• Overview
• Nanoflow HPLC »
  • Autosampler Injection
  • Nanoflow HPLC »
    • RP Separation (1D)
    • Switching
    • Coupling LC to MS
• Mass Spectrometry »
  • Introduction »
    • Spectrum
    • Chromatogram
    • Isotopes, Mass Resolution, Mass Accuracy
    • m/z, Charge State
  • Peptide Ionisation »
    • Electrospray
    • MALDI
  • Mass Spectrometry Modes »
    • MS/MS Modes
    • Hybrid Modes
  • Peptide Fragmentation »
    • Overview
    • Collisional Activation Methods
    • Mobile Proton Model

Peptide samples are placed into vials with a conical bottom to enable efficient injection of small sample volumes. Each vial is capped to minimise sample evaporation/contamination, and the cap has a penetrable lid to allow for robotic sample injection without any need to physically uncap the vial. The instrument used for automated sample injection is called an autosampler which can be programmed by a computer to inject a specific volume from a vial in a specific position in the autosampler's vial tray. Control of all components including the syringe, injection needle and valve are fully automated and user programmable. In order to penetrate the cap of each vial a stainless steel wide-bore needle first punches through the lid, subsequently a fine fused silica needle is lowered through the centre of the wide-bore needle to the bottom of the vial (bottom of the sample solution). For most samples an injection volume of 1-10 microL is used and to minimise sample waste a mode of injection called "microL Pick-Up" is used. In this mode the autosampler first aspirates some transport buffer from the transport vial which is usually aqueous buffer A. The autosampler then injects the required volume from the sample vial. The volume of sample must be approximately 25-50% of the size of the sample loop and Hamilton syringe volume. The system then washes the injection needle and aspirates more transport buffer to move the sample up the injection needle into the middle of the sample loop. For more quantitative injection a "full-loop" injection may be performed which usually uses approximately double the amount of sample that is to be injected with the unused sample solution going to waste. This ensures that the entire sample loop (whose volume has been precisely determined) is quantitatively filled with sample.