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HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
PRESENTED BY: MISS. SHIVANI JAGNNATH PATIL.
M.PHARMACY (PHARMACEUTICAL QUALITY ASSURANCE)
GUIDED BY: SMT. ARCHANA R. DHOLE
1
CONTENT
• INTRODUCTION
• PRINCIPAL
• TYPES OF HPLC CHROMATOGRAPHY
• INSTRUMENTATION
• APPARATUS
• SYSTEM SUITABILITY
2
INTRODUCTION
CHROMATOGRAPHY:
CHROMATOGRAPHY IS ONE OF THE MOST IMPORTANT ANALYTICAL
TECHNIQUES. IT ALLOWS THE SEPARATION AND SUBSEQUENTLY THE
QUALITATIVE AND QUANTITATIVE ANALYSIS OF COMPLEX MIXTURES, AS
LONG AS THE SAMPLES ARE VOLATILE OR SOLUBLE IN A SUITABLE
SOLVENT.
IT IS A SEPARATION TECHNIQUE WHICH IS USED TO SEPARATE THE
MIXTURE OF COMPOUNDS INTO ITS INDIVIDUAL COMPONENTS BASED ON
CERTAIN PHYSICAL AND CHEMICAL PROPERTIES.
INVENTION- THE RUSSIAN BOTANIST ‘MIKHAIL TSVET’ IS CONSIDERED TO
HAVE INVENTED THE TERM CHROMATOGRAPHIC TECHNIQUE. HE CALLED
THIS TECHNIQUE ‘CHROMATOGRAPHY’. IT IS DEVELOPED IN 1930S &
1940S.
3
• ADSORPTION CHROMATOGRAPHY:
THE AFFINITY OF MOLECULE TOWARDS STATIONARY PHASE IS
CALLED ADSORPTION CHROMATOGRAPHY.
• PARTITION CHROMATOGRAPHY:
THE MOLECULE CAN MOVE IN TWO PHASES OF LIQUID IS
KNOWN AS PARTITION CHROMATOGRAPHY.
TYPES OF CHROMATOGRAPHY ON SEPARATION BASIS:
1) THIN LAYER CHROMATOGRAPHY
2) PAPER CHROMATOGRAPHY
3) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
4) GAS CHROMATOGRAPHY
4
PRINCIPAL
• HPLC WORKS ON THE PRINCIPAL OF ADSORPTION CHROMATOGRAPHY & IT IS A
SEPARATION TECHNIQUE BASED ON A SOLID STATIONARY PHASE AND LIQUID
MOBILE PHASE.
• DEVELOPING AN HPLC SEPARATION REQUIRES FINDING A COMBINATION OF
COLUMN PACKING AND MOBILE PHASE THAT PROVIDES JUST THE RIGHT
BALANCE OF AFFINITY AND SOLUBILITY TO MAKE COMPOUNDS OF INTEREST
MOVE AT DIFFERENT SPEEDS.
• COMPOUNDS THAT HAVE LOW AFFINITY FOR COLUMN PACKING AND/OR HIGH
SOLUBILITY IN MOBILE PHASE ARE WASHED THROUGH THE COLUMN QUICKLY.
• COMPOUNDS THAT STICK TIGHTLY TO THE COLUMN PACKING AND/OR HAVE A
LOW SOLUBILITY IN THE MOBILE PHASE SOLVENT MOVE MORE SLOWLY.
5
TYPES OF HPLC
CHROMATOGRAPHY
• REVERSED-PHASE:
- IF YOUR MOBILE PHASE
CONTAINS WATER IN IT
MEANS POLAR MOBILE PHASE
AND NON POLAR COLUMN IS
USED FOR ANALYSIS THAT
CHROMATOGRAPHY IS
CALLED REVERSED PHASE
CHROMATOGRAPHY.
-MOST COMMON TYPE OF
HPLC SEPARATION IN USE
TODAY.
• NORMAL PHASE:
- IF YOUR MOBILE PHASE
CONTAINS ONLY ORGANIC
SOLVENTS MEANS NON-
POLAR MOBILE PHASE AND
POLAR COLUMN IS USED FOR
ANALYSIS THAT
CHROMATOGRAPHY IS
CALLED NORMAL PHASE
CHROMATOGRAPHY.
6
INSTRUMENTATION
• RESERVOIR
• DEGASSER
• PUMP
• INJECTOR
• TUBING’S AND FITTINGS
• DETECTOR
7
APPARATUS
 A LIQUID CHROMATOGRAPH CONSIST OF A RESERVOIR
CONTAINING MOBILE PHASE, A PUMP TO FORCE THE
MOBILE PHASE THROUGH THE SYSTEM AT HIGH PRESSURE,
AN INJECTOR TO INTRODUCE THE SAMPLE INTO THE
MOBILE PHASE, A CHROMATOGRAPHIC COLUMN, A
DETECTOR, AND A DATA COLLECTION DEVICE SUCH AS A
COMPUTER, INTEGRATOR, OR RECORDER.
8
• RESERVOIR:
-A RESERVOIR MUST BE PROVIDED TO ASSURE A CONSISTENT SUPPLY OF MOBILE PHASE.
• DEGASSER:
-IT IS USE TO DEGAS THE MOBILE PHASE AND FLOW SHOULD BE CONSTANT THROUGHOUT
THE SYSTEM. IT MEANS ALL AIR BUBBLES ARE REMOVED AND CONSTANT FLOW OF MOBILE
PHASE MAINTAINED THROUGHOUT THE SYSTEM.
• PUMP:
-GRAVITY ALONE CANNOT MAKE LIQUID FLOW AT A REASONABLE VELOCITY THROUGH A
COLUMN PACKED WITH VERY SMALL PARTICLES ,SO WE MUST USE A CONSTANT FLOW
PUMP.
-THE PUMPING SYSTEMS DELIVER METERED AMOUNT OF MOBILE PHASE FROM THE
SOLVENTS RESERVOIRS TO THE COLUMN THROUGH HIGH PRESSURE TUBING AND FITTINGS.
-IT CONSISTS OF ONE OR MORE COMPUTER CONTROLLED METERING PUMPS CAPABLE OF
DELIVERING MOBILE PHASE EITHER AT THE FIXED RATIO OF SOLVENTS (ISOCRATIC
ELUTION) OR VARYING THE RATIO OF SOLVENTS (GRADIENT ELUTION)
-PUMPS MAY BE PROVIDED WITH MECHANISM FOR ‘PURGING’ THE SYSTEM OF ANY
ENTRAPPED AIR BUBBLES.
• INJECTOR:
-BECAUSE THE SYSTEM IS PRESSURIZED, SOME PROVISION MUST BE MADE FOR
INTRODUCING SAMPLES FROM THE OUTSIDE WORLD INTO THE SYSTEM. THIS IS MOST
COMMONLY DONE USING A ROTARY INJECTION VALVE.
9
• TUBING’S AND FITTINGS:
-FLOW RESISTANCE IN THE COLUMN CAN GENERATE A CONSIDERABLE BACK
PRESSURE, SO THE ENTIRE SYSTEM, INCLUDING THE CONNECTING TUBING’S AND
FITTINGS MUST BE MADE OF PRESSURE RESISTANT MATERIAL SUCH AS
STAINLESS STEEL OR SPECIALLY FORMULATED PLASTICS.
• DETECTOR:
- COLLECTING AND HAND ANALYZING FRACTION IS TEDIOUS, SO SOME TYPE OF
FLOW THROUGH DETECTOR IS USED TO MONITOR FOR THE PRESENCE OF
SAMPLE COMPOUNDS IN THE COLUMN EFFLUENT.
- HPLC DETECTORS-
1. REFRACTIVE INDEX
2. UV-VISIBLE
3. FLUORESCENCE
4. CONDUCTIVITY
5. MASS SPECTROMETRY
6. PHOTO DIODE ARRAY (PDA)
10
SYSTEM SUITABILITY
• THE PURPOSE OF SYSTEM SUITABILITY TEST IS TO ENSURE THAT
COMPLETE TESTING SYSTEM (INCLUDING INSTRUMENT, REAGENTS,
COLUMNS, ANALYSTS) IS WORKING IN THE PROPER WAY AND
REPRESENTING CORRECT DATA OR NOT.
• SYSTEMS SHOULD SATISFY THE REQUIREMENTS OF SYSTEM SUITABILITY.
• SYSTEM SUITABILITY TEST CONSTITUTE SIGNIFICANT PART OF
CHROMATOGRAPHIC ANALYSIS.
• THE USP IS AN AUTHORATIVE SOURCE FOR GUIDELINES ON
CHROMATOGRAPHY OF THE DRUG SUBSTANCES. SECTION 621 USP STATES
THAT SYSTEM SUITABILITY TESTS ARE AN INTEGRAL PART OF GAS AND
LIQUID CHROMATOGRAPHYANALYSIS.
• SYSTEM SUITABILITY MUST BE PERFORMED BEFORE AND THROUGHOUT
ALL ANALYSIS.
11
• FOLLOWING PARAMETERS ARE CHECKED UNDER SYSTEM
SUITABILITY CRITERIA (SST) AS PER USP,
A) THEORETICAL PLATES- MUST BE MORE THAN 2000
B) USP TAILING- MUST BE NOT MORE THAN 2.0
C) RESOLUTION BETWEEN TWO PEAKS-MUST BE MORE
THAN 1.0
D) % RSD OF 5 INJECTIONS-BELOW 2%
12
THANK YOU
13

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High performance liquid chromatography

  • 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY PRESENTED BY: MISS. SHIVANI JAGNNATH PATIL. M.PHARMACY (PHARMACEUTICAL QUALITY ASSURANCE) GUIDED BY: SMT. ARCHANA R. DHOLE 1
  • 2. CONTENT • INTRODUCTION • PRINCIPAL • TYPES OF HPLC CHROMATOGRAPHY • INSTRUMENTATION • APPARATUS • SYSTEM SUITABILITY 2
  • 3. INTRODUCTION CHROMATOGRAPHY: CHROMATOGRAPHY IS ONE OF THE MOST IMPORTANT ANALYTICAL TECHNIQUES. IT ALLOWS THE SEPARATION AND SUBSEQUENTLY THE QUALITATIVE AND QUANTITATIVE ANALYSIS OF COMPLEX MIXTURES, AS LONG AS THE SAMPLES ARE VOLATILE OR SOLUBLE IN A SUITABLE SOLVENT. IT IS A SEPARATION TECHNIQUE WHICH IS USED TO SEPARATE THE MIXTURE OF COMPOUNDS INTO ITS INDIVIDUAL COMPONENTS BASED ON CERTAIN PHYSICAL AND CHEMICAL PROPERTIES. INVENTION- THE RUSSIAN BOTANIST ‘MIKHAIL TSVET’ IS CONSIDERED TO HAVE INVENTED THE TERM CHROMATOGRAPHIC TECHNIQUE. HE CALLED THIS TECHNIQUE ‘CHROMATOGRAPHY’. IT IS DEVELOPED IN 1930S & 1940S. 3
  • 4. • ADSORPTION CHROMATOGRAPHY: THE AFFINITY OF MOLECULE TOWARDS STATIONARY PHASE IS CALLED ADSORPTION CHROMATOGRAPHY. • PARTITION CHROMATOGRAPHY: THE MOLECULE CAN MOVE IN TWO PHASES OF LIQUID IS KNOWN AS PARTITION CHROMATOGRAPHY. TYPES OF CHROMATOGRAPHY ON SEPARATION BASIS: 1) THIN LAYER CHROMATOGRAPHY 2) PAPER CHROMATOGRAPHY 3) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 4) GAS CHROMATOGRAPHY 4
  • 5. PRINCIPAL • HPLC WORKS ON THE PRINCIPAL OF ADSORPTION CHROMATOGRAPHY & IT IS A SEPARATION TECHNIQUE BASED ON A SOLID STATIONARY PHASE AND LIQUID MOBILE PHASE. • DEVELOPING AN HPLC SEPARATION REQUIRES FINDING A COMBINATION OF COLUMN PACKING AND MOBILE PHASE THAT PROVIDES JUST THE RIGHT BALANCE OF AFFINITY AND SOLUBILITY TO MAKE COMPOUNDS OF INTEREST MOVE AT DIFFERENT SPEEDS. • COMPOUNDS THAT HAVE LOW AFFINITY FOR COLUMN PACKING AND/OR HIGH SOLUBILITY IN MOBILE PHASE ARE WASHED THROUGH THE COLUMN QUICKLY. • COMPOUNDS THAT STICK TIGHTLY TO THE COLUMN PACKING AND/OR HAVE A LOW SOLUBILITY IN THE MOBILE PHASE SOLVENT MOVE MORE SLOWLY. 5
  • 6. TYPES OF HPLC CHROMATOGRAPHY • REVERSED-PHASE: - IF YOUR MOBILE PHASE CONTAINS WATER IN IT MEANS POLAR MOBILE PHASE AND NON POLAR COLUMN IS USED FOR ANALYSIS THAT CHROMATOGRAPHY IS CALLED REVERSED PHASE CHROMATOGRAPHY. -MOST COMMON TYPE OF HPLC SEPARATION IN USE TODAY. • NORMAL PHASE: - IF YOUR MOBILE PHASE CONTAINS ONLY ORGANIC SOLVENTS MEANS NON- POLAR MOBILE PHASE AND POLAR COLUMN IS USED FOR ANALYSIS THAT CHROMATOGRAPHY IS CALLED NORMAL PHASE CHROMATOGRAPHY. 6
  • 7. INSTRUMENTATION • RESERVOIR • DEGASSER • PUMP • INJECTOR • TUBING’S AND FITTINGS • DETECTOR 7
  • 8. APPARATUS  A LIQUID CHROMATOGRAPH CONSIST OF A RESERVOIR CONTAINING MOBILE PHASE, A PUMP TO FORCE THE MOBILE PHASE THROUGH THE SYSTEM AT HIGH PRESSURE, AN INJECTOR TO INTRODUCE THE SAMPLE INTO THE MOBILE PHASE, A CHROMATOGRAPHIC COLUMN, A DETECTOR, AND A DATA COLLECTION DEVICE SUCH AS A COMPUTER, INTEGRATOR, OR RECORDER. 8
  • 9. • RESERVOIR: -A RESERVOIR MUST BE PROVIDED TO ASSURE A CONSISTENT SUPPLY OF MOBILE PHASE. • DEGASSER: -IT IS USE TO DEGAS THE MOBILE PHASE AND FLOW SHOULD BE CONSTANT THROUGHOUT THE SYSTEM. IT MEANS ALL AIR BUBBLES ARE REMOVED AND CONSTANT FLOW OF MOBILE PHASE MAINTAINED THROUGHOUT THE SYSTEM. • PUMP: -GRAVITY ALONE CANNOT MAKE LIQUID FLOW AT A REASONABLE VELOCITY THROUGH A COLUMN PACKED WITH VERY SMALL PARTICLES ,SO WE MUST USE A CONSTANT FLOW PUMP. -THE PUMPING SYSTEMS DELIVER METERED AMOUNT OF MOBILE PHASE FROM THE SOLVENTS RESERVOIRS TO THE COLUMN THROUGH HIGH PRESSURE TUBING AND FITTINGS. -IT CONSISTS OF ONE OR MORE COMPUTER CONTROLLED METERING PUMPS CAPABLE OF DELIVERING MOBILE PHASE EITHER AT THE FIXED RATIO OF SOLVENTS (ISOCRATIC ELUTION) OR VARYING THE RATIO OF SOLVENTS (GRADIENT ELUTION) -PUMPS MAY BE PROVIDED WITH MECHANISM FOR ‘PURGING’ THE SYSTEM OF ANY ENTRAPPED AIR BUBBLES. • INJECTOR: -BECAUSE THE SYSTEM IS PRESSURIZED, SOME PROVISION MUST BE MADE FOR INTRODUCING SAMPLES FROM THE OUTSIDE WORLD INTO THE SYSTEM. THIS IS MOST COMMONLY DONE USING A ROTARY INJECTION VALVE. 9
  • 10. • TUBING’S AND FITTINGS: -FLOW RESISTANCE IN THE COLUMN CAN GENERATE A CONSIDERABLE BACK PRESSURE, SO THE ENTIRE SYSTEM, INCLUDING THE CONNECTING TUBING’S AND FITTINGS MUST BE MADE OF PRESSURE RESISTANT MATERIAL SUCH AS STAINLESS STEEL OR SPECIALLY FORMULATED PLASTICS. • DETECTOR: - COLLECTING AND HAND ANALYZING FRACTION IS TEDIOUS, SO SOME TYPE OF FLOW THROUGH DETECTOR IS USED TO MONITOR FOR THE PRESENCE OF SAMPLE COMPOUNDS IN THE COLUMN EFFLUENT. - HPLC DETECTORS- 1. REFRACTIVE INDEX 2. UV-VISIBLE 3. FLUORESCENCE 4. CONDUCTIVITY 5. MASS SPECTROMETRY 6. PHOTO DIODE ARRAY (PDA) 10
  • 11. SYSTEM SUITABILITY • THE PURPOSE OF SYSTEM SUITABILITY TEST IS TO ENSURE THAT COMPLETE TESTING SYSTEM (INCLUDING INSTRUMENT, REAGENTS, COLUMNS, ANALYSTS) IS WORKING IN THE PROPER WAY AND REPRESENTING CORRECT DATA OR NOT. • SYSTEMS SHOULD SATISFY THE REQUIREMENTS OF SYSTEM SUITABILITY. • SYSTEM SUITABILITY TEST CONSTITUTE SIGNIFICANT PART OF CHROMATOGRAPHIC ANALYSIS. • THE USP IS AN AUTHORATIVE SOURCE FOR GUIDELINES ON CHROMATOGRAPHY OF THE DRUG SUBSTANCES. SECTION 621 USP STATES THAT SYSTEM SUITABILITY TESTS ARE AN INTEGRAL PART OF GAS AND LIQUID CHROMATOGRAPHYANALYSIS. • SYSTEM SUITABILITY MUST BE PERFORMED BEFORE AND THROUGHOUT ALL ANALYSIS. 11
  • 12. • FOLLOWING PARAMETERS ARE CHECKED UNDER SYSTEM SUITABILITY CRITERIA (SST) AS PER USP, A) THEORETICAL PLATES- MUST BE MORE THAN 2000 B) USP TAILING- MUST BE NOT MORE THAN 2.0 C) RESOLUTION BETWEEN TWO PEAKS-MUST BE MORE THAN 1.0 D) % RSD OF 5 INJECTIONS-BELOW 2% 12