Caliberation and validation of High performance liquid chromatography

A brief introduction to HPLCX
its principle of working , types of column , detectors
and more importantly, the caliberation and validation of HPLC its column, detector , volume of injection , oven tempreture etc

1. GROUP Members Rashid ullah Saba sabir Aiman mohsin Maryam anwar Zahra batool Asmat naz HIGH PERFORMANCE LIQUID CHROMATOGRAPHY STUDENTS OF BAQAI MEDICAL UNIVERSITY PHARM-D FINAL PROFESSIONAL(BATCH 18) KARACHI PAKISTAN

2. LEARNING OUTCOME AFTER STUDYING THIS TOPIC STUDENT SHOULD BE ABLE TO : DEFINE HPLC DESCRIBE HPLC PRINCIPLE EXPLAIN MAJOR COMPONENTS OF HPLC AND THEIR FUNCTION EXPLAIN APPLICATION OF HPLC STEPS INVOLVED IN VALIDATION AND CALIBRATION OF HPLC DESCRIBE ADVANTAGES & DISADVANTAGES OF HPLC

3. Chromato-graphy / -graph / -gram / - grapher CHROMATOGRAPHY: ANALYTICAL TECHNIQUE CHROMATOGRAPH: INSTRUMENT CHROMATOGRAM: OBTAINED “PICTURE” CHROMATOGRAPHER: PERSON

4. Common terms for hplc THE  ANALYTE  IS THE SUBSTANCE TO BE SEPARATED DURING CHROMATOGRAPHY. IT IS ALSO NORMALLY WHAT IS NEEDED FROM THE MIXTURE. THE  ELUENT  IS THE SOLVENT THAT CARRIES THE ANALYTE. AN  IMMOBILIZED PHASE  IS A STATIONARY PHASE THAT IS IMMOBILIZED ON THE SUPPORT PARTICLES, OR ON THE INNER WALL OF THE COLUMN TUBING. THE  MOBILE PHASE MOVES IN A DEFINITE DIRECTION. IT IS THE SOLVENT THAT MOVES THE SAMPLE/ ANALYTE THROUGH THE COLUMN.

5. Common terms for hplc PREPARATIVE CHROMATOGRAPHY  IS USED TO PURIFY SUFFICIENT QUANTITIES OF A SUBSTANCE FOR FURTHER USE, RATHER THAN ANALYSIS. THE  RETENTION TIME  IS THE CHARACTERISTIC TIME IT TAKES FOR A PARTICULAR ANALYTE TO PASS THROUGH THE SYSTEM (FROM THE COLUMN INLET TO THE DETECTOR) UNDER SET CONDITIONS.  THE THEORITICAL PLATES IS THE EFFICIENCY OF COLUMN RESOLUTION PROVIDES A QUANTITATIVE MEASURE OF THE ABILITY OF A COLUMN TO SEPARATE TWO ANALYTES

6. LIQUID CHROMATOGRAPHY CHROMATOGRAPHY IN WHICH THE MOBILE PHASE IS A LIQUID. THE LIQUID USED AS THE MOBILE PHASE IS CALLED THE “ELUENT”. THE STATIONARY PHASE IS USUALLY A SOLID OR A LIQUID. IN GENERAL, IT IS POSSIBLE TO ANALYZE ANY SUBSTANCE THAT CAN BE STABLY DISSOLVED IN THE MOBILE PHASE.

7. From Liquid Chromatography to High Performance Liquid Chromatography Higher degree of separation!  Refinement of packing material (3 to 10 µm) Reduction of analysis time!  Delivery of eluent by pump  Demand for special equipment that can withstand high pressures The arrival of high performance liquid chromatography!

8. introduction HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) IS BASICALLY A HIGHLY IMPROVED FORM OF COLUMN LIQUID CHROMATOGRAPHY. THE MAIN PURPOSES FOR USING HPLC ARE FOR SEPERATION, IDENTIFYING, QUANTIFYING AND PURIFYING THE INDIVIDUAL COMPONENTS OF THE MIXTURE.

9. HPLC PRIINCIPLE PRINCIPLE : HPLC OPERATE UNDER THE SAME BASIC PRINCIPLE; SEPARATION OF A SAMPLE INTO ITS CONSTITUENT PARTS BECAUSE OF THE DIFFERENCE IN THE RELATIVE AFFINITIES OF DIFFERENT MOLECULES FOR THE MOBILE PHASE AND THE STATIONARY PHASE USED IN THE SEPARATION.

10. BLOCK DIAGRAM OF HPLC .

11. Classification of hplc technique BASED ON TYPES OF ANALYSIS QUANTITATIVE ANALYSIS QUALITATIVE ANALYSIS

12. BASED ON PRINCIPLE OF SEPARATION : ADSORPTION CHROMATOGRAPHY ADSORPTION CHROMATOGRAPHY IS BASED ON THE INTERACTION BETWEEN THE SOLUTE MOLECULES AND ACTIVE SITES ON THE STATIONARY PHASE. THIS ATTACHMENT OR INTERACTION DEPENDS ON THE POLARITY OF SOLUTES.

13. BASED ON PRINCIPLE OF SEPARATION : BASED ON ION EXCHANGE THE PRINCIPLE OF SEPERATION IS ION EXCHANGE WHICH IS REVERSIBLE EXCHANGE OF FUNCTIONAL GROUP

14. BASED ON PRINCIPLE OF SEPARATION : SIZE EXCLUSION CHROMATOGRAPHY SEPERATION BASED ON DIFFERENCE IN MOLECULAR SIZE USING GEL THAT ACT AS MOLECULAR SEIVE

15. BASED ON PRINCIPLE OF SEPARATION : AFFINITY CHROMATOGRAPHY AFFINITY OF SAMPLE WITH SPECIFIC STATIONARY PHASE

16. BASED ON PRINCIPLE OF SEPARATION : CHIRAL CHROMATOGRAPHY CHIRAL CHROMATOGRAPHY REFERS TO THE SEPARATION OF ENANTIOMERS USING A CHIRAL HPLC COLUMN

17. Based on mode of chromatography Normal phase chromatography STATIONARY PHASE POLAR EXAMPLE SILICA, ALUMINA MOBILE PHASE NON POLAR EXAMPLE HEXANE ; HEPTANE mixed with slightly polar solvent like CHLOFORM OR ISOPROPANOL etc Reverse phase chromatography Widely used in pharmaceutical analysis STATIONARY PHASE NON POLAR EXAMPLE ODS SILICA GEL C18, C8 MOBILE PHASE POLAR EXAMPLE WATER ; METHANOL; ACETONITRILE; TETRAHYDROFURAN (THF)

18. Based on scale of operation Analytical hplc ONLY ANALYSIS OF SAMPLE IS DONE RECOVERY OF SAMPLE FOR RE USING IS NOT DONE, SINCE THE SAMPLE USED IS VERY LOW. Preparative hplc INDIVIDUAL COMPONENTS OF PURE COMPOUNDS CAN BE COLLECTED USING FRACTIONAL COLLECTOR. THE COLLECTED SAMPLE CAN BE REUSED

19. Based on elution technique Isocratic A SEPARATION IN WHICH THE MOBILE PHASE COMPOSITION REMAINS CONSTANT THROUGHOUT THE PROCEDURE IS TERMED  ISOCRATIC. PEAK WIDTH INCREASES WITH RETENTION TIME LINEARLY SO LATE-ELUTING PEAKS GET VERY FLAT AND BROAD. gradient   A SEPARATION IN WHICH THE MOBILE PHASE COMPOSITION IS CHANGED DURING THE SEPARATION PROCESS IS DESCRIBED AS A  GRADIENT ELUTION . GRADIENT ELUTION DECREASES THE RETENTION OF THE LATER-ELUTING COMPONENTS SO THAT THEY ELUTE FASTER, GIVING NARROWER (AND TALLER) PEAKS FOR MOST COMPONENTS.

20. Instrumentation of HPLC

21. Components of HPLC SOLVENT RESERVOIR PUMPS SAMPLE INJECTION SYSTEM COLUMNS DETECTORS DATA PROCESSING WASTE

22. Solvent Reservoir RESERVOIR FOR MOBILE PHASE THAT CARRY SAMPLE INTO THE COLUMN

23. DEGASSER PROBLEMS CAUSED BY DISSOLVED AIR IN THE ELUENT UNSTABLE DELIVERY BY PUMP MORE NOISE AND LARGE BASELINE DRIFT IN DETECTOR CELL IN ORDER TO AVOID THESE PROBLEMS, THE ELUENT MUST BE DEGASSED.

24. Pumps TO PRODUCE AN APPROPRIATE PRESSURE TO PUSH SOLVENT INTO THE SAMPLE. A PUMP CAPABLE OF PUMPING SOLVENT UP TO A PRESSURE OF 4000-6000 PSI AND AT FLOW RATE RANGING FROM 0.1- 10 ML/MIN. MOSTLY PHARMACEUTICAL ANALYSIS IS CARRIED OUT AT 5000PSI AND FLOWRATE OF 0.5-3ML/MIN.

25. Sample Injection System BY SYRINGE :, IT MAY BE MANUAL AUTOINJECTOR A FIXED-VOLUME LOOP OF BETWEEN 1 – 100 L (20 L IS OFTEN USED AS STANDARD) IS INJECTED VIA SAMPLE VALVE .

26. GUARD COLUMN GUARD COLUMNS ARE INSTALLED BETWEEN THE INJECTOR AND THE ANALYTICAL COLUMN OF A HPLC SYSTEM, MAINLY TO PROTECT ANALYTICAL COLUMNS. GUARD COLUMNS ARE WIDELY USED AS A COST EFFECTIVE FOR PROLONG HPLC COLUMN LIFE.

27. Columns STRAIGHT, 100 TO 250 MM IN LENGTH 2 TO 6 MM INNER DIAMETER. PACKING - SILICA GEL, ALUMINA, CELITE ETC COLUMN ARE SELECTED ACCORDING TO THE NATURE OF COMPOUND TO BE ANALYZED AND THE MOBILE PHASE. COLUMN PERFORMANCE SHOULD BE EVALUATED TIME TO TIME. C8 AND C18 COLUMNS ARE USED C-18 IS USUALLY BETTER FOR SEPARATING OUT COMPOUNDS LIKE LONG CHAIN FATTY ACIDS (SAY OLEIC ACID), RETAIN NON POLAR COMPOUNDS AS COMPARED TO A RELATIVELY SMALL ORGANIC COMPOUND (SAY BUTYRIC ACID).

28. HPLC Detectors SPECIFIC DETECTORS UV/VIS FLUORESCENCE MASS DETECTOR DIODE ARRAY DETECTOR IR DETECTOR BULK PROPERTY ELECTROCHEMICAL DETECTOR OPTICAL ROTATION DETECTOR REFRACTIVE INDEX

29. Data Processing PROCESSING IS DONE USING SPECIFIC SOFTWARE THAT IS CONNECTED TO HPLC MACHINE. RECEIVE THE INFORMATION FROM HPLC MACHINE AND PRESENT IT AS A GRAPH. THE GRAPH DESCRIBES ABOUT QUALITATIVE DATA (RETENTION TIME) AND QUANTITATIVE DATA (AREA UNDER CURVE).

30. validation DEFINITION VALIDATION IS ESTABLISHING A DOCUMENTED EVIDENCE WHICH PROVIDES A DEGREE OF ASSURANCE THAT A SPECIFIC PROCESS WILL CONSISTENTLY PRODUCE A PRODUCT MEETING ITS PRE-DETERMINED SPECIFICATIONS AND QUALITY ATTRIBUTE.

31. PURPOSE OF VALIDATION THE PRINCIPLE PURPOSE OF ANALYTICAL VALIDATION IS TO ENSURE THAT THE SELECTED ANALYTICAL PROCEDURE WILL GIVE REPRODUCIBLE AND RELIABLE RESULTS THAT ARE ADEQUATE FOR THE INTENDED PURPOSE.

32. Validation parameters

33. System validation

34. calibration DEFINITION (ICH) THE DEMONSTRATION THAT A PARTICULAR INSTRUMENT OR DEVICE PRODUCES RESULTS WITH IN SPECIFIED LIMITS BY COMPARISION WITH THOSE PRODUCED BY A REFERENCE OR TRACEABLE STANDARD OVER AN APPROPRIATE RANGE OF MEASUREMENTS.

35. Calibration parameters of hplc

36. Calibration parameters of hplc

37. APPLICATIONS OF HPLC

38. Advantages and disadvantages ADVANTAGES SEPARATION OF VOLATILE AND NON VOLATILE COMPONENTS QUICK ANALYSIS HIGH RESOLUTION MORE REPRODUCIBILITY DISADVANTAGES HIGH COST COMPLEX TO OPERATE