Hplc qualification copy
- 1. Dadasaheb Balpande college of pharmacy, Besa, Nagpur. Short seminar Prepared by : Himanshu M. Bhogekar B-Pharm IV Year
- 3. Mobile Phase Degasser Pump Column Thermostat Auto-sampler (Injector) Detector Control Module HPLC Instrument HPLC: • Analytical technique. • Separate, Identify and quantify components in the mixture.
- 4. Reservoir 1 Reservoir 2 Pump 1 Gradient Controller Pump 2 Mixing Chamber Solvent Conditioning Column Injector Pre-column Analytical Column Detector Recorder Instrumentation of HPLC
- 5. • Technique to collect the documented data evidence. • Process to provide a result for sample to accepted range. Objectives of Qualification • Mainly perform to check the accuracy. • Measure uncertainty in the result and deviation. • Reduces the failure rate of analysis. • Helpful for the build-up the confidence of analytes. • Time saving process.
- 6. Instrument module Parameter to be checked Typical acceptance range Solvent delivery system (Pump) Flow rate accuracy Isocratic Gradient Isocratic Gradient ±5.0% ±2.0% Injector Volume precision RSD ≤ 1.0% Carry-over ≤ 0.2% Multi-wavelength detector Linearity 𝑟2 ≥0.99900 Wavelength accuracy ±2nm Drift According to Manufacturer
- 7. Requirements: 1. Beakers. 2. Volumetric flask. 3. Calibrated analytical balance. 4. Calibrated chronometer. Mobile phase used: De-gassed water. Flow rate: 0.5ml/min, 1ml/min, 1.5ml/min Time: 1200sec (20min), 598sec (9.9min), 395sec (6.5min), Procedure 1. •Weigh the empty collecting vessel accurately and note the reading 2. •Set the flow rate properly say 0.5ml/min. 3. •Allow water to pass and collect in collecting vessel. 4. •Weigh the vessel again and note the reading. 5. •Repeat the whole procedure for 3 times by changing time and flow rate.
- 8. Sr. no. Flow rate ml/min Wt. of empty volumetric flask Wt. of volumetric flask with water Wt. of water (W2-W1) Time (sec) Flow rate of pump Acceptance criteria 1. 0.5 14.50 24.49 9.99 1200 0.49 0.4802-0.4998 Observation table 𝑉 = 𝑚 𝜌 ∴ 𝑉 = 9.99 1 ∴ 𝑉 = 9.99 𝑚 − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑡𝑖𝑚𝑒. 𝜌 − 𝐷𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 𝑎𝑡 𝑟𝑜𝑜𝑚 𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑓 = 𝑉 𝑡 ∴ 𝑓 = 9.99 20 ∴ 𝑓 = 0.49 𝑓 − measured flow rate 𝑡 − 𝐸𝑙𝑎𝑝𝑠𝑒𝑑 𝑡𝑖𝑚𝑒 (min) 𝑉 − 𝑣𝑜𝑙𝑢𝑚𝑒 𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑖𝑛 𝑚𝑙 𝐷 = 100 × 𝐹 − 𝑓 𝐹 ∴ 𝐷 = 100 × 0.5 − 0.49 0.5 ∴ 𝐷 = 2% 𝐷 − 𝐷𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 (%) 𝐹 − 𝑁𝑜𝑚𝑖𝑛𝑎𝑙 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒 𝑓 − 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒
- 9. Flow Rate accuracy (gradient method) Procedure Requirements: 1. Detector – 256nm Mobile phase A : Degassed water Mobile phase B : Degassed water + 0.5% acetone. Flow rate : 1.0 ml/min Time %mobile phase A (water) %mobile phase B (water+0.5% acetone) 0.0 100 0 10 90 10 10.1 50 50 20 50 50 20.1 10 90 30 10 90 30.1 0 100 Ripple gradient composition: % of noise of the 50% line from gradient chromatogram. %𝑅 = 𝑁 ℎ50 × 100 %𝑅 = 𝑟𝑖𝑝𝑝𝑙𝑒 ℎ50 = ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 50% 𝑙𝑖𝑛𝑒 𝑁 = ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑛𝑜𝑖𝑠𝑒 Limit: ≤ 0.2%
- 10. Observation table Sr.no. Name Height of AB Gradient accuracy (AB) 1. Height at 10% 108124 9.86 2. Height of 50% 551808 50.3 3. Height of 90% 994505 90.7 4. Height at 100% 1096183 100.0 %𝐻 = ℎ 𝐻 × 100 ∴ %𝐻 = 108124 1096183 × 100 ∴ %𝐻 = 9.86 %𝐻 − 𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑐𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 ℎ − ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑙𝑖𝑛𝑒 𝐻 − ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 100% 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 𝐵 𝑑 = 10 − 9.86 ∴ 𝑑 = 0.14 𝑑 − deviation Time %mobile phase A (water) %mobile phase B (water+0.5% acetone) 0.0 100 0 10 90 10 10.1 50 50 20 50 50 20.1 10 90 30 10 90 30.1 0 100
- 11. Volume precision and carry-over Solutions: Solvent A: Degassed water : methanol (40:60)V/V Reference solution (a) : dissolve 15.0mg methyl- paraben and 15mg propyl-paraben in solvent A and dilute to 100 ml with the same solvent. Reference solution (b) : Dilute 1.0ml of reference solution (a) to 10 ml with solvent A. Requirements: 1. Column 2. Flow rate: 1.0 ml/min 3. Detection: 254 nm 4. Injection volume: • (“Short pathway” flow-cell, for example 5 to 10 mm): 20µL • (“long pathway” flow-cell, for e.g. 60 to 85mm): 2µL Procedure 1 × Solvent A (blank injection 1) 6 × reference solution (b) 1 × reference solution (a) 1 × solvent A (blank injection 2) 1 × reference solution (b) Limits: Repeatability of peak area: The RSD of peak area of methyl- paraben and propyl-paraben in chromatogram of six consecutive injections of reference solution (b) should be ≤ 1.0%. Carry-over: Peak area of propyl-paraben obtain from blank injection 2 and peak obtain from reference solution (b) not exceed 2 %. Corresponds to 0.2%.
- 12. Multi-wavelength detector (linearity) Stock solution: 10 mg caffeine + 20ml water (conc. 0.5mg/ml). Solution Process Concentration A 10ml + 100ml water 50µg/ml B 1ml + 100ml water 0.5µg/ml C 2ml + 200ml water 1µg/ml D 1ml + 10ml water 5µg/ml E 2ml + 20ml water 10µg/ml F 5ml + 50ml water 25µg/ml Requirements: 1. Column 2. Mobile phase – Acetonitrile: degassed water (15:85 V/V) 3. Oven temperature -- 40°C 4. Flow rate – 1.0ml/min. Run time : 6 min 5. Detection – 273 nm Working Solution Limit : 𝑟2≥0.99900 Procedure 2 × Blank 1× B 1× C 1× D 1× E 1× F y = 53956x - 2423.2 R² = 0.9998 0 500000 1000000 1500000 2000000 2500000 3000000 0 10 20 30 40 50 60 AREA CONCENTRATION (ΜG/ML)
- 13. Multi-wavelength detector (Accuracy) Solution: Caffeine 25µg/ml used. Inject Caffeine solution. Record the spectrum by scanning from 202 – 208 (Maxima), 270 – 276 (Maxima) 242 -248 (Minima) Limit : ±2nm