Hypercarb is a 100% porous graphitic carbon (PGC) stationary phase and possesses a flat, highly crystalline surface. The permanent dipole of polar molecules can induce a dipole on the polarizable surface of the graphite as they approach it, increasing the interaction between the analyte and the graphite surface. This dipole–dipole interaction results in excellent retention for polar compounds. Hypercarb columns show greater retention and resolution than silica based C18 columns, without an ion pair reagent in the mobile phase.
ExperimentalThe analyses of glyphosate and AMPA were performed using a Finnigan LC–MS system (Thermo, San Jose, California, USA). The separations were performed using 50 × 2.1 mm columns (Thermo, Bellefonte, Pennsylvania, USA); Hypersil BDS C18 5 µm (part number 28105-052130) and Hypercarb 5 µm (part number 35005-052130) with the following conditions:
Eluent: A: H2O + 0.1% formic acid
B: ACN + 0.1% formic acid
Gradient: 5–100% B in 10 min
Flow-rate: 0.3 mL/min
Detection: MS +ESI SIM [M+H]+ (m/z 112, 170).
Results and Discussion
Conversely, Hypercarb is observed to retain and resolve AMPA and glyphosate as shown in the left hand chromatogram. This ensures that a robust quantitative method can be developed. The capacity factor for glyphosate is 2.2. Baseline resolution is readily achievable between the two compounds, allowing for maximum sensitivity when quantifying them together.
The method developed on Hypercarb uses a mobile phase containing 0.1% formic acid, which promotes protonation of the amino/NH groups and further enhances sensitivity when +ESI is used as the detection method.
Because Hypercarb is able to retain polar compounds, it can also be used for solid-phase extraction applications where its strong retention properties allow the use of low bed weights. Hypercarb solid-phase extraction formats (HyperSep Hypercarb) have proven successful for trace enrichment of polar pesticides and metabolites,2 and may provide a solution for glyphosate and AMPA applications.
The combination of retention, ease of method development and increased sample throughput makes Hypercarb ideal for accurate routine measurement of glyphosate and AMPA.
1. C. Cox, Herbicides Fact Sheet, Glyphosate (Roundup), Journal of Pesticide Reform, 18, 3–17 (1989).
2 M.C. Hennion, J. of Chromatogr. A, 885, 73–95 (2000).