence of concentration of the interrogated ions on peptides migration, the association between positively charged protonated sites of peptides and various anions was proved. Especially a strong interaction with phosphate, sulphate and sulphonate groups was established. Conditional stability constant of the [Pep(z+), (H(2)PO(4)(-))(n)](z - n) ion associate (n = 1) for [G-14]-HN equals to log K approximately 1.78.
- MeSH
- Alkanesulfonates chemistry MeSH
- beta-Cyclodextrins chemistry MeSH
- Electrophoresis, Capillary methods MeSH
- Financing, Organized MeSH
- Phosphates chemistry MeSH
- Intracellular Signaling Peptides and Proteins chemical synthesis chemistry isolation & purification MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- Neuropeptides chemical synthesis chemistry isolation & purification MeSH
- Peptide Fragments chemical synthesis chemistry isolation & purification MeSH
- Buffers MeSH
- Solutions MeSH
- Amino Acid Sequence MeSH
- Sulfates chemistry MeSH
- Amino Acid Substitution MeSH
- Check Tag
- Humans MeSH
The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.
- MeSH
- Biotechnology methods MeSH
- Time Factors MeSH
- Cryptococcus enzymology metabolism MeSH
- Cells, Immobilized enzymology metabolism MeSH
- beta-Fructofuranosidase isolation & purification MeSH
- Hydrogen-Ion Concentration MeSH
- Culture Media chemistry MeSH
- Buffers MeSH
- Sucrose MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
The paper deals with the characterisation of the bioactive phenomena of glass-ceramic scaffold derived from Bioglass® (containing 77 wt.% of crystalline phases Na(2)O·2CaO·3SiO(2) and CaO·SiO(2) and 23 wt.% of residual glass phase) using simulated body fluid (SBF) buffered with tris-(hydroxymethyl) aminomethane (TRIS). A significant effect of the TRIS buffer on glass-ceramic scaffold dissolution in SBF was detected. To better understand the influence of the buffer, the glass-ceramic scaffold was exposed to a series of in vitro tests using different media as follows: (i) a fresh liquid flow of SBF containing tris (hydroxymethyl) aminomethane; (ii) SBF solution without TRIS buffer; (iii) TRIS buffer alone; and (iv) demineralised water. The in vitro tests were provided under static and dynamic arrangements. SBF buffered with TRIS dissolved both the crystalline and residual glass phases of the scaffold and a crystalline form of hydroxyapatite (HAp) developed on the scaffold surface. In contrast, when TRIS buffer was not present in the solutions only the residual glassy phase dissolved and an amorphous calcium phosphate (Ca-P) phase formed on the scaffold surface. It was confirmed that the TRIS buffer primarily dissolved the crystalline phase of the glass-ceramic, doubled the dissolving rate of the scaffold and moreover supported the formation of crystalline HAp. This significant effect of the buffer TRIS on bioactive glass-ceramic scaffold degradation in SBF has not been demonstrated previously and should be considered when analysing the results of SBF immersion bioactivity tests of such systems.
We studied the disinfection efficacy of boron-doped electrodes on Escherichia coli-contaminated water-based solutions in three different electrolytes, physiological solution (NaCl), phosphate buffer (PB), and phosphate buffer saline (PBS). The effect of the electrochemical oxidation treatment on the bacteria viability was studied by drop and spread plate cultivation methods, and supported by optical density measurements. We have found that bacterial suspensions in NaCl and PBS underwent a total inactivation of all viable bacteria within 10 min of the electrochemical treatment. By contrast, experiments performed in the PB showed a relatively minor decrease of viability by two orders of magnitude after 2 h of the treatment, which is almost comparable with the untreated control. The enhanced bacterial inactivation was assigned to reactive chlorine species, capable of penetrating the bacterial cytoplasmic membrane and killing bacteria from within.
Bicarbonate and phosphate constitute major salivary buffering components, and their importance consists in the neutralization of acidic gastric contents during reflux episodes. In this work, capillary electrophoresis with capacitively coupled contactless conductivity detector was applied for the analysis of bicarbonate, phosphate, and another inorganic (chloride, nitrite, nitrate, sulfate, thiocyanate) and organic anions (acetate, butyrate) to evaluate their levels in saliva. The background electrolytes of different composition and pH between 6.02-9.41 were assessed for the bicarbonate and phosphate determination by comparison of the real analyses of a model solution with the simulation by PeakMaster software. The optimized background electrolyte was composed of 10 mM 2-(N-morpholino)ethanesulfonic acid, 20 mM arginine, and 30 µM cetyltrimethylammonium bromide, pH 8.95. Using this BGE, the anion levels were compared in saliva from 20 patients suffering from gastroesophageal reflux disease (GERD) and saliva from 12 healthy subjects. Bicarbonate levels were significantly elevated in saliva from GERD patients suggesting the possible applicability of bicarbonate as a biomarker in non-invasive diagnostics of GERD by CE-C4 D.
- MeSH
- Anions analysis MeSH
- Electric Conductivity MeSH
- Electrophoresis, Capillary methods MeSH
- Phosphates analysis MeSH
- Gastroesophageal Reflux diagnosis MeSH
- Bicarbonates analysis MeSH
- Humans MeSH
- Saliva chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
