How can I prevent non-specific adsorption of my analyte to my reverse-phase column? - WKB242579

ENVIRONMENT

• Non-specific adsorption
• Saturation
• Stainless steel
• Low sensitivity

ANSWER

Make repeated injections of a higher concentration of the analyte of interest.

ADDITIONAL INFORMATION

Non-specific adsorption of analytes occurs in two ways:

• Adsorption to the stainless steel surfaces (positive charges on steel) through anion exchange.
• Adsorption to the particle surface active sites (e.g., silanol groups, trace metals if present, etc.)

If you make high-concentration injections (e.g., 1 mg/mL concentration for small molecules or 4 mg/mL concentration for biomolecules) of the analyte of interest that is adsorbing, it can saturate "active sites" on the stainless steel surfaces of the column and/or particles. When these active sites become fully saturated, analyte recovery can increase and tailing can decrease. Retention time may also decrease when active sites are saturated properly.

For conditioning the column for biomolecules, injections of a thyroglobulin are found to be effective. 

For Insulin testing, injection of protein-precipitated rat plasma is found to be effective.

NOTE: Waters has introduced MaxPeak Premier LC Systems (ACQUITY Premier, Arc Premier) and columns (ACQUITY Premier BEH, and others), which have MaxPeak High Performance Surfaces that are designed to decrease analyte adsorbance to metal surfaces.