Calibaration and validation of hplc
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Calibration and validation of HPLC methods involves establishing that the instrument produces accurate and reproducible results. Key aspects include: 1. Calibration involves checking parameters like flow rate accuracy, wavelength accuracy, and detector linearity using reference standards. 2. Validation establishes method performance meets requirements. It involves determining accuracy over 3 concentration levels, precision through repeatability studies, and linearity through correlation coefficients. 3. Qualification includes design, installation, operational, and performance qualification to prove equipment works correctly and leads to expected results.
Calibaration and validation of hplc
- 1. CALIBARATION AND VALIDATION OF HPLC PRESENTED BY : ANANT NAG ID: 17BPH005 BATCH : 17-2021 4TH YEAR
- 3. INTRODUCTION •HPLC stands for “High-performance liquidchromatography”(sometimes referred to as High-pressure liquid chromatography). •High performance liquid chromatography is apowerful tool in analysis, it yields high performanceand high speed compared to traditional columnschromatography because of the forcibly pumpedmobile phase. •HPLC is a chromatographic technique that canseparate a mixture of compounds •It is used in biochemistry and analytical chemistry toidentify, quantify and purify the individualcomponents of a mixture. •Chromatography : physical method inwhich separation of components takesplace between two phases-a stationaryphase and a mobile phase •Stationary phase : The substance onwhich adsorption of the analyte (thesubstance to be separated duringchromatography) takes place . It can be asolid, a gel, or a solid liquid combination •Mobile phase : solvent which carries theanalyte (a liquid or a gas) Chromatographic techniques are divided into differenttypes based on : The type of chromatographic bed usedi.e. column chromatography (gas chromatography) andplanar chromatography(paper and thin layer)The physical state of mobile phasei.e. gas chromatography and liquid chromatographyThe separation mechanismi.e. ion-exchange and size exclusion HPLC is a type of liquid chromatography where thesample is forced through a column that is packed with astationary phase composed of irregularly or sphericallyshaped particles, a porous monolithic layer , or a porousmembrane by a liquid (mobile phase) at high pressure.
- 4. PRINCIPLE To understand the principle of HPLC , we must first look at theprinciple behind liquid chromatographyLiquid chromatography is a separation technique that involves: •the placement (injection) of a small volume of liquid sample •into a tube packed with porous particles (stationary phase) •where individual components of the sample are transported along thepacked tube (column) by a liquid moved by gravity.The main principle of separation is adsorption . •When a mixture of components are introduced into the column . variouschemical and/or physical interactions take place between the samplemolecules and the particles of the column packing . •They travel according to their relative affinities towards the stationaryphase. The component which has more affinity towards theadsorbent, travels slower.The component which has less affinity towards the stationary phasetravels faster .•Since no two components have the same affinity towards the stationaryphase, the components are separated
- 5. INSTRUMENTATION . Solvent delivery system(mobile phase) •The mobile phase in HPLC refers to the solvent beingcontinuously applied to the column or stationary phase •The mobile phase acts as a carrier to the sample solution •A sample solution is injected into the mobile phase of an assaythrough the injector port •As a sample solution flows through a column with the mobilephase,the components of that solution migrate according to thenon-covalent interaction of the compound with the column •The chemical interaction of the mobile phase and sample , with thecolumn , determine the degree of migration and separation ofcomponents contained in the sample •The solvents or mobile phases used must be passed through the columnat high pressure at about 1000 to 3000 psi. this is because as the particlesize of stationary phase is around 5-10µ, so the resistance to the flow ofsolvent is high.
- 6. INSTRUMENTS
- 7. CALIBRATION METHOD The demonstration that a particular instrument or device produces results within specified limits by comparison with those produced by a reference or traceable standard over an appropriate range of measurements. Various Calibration parameters for HPLC are: Flow rate accuracy (Pump Module) Injector accuracy Carryover System Precision Wavelength accuracy Detector linearity Injector linearity Gradient Performance Check Column oven temperature accuracy
- 8. FLOW RATE ACCURACY 1. Prime all the solvent lines with water. 2. Set the flow rate to 0.500 ml/m. 3.Wait for about 15 m to stabilize the system and ensure that the pressure is stable. 4.Insert the outlet tubing into a 10 ml volumetric flask and start the stop watch simultaneously. 5. Stop the stopwatch when the lower meniscus reaches the 10 ml mark on the flask. 6. Record the elapsed time. 7. Similarly check the flow for 1.0 ml/m and 2.0 ml/m.
- 9. WE CAN CALIBRATE THE PDA DETECTOR FOR 5 PARAMETERS 1-Baseline Noise and PDA Drift. 2- Your Lamp Intensity. 3- You have to check for the Wavelength Accuracy. • You can inject Pyrene with Methanol as eluent at a flow of 1 ml/min. • The characteristically maximum of pyrene is determined at 333 nm and compared to a theoretical value. 4- Lastly- You must check your PDA's Linearity you can inject 5 caffeine solutions at different concentrations. Concentrations and peak heights can be represented in a graph. The regression coefficient of the resulting line and the deviations from it indicate indicates the linearity- Usually you R square value should be above 99.90% .
- 10. COLUMN HEATING MODULE •The efficiency of a HPLC column varies with column temperature. •capacity factor k’ decreases with temperature, and hence the retention of the analysis decreases with temperature . •Retention drops by 1 to 3% for each increase of 1◦C. How To Check?? •The temperature accuracy of the column heater is evaluated by placing a calibrated thermometer in the column compartment to measure the actual compartment temperature. •The thermometer readings are compared to the preset temperature at 40 ◦C and 60◦C. 33
- 12. VALIDATION METHOD VALIDATION USP: “Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications.” VALIDATION PROTOCOL: A written plan stating how validation will be conducted and defining acceptance criteria. For example, the protocol for a manufacturing process identifies processing equipment, critical process parameters/operating ranges, product characteristics, sampling, test data to be collected, number of validation runs, and acceptable test results. EQUIPMENT VALIDATION: Demonstrate that equipment used in validation studies is suitable for use and is comparable to equipment used for routine analysis.
- 13. QUALIFICATION Action of proving and documenting that equipment or ancillary systems are properly installed, work correctly, and actually lead to the expected results. •Qualification is part of validation, but the individual qualification steps alone do not constitute process validation. PARTS OF QUALIFICATION: The entire process typically consists of four parts: •Design qualification (DQ) •Installation qualification(IQ) •Operational qualification (OQ) •Performance qualification (PQ).
- 15. Basic parameters for the validation of method:
- 16. ACCURACY Definition: The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a conventional true value or as an accepted reference value and the value found. How To Determine?? • Known amounts of Related substances and the drug substance in placebo are spiked to prepare an accuracy sample of known concentration of Related substance. • According to the ICH, accuracy should be determined using a minimum of nine determinations over a minimum of three concentration levels covering the range(from 50% of the ICH reporting level to 150% of the proposed shelf life specification of the related substances) specified. e.g. Level Accuracy 125% 99.6 +/- 0.2% 75% 100.3 +/- 0.8% 25% 99.2 +/- 0.7% 7 (Reference: IJPSR Determination of Voglibose by HPLC and validation of method)
- 17. INTRINSIC ACCURACY: Intrinsic accuracy indicates the bias caused by sample matrix and sample preparation. OVERALL ACCURACY: : oMatrix effect o Sample preparation o Calculation error %relative substance(calculated) •Overall accuracy = %RELATIVE SUBSTANCES(CALCULATED) %RELATIVE SUBSTANCES(THEORY)
- 18. PRECISION Definition : The Precision is a measure of the ability of the method to generate reproducible results. The precision of a method is evaluated for repeatability, intermediate precision, and reproducibility. •Repeatability is a measure of the ability of the method to generate similar results for multiple preparations of the same homogeneous sample by one analyst using the same instrument in a short time duration (e.g., on the same day). For instance, method repeatability for pharmaceutical assays may be measured by making six sample determinations at 100% concentration, or by preparing three samples at 80, 100, and 120% concentration levels each. •Intermediate precision is a measure of the variability of method results where samples are tested and compared using different analysts, different equipment, and on different days, etc. This study is a measure of the intra-laboratory variability and is a measure of the precision that can be expected within a laboratory. •Reproducibility is the precision obtained when samples are prepared and compared between different testing sites. Method reproducibility is often assessed during collaborative studies at the time of technology or method transfer. %RSD= (s/µ)*100
- 19. LINEARITY Definition : Linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration of analytes in the sample. •If there is a linear relationship test results should be evaluated by appropriate statistical methods like, •Correlation coefficient •Y- intercept •Slope of regression line •Plot of the Data •Method To Determine Linearity •Direct Weigh Method •Dilution Method
- 21. ADVANTAGES OF HPLC 1. Separations fast and efficient (high resolution power) 2. Continuous monitoring of the column effluent 3 It can be applied to the separation and analysis of very complex mixtures 4. Accurate quantitative measurements. 5. Repetitive and reproducible analysis using the same column 6. Adsorption, partition, ion exchange and exclusion column separations are excellently made.
- 22. REFERENCES 1. Morden HPLC by Michel Dong, Chapter 9. 2. Analytical Method Validation and Instrument Performance Verification by Herman Lam and Y.C. Lec, Chapter 3 and Chapter 11. 3. Net surf. •Scientist solution.com •Chemicalforum.com •Chemistpub.org 4. Pharmaceutical Journals, •IJPSR •JBSR