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HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMATOGRAPHY,  BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE  AND RP- HPLC , ST
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HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMATOGRAPHY, BRIEF HISTORY, DEFINITION, WHAT IS HPLC? USES OF HPLC, SEPARATION MECHANISM, CLASSIFICATION, ELUTION TEXHNIQUES, GRADIENT AND ISOCRATIC, NORMAL PHASE AND RP- HPLC , ST

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GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF …

GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.

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  • 1. HPLCProf. RavisankarVignan Pharmacy collegeValdlamudiGuntur Dist.Andhra PradeshIndia.banuman35@gmail.com00919059994000
  • 2. Brief History and DefinitionLiquid chromatography was defined in the early 1900s by the work of the Russianbotanist, Mikhail S. Tswett. His studies focused on separating compounds [leafpigments], extracted from plants using a solvent, in a column packed with particles.Tswett filled an open glass column with particles. Two specific materials that he founduseful were powdered chalk [calcium carbonate] and alumina. He poured his sample[solvent extract of homogenized plant leaves into the column and allowed it to pass into theparticle bed.. The compounds that were more strongly attracted to the particles sloweddown, while other compounds more strongly attracted to the solvent moved faster. Thisprocess can be described as follows: the compounds contained in the sample distribute, orpartition differently between the moving solvent, called the mobile phase, and theparticles, called the stationary phase. This causes each compound to move at a differentspeed, thus creating a separation of the compounds.Tswett coined the name chromatography[from the Greek words chroma, meaning color,and graph, meaning writing—literally, color writing]to describe his colorful experiment.Curiously, the Russian name Tswett means color. Tswetts ExperimentThe concept of the first HPLC was put forth by Kirklandand Huberb/n 1967 and 1969.
  • 3. History of HPLC:Prior to the 1970s, few reliable chromatographic methods were commerciallyavailable to the laboratory scientist were carried out using a variety of techniquesincluding open-column chromatography, paper chromatography, and thin-layerchromatography.However, these chromatographic techniques were inadequate for quantification ofcompounds and did not achieve sufficiently high resolution to distinguish between similarcompounds.During this time, pressure liquid chromatography began to be used to decrease flow throughtime, thus reducing purification times of compounds being isolated by columnchromatography. However, flow rates were inconsistent, and the question of whether it wasbetter to have constant flow rate or constant pressure was debated.High pressure liquid chromatography was developed in the mid-1970s and quickly improvedwith the development of column packing materials and the additional convenience of on-linedetectors. In the late 1970s, new methods including reverse phase liquid chromatographyallowed for improved separation between very similar compounds.
  • 4. • By the 1980s HPLC was commonly used for the separation of chemicalcompounds. New techniques improvedseparation, identification, purification and quantification far above thoseobtained using previous techniques. Computers and automation addedto the convenience of HPLC. Additional column types giving betterreproducibility were introduced and such terms as micro-column, affinitycolumns, and Fast HPLC began to immerge.• The past decade has seen a vast undertaking in the development ofmicro-columns, and other specialized columns. The usual diameter ofmicro-columns, or capillary columns, ranges from 3 µm to 200 µm. FastHPLC utilizes a column that is shorter than the typical column. A FastHPLC column is about 3 mm long and is packed with smaller particles.• Although HPLC is widely considered to be a technique mainly forbiotechnological, biomedical, and biochemical research as well as for thepharmaceutical industry,in actual fact these fields currently compriseonly about 50% of HPLC users. Currently HPLC is used in a variety offields and industries including the cosmetics, energy, food, andenvironmental industries.
  • 5. Introduction: HLC is basically a high improved form of column chromatography.Instead of solvent being allowed to drip through a column under gravity, it isForced through under high pressure up to 400 atmospheres.That makes it much faster.In HPLC it is also allows you to use a very much smaller particle size (3-5µm)for columnpacking. Material which gives a much greater surface area for interactions b/nthe stationary phase And the mobile phase it. This allows the better separationof the components of the complex mixture.This in turn Increase the elution by over 100 folds.The other major improvement over column chromatography concerns theDetection methods which can be used.These methods are highly automated and extremely sensitive.What Is UltraPerformance Liquid Chromatography (UPLC Technology)?In 2004, further advances in instrumentation and column technology weremade to achieve very significant increasesin resolution, speed, and sensitivity in liquid chromatography. Columns withsmaller particles [1.7 micron]and instrumentation with specialized capabilitiesdesigned to deliver mobile phase at 15,000 psi [1,000 bar]were needed to achieve a new level of performance. A new system had to be holisticallycreated to perform ultra-performance liquid chromatography, now known asUPLC technology.
  • 6. HPighressureL iquidC hromatographySince high pressure is used when compared to classical chromatographySeparation of the components from a mixture is achieved by pumpingmobile phase at High pressure using appropriate displacement pumpsor gas pressure. (Due to the small particle size (3-5 um).
  • 7. HPigherformanceL iquidC hromatographyMore ever it is improved performance when compared to otherconventional column Chromatographic techniques.
  • 8. (advantages)
  • 9. DeterminationPeak area (or height) is proportional to the concentration(or amount) of the component.The concentration of component A(caffeine) is determined bycomparing the peak area with that of the standard caffeinepeak.What is the concentration of component A?ABCCaffeine (1mg/ml)5ul injection (5ug)
  • 10. ABCResults obtained by HPLCChromatogram containing three peaksQualitative analysis (identification) andQuantitative analysis (determination)Can be performed using the information contained in thechromatogramChromatography : MethodChromatogram : ResultsChromatograph : Instrument
  • 11. Separation MechanismABCtime.CBAColumnPackingmaterial↓ ↓ ↓ ↓Mobile phase (solvent)C > B > A
  • 12. CLASSIFICATION OF HPLC:BASED ON MODE OF RETENTIONBASED ON OPERATING METHODBASED ON MODE OF SEPARATION
  • 13. BASED ON MODE OF RETENTIONADSORPTION PARTITIONSIZE EXCLUSIONAFFINITYIONEXCHANGELiquid mobilephase and solidadsorbentLiquidmobilephase andionicstationaryphaseLiquid mobilephase andliquid stationaryphaseMolecularmodellingLock & key
  • 14. (A) uses charge, (B) usespores, and (C) usescovalent bonds to createthe differential affinitiesamong the mixturecomponents for thestationary phase.
  • 15. BASED ON OPERATING METHODELUTION DISPLACEMENTISOCRATIC GRADIENTFRONTAL
  • 16. Based on elution TechniqueThere are 2 types of elution :1- Isocratic elution : fixed ratio of mobile phasecomponents during the analysis.2- Gradient elution (mobile phase programming) :thecomponents of mobile phase ratio is changedthrough out the analysis for separation of components veryclose in polarity.
  • 17. BASED ON MODE OF SEPARATION;MODE OFSEPARATIONSTATIONARYPHASEMOBILEPHASENORMAL PHASE POLAR NON POLARREVERSE PHASE NON POLAR POLAR
  • 18. 1.Based on Modes of chromatography:There are 2modes (Normal phase mode and Reverse phase mode)depending on the polarity of the stationary and mobile phase.Polar- polar --- interaction (or) affinity is more.Nonpolar- Nonpolar– interaction (or) affinity is more.Polar- Nonpolar --- Interaction is less.Remember, "Like attracts like” in polarityThose with dissimilar polarity exhibit muchweaker attraction, if any, and may even repel one another.
  • 19. Normal Phase Mode HPLC or NP- HPLC or Normal phase HPLC:This is essentially just the same as you will already have read about in TLC orColumn chromatography.Although it is described as “Normal” it is not most commonly used form of HPLC.In This separations of plant extracts, Tswett was successful using a polar stationaryphase [chalk in a glass column; see Figure A] with a much less polar [non-polar]mobile phase. This classical mode of chromatography became known as normal phase.normal-phase chromatographic separation of our three-dye test mixture.,The stationaryphase is polar and retains the polar yellow dye most strongly. The relatively non-polarblue dye is won in the retention competition by the mobile phase, a non-polar solvent,and elutes quickly. Since the blue dye is most like the mobile phase [both are non-polar],it moves faster. It is typical for normal-phase chromatography on silica that the mobilephase is 100% organic; no water is used.The commonly used columns for NP-HPLC include Cyano, amino and diol bonded phasesAs well as base silica or its derivatives.Stationary phase(Polar)Mobile phase (Non-Polar)Non polar materialelutes first.
  • 20. Normal Phase ChromatographyInteraction : AdsorptionPacking materials : Polar ex. Silica gelSilica-NH2Silica-CNSilica-OHMobile phase : Non-polar ex. n-Hex/CH2CL2iso-Oct/IPAiso-Oct/AcOEtSample : Fat-solubleDifferent polarity
  • 21. Stationary phases for Normal-Phasechromatography:Common porous adsorbents such as silica & Alumina,that have polar hydroxyl groups on surface.In addition to porous adsorbents, a variety of polar bonded phasesexist in some functional groups, such ascyano [-(CH2)3CΞN],diol [-(CH2)3OCH2CH(OH)CH2OH]Amino groups [-(CH2)n NH2, where n is 3 or 4],
  • 22. Solvent Characteristicsn-Hexane Mobile phasen-Heptane Mobile phaseIsooctane Mobile phase1-chlorobutane Large dipoleChloroform Proton donorMethylene chloride Large dipoleEthylene acetate Proton donorTetrahydrofuron Proton acceptorPropylamine Proton acceptorAcetonitrile DipoleMobile phases for Normal-phase chromatography:
  • 23. Applications of Normal phasechromatography:Analysis of samples that are soluble innon polar solvents.Separation of isomers.Fat and water soluble vitamins,Hydrocarbons and Pesticides have beenseparated using Hexane as mobile phase.Separation of saccharides in food andbiological products.
  • 24. CH3 CH2COOCH3CH3 CH2COOCH3Stationary phase (Non polar)Mobile phase (Polar)P0lar material elutes first.
  • 25. 3. Separation modeReversed Phase ChromatographySiSiO-Si-CH2(CH2)16CH3CH3CH3O-Si-CH2(CH2)16CH3CH3CH3O-Si-CH2(CH2)16CH3CH3CH3CH3CH3O-Si-CH3Silica-C18 Packing materialsCommonly used packing materials arehydrocarbons having 18 carbon atoms(called the Octadecyl radical) which arechemically bonded to silica gel (Silica-ODS).Since the surface of the Silica-ODS is covered with hydrocarbon, thepolarity of the packing material itself isvery low.Today, because it is more reproducible and hasbroad applicability, reversed-phasechromatography is used for approximately 75% ofall HPLC methodsIn this technique the silica is modified to make it non-polar by attaching long hydrocarbonChain to its surface typically with either 8 or 18 carbon atoms in them.
  • 26. Stationary phases for Reverse phase chromatography: Functional group chemically attached to asilica support- Bonded phase. Popular bonded phases;•Alkyl-C4H9,C8H17,C18H37•Phenyl-C6H5
  • 27. solvent Solvent polarity(P’)WaterDimethyl sulfoxideEthylene glycolAcetonitrileMethanolAcetoneDioxaneEthanolTetrahydrofuran2-Propanol10.27.26.95.85.15.14.84.34.03.9Mobile phases for Reverse-phasechromatography:
  • 28. Applications of Reverse phase chromatography:Pharmaceutical industry; steroids,vitamins,beta blockersClinical laboratories; catecholaminesChemical industry; polymer additivesEnvironmental pesticides and herbicidesFood and beverage industy; sweetners and additives
  • 29. ofCStationary phases Mobile phases Applicationsal phaseCBonded phase silica gel with functional groupssuch asCyano [-(CH2)3 C=N]Diol [-(CH2)3 OCH2-CH-OH CH2OHAmino [-(CH2)2NH2] (n=3 or 4)n-Hexane, n-heptane,isooctane,1-chlorobutane, CHCL3,CH2CL2, Ethyl acetate, THF,propylamine, acetonitrile andMeOH- Fat & oil solublevitamins- Hydrocarbons- Pesticides- 8,9-oxide of avermectinB1sedHPLCBonded phase silica gel with functional groups(-C4 H8) – butyl(-C8 H17) – octyl(-C6 H5) – phenylC18 – octadecylWater, dimethyl sulfoxide,ethylene glycol, ACN,MeOH, acetone, dioxane,EtOH, THF &2-propanol.- Most popular mode for theseparation of low molecular weightneutral species that are soluble inH2O- Steroids- B Vitamins- β-blockers- Catecholamines- Polymer additives- Pesticides & herbicides- Carbohydrates- Sweeteners- Food additives- Aminoacids, peptides,nuclesedes,oligosaccharidesxchangeCOrganic ion-exange resins containingexchange sites. The resin is composed ofpolystyrene- divinyl benzeneFunctional groups present in different ionexchange resins:Acids, Alkali, Buffers,Acetate buffer, Borate bufferAnalyses of amino acids,nucleotides, (carbohydrates at PH>12) & proteinsInorganic anions, organic acids,inorganic cations etc.Fractionation of cations inorganiclanthanides peptides, amino acids,B.vitaminsStrong Cation Exchange Resin:Sulfonic acids (-SO-3H+)(sulfonated polystyrene)Weak Cation Exchange Resins:(polystyrene with functional groups)+Fractionation of cations inorganiclanthanides peptides, amino acids,B.vitaminsFractionation of cationsBiochemical separations organic
  • 30. GPC : Gel Permeation ChromatographyGFC : Gel Filtration ChromatographySeparation modes and featuresMode Stationary phase Mobile phase Interaction FeatureNormal phase Silica gel Organic solvent Adsorption Fat-solublechromatography (n-Hexane/IPE)Reversed phase Silica-ODS MeOH/Water Hydrophobic Most widely usedchromatography (Silica-C18)Size exclusion ChromatographyNon-aqueous (GPC) Porous Polymer Organic solvent (THF) Gel permeation Molecular weight distributionAqueous (GFC) Aqueous porous Polymer Buffer solution Gel permeation Protein SeparationIon exchange Ion exchange gel Buffer solution Ion exchange Separation ofChromatography ionic substancesAffinity Packing with ligand Buffer solution Affinity Purification ofChromatography enzymes and proteins
  • 31. High performance (or high pressure) liquid chromatography (HPLC) is a separationtechnique in which a sample mixture is introduced into a stream of solvent (themobile phase) which then flows over a surface (the stationary phase) consisting ofeither spherical particles or a film coated onto such particles. Separation resultsfrom differences between sample components in their relative affinity for thestationary and mobile phases
  • 32. oSolvent reservoirs (mobileoPhase reservoirs)oPumpoInjectoroColumnoDetectoroRecorderoPulse DamperoDegasseroColumn HeaterTypical HPLC system consists of followings
  • 33. A Modern HPLC instrument contains Glassor stainless steel reservoirs of capacity of500mL,1000ml,2000ml,5000ml reservoir kit.for storing the mobile phase.Provisions are included to remove dissolvedgases and dust from the liquid.Mobile phase reservoirs and Solventpretreatment systems:1000 ml HPLC SolventReservoir Kit. Comeswith 1L solventreservoir, cap andteflon insert for 1/8"OD tubing, 1 meter1/8" OD PTFE tubingand 10 um 316stainless steel frit.$60.00
  • 34. •The reservoir and its attachment to the pump should bemade of materials that will not contaminate themobile phase: Teflon, glass, or stainless steel.•The vessel should have some sort of cap to prevent particulatematter from contaminating the mobile phase.•If you are using a solvent bottle as a reservoir, the top of the bottlecan be wrapped in aluminium foil to keep dust out or the bottlecap can be drilled to allow inserting the inlet line through the cap.•Dont close the bottle too tightly or removal of mobile phase bythe pump will create a vacuum. This prevents mobile phase fromflowing the pump, creating a "vapour lock" within the pump.•The material chosen for constructing mobile phase reservoirsshould not be reactive towards the mobile phase.Examples:Acidic or alkaline solvents can not be stored in metallic reservoirs.Non-polar solvents (chloroform) cannot be stored in polymeric reservoirs.
  • 35. It is important that particulate matter be kept out of the mobile phase sinceparticulates can damage the pump and injector as well as plug the column. Mobilephases are often filtered before adding them to the reservoir. In addition, a 10-micron frit or inlet filter should be connected to the end of the inlet line that dips intothe reservoir. This inlet frit serves more than one purpose. Besides providing extraprotection against particulates entering the pump, the inlet filter serves to hold theinlet line at the bottom of the reservoir. The stiffness of the inlet line tends to allowthis Teflon tubing to creep out of the reservoir, preventing use of all the mobilephase unless an inlet filter is used to weigh down the end of the tubing. Forthis reason, inlet filters are often called "sinkers".Sinker" frits help keep theinlet line submerged andprovide a final line of defenseagainst particulatecontamination.The sinker frit is not a substitute for filteringthe mobile phase. The typical pore sizeused in a sinker frit is on the order of 5 - 10microns frits of smaller pore size are toolikely to plug. In general, the mobilephase should be filtered through a 0.3to 0.5-micron frit after the solventsand buffers are mixed in order toremove particulate matter. The sinker fritprotects against the occasional larger dustparticle encountered after the mobilephase has been filtered.frit or inlet filter
  • 36. Filter systems:Micro filters are (1-5µm) are placed before the solvent reservoirs in modernHPLC Instruments for removing the particulate matter as well asdust under vacuum.purity of the solvents is utmost important because the particulate matterEven in trace amounts interferes with the detector performance.
  • 37. SOLVENT DEGASSING IN HPLC:Solvent De-gassingExternal vacuumDegassingHeliumDegassingOn-lineDegassingSource:Phyllis R. Brown Kingston, Rhode island Pg. No:78-79
  • 38. Sparging:It is important to remove any dissolved gas from the mobile phase before it ispumped into the system.H PLC analysis may contain gases such as oxygen that are non-visible to our eyes.When gas is present in the eluent, this is detected as a noise and causes unstablebaseline.This prevents bubble formation that might damage the column and degrade systemperformance.Most systems provide a means to pass helium through the solvent reservoirs in aprocess called “sparging”.This strips out any dissolved gas, but helium’s low solubility and inert nature preventit from being a problem itself. The image above shows this procedure being performedon all four reservoirs.
  • 39. Generally used method includes sparging (bubbling of inert gas), use ofaspirator, distillation system, and/or heating and stirring.However, the method is not convenient and also when the solvent is left for a certain timeperiod (e.g., during the long analysis), gas will dissolve back gradually. Degasser usesspecial polymer membrane tubing to remove gases. The numerous very small pores onthe surface of the polymer tube allow the air to go through while preventing any liquid togo through the pore.By placing this tubing under low pressure container, it created pressure differences insideand outside the tubing (higher inside the tubing). This difference let the dissolved gas tomove through the pores and remove the gas.Compared to classical batch type degassing, the degasser can be used on-line, it is moreconvenient and efficient. Many of new HPLC unit system contain a degasser.
  • 40. Solvents used :The mobile phase is used to transport the sample components through the columnover the surface of the stationary phase.The exact choice of mobile phase depends on the nature of both the samplecomponents to be separated and the column stationary phase.organic compounds are commonly separated using water-methanol or water-acetonitrile mixtures.For Normal phase HPLC ( or) NP_HPLC(or) Straight -phase HPLC.There are 4 commonly used solvents.In HPLC pure analytical gradeOrganic solvents are used(known as HPLC solvents)It has been duly establishedThat “the stronger the solventOr solvent mixture, the moreQuickly it will elute an analyte(i.e., an organic compound) fromA specific HPLC column)
  • 41. Solvent choice:The mobile phase is used to transport the sample components through thecolumn over the surface of the stationary phase. In normal phasehplc, it would typically be a non-polar solvent or mixture ofsolvents, such as hexane, cyclohexane, toluene, or dichloromethane.In reverse phase hplc, it is typically either one of, or a mixture of two or more of,the following: water, an aqueous buffer, methanol, acetonitrile, ortetrahydrofuran (THF).The exact choice of mobile phase depends on the nature of both the samplecomponents to be separated and the column stationary phase. Ionchromatography, for example, often uses an aqueous buffer, while organiccompounds are commonly separated using water-methanol or water-acetonitrilemixtures.
  • 42. HPLC Pump Classification based on the Flow RateMicrobore Standard bore Preparative(1-250µL/min) (10µL/min—100 µL/min) (>10 mL/min)SOLVENT DELIVERY SYSTEM (PUMPS):
  • 43. Classification According to mechanism of EluentDisplacementSolvent delivery systemSyringe pumps Reciprocating-piston pumpsSingle head Multi headSOLVENT DELIVERY SYSTEMS(PUMPS)
  • 44. HPLC pumps can be single, binary, orternary, meaning that the can make use ofone, two, or more different solventreservoirs. (Most ternary pumps actuallyhave four solvent reservoirs.) The image onthe right shows a fairly typical ternarypump, which can be controlled viaeither thefront panel or computer control. The keyparts of such a pump system are theproportionating valve, the mixingchamber, the purge valve, and the actualpump mechanism.
  • 45. Solvent mixing:The proportionating valve (lower-front of image) allows the desired volume ratio ofthe different solvents to be drawn from the reservoirs. For example, if reservoirA contained water and B methanol, the following pump settings would give a 50:50water:methanol mixture – A=50%, B=50%, C=0%, D=0%. To ensure a uniformcomposition, a mixing chamber (middle-right of image) is used. This particularpump uses a reciprocating piston (upper-centre of image) to actually deliver themobile phase mixture at the required flow rate.
  • 46. Pump assemblyThis view shows the pump dis assembled. Theglass rod in the centre of the image is the piston;the pump chamber has been removed. Thesolenoid valve to the upper-left of the piston isone of the proportioning valves. Routinemaintenance requires that the seals preventingleaks around the piston be replaced periodicallyThis view shows the pump body removed from thepiston assembly. The piston assembly connects to thelarge collar on the left of the unit. The mobile phaseenters through the top-right connector and leavesthrough the bottom-left connector. The nuts adjacentto these on the upper and lower sides actually retainthe check valves, which are essential to the working ofthe pump; they basically determine the directionthrough the pump taken by the mobile phase.Check valves:
  • 47. Reciprocating pistonpumps do not work if thereis air in the lines, which canbe the case after changing asolvent reservoir, orleaving the pump off.Opening the purge valveallows the contents of thelines to be flushedout without the contentsentering the injector orcolumn. It’s important toremember to close thepurge valve before trying toperform an experiment!Purge valve:
  • 48. Classification According To Materials Of ConstructionSolvent Delivery systemMetallic Non-metallicSteel TitaniumPEEK Teflon CeramicSOLVENT DELIVERY SYSTEMS (PUMPS)
  • 49. PumpsIn the earlier state of HPLC development, the pump was the most important part ofthe system.The development of HPLC can be said that it was a development of pump system.However, nowadays, the high pressure generation is a “standard” requirement andwhat is more concerned nowadays is to be able to provide a consistent pressure atany condition,Most pumps used in current HPLC systems generate the flow by back-and-forthmotion of a motor-driven piston (reciprocating pumps).The pump is a heart of the instrument. It can deliver the solvent through thechromatograph with a pressure not less than 3000 Psig and 6000 Psig.
  • 50. SYRINGE PUMPS:Syringe Pumps: These are also called as positive displacement pumps. These are popularas pulse less solvent delivery systems in HPLC. The syringe pump contains a large barrelsyringe with the plunger connected to a motor. As the plunger moves forward, it drives theeluent through the chromatograph with a pulse less flow; the main advantage of syringepumps is that it generates stable flow.
  • 51. WORKING OF A RECIPROCATING PUMP:
  • 52. Isocratic pumps versus Gradient pumpingsystems:Isocratic Pumping Systems Gradient Pumping Systems:An Elution with singlesolvent or mixture of solventof constant composition istermed as Isocratic elution.Two or more solvents areused for the elution ofanalytes is termed as GradientElution.
  • 53. High pressure mixing:In high pressure mixing the eluents are blended on the injector side, or high pressure side ofthe pump. For this purpose two isocratic pumps are required. High pressure mixing is widelyused in HPLC-MS and capillary-LC.Low pressure mixing:Low pressure mixing is a common design for modern gradient HPLC systems. In this thesolvents are blended at atmospheric pressure. By means of a small Teflon block with 3 or 4proportioning values, a mixture of any combination of the four solvents is delivered to thelow pressure intake side of the pump.
  • 54. Based on the elution technique there are 2 types of HPLC techniques:There are 2 types of elution :1- Isocratic elution : fixed ratio of mobile phase components during the analysis.2- Gradient elution (mobile phase programming) :the components of mobile phaseratio is changed through out the analysis for separation of components very close in polarity.1. Isocratic elution(separation) technique:Isocratic elution is where the composition of the mobile phaseis unchanged during the entire elution process.a. In this elution technique in which the components of mobile phase are maintainedconstant throughout the process of separation is known as isocratic elution.b. In this technique the mobile phase may contains single or two or more solvents ofsimilar polarity to elute the sample through the column.Examples of mobile phases for isocratic elution are Chloroform,Benzene:petether(1:1)In this technique changing the length/internal diameter of the column, the elutionorder of the peaks remains same i.e., selectivity does not change.In gradient elution any change in the dimensions of the column immediately affectsthe elution order.An isocratic elution cannot separate a complex mixture with adequate resolution andgood detectability.Hence it is used only in the following cases…1. When sample mixture contains less than 10 weakly retained components.2. When gradient base line negatively effects trace analysis.
  • 55. Advantages of isocratic elution technique:1. Components of mobile phase remains constant through the process. Hence easyto handle and does not require continuous monitoring.2. Any change in column dimensions does not change the elution order (peaksare of same order.Disadvantages:1. Late eluting peaks are flat and broad.2. Takes longer time than gradient elution for complex mixtures.
  • 56. HPLC analysis is a prominent analytical tool in pharmaceutical laboratories.Once a method for the separation is developed, students can conduct both qualitativeand quantitative analysis of an analgesic mixture. These experiments indicated thatthe analgesic compounds could be divided into two groups of polar and non-polarmolecules.a) Isocratic elution of polar analgesics:he group of polar molecules included Aspirin,Acetaminophen and caffeine which could be eluted on an octylsiloxane, C-8, column.Furthermore, the mobile phase was composed of 67% H 2O, 3% 5 mM KH 2PO 4 (pH6.1) and 30% methanol (volume by volume). Note that the presence of thepotassium phosphate buffer was necessary in order to obtain reproducible results,particularly with respect to each component’s retention time. The optimizedexperimental conditions were as follows:
  • 57. A multicomponent mixture of six analgesic drugs commonly found in over-the-counterpain-relieving formulations: Aspirin, Acetaminophen, caffeine, Naproxen, Ibuprofen andsalicylamide.
  • 58. Once a method for the separation is developed, students can conduct both qualitativeand quantitative analysis of an analgesic mixture In order to develop the gradient elutionanalysis, it was first necessary to conduct many preliminary trial-and-errorruns. These experiments indicated that the analgesic compounds could be divided intotwo groups of polar and non-polar molecules..
  • 59. a) Isocratic elution of polar analgesicsThe group of polar molecules included Aspirin, Acetaminophen and caffeine whichcould be eluted on an octylsiloxane, C-8, column. Furthermore, the mobile phase wascomposed of 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30% methanol (volumeby volume). Note that the presence of the potassium phosphate buffer was necessaryin order to obtain reproducible results, particularly with respect to each component’sretention time. The optimized experimental conditions were as follows:
  • 60. The following HPLC chromatogram was obtained by elution of a mixture of the threepolar analgesics according to the conditions specified above
  • 61. b) Isocratic elution of non-polar analgesicsThe remaining compounds, namely, Naproxen, salicylamide, and Ibuprofen arerelatively non-polar and therefore could be eluted from a C-8 column using a mobilephase (solvent) that was predominantly made up of methanol. The optimizedconditions for the separation of the non-polar analgesic group were as follow
  • 62. The following HPLC chromatogram was obtained by elution of a mixture of non-polar analgesicsNote that under these conditions, it was possible to resolve Naproxen andIbuprofen only. When a mixture of all three components waschromatographed, considerable overlap between Ibuprofen and salicylamide took place.The latter was probably due to the relatively polar nature of the octylsiloxane, C-
  • 63. Gradient elution of polar and non-polar analgesicsAccording to the preceding isocratic elution experiments, the polar analgesiccompounds Aspirin, Acetaminophen and caffeine required a relatively polar solvent.The mobile phase was made up of 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30%methanol (volume by volume). By contrast, the non-polar analgesics Naproxen andIbuprofen required a non-polar solvent. The mobile phase was made up of 7% H 2O,3% 5 mM KH 2PO 4 (pH 6.1) and 90% methanol (volume by volume).Because of the singular discontinuity in the polarity, the solvent ramp to resolve theanalgesic mixture involved two time-dependent steps of decreasing polarity: in step 1the mobile phase contained 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30%methanol(volume by volume), for a period of two minutes. In step 2 the mobile phasewas made up of 100% methyl alcohol.Thus, in the first transient, the mobile phase is predominantly aqueous and thereforehighly polar -- it elutes the polar analgesics. Conversely, in the second transient, thesolvent is predominantly methanol which is relatively non-polar -- it elutes the non-polaranalgesics.
  • 64. The gradient method operated as follows. When the sample was first loaded onto thecolumn, the mobile phase was predominantly water. Consequently, the more polarAspirin, Acetaminophen and caffeine traversed the stationary phase. The relatively non-polar Naproxen and Ibuprofen remained tightly adsorbed at the column’s gate.After two minutes, as shown by the vertical white marker, switching to absolutemethanol decreased the polarity of the mobile phase. As a result, the non-polaranalgesics, Naproxen and Ibuprofen, were successfully dislodged from the non-polarmatrix and eluted by the solvent.A mixture containing Aspirin, Acetaminophen, caffeine, Naproxen and Ibuprofen wasthen analysed under gradient elution conditions. As the chromatogram below shows, allfive analgesics were successfully resolved.
  • 65. Isocratic elution for simple mixtures.Gradient elution for complex mixture.Isocratic and Gradient(techniques) HPLC System OperationTwo basic elution Techniques (modes) are used in HPLC. The first iscalled isocratic elution. In this technique (mode), the mobile phase,either a pure solvent or a mixture, remains the same throughoutthe run.Isocratic HPLC System
  • 66. Gradient elution Technique:Gradient elution is where the solvent strength of the moblie phase inincreased during the elution starting with a solvent of relatively lowsolvent strength.Steady changes of the mobile phase composition during the chromatographic run iscalled gradient elution. It may be considered as an analogy to the temperatureprogramming in gas chromatography.The main purpose of gradient elution is to move strongly retained components of themixture faster, but having the least retained component well resolved.Gradient HPLC also known as solvent programming is a technique employed in HPLC.Starting with the low content of the organic component in the eluent we allow the leastretained components to be separated. Strongly retained components will sit on theadsorbent surface on the top of the column, or will move very slowly.When we start to increase an amount of organic component in the eluent (acetonitrile)then strongly retained components will move faster and faster, because of the steadyincrease of the competition for the adsorption sites.For example in a HPLC technique mobile phase as a mixture of A and B components.The initial concentration of the component A(organic solvent) is say 10%,then afterA time period of about 20 min. the concentration is steadily increased to about 90%.The other component B, is water which is regarded as the weak solvent that allows theElution of solutes very slowly, while the organic solvent is considered as the strongsolvent that rapidly elutes analytes from the column.The commonly employed organic solvents includemethonol,acetonitrile,isopropanol,THF etc.
  • 67. Performance of the gradient elution is strongly dependent on the instrumentation.Two main points the chromatographer needs to know about his instrument:How large the volume between the component mixing point and column inlet is. Forthe low-pressure gradient systems this volume usually correspond to the pumpvolume, and about 2 - 3 ml.How well does the system mix eluent components. If the system does not mixcomponents well then it will supply for the certain time one component then anotherand so on. Chromatographic performance of such system will be very lowespecially for the least retained components.Advantages:-The technique increases the efficiency of columns-Complex sample mixtures with wide retention range can be separated.-Samples which contain a unknown composition can be screened.-Retention of late eluting components is decreased. Hence they elute faster andgive narrower peaks.Disadvantages:-Requires continuous monitoring.-Maintenance cost is more.-The additional mixing of liquids causes a delay in the formation of gradient.-Any change in column dimension changes the elution order (peaks are of differentorder)
  • 68. The second type is called gradient elution technique, wherein, as its nameimplies, the mobile phase composition changes during the separation. This modeis useful for samples that contain compounds that span a wide range ofchromatographic polarity. As the separation proceeds, the elution strength of themobile phase is increased to elute the more strongly retained sample components.High-Pressure-Gradient System
  • 69. Column heaterThe LC separation is often largely influenced by the column temperature. In order to obtain repeatable results, it is important to keep the consistent temperatureconditions.Also for some analysis, such as sugar and organic acid, better resolutions can beobtained at elevated temperature (50~80 °C). Thus columns are generally kept insidethe column oven (column heater).
  • 70. The function of injector in HPLC is to introduce the sample in theflowing solvent so that it may be carried to the column.Injector may be operated either manually or automaticallyManual has two different modes for injecting samplesSample Injector Device:1.Septum mode injection2.Valve injection
  • 71. 1. Septum injection:• This allows injecting sample into pressurized solventstream using a self-elastic septum with a micro syringe• It is the simplest form of injector in the septum device(similar to one used in GC).Greatest Drawback:Limited to max operating pressure of 1500psi. Therefore, not widely used in HPLC anymore.
  • 72. Widely used in HPLC, Allows reproducible introductionof sample into the pressurized M.P without interruption offlow even at high temperature.Six-port Valve Type Injector One of the simplest and most common valve injector inHPLC is the “six-port valco”(or Rheodyne model) injector.Inject valve has two position:(a) Load(b) Inject2. Valve-Type Injection in HPLC:
  • 73. COLUMNS FOR HPLC;Empty Stainless steel HPLC columnhardware come with the threadedtube, two 2um frits, and twostainless steel end fitting nuts.2.1, 3.0, 4.0, 4.6, 7.8, 10, 22,and 30mmI.D.and 50mm,100mm.150mm,250mm and 300mm length
  • 74. . For normal phase, SIL or DIOL is usually the best choice while C18, C8, C4 orPhenyl is the best choice for reversed phase applications
  • 75. Stationary phases in column:Silica gelAluminaZirconium Carbon Hydroxyapatite.Polymeric adsorbents such as polystyrene-divinyl benzene.Packed with 3-10µm particles.Resin based packings such as Polystyrene-divinylbenzene& Acrylic based polymers are used in HPLC columns.:
  • 76. COLUMNS FOR HPLC:A column is a heart of the chromatograph for separating a mixtureinto components.ColumnPackingmaterialcontainerNature ofparticleParticlesizeLengthDiameterMaterial
  • 77. Pore size:Porous microparticles are most common particles used inHPLC.Particles with small pores exhibit a high surface area & greaterretention.Pore sizePorousparticlePellicularparticlePorousmicroparticleStationary phases in column:
  • 78. Particle size:Small particle sizes result in high efficiencies with increasedback- pressure, results decreased column permeability.Porous particle ranges from ~20-40µm.Porous micro particles are chosen for high efficiency, rangesfrom 3-10µmInternal diameter of column:Internal diameter will effect the sample load, peak dilution, flowrate.Larger the diameter, greater the sampling load, higher the flowrate.Internal diameter for analytical columns ranges from 2-5mmStationary phases in column :
  • 79. Column length:Effects both efficiency & speed of separation.Column efficiency tends to increase with column length.Longer columns tends to longer time analysis.Ranges from 30-300mm in length.Construction materials of column:316stainless steel for rigidity & mechanical strength at highpressures.Glass, Teflon, & PEEK Columns are used for aggressivemobile phases, such as hydrochloric acid, or with solutes suchas proteins may adsorb to stainless steel.Polymeric columns for ion-exchange packings, glass forprotein separation
  • 80. One common stationary phase is asilica which has been treated withRMe2SiCl, where R is a straight chainalkyl group such as C18H37 or C8H17.With these stationaryphases, retention time is longer formolecules which are more non-polar, while polar molecules elutemore readily.
  • 81. SiliaChrom AQ C18 columns use a new generationadsorbent based on high purity spherical silica and uniquebonding technology. SiliaChrom AQ C18 is a slightly polarC18 column, and is the most versatile reversed phasecolumn offered by SiliCycle for highly aqueous mobilephases. SiliaChrom AQ C18 is considered as the mostuniversal HPLC column for any type of analytes (acidic,basic and neutral). Furthermore, SiliaChromAQ C18 isapplicable to a wide range of sample types: plasma, urine,drug formulation, food extraction, forensic, clinical andbioanalytical.Main Characteristics•Exceptional stability at pH 1,5 to 9,0•Inertness for acidic and basic analytes•Compatible from 100% aqueous mobilephase to 100% organic•Rapid equilibration•Reduced need for mobile phase modifiers•Partially endcapped
  • 82. Reversed-Phase Column1.1 ODS columnThe packing material used for reversed-phase column is often made of silica gelmodified with functional grouphe most often used functional group is octadecyl.Octadecyl functional group is a straight carbon chain of 18. Its molecular structure isshown below..Ideally, entire surface of ODS gel is modified with C18 functional group; howeverthere will be remaining spaces that are not modified. Those part are called “residualsilanol” and the presence of residual silanols can influence the separation. Often “endcapping” is applied to the gels to immobilize the residual silanol. Almost all ODScolumns nowadays are end-capped, however depending on the type ofanalyte, presence of silanol may provide better separation results.
  • 83. Other Silica-Based ColumnsODS is most popularly used RP column, however since C18 is a long chain, it mayretain compounds too much and consequently results in long analysis time. Referring toour story, this maybe a case that promotion was “too effective” making the customer stayat the bar forever, which is not a good management for the bar. So in those cases, it isbetter to use functional group with shorter chain, such as C8, C4, and C3.C8:Octyl functional group -CH2CH2CH2CH2CH2CH2CH2CH3C4:Butyl functional group -CH2CH2CH2CH3C3:Trimethyl functional group -CH2CH2CH3Also there are silica gels modified with phenyl and cyanopropyl functional groups.The most popular Shodex TM polymer column is ODP series. As you may guess, thename of ODP came from OctaDecyl Polymer. We believe it is easy to remember, as S(silica) in ODS was simply replaced with P (polymer)Polymer-Based ColumnsLong column lifeIn general, polymer gel is chemically morestable than silica gel and deteriorates slow
  • 84. •Despite that the column life of silica column is about 3 months whereas it is notsurprising to see a polymer column provides stable analysis over 1 year.•Good repeatability•Usability under alkaline conditionsSilica column cannot be used under alkaline conditions where polymer column can.Some basic samples (often pharmaceutical compounds) require alkaline mobile phaseto obtain good separations, thus polymer columns are suitable for such analysis.Resolution and priceGeneral consensus is that polymer columns compared to silica columns are moreexpensive and provide less resolution. Therefore even though polymer columns haveseveral advantages over silica columns, former is less often used. The price of thepolymer column is becoming lower as the technology has been improved. Alsoconsidering the longer column life, from a long term point of view, polymer column is notextremely more expensive than silica columns. The biggest concern will be theresolution. However, again the performance of newer polymer column has beenimproved and is becoming very comparable to silica columnsThe most popular Shodex TM polymer column is ODP series. As you may guess, thename of ODP came from OctaDecyl Polymer. We believe it is easy to remember, as S(silica) in ODS was simply replaced with P (polymer).The next popular polymer-based RP column is DE-413. The packing gel is made ofpolymethacrylate.
  • 85. Normal-Phase ColumnSilica gel without modification has large polarity, but when C18 functional group ismodified, the polarity becomes small. The larger polarity of silica gel is due to theseoxygen and hydrogen; the composition of silica is SiO2, but it has hydroxide group(oxygen + hydrogen) on its surface. RP mode uses gel with small polarity (e.g., ODS)and mobile phase with large polarity (e.g., water, acetonitrile). Normal-phase modeuses gel with large polarity (e.g. silica) and mobile phase with small polarity (e.g.hexane, chloroform). Instead of using the word small or large polarity, it is also commonto use words, hydrophilic or hydrophobic. Something easy to dissolve in water(i.e., large polarity) is called hydrophilic and something easy to dissolve in oil (i.e., smalpolarity) is called hydrophobic.At the very early stage of HPLC development, silica gel without any functional groupwas only used. Thus, historically normal-phase mode was developed first and sonamed “Normal”. Then the separation mode which uses opposite separation theory tonormal phase was developed and named “Reversed”. RP mode is much morepopularly used than normal phase nowadays, but we cannot change the historicalbackground, and thus they are still called normal and reversed-phase modes.
  • 86. The matrix of the primary silica gel particle consists of a core of silicon atoms joinedtogether with oxygen atoms by siloxane bonds (silicon-oxygen-silicon bonds). On thesurface of each primary particle some residual, uncondensed hydroxyl groups from theoriginal polymeric silicic acid remain. These residual hydroxyl groups confer upon silica gelits polar properties. These hydroxyl groups react with the silane reagents to form bondedphasesThere are three types of hydroxyl group.The first is a single hydroxyl group attached to a silicon atom which has three siloxanebonds joining it to the gel matrix.The second is one of two hydroxyl groups attached to the same silicon atom which, inturn, is joined to the matrix by only twosiloxane bonds. These twin hydroxyl groups arecalled Geminal hydroxyl groups.The third is one of three hydroxyl groups attached to a silicon atom which is now onlyjoined to the silica matrix by only a single siloxane bond. An example of each type ofhydroxyl bond is ….adsorption could also take place the surface of siloxane bonds as well.
  • 87. Silica gel is manufactured by releasing silicic acid from a strong solution of sodium silicateby hydrochloric acid. (Sodium silicate is prepared by heating sand at a high temperature incontact with caustic soda or sodium carbonate).Initially, silicic acid is released,Na2SiO3 +H2O + 2HCl = Si(OH)4 + 2NaCland then the free acid quickly starts to condense with itself with the elimination ofwater to form dimers, trimers and eventually polymeric silicic acid.The polymer grows, initially forming polymer aggregates and then polymer spheres, afew Angstrom in diameter. Thesepolymeric spheres are called primary silica particles.These primary particles continue to grow until, at a particular size, the surface silanolgroups on adjacent primary polymer particles, condense with the elimination of water.This condensation causes the primary particles to adhere to one another and at thisstage the solution begins to gel. During this process, the primary particles of silica gelwill have diameters ranging from a few Angstrom to many thousands of Angstromdepending on the conditions of formation.
  • 88. his unique ultra-solvent-resistant computer-printable label range for HPLC columns isdesigned to permanently adhere to allsurfaces and withstand aggressive HPLCcleaning agents. when exposed tomethanol, ethanol, acetone, IPA, andTHF, providing lab managers with theperfect HPLC identification solution.variable data (batchnumbers, barcodes, etc
  • 89. PRP Polymeric Reversed Phase Poly(styrene-divinylbenzene) HPLC Columnsare used in quality assurance as well as research and development laboratoriesbecause they last significantly longer than silica-based reversed phase columns. This isparticularly true for difficult analyses like: high pH samples (pH 8-13); labile orreactive samples (irreversible adsorption); high aqueous purifications (80-100%water); and separations with ion pairing reagents. HxSil silica-based columns are idealfor applications where a higher efficiency reversed phase C8 or C18 column is needed.Hamilton PRP-1 reversed phase columnsPolymeric columns for generalseparations.•pH Stable from 1 to 13•Better sample recovery than silicabased columns.•Excellent durability (stable to anyconcentration of water or organicsolvent).Product Features•Four particle sizes: 5, 7, 10 and 12-20 µm.•Ten column internal diameters: 1.0to 101.6 mm.•Two column materials: 316 stainlesssteel and PEEK
  • 90. APPLICATIONS of PRP columns (polymeric Reverse phase columnsPeptide StandardsResveratrolFolic AcidPregnenoloneBiotin, Folic Acid, Vitamin B12Coenzyme Q10Ginkgo BilobaSoy IsoflavinsFat Soluble VitaminsPhenobarbital, Scopolamine,Hyoscyamine, ErgotaminePregnenolone, progesteroneHormonesErythromycinsPyridinolineDiphenylguanidineseparations up to 9 times faster if required
  • 91. Special fitting style guard column forprotect main column!For 4.6mmI.D. HPLC column.Features・Special fitting style guard column・Easy hand tight connectionGuard Column 3mm (Cartridge type)
  • 92. End-CappingA reversed-phase HPLC column that is end-capped has gone through asecondary bonding step to cover unreacted silanols on the silica surface.End-capped packing materials eliminate unpredictable secondaryinteractions. Basic analytes tend to produce asymmetric tailed peaks onnon end-capped columns, requiring the addition of modifiers to themobile phase. Non end-capped materials exhibit different selectivity thanend-capped columns. This selectivity difference can enhance separationsof polar analytes by controlling the secondary silanol interactions. Acolumn is said to be endcapped when a small silylating agent, such astrimethylchlorosilane, is used to bond residual silanol groups on a packingsurface. It is most often used with reversed-phase packings and may cut down onundesirable adsorption of basic or ionic compounds.
  • 93. what is the specific advantage in using endcapped columns against nonendcapped one?Because of steric hindrance and other factors, it is not possible to react allof the silanols with C8 or C18 alkyl groups. Because of this, after the initialbonding, additional bondings are sometimes performed, attaching smalleralkyl groups, quite often propyl or isopropyl groups, to the residualsilanols. There is no endcapping process that is complete. There arealways unreacted silanols.The resulting stationary phase, having fewer residual silanols, will displayfewer secondary interactions characteristic of silanols than a non-endcapped stationary phase.The result is less peak broadening and reduced tailing -- especially withorganic acids and bases.The common endcapping group is a trimethylsilyl group
  • 94. The silica is first dried at 250oC to remove the majority of the stronglybound water from the surfacehe silica is suspended in toluene that iscontained in a flask fitted with a reflux condenser and then treated with anexcess of dimethylchlorsilane (all the adsorbed water on the silica gelsurface my not have been completely removed and the excess reagent willscavenge any residual water) The mixture is refluxed for about three hoursand finally any remaining reagent or hydrochloric acid is The surface isnow covered with a layer of dimethylsilane groups. Squalene is then addedto the suspension, accompanied by the catalyst chlorplatinic acid, and themixture is next refluxed for a further 6 hours. Squalene contains six doublebonds along its chain, each double bond being separated by two methylenegroups and thus, in the presence of the catalyst, any double bond comingin contact with the silane hydrogen on the silica surface will open andattach the squalene chain to that silyl group.reacted with trimethylchlorsilane to endcap the finalmethyloctylmonohydroxysilane groups thatwere generated on the surface
  • 95. An end-cappedC8 sorbent thatoffers moderatehydrophobicretention withnegligiblesecondary polarinteractions fromactive silanolgroups.Description:Octadecyl (non end-capped) functionalizedsilica, manufactured using trifunctional silane.Average Particle Size: 50 µm (irregular shapedparticles)Nominal Porosity: 60 ÅApplications: Matrix – AqueousAnalytes – Wide range polarityRetention mechanism – Non-polar [polar, cation exchange]Comments: Strong non-polar (hydrophobic) phase.Non end-capped to provide additional silanol interactions.

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