Arsenazo III calcium-sensitive dye

Arsenazo III is a metallochromic indicator dye that forms colored complexes with calcium and other metal ions, enabling quantitative determination by spectrophotometry.

Primary applications:

• Clinical chemistry: Serum and plasma calcium measurement for diagnostic purposes
• Biochemical research: Calcium flux studies, enzyme assays, cellular calcium measurements
• Water analysis: Hardness determination and calcium content in environmental samples
• Pharmaceutical QC: Calcium content verification in formulations and supplements
• Food analysis: Nutritional calcium determination in food products

How it works: Arsenazo III binds calcium in a 1:1 stoichiometry, causing a spectral shift from 600 nm (free dye) to 650-660 nm (calcium complex). The absorbance change at 650-660 nm is directly proportional to calcium concentration, allowing accurate quantification using Beer’s Law.

Advantages: High sensitivity, rapid reaction, minimal interference from other ions at physiological pH, compatible with automated analyzers, and suitable for both manual and high-throughput applications.

Arsenazo III provides excellent sensitivity for calcium determination with well-defined spectral properties.

Spectral characteristics:

• Free dye absorption: Maximum at ~600 nm (purple-blue color)
• Calcium complex absorption: Maximum at 650-660 nm (blue-purple color)
• Measurement wavelength: 650-660 nm (primary) or 600 nm (reference)
• Molar absorptivity: Approximately 1.5 × 10⁴ M⁻¹cm⁻¹ at 650 nm

Sensitivity and detection range:

• Detection limit: 10-50 µM calcium (0.4-2 mg/dL)
• Linear range: 0.1-10 mM (4-400 mg/dL)
• Optimal working range: 0.5-5 mM for best precision
• Coefficient of variation: Typically <2% for replicate measurements

Measurement modes:

• Single wavelength: 650-660 nm direct measurement
• Dual wavelength: 650 nm minus 600 nm for improved accuracy
• Kinetic mode: Continuous monitoring of calcium binding

Standard protocol for calcium determination using Arsenazo III is straightforward and reproducible.

Reagent preparation:

• Arsenazo III working solution: Dissolve 0.05-0.2 mM in buffer (typically 0.1 mM)
• Buffer: 50 mM HEPES or PIPES, pH 6.5-7.5
• Calcium standards: 0, 0.5, 1, 2, 5, 10 mM in same buffer
• Storage: Working solution stable for several weeks at 4°C, protected from light

Basic assay procedure:

• Step 1: Add sample or standard to cuvette or microplate well
• Step 2: Add Arsenazo III working solution (typically 1:10 or 1:20 dilution)
• Step 3: Mix thoroughly and incubate 5-10 minutes at room temperature
• Step 4: Measure absorbance at 650-660 nm
• Step 5: Calculate calcium concentration from standard curve

Protocol optimization:

• pH: Maintain pH 6.5-7.5 for optimal calcium binding
• Temperature: Room temperature (20-25°C) is standard
• Time: Color develops within 5 minutes, stable for 30+ minutes
• Blank: Use reagent blank (no calcium) to zero spectrophotometer

Understanding potential interferences is important for accurate calcium determination.

Metal ion interferences:

• Magnesium: Minimal interference at physiological pH (6.5-7.5), significant at higher pH
• Strontium: Similar binding affinity to calcium, will be measured if present
• Barium: Also binds but less common in biological samples
• Heavy metals: Copper, zinc, iron may interfere at high concentrations
• Rare earths: Lanthanides bind strongly but rare in typical samples

Sample matrix effects:

• Protein: Minimal interference, but high protein may require dilution
• Lipids: Turbidity from lipemic samples affects measurement
• Hemolysis: May cause interference; use clear serum/plasma
• pH: Outside optimal range (6.5-7.5) affects binding stoichiometry

Minimizing interferences:

• Maintain pH 6.5-7.5 to minimize magnesium interference
• Dilute turbid or lipemic samples appropriately
• Use chelating agents (EDTA, EGTA) in blank for specificity testing
• Pre-treat samples to remove protein if necessary
• Compare results with ion-selective electrode methods if interference suspected

Yes, Arsenazo III is a versatile metallochromic indicator that forms complexes with many metal ions beyond calcium.

Metal ions detected by Arsenazo III:

• Alkaline earth metals: Calcium, magnesium, strontium, barium
• Transition metals: Copper, zinc, nickel, cobalt
• Rare earth elements: Lanthanides (La, Ce, Nd, etc.)
• Actinides: Thorium, uranium
• Other metals: Aluminum, iron (III), lead

Selectivity by pH adjustment:

• pH 3-4: Rare earth elements, thorium, uranium
• pH 5-6: Transition metals
• pH 6.5-7.5: Calcium (minimal magnesium interference)
• pH >8: Magnesium, other alkaline earth metals

Applications for other metals:

• Magnesium: Clinical chemistry (at pH >8)
• Rare earths: Analytical chemistry, separation processes
• Uranium/thorium: Nuclear industry, environmental monitoring
• Heavy metals: Environmental and toxicology studies

The spectral characteristics change depending on the metal ion, allowing some degree of selectivity through wavelength selection and pH control.

Proper storage ensures long-term stability and consistent performance.

Dry powder storage:

• Temperature: Room temperature (15-25°C) is suitable
• Protection: Store in amber bottle, protected from light
• Moisture: Keep dry; desiccator storage recommended
• Container: Tightly sealed to prevent moisture uptake
• Shelf life: 36 months when stored properly

Working solution storage:

• Temperature: Store at 4°C refrigerated
• Protection: Amber bottle or foil-wrapped
• Stability: Several weeks to months at 4°C
• pH: Maintain at optimal pH (6.5-7.5) for best stability
• Contamination: Avoid metal ion contamination from containers

Handling recommendations:

• Use clean, metal-free spatulas and containers
• Prepare in deionized or distilled water
• Filter solutions if particulates appear
• Protect from prolonged light exposure during use
• Discard if significant color change or precipitation occurs

Quality checks:

• Verify spectral properties periodically (λmax at 600 nm for free dye)
• Test with calcium standards to confirm activity
• Compare absorbance to fresh solution if results seem inconsistent

We offer multiple pack sizes to meet diverse laboratory requirements.

Available quantities (in stock):

• 1 g – For initial method development and small-scale testing
• 5 g – Standard research quantity
• 10 g – For routine clinical or research use
• 25 g – Clinical laboratory or high-volume research
• 100 g – Commercial/industrial applications
• Custom quantities: Available for large-scale operations

Usage estimates:

• 1 g = sufficient for ~10,000-50,000 assays (depending on protocol)
• Typical working solution: 0.1 mM (77.6 mg/L)
• 1 g makes ~13 liters of 0.1 mM working solution

Delivery times:

• Stock quantities: Ships within 24-48 hours
• Domestic USA: 2-3 business days (FedEx/UPS)
• International: 5-7 business days (DHL/FedEx)
• Custom quantities (>100g): 2-3 weeks

Packaging:

• Amber glass bottle to protect from light
• Sealed under nitrogen atmosphere
• Certificate of Analysis included
• Safety Data Sheet included
• Handling and storage instructions provided