Titration Curves Research Articles

EXPLORING OF ACID-BASIC PROPERTIES, DEVELOPMENT AND VALIDATION OF THE METHOD OF QUANTITATIVE DETERMINATION OF ORIGINAL PHARMACEUTICAL SUBSTANCE

Introduction. The object of analysis was a newly synthesized organic substance, a derivative of 4-aminobenzoic acid and malonic ester – 4,4’-(propandiamido) sodium dibenzoate, which has an antisteatotic effect. For the quantitative determination of pharmaceutical substances, it is preferable to use titrimetric methods of analysis, since they are absolute and allow us to estimate the content of the active compound in the substance without using standard samples. Aim. The objective of this work was to develop and validate a quantitative determination method for 4.4’-(propandiamido) sodium dibenzoate. Materials and methods. The analyzed compound is a salt of a weak dibasic acid, which exhibits basic properties in an aqueous solution; therefore, for its quantitative determination, a direct acid-base titration method (acidimetry) was proposed. The method of quantitative determination of the active compound in the substance 4,4’-(propandiamido) sodium dibenzoate will be used in future studies for routine analysis, therefore a visual (indicator) method for determining the end point of the titration has been chosen. To prove the suitability of the developed method and the possibility of obtaining reliable results of the quantitative content of the active compound in the newly synthesized pharmaceutical substance the used analytical method was validated. Results and discussion. The study of the acid-base properties of the newly synthesized substance was carried out using the method of potentiometric titration, on the basis of which the values of the dissociation constants were calculated. Using spreadsheets, the pKa1 and pKa2 values were calculated using a titration model of a multi-basic acid, the pKa values of which are 7.2 and 5.0. The titration curve that was constructed from the experimental data and the curve constructed from the calculated values of the dissociation constants almost completely overlapped with each other. Based on the data obtained, a 0.1% water-alcohol solution of bromocresol green (blue) with pT=4.6 was selected for titration, the color transition interval of which falls into the titration jump of the analyte. The developed methodology was validated according to the requirements of the XIV edition State Pharmacopoeia of the Russian Federation by the following indicators: specificity, linearity, accuracy, precision (convergence and internal reproducibility), robustness, analytical domain. Conclusion. As a result of the performed studies, the values of acid dissociation constants of the newly synthesized pharmaceutical substance 4,4’-(propandiamido) sodium dibenzoate were determined, and a method for quantitative determination was developed and validated using acidbase titration.

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Single-Molecule Studies of ATP Binding to the Sodium Pump

The sodium pump uses energy from the hydrolysis of 1 ATP to move 3Na+ out of and 2K+ into the cell via transitions between phosphorylated and unphosphorylated forms of two major conformations (E1 and E2). In earlier experiments, we had found that ATP binds with low and high affinity to the E1 conformation of pump isolated from nasal glands of juvenile ducks. We attributed this heterogeneity to interactions between ATP binding sites on different protomers within multiprotomeric aggregates. We report here on experiments designed to examine ATP binding to pump protomers using single-molecule fluorescence measurements. Pump labeled with Cy3-maleimide was solubilized with C12E8. Protomers were separated from aggregates using FPLC and reconstituted into lipid vesicles at an average concentration of 1 protomer/vesicle. The fluorescence of vesicles tethered to a glass cover slip using a biotin-streptavidin linkage was measured as a function of time using single-molecule total internal reflection fluorescence microscopy under conditions where the pump was in the E1 conformation. Vesicles where fluorescence photobleached in a single-step were identified as containing single protomers. These traces were either dynamic (fluorescence fluctuated between two states) or nondynamic (no fluctuations). The fraction of protomers that showed dynamic behavior increased saturably with ATP from ∼40% at 0 ATP to ∼70% at 1 mM (saturating) ATP. This titration curve could not be fitted with a single binding isotherm. A fit to a model that assumed a heterogeneous mixture of pump molecules with high- and low-affinity ATP binding showed: (a) high- and low-affinity apparent dissociation constants of 0.1 and 40 μM; and (b) that the preparation had equal amounts of high- and low-affinity forms of the pump. Our data suggest that binding heterogeneity is an intrinsic property of the protomer and is not attributable to aggregation.

Heat-induced gelation of mixtures of casein micelles with whey protein aggregates

Gelation of aqueous solutions of casein micelles (MC) in mixtures with fractal aggregates of whey protein isolate (WPI) has been studied first by addition of different amounts of aggregates at a fixed concentration of micelles and then by substitution of micelles with aggregates at a fixed total protein concentration. Addition of small amounts of aggregates (1–5 g/L) to suspensions of micelles (20–60 g/L) at a fixed pH led to a strong decrease of the gelation temperature (Tg) and to an increase of the elastic modulus (G′) of the mixed gels. A minimum in Tg and a maximum in G′ as a function of the WPI fraction were obtained in mixtures with fixed total protein concentration and fixed pH and explained by the opposing influence of increasing WPI aggregate concentration and decreasing concentration of micelles. Increasing the total protein concentration at the same WPI fraction caused a decrease of Tg and increase of G′. Decreasing the pH at the same protein composition led to a decrease of Tg but caused an increase of G′. Tg decreased sharply if a small amount of CaCl2 was added, but if more than about 30 mM of calcium was added, Tg increased again. Gelation of mixtures did not depend significantly on the size of the WPI aggregates, but if fractal aggregates were replaced by microgels, weaker gels were produced and the temperature of gelation was higher. Potentiometric titration curves showed that protonation of the micelles and of the WPI aggregates in the mixtures was independent. It is concluded that MC and WPI aggregates co-aggregate during gelation and there is a transition between gelation at a critical temperature for pure MC suspensions and gelation controlled by an activation energy for pure WPI aggregate suspensions.

Estimation of Hypochlorites in Commercial Bleaching Solutions by Cl-ISE and Platinum Electrode

Chloride ion-selective and platinum electrodes were successfully applied in the determination of hypochlorites in commercial bleaching solutions. The results obtained with Cl-ISE are compared to those obtained with Pt electrodes since the reactions are oxidation-reductions where the Pt responds to their potential change. A clear change and jump at equivalence points were found and titration curves are in S form which were approved by first and second derivatives. The percentages obtained with Cl-ISE are relatively larger than results with Pt electrode which are due to practical errors, interference of I-ions and little Cl-by-Product of hypochlorite manufacture. In general, almost both electrodes offered results in the range of 3–8% solutions of hypochlorites within the period of storage. Low results may by due to wasting and decomposition by temp, transferring, dilution and stirring the solution.

The multiple dissociation constants of glutathione disulfide: interpreting experimental pH-titration curves with ab initio MD simulations.

The hexapeptide glutathione disulfide (GSSG) has six ionizable groups with six associated dissociation constants. The experimentally measured pH-titration curve, however, does not exhibit the six corresponding equivalence points and bears little resemblance to standard textbook examples of acid-base pH-titration curves. The curve highlights the difficulties in determining multiple pKa values of polyprotic acids - typically proteins and peptides - from experiment. The six pKa values of GSSG can, however, be estimated using Car-Parrinello molecular dynamics (CPMD) simulations in conjunction with metadynamics sampling of the underlying free energy landscape of the dissociation reactions. Ab initio MD simulations were performed on a GSSG molecule solvated by 200 water molecules. Using the estimated pKa values the theoretical titration curve was calculated and found to be in good agreement with experiment. The results clearly highlight how dissociation constants estimated from ab initio MD simulations can facilitate the interpretation of the pH-titration curves of complex chemical and biological systems.

Diketopyrrolopyrrole-based fluorescence probes for the imaging of lysosomal Zn2+ and identification of prostate cancer in human tissue.

A series of diketopyrrolopyrrole-based fluorescent probes (DPP-C2, LysoDPP-C2, LysoDPP-C3, and LysoDPP-C4) have been developed for the detection of low pH and Zn2+ in an AND logic fashion. The chelation of Zn2+ or the protonation of a morpholine moiety within these probes results in a partial increase in the fluorescence intensity, an effect ascribed to suppression of one possible photo-induced electron transfer (PET) pathway. In contrast, a large increase in the observed fluorescence intensity is observed at low pH and in the presence of Zn2+; this is rationalized in terms of both possible PET pathways within the probes being blocked. Job plots, fluorescence titration curves, and isothermal titration calorimetry proved consistent with a 1 : 1 Zn2+ complexation stoichiometry. Each probe demonstrated an excellent selectivity towards Zn2+ and the resulting Zn2+ complexes demonstrated pH sensitivity over the 3.5-9 pH range. Fluorescence imaging experiments confirmed that LysoDPP-C4 was capable of imaging lysosomal Zn2+ in live cells. Little evidence of cytotoxicity was seen. LysoDPP-C4 was successfully applied to the bioimaging of nude mice, wherein it was shown capable of imaging the prostate. Histological studies using a human sample revealed that LysoDPP-C4 can discriminate cancerous prostate tissue from healthy prostate tissue.

Copolymers of sodium 4-styrene sulphonate and vinyl derivatives of nitrogen-containing heterocycles

The formation of ion-exchange composite materials based on high molecular weight precursors appears to be an intensively developing area in the synthesis of proton-conducting membranes for fuel cells. In such membranes, proton transfer is often provided by functional polymers simultaneously containing sulphonic acid groups in their composition units along with fragments of vinyl derivatives of nitrogen-containing heterocyclic bases. Proton exchange activity in the latter is determined by the possibility of doping with inorganic acids. In the framework of this study, for the further formation of hybrid composite membranes under conditions of radical initiation, copolymers of sodium 4-styrene sulphonate (SSt) with 4-vinylpyridine (VP) and 1-vinylimidazole (VIM) were obtained. The monomodal nature of the turbidimetric titration curves for solutions of copolymerisation reaction products indicates the presence of true copolymers during the process of formation. The composition and structure of the copolymers were characterised using data from elemental analysis, as well as from IR and 13 C NMR spectroscopy. Constants of the relative activity for the monomers and the microstructure parameters of the polymer chains are calculated according to the non-linear leastsquares method using the MathCAD package. The calculated copolymerisation constant values indicate a greater reactivity of SSt in comparison with nitrogen-containing monomers. The lengths of the monomer unit blocks depend on the composition of the initial mixture and vary over a wide range from 1 to 18. The possibility of varying the length of the unit blocks in the composition of the copolymers will affect the ion-conducting properties of hybrid composites formed on their basis. The stability of the copolymers to thermal oxidative degradation by heating in air was studied using the differential scanning calorimetry (DSC) method. The copolymers demonstrated significant thermo-oxidative stability. Decomposition temperatures were 350 °С and 400 °С for SSt-VIM and SSt-VP copolymers, respectively.

Analysis of the Fibroin Solution State in Calcium Chloride/Water/Ethanol for Improved Understanding of the Regeneration Process

Shaping of fibroin protein from Bombyx mori from calcium chloride/water/ethanol solution is of high interest for the manufacturing of biocompatible structures. Potentiometric titration experiments of the dissolved state permit new insight into the solution state of fibroin as a basis for improved regeneration. Titration experiments and infrared spectroscopy of the solution state support the model of an ion-rich hydration layer and interaction of the solvent with charged and polar groups of the fibroin, rather than through formation of defined calcium complexes. The potentiometric titration curves indicate the formation of calcium complexes at pH values above 9, most probably through involvement of basic amino acids and phenolic groups of tyrosine. After regeneration by the addition of methanol, the regenerated protein was characterised by FTIR, nitrogen and calcium analysis. The analysis of supernatants for their residual protein content indicated minor losses of protein, most probably low molecular weight proteins. The results contribute to an improved understanding of the solution state as a basis for larger scale regeneration, e.g. for the coating of textile fibres with regenerated fibroin.

PH dependence of the charge recombination kinetics in bacterial RC reconstituted in liposomes

ABSTRACT:The photosynthetic Reaction Center from the carotenoidless mutant strain of the purple non sulphur bacterium Rhodobacter (R.) sphaeroides was reconstituted in artificial phospholipid vesicles (liposomes) to mimic the physiological membrane environment. The pH dependence in the interval 5 – 10 of the rate of the charge-recombination reactions from the final electron acceptors QA and QB to the primary electron donor (namely kAD and kBD) have been investigated. The liposomes were constituted of either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG), two of the main phospholipids found in the photosynthetic membrane of the bacterium. In both cases, the kAD has no pH dependence similarly to the detergent case. The kBD also has a pH dependence similar to the detergent case, having two distinct regions below pH 7 and above pH 9. Fitting of the titration curve to a function involving two protonation sites results in a marked shift of the pKAs between the different solubilizing environments. These differences are discussed in the frame of possible physiological implications.

Microfluidic ballistic regime for the generation of linear gradients inside a capillary column: Proof-of-concept and application to the miniaturized acid-base volumetric titration

This work details a simple and original approach for the generation of linear gradients inside straight cylindrical microchannels such as a capillary column. The concept takes advantage of an oft-overlooked regime of dispersion of flowing liquids inside narrow channels: the ballistic regime. The ballistic regime is a pure convective regime and is produced by imposing a high velocity flow in a pre-filled capillary thus limited diffusion takes place. This is obtained by forcing the injection of a plug of solution on a short time scale t, much shorter than t<110×D/r2, D is the diffusion coefficient and r the capillary radius. The result is a stretched solution of a given length or depth of penetration, inside the capillary column. This leads to a linear mean concentration field through the mixing zone forming a linear gradient. In miniaturized systems, this transient regime is followed by mainly radial diffusion of the solution inside the capillary due to the short characteristic diffusion time of narrow channels. A convection-diffusion simulation was used to model the gradient formed under this ballistic regime. A specific experimental prototype set-up was designed to investigate this ballistic regime and the formation of a linear gradient of titrant NaOH solution inside a capillary tubing of 500 µm inner diameter and 35-cm total length pre-filled with nitric acid solution. With this prototype, the linear gradient was then pushed in a non-ballistic regime over a confocal fluorescence point detection system in order to measure the fluorescence emission of a fluorescent dye added to the solutions. Considering strong acid-base reaction, fluorescein was used due to its strong fluorescence dependency with pH near its pKa, i.e 6.4. A first set of experiments was realized to demonstrate the validity of such an approach and to determine the optimal condition for the formation of a linear gradient over 300 mm of the 350-mm capillary length. An injection pressure of 1000-mbars over 0.75 s was chosen. The first result was the stability of the system in its ability to produce reproducible linear gradients. As further proofs of feasibility, samples of different nitric acid concentrations were titrated with a 0.25 M NaOH solution. The result was rapid and reproducible titration curves obtained with a fully automated system that consumes less than approximately 70 µL of sample solution.

Formation and Composition of Arachin/Carrageenan Complexes: Effects of Protein/Polysaccharide Ratio

The formation and composition of arachin/carrageenan complexes at various protein/polysaccharide ratios (r) have been studied by a combination of turbid metric titration and composition analysis. Our results suggest that an increase in r favors the formation of complex coacervates. Unlike pectin/β-lacto globulin, due to the strong polyelectrolyte nature of carrageenans, no pHφ2 was observed in the pH titration curves of arachin/carrageenan even at pH as low as 2.0. Our composition analysis results indicated that at higher r, the coacervate was more concentrated.

A portable eight-channel titrator based on high-throughput capacitively coupled contactless conductivity measurements

Analytical chemists require multichannel and automatic tools for batch measurements. Herein, an eight-channel electronic titrator (8-CET) system was constructed to facilitate high-throughput titrations. In this system, multichannel piston pumps were used for delivery, and an 8-channel capacitively coupled contactless conductivity detector (8-C4D) was used to monitor the conductivity of the titration solutions in arrays of disposable reaction cells. The delivery and the monitoring were both fully software-controlled allowing a simultaneous formation of eight titration curves. Its capacity for classical acid-base and precipitation titration was characterized with satisfactory results. It was also used to measure the content of CH3COOH in batched vinegar samples. The RSD between each reaction cell was 1.9%. There are two outstanding features of this new system: 1) Up to eight samples can be measured simultaneously; and 2) simple operation is realized by avoiding electrode switching and cleaning after each measurement. Therefore, it is reasonable to believe that this 8-CET system is a prospective alternative for developing more automatic and efficient titration platforms.

Electrostatically Driven Protein Adsorption: Charge Patches versus Charge Regulation.

The mechanisms of electrostatically driven adsorption of proteins on charged surfaces are studied with a new theoretical framework. The acid-base behavior, charge distribution, and electrostatic contributions to the thermodynamic properties of the proteins are modeled in the presence of a charged surface. The method is validated against experimental titration curves and apparent p Kas. The theory predicts that electrostatic interactions favor the adsorption of proteins at their isoelectric points on charged surfaces despite the fact that the protein has no net charge in solution. Two known mechanisms explain adsorption under these conditions: (i) charge regulation (the charge of the protein changes due to the presence of the surface) and (ii) charge patches (the protein orients to place charged amino acids near opposite surface charges). This work shows that both mechanisms contribute to adsorption at low ionic strengths, whereas only the charge-patch mechanism operates at high ionic strength. Interestingly, the contribution of charge regulation is insensitive to protein orientation under all conditions, which validates the use of constant-charge simulations to determine the most stable orientation of adsorbed proteins. The present study also shows that the charged surface can induce large shifts in the apparent p Kas of individual amino acids in adsorbed proteins. Our conclusions are valid for all proteins studied in this work (lysozyme, α-amylase, ribonuclease A, and β-lactoglobulin), as well as for proteins that are not isoelectric but have instead a net charge in solution of the same sign as the surface charge, i.e. the problem of protein adsorption on the "wrong side" of the isoelectric point.

Protein p Ka's from Adaptive Landscape Flattening Instead of Constant-pH Simulations.

Protein acid/base constants, or p Ka's are often computed from Monte Carlo or molecular dynamics simulations at a series of constant pH values. Instead, we propose to adaptively flatten the free energy landscape in the space of protonation states. The flattening is achieved by a Wang-Landau Monte Carlo, where a bias potential is constructed adaptively during an initial phase, such that all protonation states achieve comparable probabilities. Biased ensembles of states are then reweighted by subtracting out the bias and adding a pH-dependent free energy term. Titration curves constructed for three test proteins agreed, within the small numerical uncertainty, with those obtained earlier from the constant-pH approach.

Determination of individual stability constants of coexisting molecular complexes formed by unresolvable isomers

ABSTRACTSome biogenic substances undergo steric isomerism or tautomerism in which the isomers are unresolvable. Naturally, the isomers are expected to form molecular complexes with distinct stabilities. This paper proposes the method of determining individually the stability constants of complexes formed under isomerisation and complexation equilibria: the NMR titration curves are modelled and formulated for fast and slow isomeric equilibria, the process of least-squares fittings is explained, and the application to supramolecular complexation of unresolvable isomers is discussed as compared with NMR titration data reported for α- and β-D-glucosamine anomers.

Improving biocatalytic microenvironment with biocompatible ε-poly-l-lysine for one step gluconic acid production in low pH enzymatic systems

Surface amine modification could not only improve the microenvironment near the active sites of enzyme, but also enhance the multi-point chemical crosslinking between the enzyme and carrier. The linear structured polymer of ε-poly-l-lysine (EPL) is an ideal donor of amino with much more exposed on the surface for enzyme attachment. Analysis of the result of dynamic light scattering (DLS) and circular dichroism (CD) demonstrated the favorable electrostatic interactions and negligible impact on the conformation of enzymes, Glucose Oxidase and Catalase (GOx&CAT). Titration and dissociation curve together with Zeta-potential characterization revealed that enzymes ([email protected]&CAT) under the protection of EPL had more stable structure and better activity and stability in acidic reaction environment. Meanwhile, the activity recovery of immobilized [email protected]&CAT increased to 1.56 times higher than that of GOx&CAT, and the acid resistance was improved by 1.44 times with the optimum pH shifting to acidic by 0.50 unit. The substrate affinity was raised with the decrease of Km from 5.98 to 4.35 mg mL−1. Thus, the gluconic acid production in low pH system could be increased by pH-engineering of the enzyme microenvironment via conjugation with positively charged polyelectrolyte.

PH sensing using whispering gallery modes of a silica hollow bottle resonator

The limitations of electrochemical pH sensors have stimulated the development of optical pH sensing methods. In the method reported here, swellable pH-sensitive polymer particles are deposited on the interior surface of a silica hollow bottle resonator. As the pH of the buffer solution in contact with the particles increases, the refractive index of the particles decreases. As a result, whispering gallery modes with internal evanescent components shift in frequency as a function of pH. This shift is monitored by the throughput of tunable diode laser light coupled into the whispering gallery modes using a tapered fiber. Plots of selected mode frequencies vs. pH yielded sigmoid shaped titration curves similar to those obtained using turbidity to monitor refractive index changes of the particles as a function of pH. The response time of 10–15 s and best resolution of 0.06 pH unit represent improvements over previous optical pH sensing methods.

Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma

BackgroundAnaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. The microfluidic tissue processor (MTP) device is based on a chip-confined low-volume technology allowing for rapid immunohistochemistry/immunofluorescence (IHC/IF) stainings of formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples.MethodsA novel ALK IF protocol was developed for the MTP device using the primary mouse anti-human ALK antibody clone 5A4. FFPE tumor whole sections from 14 resected lung adenocarcinoma patients documented to be ALK positive (ALK+) by automated chromogenic IHC and/or FISH were used. MTP-derived IF immunoreactivity was measured by computerized analysis of digitalized images on individual frames of tumor epithelia and surrounding stroma, using an ImageJ plug-in.ResultsThe 5A4 antibody yielded saturated immunoreactivity at an incubation time of 4 min on a titration curve ranging from 2 to 32 min. Total staining time on the MTP device was 18 min including secondary IgG Alexa Fluor 647. MTP-based ALK IF confirmed all 12 cases; with epithelial signal above stromal staining based on computerized pixel-based measurement. MTP-IF (mean intensity levels 458 to 1301) and chromogenic IHC (H-score 120 to 300) showed an equal range of variation of 2.8 and 2.5 folds, respectively, and a trend for direct correlation (p-value 0.051).ConclusionThe newly developed protocol for immunofluorescent detection of ALK protein with the MTP device confirms chromogenic IHC results on FFPE lung adenocarcinoma specimens. MTP-based IF is fast and reliable. We foresee this study to be a first step opening the road for further realization of microfluidic-based assays for rapid simultaneous detection of targetable oncogenic and immune-system related markers in their topographical context to investigate tumour heterogeneity and micro-environmental interactions.

Influence of solution pH on the formation of iron oxide nanoparticles

Iron oxide phase orientation were highly influenced by solution pH, crystalline structure, purity, surface enrichment, particle size, and morphology. This paper investigates the influence of pH changes by varying the precursor anions of chloride (FeCl2) and sulphate (FeSO4) on the formation of iron oxide nanoparticles using one step controlled precipitation technique. The obtained titration curve provided vital information on the reaction mechanism whereby different hydrolysis rate of precursor leads to different iron oxide phases. It was determined that at pH 4, goethite (α-FeOOH) was obtained. Continuous addition of hydroxyl ions (OH−) then forms iron hydroxides (Fe(OH)2) which will then subsequently react with the goethite precipitating magnetite (Fe3O4) nanoparticles (35–45 nm) at pH 10 with ferromagnetic behavior. By the use of Cl− anion, the slower hydrolysis process induced requires more hydroxyl ions to reach pH equilibrium leading to larger maghemite nanoparticles (50–60 nm). Spectroscopic analysis via Raman and FTIR analysis confirms the phases obtained. SEM and TEM imaging shows the correlation of structure orientation anisotropy which influenced the magnetic properties. Spherical magnetite nanoparticles strong dipolar interaction have higher coercivity (117G) and remanence (12 emu g−1) compared to the synthesised maghemite spinel needle-like structure nanoparticles. The understanding of the iron oxide structure directing effect by complex pH solution mechanism was essential for not only to prepare different forms of iron oxide and hydroxides but also controlled synthesis reproducibility.