This year has been very special for everyone, in order to adapt, Interchim® has set up a laboratory to be able to perform online demonstrations. These demonstrations can be articulated around purifications of “standard” products in order to demonstrate the potential of our instruments. However, when one wishes to acquire a purification system, it is legitimate to want to purify one’s product. This is exactly what we can do in our laboratory and we would like to share an example with you through this article.

Installation

For this online demonstration, we had to purify a peptide resulting from a chemical synthesis. The selected device was therefore naturally the puriFlash® 5.250P. To follow this purification, we will use a UV detector as well as our puriFlash® MS mass spectrometer.

The detection of peptides by mass spectrometry is done with an ESI source ideal for the detection of large molecules such as peptides. In order to allow this detection, a very small amount of product is “diverted” by our MS interface to be passed on this detection mode.

 

Direct injection of the crude sample in the mass spectrometer puriFLash® MS (FIA), sources parameters selection.

  • Source type: ESI
  • Source parameters:
    Mode : Positif
    Capillary temperature : 200°C
    Capillary voltage : 160V
    Source voltage offset : 30 V
    Source voltage span : 5 V
    Source gas temperature : 250°C
    ESI voltage : 3500V

Here the result:

Resultat injection directe du mélange à purifier sur le spectromètre de masse puriFlash® MS

We observe a peak at m/z 643, which corresponds to the molecule of interest charged 3 times (M+3H)3+ as well as a peak at m/z 964.3 which corresponds to the molecule of interest charged 2 times (M+2H)2+.

 

HPLC conditions

  • Solvants:
    A: Water + 0,1%FA
    B: ACN
  • HPLC column: Onyx Monolithic C18 50x2mm
  • Flow-rate: 0,6mL/min
  • Injected volume: 5µL (Concentration 1mg/mL)
  • Elution:
    T (min) A % B %
    0 95.0 5.00
    3.8 0.0 100
    3.81 0.0 100
    4 0.0 100
    4.07 95.0 0.0
    4.2 95.0 0.0
    4.3 95.0 0.0

     

HPLC results:

Chromatogrammes

 

Transposition & purification

We chose to use a peptide column with the preparative column PFB5C18T-150/212.

As a first step, we carried out the transposition of the analytical HPLC method to a preparative HPLC method.

  • Device: puriFlash® 5.250P + MS
  • Solvents:
    A: Water + 0,1%FA
    B: Acetonitrile
  • Column: PFB5C18T-150/212
  • Flow rate: 16mL/min
  • Injection mode: Liquid
  • Injection volume: 1mL
  • Concentration: 1mg/mL
  • Crude sample: 1mg
  • Detection: UV 214nm (purple)
T (min) A % B %
00:00 95.0 5.00
20:00 0.00 100

Transposition de la méthode HPLC analytique vers une méthode HPLC préparative

Then increase the concentration of the injected sample in order to be able to purify a maximum of product in a single run.

Quantity injected: 1mL (concentration 25mg/mL)

  • Device: puriFlash® 5.250P + MS
  • Solvents:
    A: Water + 0,1%FA
    B: Acetonitrile
  • Column: PFB5C18T-150/212
  • Flow rate: 16mL/min
  • Injection mode: Liquid
  • Injection volume: 1mL
  • Concentration: 25mg/mL
  • Crude sample: 25mg
  • Detection: UV 214nm (purple),
    UV 254nm (green)
    Mass XIC 644m/z (Orange),
    Mass XIC 964m/z (Blue)
  • MS split parameters
  • Valve rotation: 0,5s
  • Make-up flow-rate: 0,5mL/min
  • Dilution flow-rate: 0,5mL/min
  • Solvents for make-up and dilution: Acetonitril + 0,1% FA
  • Pressure:60bar
  • Elution conditions:
T (min) A % B %
00:00 95.0 5.00
20:00 0.00 100

Transposition de la méthode HPLC analytique vers une méthode HPLC préparative

Thanks to the puriFlash® PF-5.250P system coupled with the puriFlash® MS we were able to purify and collect the product of interest based on its mass.

 

Lock down? Need a demonstration but no possibility to bring suppliers? Interchim® sets up demonstration systems by videoconference.

In our laboratories, we can set up our systems in order to purify your products during online demonstrations. You will then be able to benefit from a privileged expertise during the demonstration.

 

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