Sample Preparation and Injection Techniques
Sample Preparation and Injection Techniques
This domain tests what happens before the sample reaches the column: diluent, clean-up, concentration, and the injection itself. Many so-called chromatographic problems - split peaks, carryover, poor precision - originate here.
Preparing the sample
- Match diluent strength to the mobile phase. Injecting a strong solvent (high organic on a reversed-phase method) focuses poorly and gives split or broadened peaks; inject in mobile phase or weaker.
- Filter or centrifuge to protect the column. Choose the membrane by compatibility: nylon adsorbs some analytes and is unsuitable for strong acids; PTFE resists organic solvents; PVDF and cellulose suit aqueous samples. Verify recovery for your analyte.
- Clean-up options: protein precipitation (ACN or acid), liquid–liquid extraction, and SPE (reversed-phase, ion-exchange, mixed-mode). SPE concentrates and cleans up but needs recovery and breakthrough checks.
- Add an internal standard before extraction to correct for volume loss and injection variability; stable-isotope-labeled standards are best for MS.
Injection
- Full-loop injection gives the best precision; partial-loop saves sample but precision depends on the volume drawn.
- Control carryover with a strong needle wash and validate it using a blank after a high standard (commonly ≤ 20% of the LOQ response).
Worked example
A method injecting 10 µL of sample dissolved in 100% methanol into a 40:60 ACN:water gradient shows split, fronting peaks for the early analyte. The diluent is far stronger than the mobile phase at injection. Diluting the sample 1:1 with water, or reducing injection volume to 2 µL, restores peak shape; best practice is to match the diluent to the initial mobile phase.
Traps
Assuming filters are inert; using a 0.45 µm filter ahead of a UHPLC column; overfilling autosampler vials so the septum contacts liquid; ignoring sample stability in the autosampler.