JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY HYDERABAD Pharm. D III Year PHARMACEUTICAL ANALYSIS Previous Question Paper (January/February - 2021 - R08)

Calibration is the process of comparing an instrument's output against a known standard to ensure accuracy and precision. Two common analytical instruments requiring regular calibration are the pH meter and the UV-Visible Spectrophotometer.

1. Calibration of a pH Meter:

1. Prepare at least two standard buffer solutions with known pH values (e.g., pH 4.0, 7.0, and 10.0) that bracket the expected sample range.
2. Rinse the electrode thoroughly with deionized water and blot dry with lint-free tissue.
3. Immerse the electrode in the first buffer (usually pH 7.0) and allow the reading to stabilize. Adjust the meter to the exact value of the buffer using the calibration control.
4. Repeat the process with the second buffer (e.g., pH 4.0 or 10.0) to set the slope of the electrode.
5. Verify the calibration by checking the electrode in one of the buffers again; the reading should match the expected value within specified tolerance limits.

2. Calibration of a UV-Visible Spectrophotometer:

1. Wavelength Accuracy: Use a holmium oxide filter or a didymium glass filter. Measure the absorbance spectrum and compare the observed peak maxima with the certified values provided by the manufacturer. The deviation should be within ±1 nm.
2. Photometric Accuracy: Use certified neutral density filters or standard solutions of potassium dichromate in perchloric acid. Measure the absorbance at specific wavelengths and compare the results with the theoretical absorbance values.
3. Stray Light: Measure the transmittance of a solution that absorbs completely at a specific wavelength (e.g., sodium iodide or sodium nitrite). A reading greater than zero indicates the presence of stray light.
4. Resolution: Measure the spectral bandwidth using a standard such as a toluene-in-hexane solution to ensure the instrument can distinguish between closely spaced peaks.

High-Performance Liquid Chromatography (HPLC) is an advanced analytical technique based on the principle of differential migration of analytes between a stationary phase (typically a chemically modified silica column) and a mobile phase (solvent system). Separation occurs due to differences in the partition coefficients of the components in the mixture as they are forced through the column under high pressure.

Instrumentation:

1. Solvent Reservoir: Contains the mobile phase, which is degassed to prevent air bubbles.
2. High-Pressure Pump: Delivers the mobile phase at a constant, precise flow rate, overcoming the resistance of the tightly packed stationary phase.
3. Sample Injector: Introduces the sample into the high-pressure stream, typically via a six-port injection valve with a fixed-volume loop.
4. Column: The heart of the system, containing the stationary phase (e.g., C18 silica particles). Separation occurs here based on polarity or size.
5. Detector: Monitors the eluent continuously. Common types include UV-Visible spectrophotometers, refractive index detectors, and fluorescence detectors.
6. Data System: Converts the electrical signal from the detector into a chromatogram, allowing for qualitative identification (retention time) and quantitative analysis (peak area/height).

Applications:

• Pharmaceutical Analysis: Used for assay of drug substances, purity testing, and detection of degradation products or impurities in formulations.
• Pharmacokinetics: Determination of drug concentrations in biological fluids like plasma or urine.
• Quality Control: Ensuring compliance with pharmacopoeial standards for batch-to-batch consistency.
• Biochemical Research: Separation and purification of proteins, peptides, and nucleic acids.
• Environmental Analysis: Detection of trace contaminants or pesticides in water and soil samples.