QC LEC 12

• Column chromatography


  Open Column Chromatography (OCC): column consists of glass or Teflon tube, bottom is fitted with stopcock to control flow rate of mobile phase, packing material is supported by fritted glass disk or glass wool to prevent leakage of stationary phase

  
• Liquid chromatography (LC)


  Simple, versatile, high-resolution separations, stationary phase is packing material, mobile phase is liquid

  
• Eluotropic series


  Rating of solvents in order of their strength of adsorption on a given adsorbent material, solvents near top have less affinity for adsorption sites of adsorbent than those at bottom, alumina & silica gel interact strongly with polar solutes
• Eluotropic series for alumina (from least to most likely to interact)


  • Petroleum ether
  • Carbon tetrachloride
  • Benzene
  • Chloroform
  • Diethyl ether
  • Ethyl acetate
  • Pyridine
  • Acetone
  • N-propanol
  • Ethanol
  • Methanol
  • Water
• Open Column Chromatography


  Mobile phase is allowed to flow through packed column under influence of gravity

  
• HPLC (High Pressure / High Performance Liquid Chromatography)


  Mobile phase flows through column under high pressure (1000 to 3000 psi required), most drugs are analyzed using 1400 psi, less than 2000 psi

  
• Modes/Principles/Classification of Liquid Chromatography

  • Adsorption chromatography
  • Partition chromatography
  • Size exclusion chromatography
  • Affinity chromatography
  • Ion exchange chromatography

  
• HPLC


  Form of liquid chromatography used to separate compounds that are dissolved in solution, instruments consist of reservoir, mobile phase, pump, injector, column, detector

  
• HPLC Principle


  A liquid mobile phase is pumped under pressure through a stainless-steel column containing particles of stationary phase, analyte is loaded onto head of column via a loop valve and separation of a mixture occurs according to relative length of time spent by its components in stationary phase, monitoring of column effluent can be carried out with a variety of detectors

  
• HPLC Strengths


  • Most intensive development in recent years, leading to improved columns and detectors, variety of columns and detectors means that selectivity of method can be readily adjusted, less risk of sample degradation because heating is not required
• HPLC Limitations


  • Drugs have to be extracted from formulations prior to analysis (pretreatment procedures), increased amounts of organic solvent waste are generated, which are expensive to dispose of
• HPLC Compartments


  • Solvent reservoirs
  • Injection device
  • Pump
  • Precolumn
  • Analytical Column
  • Detectors

  
• HPLC Detectors


  Optical detectors (UV/VIS, IR/Raman, optical activity, refractive index, luminescent, fluorescence), Mass Spectrometry detectors, Elemental Detectors (AA/Emission, ICP-MS), Electrochemical Detectors (conductivity, coulometry, polarography)
• HPLC Detector Categories


  • Bulk Property Detectors
  • Solute Property Detectors
  • Electrochemical Detectors
  • Multipurpose Detectors
• Ion exchange chromatography


  Process that allows separation of ions and polar molecules based on their charge, uses ion exchange resins to separate atomic or molecular ions depending on charge surface desired
• Electrophoresis


  Migration of charged molecules under influence of an external electric field, for separation of complex mixtures of biological substances such as protein, nucleic acids and polysaccharides
• Electrochromatography


  Technique that shares attributes of both chromatography and electrophoresis, separation based on electroosmotic flow (EOF)

  
• Supercritical Fluid Chromatography (SFC)


  Mobile phase is supercritical fluid, most common mobile phase is carbon dioxide gas CO2 which has critical temperature of 31°C at 73 atm, provides rapid separation without use of organic solvents
• Analyte


  Will separate based on EOF
• Analyte separation based on EOF


  • +charge move faster than EOF
  • -charge move slower than EOF
  • Neutral move at same speed as EOF
• Supercritical Fluid Chromatography (SFC)


  Mobile phase is a supercritical fluid

  
• Supercritical fluid


  • Has characteristics midway between its gaseous and liquid state (critical temperature)
  • Non-compressible
  • High-density fluid
  • Temperature and pressure above critical temperature and critical pressure
  • Mesophase between liquid and gas
• Most common mobile phase in SFC

  Carbon dioxide gas (CO2), has critical temperature of 310°C at 73 atm
• Reasons why carbon dioxide is used in SFC

  • Cheaper than organic solvents
  • Similar efficiency to organic solvents
  • Less toxic
  • Non-flammable
  • Many disposal methods (dry ice, purify and use for carbonation)
  • Excellent solvent for organic compounds
• SFC Extraction


  Rapid separation without use of organic solvents
• SFC Advantages


  • Faster separating time than HPLC and GC
  • Can handle non-volatile and thermally or chemically labile drug substances that are difficult to analyse by GC
• No universal procedure that can be adopted for different types of samples in method development
• Suggested characteristics of optimized separation methods


  • Resolution: precise
  • Separation time: less than 5-10 minutes
  • Pressure: less than 2000 psi (most drugs = 1400 psi)
  • Peak height: narrow peaks
  • Solvent composition: as close to 1 as possible, minimum mobile phase use
• Sample pretreatment and special procedures


  • Derivatization
  • Ensure analyte in samples can be detected
  • Add a chromotag (ninhydrin) or fluorotag
  • Modify functional groups to absorb UV or become fluorescent
  • Pre-column derivatization: sample pre-treatment
  • Post-column derivatization: sample post-treatment
  • Solid phase extraction
  • Liquid-liquid extraction
  • Dilution
  • Evaporation
  • Distillation
  • Lyophilization / Freeze-drying / Cryodesiccation
• Lyophilization / Freeze-drying / Cryodesiccation


  • Aqueous sample is frozen, water removed by sublimation under vacuum
  • Dehydration process used to preserve perishable materials and make them more convenient for transport
  • Works by freezing material and reducing surrounding pressure for frozen water in material to sublimate directly from solid phase to gas phase
  • Used for extracts of animal origin
• Some lyophilized materials have a tendency to become hygroscopic and form large chunks of powder
• Gas Chromatography (GC)


  • Mobile phase is always a gas
  • Used for volatile materials / stable in vapor configuration
  • Sample is vaporized and injected onto chromatographic columns and separated into many components

  
• GC Types


  • GSC (gas-solid chromatography)
  • GLC (gas-liquid chromatography)
  • Liquid = liquid substrate
• GC Advantages


  • Fast analysis
  • Efficient, high resolution
  • Sensitive, detect minute concentrations (ppm or ppb)
  • Non-destructive, does not require derivatization
  • Highly accurate quantitative analysis
  • Requires small sample volumes (microliters)
  • Reliable and simple
• GC Disadvantages


  • Limited to volatile samples (e.g. steroids cannot be analyzed)
  • Not suitable for thermally labile samples
  • Difficult for large preparative samples
  • Requires mass spectroscopy for confirmation of peak identity
• GC Components


  • Carrier gas
  • Pressure regulator
  • Flow controller
  • Injection Port
  • Column
  • Detector
  • Signal processor
• Carrier gas


  • In steel tanks under high pressure
  • Helium or Nitrogen (occasionally Hydrogen, Argon or CO2)
  • Helium is the most prevalent carrier gas due to excellent thermal conductivity and inertness
  • Nitrogen is cheaper but less sensitive
  • Hydrogen has the highest thermal conductivity but is hazardous
• Stationary phases in GSC

  • Activated charcoal
  • Silica gel
  • Alumina
  • Glass beads
• Stationary phases in GLC


  • Diatomaceous earth (kieselguhr)
• Injection Port


  • Small chamber where the sample is vaporized rapidly before entering the column
  • Injection may be manual or automatic