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HPLC Column

HPLC Column:

Normal phase chromatography: Polar stationary phase and non polar mobile phase.

Reverse phase chromatography: Non polar stationary phase and polar mobile phase.

  1. Modern reverse phase liquid chromatography consists of an organic phase chemically bonded to silica or other material.
  2. Packaging material consist of silica gel particles roughly spherical or irregular porous material with large surface area.
  3. Column polarity depends on the polarity of the bound function groups. 
  4. Such packaging material are robust variable and most preferred.

  • C8 - Octyle
  • C18 - Octadecyl
  • Phenyl- C6H5
  • Cynopropyl - CN
  • Aminopropyle - NH2

Column Regeneration:

Column regenerate procedure for C8, C18, ODS, L2:
Octadecyl silane chemically bonded to silica gel of a controlled surface porosity that has been bonded to a solid spherical core 30 to 50 mm in diameter. 
  • Fist washing water 1.0 ml/min up to 30 min.
  • Second washing Acetonitrile 1.0 ml/min up to 30 min.
  • Third washing Tetrahydrofurane 1.0 ml/min up to 30 min.
  • Fourth washing Methanol 1.0 ml/min up to 30 min.
Column regenerate procedure for  L3:
Porous silica particles 5 to 10 mm in diameter use for revers phase.
  • Fist washing with 0.05M phosphoric acid at 1.3 ml/min up to 15 min.
  • Second washing with water at 1.3 ml/min up to 15 min.
  • Third  washing with Acetonitrile at 1.3 ml/min up to 15 min.
  • Fourth washing with Methanol at 1.3 ml/min up to 15 min.
Column regenerate procedure for  L16:
Dimethylsilane chemically bonded to porous silica particles 5 to 10 mm in diameter.
  • First flushing with neat organic solvent.It is resolve the problem shifting retention time or resolution and contamination of column. (Note: Dose not solve the problem wash a column with a sequence.)
  • Fist washing with water  at 1.0 ml/min up to 15 min.
  • Second washing Tetrahydrofurane (THF) at 1.0 ml/min up to 15 min.
  • Third washing with methylene chloride at 1.0 ml/min up to 15 min.
  • Fourth washing Tetrahydrofurane (THF) at 1.0 ml/min up to 15 min.
  • Fifth washing with water  at 1.0 ml/min up to 15 min.

Reverse phase: Heat water at 55°c & pass for one hours in the column at 1.0 ml/min flow rate.
                          Inject: 5 Injection 100 µl for 15 min RT.
Then run methanol for 50 min. ------> Chloroform for 50 min.
Run methanol for 50 min. (Second time) than checked column efficiency.
 Column efficiency:(C8, C18,ODS)
Column:           C8, C18, ODS
Flow rate:         1.0 ml/min
Mobile phase:   ACN:Water (700:500)
Wavelength:      254 nm,
Inj. Vol.:            20 µl

Sample preparation: 1 ml Benzene + 1 ml Toluene in 100 ml volumetric flask up to mark with                                                mobile phase
Checked system suitability:

  • Theoretical plate (NLT=3000)
  • Tailing factor (NMT=1.5)   
    Normal phase: Wash with IPA ,Methanol, N-Hexane 
Column efficiency:(Silica, CPS, Cyano)
Flow rate:       1.0 m/min.
Mobile phase: Hexane:Ethyl acetate (980:20)
Wavelength:    254 nm.
Inj Vol.;           20   µl   

Sample preparation: 1 ml Benzene + 1 ml Toluene in 100 ml volumetric flask up to mark with                                                mobile phase
Checked system suitability:

  • Theoretical plate (NLT=3000)
  • Tailing factor (NMT=1.5) 
Washing Procedure: 
  • Reverse phase: Water ----> ACN----> DCM----> ACN---->Methanol---->Water
  • Normal phase: Heptane---->Chloroform---->Ethyl acetate---->Acetone---->Ethanol->Heptane

Additional information:

 Peak Type for chromatogram:

  • B.V = Base to valley
  • B.B = Base to base 
  • V.V = Valley to Valley
  • P.    =  Peak started on a penetration. (Penetration mean entry of peak)
  • T    =  Tangent or baseline
  • S    =  Shoulder peak.
Type of UV Detector: Generally three type of UV Detector used in HPLC instrument. In these three type of detector, mostly used Multi wavelength detector in HPLC instrument. 
  • Fixed UV Detector.
  • Variable UV Detector.
  • Multi wavelength Detector.


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