Combined Glass-Calomel pH Electrode
High-impedance research-grade combined glass electrode with saturated calomel reference and low-resistance bulb glass for measuring electromotive force and pH with readability ±0.001 pH units from pH 0 to 14.
Chemistry / Isoelectric Point of Amino Acids
FREE TO EXPLORELABORATORY 40 · NATURAL SCIENCES
Isoelectric Point and Dual-Region Potentiometric Titration of Amphoteric Amino Acids
Determine the macroscopic acid dissociation constants (pKa1, pKa2) and the isoelectric point (pI = 5.970) of glycine by high-precision dual-region potentiometric titration with standardized 0.1000 M NaOH at 25.0 °C. Explore interactive analytical gravimetry, Class A volumetric glassware and density determination.
MEASURE · CALIBRATE · ANALYSE
Your virtual chemistry laboratory.
CONJUGATE MEAN BUFFER PH—
MEASUREMENT STATUS
Ready to dispense 12.5 mL of liquid.
Adjust V₁ V, then weigh the dispensed aliquot.
Mass observations are determined by the high-precision thermodynamic equation of state for pure liquid water. Readings reflect thermal equilibrium and analytical balance force restoration.
Physical chemistry assumptions and metrological limits
['Potentiometric titration requires analyte concentrations of at least 10⁻² M to obtain well-defined buffer regions and inflection points.', 'Activity coefficients vary slightly during titration due to changing ionic strength as neutral zwitterions are converted into charged ions.', 'Alkaline glass electrode error (sodium ion alkaline error) can cause slight underestimation of pH above pH 11.0 unless accounted for.'] The potentiometric model evaluates 25.00 mL of 0.1000 M glycine hydrochloride titrated with 0.1000 M NaOH at 25.00 °C. The first equivalence point occurs at V_eq1 = 25.00 mL (pure zwitterion, pH = pI = 5.970), and the second occurs at V_eq2 = 50.00 mL (pure glycinate, pH = 11.35). Across the six experimental sample volumes in the first buffer V₁ = [5.00, 7.50, 10.00, 12.50, 15.00, 17.50] mL (corresponding to fractional extents θ = [0.20, 0.30, 0.40, 0.50, 0.60, 0.70] and matched second buffer volumes V₂ = 25.00 + V₁), the conjugate mean pH values are [5.368, 5.602, 5.794, 5.970, 6.146, 6.338]. Linear regression of conjugate mean pH versus log₁₀(θ / (1 - θ)) yields an empirical slope m = 1.00000 and Y-intercept b = pI = 5.97000 with R² = 1.0000000, isolating the isoelectric point pI = 5.970 with 0.0000% difference from literature reference standards.
Analytical chemistry apparatus & tools.
Class A Automatic Digital Burette (50.00 mL)
Precision motor-driven piston burette with digital stepper dispensing resolution of 0.005 mL, calibrated to ISO 385 standards for delivering carbonate-free 0.1000 M NaOH titrant.
Jacketed Reaction Cell with Magnetic Stirrer
Water-jacketed double-walled borosilicate titration vessel connected to a circulating water bath maintaining 25.00 ± 0.02 °C with variable-speed PTFE-coated magnetic stirrer.
Carbonate-Free NaOH Reagent Delivery System
Aspirator bottle containing standardized 0.1000 M NaOH equipped with soda-lime and Ascarite II guard tubes to prevent atmospheric carbon dioxide absorption.
Analytical Balance & Class A Volumetric Flasks
Four-decimal analytical balance (readability ±0.0001 g) and 250.00 mL volumetric glassware used to prepare primary standard potassium hydrogen phthalate (KHP) and glycine hydrochloride stock solutions.
Microprocessor Millivolt / pH Interface
High-input impedance (> 10¹² Ω) digital electrometer interface logging cell potential (mV) and pH with automatic temperature compensation probe.
FOLLOW THE METHOD
A reason behind every step.
Determine the macroscopic acid dissociation constants (pKa1, pKa2) and the isoelectric point (pI = 5.970) of glycine by high-precision dual-region potentiometric titration with standardized 0.1000 M NaOH at 25.0 °C. Suggested independent volumetric settings: 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 mL.
01
Electrode Calibration with IUPAC Primary Standard Buffers
Power on the pH meter and allow 30 minutes for thermal and electronic stabilization. Perform a two-point calibration of the combined glass electrode using fresh primary IUPAC buffers (pH 4.008 potassium hydrogen phthalate and pH 9.180 sodium tetraborate decahydrate) at 25.00 °C, verifying a Nernstian slope between 98.5% and 100.5% (58.3 to 59.5 mV/pH).
02
Standardization of Carbonate-Free Sodium Hydroxide
Standardize the ~0.1 M NaOH solution by potentiometric titration against primary standard potassium hydrogen phthalate (KHP) previously dried at 110 °C for 2 hours. Accurately establish the NaOH molarity to four significant figures (0.1000 M).
03
Preparation and Thermal Equilibration of Glycine Hydrochloride
Pipette exactly 25.00 mL of 0.1000 M glycine hydrochloride (H₂Gly⁺ Cl⁻) into the jacketed reaction cell. Immerse the calibrated pH electrode and burette tip below the solution surface. Engage the magnetic stirrer at a moderate rate (300 rpm) without vortex entrainment, and circulate water at 25.00 °C for 15 minutes.
04
First Buffer Region Titration (Carboxylic Deprotonation)
Add standardized 0.1000 M NaOH in increments of 1.00 mL (reducing to 0.10 mL near the equivalence point). Allow 30 seconds for complete mixing and thermal equilibration after each addition before recording the equilibrium pH₁. At V₁ = 12.50 mL (half-neutralization), record pKa1 = 2.340.
05
First Equivalence Point & Second Buffer Region Titration
Continue adding NaOH past the first equivalence point (V_eq1 = 25.00 mL, pH = 5.970) into the second buffer region (25.00 to 50.00 mL). Record equilibrium pH₂ at matched conjugate increments V₂ = 25.00 + V₁. At V₂ = 37.50 mL (half-neutralization of the ammonium group), record pKa2 = 9.600.
06
Conjugate Mean Evaluation and Linear Regression
For each fractional titration extent θ = V₁ / 25.00, compute the conjugate mean pH = (pH₁ + pH₂) / 2 and the logarithmic ratio log₁₀(θ / (1 - θ)). Perform ordinary least-squares regression of mean pH versus log₁₀(θ / (1 - θ)) to directly extract the isoelectric point pI from the Y-intercept and verify unity slope.
YOUR NOTEBOOK · 0 OBSERVATIONS
Turn measurements into quantitative chemical insight.
Points are your recorded gravimetric observations. The regression line validates the linear mass-volume proportionality and extracts density.
ORDINARY LEAST-SQUARES FIT · m = ρ·V + m₀
\text{pH}_{\text{mean}} = 1.0000 \cdot \log_{10}\left(\frac{\theta}{1 - \theta}\right) + pI \implies pI = 5.970 \implies pK_{a1} = 2.340,\; pK_{a2} = 9.600
| # | Titrant Volume in First Buffer V₁ (mL) | Conjugate Mean Buffer pH (pH) | log(θ / (1 - θ)) | Conjugate Mean Buffer pH | Remove |
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