Preparation Of Stock Solutions Research Articles

RP-CAD for Lipid Quantification: Systematic Method Development and Intensified LNP Process Characterization.

Lipid nanoparticles (LNPs) and their versatile nucleic acid payloads bear great potential as delivery systems. Despite their complex lipid composition, their quality is primarily judged by particle characteristics and nucleic acid encapsulation. In this study, we present a holistic reversed-phase (RP)-charged aerosol detection (CAD)-based method developed for commonly used LNP formulations, allowing for intensified LNP and process characterization. We used an experimental approach for power function value (PFV) optimization termed exploratory calibration, providing a single PFV (1.3) in an appropriate linearity range for all six lipids. Followed by the procedure of method calibration and validation, linearity (10-400 ng, R2 > 0.996), precision, accuracy, and robustness were effectively proven. To complement the commonly determined LNP attributes and to evaluate the process performance across LNP processing, the developed RP-CAD method was applied in a process parameter study varying the total flow rate (TFR) during microfluidic mixing. The RP-CAD method revealed a constant lipid molar ratio across processing but identified deviations in the theoretical lipid content and general lipid loss, which were both, however, entirely TFR-independent. The deviations in lipid content could be successfully traced back to the lipid stock solution preparation. In contrast, the observed lipid loss was attributable to the small-scale dialysis following microfluidic mixing. Overall, this study establishes a foundation for employing RP-CAD for lipid quantification throughout LNP processing, and it highlights the potential to extend its applicability to other LNPs, process parameter studies, or processes such as cross-flow filtration.

TiCl4 precursor affecting the performance of HTM-free carbon-based perovskite solar cell

The presence of TiO2 used as an efficient electron transport layer is crucial to achieving high-performance solar cells, especially for a hole transport material (HTM)-free carbon-based perovskite solar cell (PSC). The hydrolysis of TiCl4 is one of the most widely used routes for forming TiO2 layer in solar cells, which includes the stock solution preparation from TiCl4 initial precursor and the thermal hydrolysis of the stock solution. The second thermal hydrolysis step has been extensively studied, while the initial hydrolysis reaction in the first step is not receiving sufficient attention, especially for its influence on the photovoltaic performance of HTM-free carbon-based devices. In this study, the role of TiCl4 stock solution in the growth process of TiO2 layer is examined. Based on the analysis of the Ti(IV) intermediate states for different TiCl4 concentrations from Raman spectra, 2 M TiCl4 precursor exhibits moderate nucleation and growth kinetics without generating too many intermediates which occurs in 3 M TiCl4 precursor, yielding ∼300 nm size spherical TiO2 agglomerates with a rutile phase. In the aspect of devices, the HTM-free carbon-based PSCs fabricated using 2 M TiCl4 precursor deliver a conversion efficiency beyond 17%, which may be attributed to the reduced defect in compact TiO2 layer.

B-129 Uncertainty Calculations for Reference Measurement Procedures

Abstract Background Quantitative results based on reference measurement procedures (RMP) require the calculation of the uncertainty of the result. In order to take the uncertainty of the full traceability chain into account a modularisation of the different execution steps is needed, starting from the primary reference material until the final concentration value. Different strategies have to be applied and combined to obtain the final uncertainty.We combine different ways for calculating the uncertainty of an isotope dilution -liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure for Cortisol, and explain the differences between Type A and Type B uncertainties and best strategies to choose from them. In addition, combinations of results from subsequent steps to a final overall uncertainty are presented. Methods An ID-LC-MS/MS based reference measurement procedure requires as a starting point a primary reference material with known purity of the analyte under consideration. Nowadays this purity assessment is often done with quantitative nuclear magnetic resonance (qNMR) spectroscopy. The qNMR uncertainty calculation is a Type A approach, based on replicate sample preparations and measurements. With this primary material a first set of primary calibrators is produced, often through multiple steps of weighings, preparation of stock solutions and dilutions. For these steps balances, volumetric flasks, or pipettes are used, all of them coming with an error budget which has to enter the uncertainty budget of the primary calibrator preparation. We will show how this can be done through Type B uncertainty calculation. Based on the primary calibrators the LC/MS instrument is calibrated and measurements of individual samples are carried out. We will introduce a hierarchical variability experiment, which takes into account the most important error sources and results in a Type A uncertainty for the LC/MS measurements.Finally, the errors of the LC/MS measurements have to be combined with those of the primary calibrator preparation to obtain the overall uncertainty of an LC/MS reference measurement procedure. Results We show the approaches and calculation strategies based on the Cortisol candidate reference measurement procedure, developed within the Roche LC/MS Reference Measurement Procedure project. The purity assessment by qNMR carries an uncertainty of 0.18%. This is incorporated into the uncertainty calculations of the primary calibrators, where in addition uncertainties from weighing and dilutions are incorporated resulting in uncertainties of the primary calibrator values between 0.6% and 1.1%. Combining these uncertainties with the variabilities of the LC/MS measurement procedure results in final uncertainties between 1.6% and 2.8%. Conclusion We have developed a sound framework for uncertainty calculation of reference measurement procedures, taking into account the different procedure steps, and included this into a process for the management of the different input variables up to comprehensive reports of the calculations.

Simultaneous Determination of Luliconazole and Salicylic Acid by Simultaneous Equation Method and Absorbance Ratio Method

The simultaneous determination of Luliconazole and Salicylic Acid in pharmaceutical formulations holds significant importance for ensuring therapeutic efficacy and patient safety. This research focuses on developing an accurate and efficient method using UV-Visible spectrophotometry, specifically the Simultaneous Equation Method (SEM) and Absorbance Ratio Method (ARM), to quantify these active ingredients in topical creams. The study begins with the preparation of stock solutions and selection of the analytical wavelength, with 298 nm chosen in reference to the isobestic point at 230 nm. Cream samples containing 10 mg Luliconazole and 30 mg Salicylic Acid are prepared, sonicated, and filtered for analysis. The UV method is validated in accordance with ICH guidelines, encompassing linearity, precision, LOD, LOQ, specificity, robustness, and accuracy. Calibration curves show excellent linearity within the concentration range of 5-30 µg/ml for both compounds. Intra-day and inter-day precision studies exhibit low % RSD values, indicating the reliability and reproducibility of the method. LOD and LOQ values demonstrate the sensitivity of the method, enabling detection and quantification at trace levels. Specificity is confirmed through overlay spectra and comparisons of analyte responses. Robustness is assessed by varying temperature and concentration, demonstrating the method's resilience to minor changes. Accuracy is evaluated through the standard addition method, yielding recoveries within acceptable limits. The proposed UV method is successfully applied to the analysis of Luliconazole and Salicylic Acid in commercial cream samples, with assay results confirming its accuracy and suitability for routine quality control.

Development and Validation of Stability Indicating Method of UV-Spectrophotometry for the Estimation of Flavoxate in Bulk and Tablet Dosage form

Literature survey reveals that few spectrophotometric, high performance liquid chromatographic (HPLC) and High Performance Thin Layer Chromatographic (HPTLC) methods were reported for the estimation of Flavoxate HCl in single and in combined dosage forms. But there is no any stability indicating UV spectroscopic study is reported for Flavoxate in single dosage form. So the present study was aimed to development of simple, accurate, rapid and economical UV-spectrophotometric stability indicating assay method were established for the determination of Flavoxate HCl in bulk and tablet formulation. The solvent was selected for stock solution preparation is water and required concentrations were prepared by using the same solvent. The λmax for detection of Flavoxate HCl is 211nm. In this method Flavoxate followed linearity in the concentrations of 1, 3, 5, 7, 9µg/ml with regression coefficient (r2) 0.999. The percent drug content was found to be 98.81±0.39 for FLV in tablet formulation. Recovery study results of FLV ranges from 99.11 to 100.49%. The method was found to be accurate and precise, as indicate by recovery studies as recoveries were close to 100% and %RSD is less than 2. Inter-day and intraday variation was found to be 0.75 and 0.45%, respectively. The LOD and LOQ values were found to be 0.719µg/ml and 2.186µg/ml, respectively. The stability study of Flavoxate was investigated when the drug was subjected to hydrolytic, peroxide, heat and photolytic (UV and sunlight) degradation. The results express that the Flavoxate is under thermal and photolytic (sunlight and UV light) conditions are more stable. In acidic, basic and hydrogen peroxide conditions Flavoxate is degraded in higher amount.

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

Role of Thermolysin in Catalytic-Controlled Self-Assembly of Fmoc-Dipeptides

In recent years, short peptide self-assembled materials, prepared under the control of the thermolysin catalyst, have been investigated extensively and shown to acquire various morphologies and fun...

Key Factor Study for Amphiphilic Block Copolymer‐Templated Mesoporous SnO2 Thin Film Synthesis: Influence of Solvent and Catalyst

AbstractAs a crucial material in the field of energy storage, SnO2 thin films are widely applied in daily life and have been in the focus of scientific research. Compared to the planar counterpart, mesoporous SnO2 thin films with high specific surface area possess more attractive physical and chemical properties. In the present work, a novel amphiphilic block copolymer‐assisted sol–gel chemistry is utilized for the synthesis of porous tin oxide (SnO2). Two key factors for the sol–gel stock solution preparation, the solvent category and the catalyst content, are systematically varied to tune the thin film morphologies. A calcination process is performed to remove the polymer template at 500 °C in ambient conditions. The surface morphology and the buried inner structure are probed with scanning electron microscope and grazing‐incidence small‐angle X‐ray scattering. Crystallinity is characterized by X‐ray diffraction. The multi‐dimensional characterization results suggest that cassiterite SnO2 with spherical, cylindrical, and vesicular pore structures are obtained. The variation of the film morphology is governed by the preferential affinity of the utilized solvent mixture and the hydrogen bond interaction between the employed cycloether and H2O molecules in the solution.

Chemical Bioconjugation of Proteins in an Undergraduate Lab: One-Pot Oxidation and Derivatization of the N-Terminus

A laboratory experiment introducing the concept of chemical bioconjugation of proteins to undergraduate students in a therapeutically relevant context was developed. Initially, students installed an aldehyde functionality into a protein via the oxidation of the N-terminal threonine residue of the cholera toxin subunit B (CTB) protein, which was followed by subsequent modification via hydrazone addition under mild conditions with a chromophore bearing a distinct UV–vis-absorption peak. Students determined the yield of the reaction to be ca. 11% by HPLC coupled to UV–vis spectroscopy and developed key skills such as preparation of stock solutions, chemical manipulation of proteins, and analysis via HPLC. The reported experiment can be readily adapted for use with other proteins and may contribute to enhancing constructive alignment in interdisciplinary degree programs at the chemistry–biology interface.

Preliminary investigation on the use of <i>Moringa oleifera</i> for the purification of lead polluted wastewater

The need for low-cost wastewater treatment alternatives especially in poor hinterlands, where synthetic coagulants or activated carbon may not be cheaply and readily available, necessitated this study. Moringa Oleifera (MO) kernel was investigated as coagulant in the purification of wastewater polluted with lead. The adsorption potential of MO was also investigated. MO with effective size of 0.14mm was used in the preparation of stock solutions with dosages of 500mg/l and 1000mg/l. Synthetic wastewater polluted with lead monoxide was prepared. The wastewater had an initial concentration of 0.216mg/l of lead. 2%, 4%, 6%, 8% and 10% of the stock solutions were added to the wastewater samples. The samples were subjected to jar test experiments. The research revealed that efficiency of lead removal increases with increase in the concentration of the MO coagulant. For one hour contact time, a maximum efficiency of 35.2% of lead removal was achieved when 10% of the stock solution (1000mg/l solution) was added. The fitting of the experimental data with the Langmuir and Freundlich adsorption isotherm models yielded R2 values of 0.784 and 0.906, respectively for the 1000mg/l dosage stock solution. Freundlich model described the lead (Pb) adsorption better than the Langmuir model. Chemisorption was suggested to be involved in lead (Pb) adsorption (adsorption intensity n<1). The utilization of the extraction method presented in this research proposes the combination of the MO coagulants with the inorganic coagulants.Keywords: Investigation; Moringa Olifera; lead; purification; wastewater

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

Waste water treatment with adsorptions by mushroom compost

High concentrations of heavy metals (up to the pollution level) can disturb the normal ecosystems of natural rivers. There are some methods for treating heavy metals in waste waters. Heavy metal treatments save the ecosystem and clean drinking water. We determined a new adsorption method for heavy metals using a special mushroom compost, which shows the adsorption capacity of this biomass. The laboratory methods were the preparation of stock solutions from heavy metals, the adsorptions of heavy metals by special mushroom compost with a new technique (shaking method), the degradation of samples and analytical control measurements by inductively coupled plasma mass spectrometry. The results show the success of the new adsorption method. The heavy metal adsorption capacity of this mushroom compost seems to be useful on waste water treatment plants because waste water contains heavy metal ions above the limit concentrations. The new absorption technology solution also includes a special economic analysis that examines material flows. In the sustainable economic analysis, as the circular economy focuses on material and energy circularity combined with natural resources (circular economic value (CEV)), the key question is how to make the systems currently operating in a linear form circular. The difference between CEV%BAU(mod) = 45.5% and CEV%Scen(mod) = 87.5% is the amount of material which could be recycled at the end of life cycle of the product, which is the same as the primary raw material, and so mushroom compost is a completely recyclable material.

Overlooked effects of organic solvents from sample preparation on reaction constants of micropollutants in UV-based advanced oxidation processes

Stock solutions of micropollutants with low water solubility are commonly prepared using organic solvents in laboratory studies on degradation of these organic compounds. Dilution of the stock solution unavoidably introduces a small amount of organic solvent into the experimental working solutions. This could possibly affect the estimation of the degradation rate constants of these organic micropollutants by UV-based advanced oxidation process such as UV/H2O2. To demonstrate this problem, the effect of organic solvents on the reaction rate constant of malathion has been investigated in the UV/H2O2 process at the concentration levels that would likely be derived from stock solutions. Several organic solvents commonly used for stock-solution preparation were selected, including acetonitrile (ACN), acetone (Ac), methanol (MeOH), ethanol (EtOH), 1-propanol (PrOH), 1-butanol (BuOH) and 1-pentanol (PeOH). The results show that the reaction rate of malathion in the UV/H2O2 process could be affected by the presence of these organic solvents, even at a concentration well below that possibly introduced during the preparation of working solutions from the organic solvent stock solutions (e.g. 0.00005%, v/v). The suppressive effect on the reaction rate constant depends on the type and concentration. With the increase of organic solvent concentration, the reaction rate constant of UV/H2O2 gradually decreases to the value for photolysis alone. The organic solvents having a stronger reaction activity with OH tend to impose a greater effect on the reaction rate constant. The findings here provide a plausible explanation for the discrepancies in the rate constants reported in the literature for some organic micropollutants during the UV-based advanced oxidation processes.

Effect of organic co-solvents in the evaluation of the hydroxyl radical scavenging activity by the 2-deoxyribose degradation assay: The paradigmatic case of α-lipoic acid

The 2-deoxyribose degradation assay (2-DR assay) is an in vitro method broadly used for evaluating the scavenging activity against the hydroxyl radical (HO). One of the major drawbacks of the assay, however, is that only water soluble compounds can be tested for their radical-scavenging activity. Lipoic acid (LA) is an excellent scavenger of HO but it exhibits a low solubility in the aqueous milieu of the 2-DR assay and a high tendency to polymerize under a variety of conditions. We used LA as a paradigmatic substrate to evaluate the effect of several organic co-solvents in increasing its solubility. Most of these solvents, however, demonstrated to be potent scavengers of HO making their use in the 2-DR assay improper. On the other hand, acetonitrile showed a remarkably low reactivity toward HO (rate constant ~8.7×106M−1s−1) which allowed us to use it as a co-solvent in the preparation of stock solutions of LA ~5mM. We therefore evaluated the radical-scavenging activity of LA by the 2-DR assay in a relatively large range of concentrations, 1–200μM. We found that the rate constant for LA+HO is diffusion-controlled (~1×1010M−1s−1 in water at 25°C) and uninfluenced by the presence of small quantities of acetonitrile. Therefore, the use of acetonitrile in the 2-DR assay does not interfere with the test and may increase the solubility of the radical scavengers.

A pipeline for developing and testing staining protocols for flow cytometry, demonstrated with SYBR Green I and propidium iodide viability staining

The increasing use of flow cytometry (FCM) for analyses of environmental samples has resulted in a large variety of staining protocols with varying results and limited comparability. Viability assessment with FCM is in this context of particular interest because incorrect staining could severely affect the outcome/interpretation of the results. Here we propose a pipeline for the development and optimization of staining protocols for environmental samples, demonstrated with the common viability dye combination of SYBR Green I (SG) and propidium iodide (PI). Optimization steps included the assessment of dye solvents, determination of suitable PI concentration, and determining the optimal staining temperature and staining time. We demonstrated that dimethyl sulfoxide (DMSO) could impair membrane integrity, when used for SGPI stock solution preparation, and TRIS buffer was chosen as an alternative. Moreover we selected 6μM as optimal PI final concentration: less than 3μM resulted in incomplete staining of damaged cells, while concentrations higher that 12μM resulted in false PI-positive staining of intact cells. Low temperatures (25°C) resulted in a slow reaction and did not enable the staining of all bacteria, while high temperatures (44°C) caused damage to cells and false PI-positive results. Hence, 35°C was selected as optimal staining temperature. We further showed that a minimum of 15min were necessary to obtain stable staining results. Moreover, we showed that addition of EDTA resulted in 1–39% more PI-positive results compared to an EDTA-free sample, and argue that insufficient evidence currently exist in favor of adding EDTA to all samples in general. Altogether, the data clearly shows the need to be careful, precise and reproducible when staining cells for flow cytometric analyses, and the need to assess and optimize staining protocols with both viable and non-viable bacteria.

Telavancin activity when tested by a revised susceptibility testing method against uncommonly isolated Gram-positive pathogens responsible for documented infections in hospitals worldwide (2011-2013).

The broth microdilution method for telavancin susceptibility testing was revised and now utilises DMSO as solvent for stock solution preparation and diluent for stock solution dilution, following CLSI guidelines for water-insoluble agents. The revised method also incorporates polysorbate 80 in the test medium to mitigate drug binding to plastics. This revised methodology provides more accurate and reproducible MIC determinations, which results in values lower than the previously established method. This study was conducted to re-establish telavancin potencies and susceptibility profiles (using updated interpretive criteria) against a collection of uncommon clinical pathogens (3821 isolates). Telavancin showed MIC50 values of 0.06mg/L against tested staphylococcal species (MIC50/90, 0.03/0.06mg/L; 98.1-100.0% susceptible), with lower results for Staphylococcus hominis (MIC50, ≤0.015mg/L), Staphylococcus lugdunensis (MIC50, ≤0.015mg/L) and Staphylococcus simulans (MIC50, 0.03mg/L). Vancomycin (MIC50, 1mg/L), daptomycin (MIC50, 0.12-1mg/L) and linezolid (MIC50, 0.25-1mg/L) had MIC50 results at least four-fold higher than telavancin against CoNS. Streptococci (99.2-100.0% susceptible) displayed telavancin MIC50 values of ≤0.015-0.03mg/L. Vancomycin (MIC50, 0.25-0.5mg/L) and linezolid (MIC50, 0.5-1mg/L) had higher MIC50 results against streptococci, whilst daptomycin MIC50 values varied from ≤0.06mg/L to 0.5mg/L. Micrococcus, Listeria and Corynebacterium spp. were inhibited by telavancin at ≤0.015, ≤0.03 and ≤0.06mg/L, respectively. Telavancin exhibited potent in vitro activity against this collection, greater than comparators (daptomycin, linezolid, vancomycin). This study provides new baseline MIC results for telavancin and confirms the spectrum and potency of telavancin against less commonly encountered Gram-positive species.

Direct analysis of trichloropyridinol in human saliva using an Au nanoparticles-based immunochromatographic test strip for biomonitoring of exposure to chlorpyrifos

A portable immunochromatographic strip-based biosensor for direct detection of trichloropyridinol (TCP), a specific biomarker of exposure to chlorpyrifos, in human saliva sample is presented. In this approach, a series of immunoreactions was performed on the test strip, where the targeted analytes (TCP) bound to the Au nanoparticles-labeled antibodies on the conjugate pad to form analyte–Au–antibody conjugates, and then free Au-labeled antibodies were captured by TCP–BSA in the test zone. Captured Au nanoparticles, increased with decreased levels of analytes, can be observed visibly without any equipment and later quantified by a colorimetric reader. Several experimental parameters were optimized including Au nanoparticle-to-TCP antibody coupling ratio, the amount of Au-labeled TCP antibody, immunoreaction time, the pretreatment of sample pad and the preparation of stock solution of Au–TCP antibody that realize sensitivity, selectivity and direct detection of TCP. Under optimal conditions, this biosensor displays a highly linear range of 0.625–20ng/mL TCP, with a detection limit of 0.47ng/mL. Moreover, the immunosensor was successfully used for direct analysis of human saliva sample without any pretreatment. These results demonstrate that this Au nanoparticles-based immunochromatographic test strip (ITS) provides a simple, accurate, and quantitative tool for TCP detection and holds a great promise for point-of-care and in-field analysis of other biomarkers.

The Big Impact of a Small Detail: Cobalt Nanocrystal Polymorphism as a Result of Precursor Addition Rate during Stock Solution Preparation

The control of nanocrystal structures at will is still a challenge, despite the recent progress of colloidal synthetic procedures. It is common knowledge that even small modifications of the reaction parameters during synthesis can alter the characteristics of the resulting nano-objects. In this work we report an unexpected factor which determines the structure of cobalt nanoparticles. Nanocrystals of distinctly different sizes and shapes have resulted from stock solutions containing exactly the same concentrations of [Co{N(SiMe(3))(2)}(2)(thf)], hexadecylamine, and lauric acid. The reduction reaction itself has been performed under identical conditions. In an effort to explain these differences and to analyze the reaction components and any molecular intermediates, we have discovered that the rate at which the cobalt precursor is added to the ligand solution during the stock solution preparation at room temperature becomes determinant by triggering off a nonanticipated side reaction which consumes part of the lauric acid, the main stabilizing ligand, transforming it to a silyl ester. Thus, an innocent mixing, apparently not related to the main reaction which produces the nanoparticles, becomes the parameter which in fine defines nanocrystal characteristics. This side reaction affects in a similar way the morphology of iron nanoparticles prepared from an analogous iron precursor and the same long chain stabilizing ligands. Side reactions are potentially operational in a great number of systems yielding nanocrystals, despite the fact that they are very rarely mentioned in the literature.

Application of LC/MS to determine specific activity variation in a series of sulfonamide tracers from the [35S]methane sulfonate reagent

The specific activities for a series of S-35 tracers were found to vary from the decay-corrected specific activity of the labeled reagent. If not known before the stock solution preparation and binding assay, this variation would have resulted in performing the assay at approximately two to three times over the targeted concentration, thereby leading to considerable error in the calculated binding and related conclusions. Copyright © 2012 John Wiley & Sons, Ltd.

How to get the magic triangle and the MAD triangle into your protein crystal

The magic triangle 5-amino-2,4,6-triiodoisophthalic acid (I3C) and the MAD triangle 5-amino-2,4,6-tribromoisophthalic acid (B3C) are two representatives of a novel class of compounds that combine heavy atoms for experimental phasing with functional groups for protein interactions. These compounds are readily available and provide easy access to experimental phasing. The preparation of stock solutions and the incorporation of the compounds into protein crystals are discussed. As an example of incorporation via cocrystallization, the incorporation of B3C into bovine trypsin, resulting in a single site with high occupancy, is described.