UHPLC Analysis of Paraquat and Diquat

William L. Maule III, Clint Corman, Michael Ye, Cory Muraco, Curtis Frantz

Merck, Bellefonte, PA

Paraquat and diquat are two toxic herbicides used to prevent the growth of invasive plant species. As both species are polar in nature, they can be difficult to resolve using conventional reversed phase chromatography. This application demonstrates the use of a Supel™ Carbon LC UHPLC column for retaining and resolving these two compounds. In addition, lot-to-lot reproducibility was investigated with two columns packed from different batches of the stationary phase.

Figure 1. Chemical Structures of Paraquat and Diquat.

Experimental Conditions for Diquat/Paraquat analysis

Table 1. Experimental Conditions for Diquat/Paraquat analysis

Column(s)	Supel™ Carbon LC, 10 cm x 2.1 mm I.D., 2.7 µm; Competitor T, 10 cm x 2.1 mm I.D., 3.0 µm
Detection	UV: 290 nm
Mobile Phases	[A] Water (0.1% (v/v) DFA); [B] Acetonitrile (0.1% (v/v) DFA)
Gradient	Time (min) %A %B 0 100 0 1 100 0 11 85 15 20 100 0
Test Mix	Diquat/Paraquat Mix, 100 µg/mL, water (0.1% DFA)
Injection Volume	2 µL
Flow rate	0.50 mL/min
Temperature	Column 55 oC; Autosampler 10 oC

Performance results and comparison

Figure 2. Analysis of Paraquat and Diquat on Supel™ Carbon LC.

Table 2. Compound Retention Parameters Seen in Figure 2.

Peak	Compound	Ret. Time (min)
1	Diquat (100 µg/mL)	4.362
2	Paraquat (100 µg/mL)	7.295

Figure 3. Analysis of Paraquat and Diquat on Competitor Column.

Table 3. Compound Retention Parameters Seen in Figure 3.

Peak	Compound	Ret. Time (min)
1	Diquat (100 µg/mL)	4.758
2	Paraquat (100 µg/mL)	7.848

Additional performance demonstrating lot-to-lot reproducibility with Supel™ Carbon LC columns

Experimental Conditions

Table 4. Chromatographic Conditions for Lot-to-Lot Reproducibility Study

Column(s)	Supel™ Carbon LC, 5 cm x 3.0 mm I.D., 2.7 µm Competitor T, 5 cm x 3.0 mm I.D., 3.0 µm
Detection	UV: 290 nm
Mobile Phases	[A] Water (0.1% (v/v) DFA); [B] Acetonitrile (0.1% (v/v) DFA)
Gradient	Time (min) %A %B 0 100 0 1 100 0 11 85 15 20 100 0
Test Mix	Diquat/Paraquat mix, 100 µg/mL, water (0.1% (v/v) DFA)
Injection Volume	5 µL
Flow rate	1.0 mL/min
Temperature	Column:  55 °C Autosampler:  10 °C

Peak	Compound	Ret. Time (min)
1	Diquat (100 µg/mL)	3.800
2	Paraquat (100 µg/mL)	6.709

Peak	Compound	Ret. Time (min)
1	Diquat (100 µg/mL)	3.762
2	Paraquat (100 µg/mL)	6.660

Peak	Compound	Ret. Time (min)
1	Diquat (100 µg/mL)	4.118
2	Paraquat (100 µg/mL)	6.517

Figure 4. Analysis of paraquat and diquat on two Supel™ Carbon LC columns and a Competitor Carbon Column. Note high degree of reproducibility between the two Supel™ Carbon LC columns. Tables below each image correspond to retention parameters of the compounds.

Conclusion

This application demonstrates the use of Supel™ Carbon LC column to give baseline resolution and excellent peak shape for the separation of the polar herbicides― diquat and paraquat. A unique and proprietary synthetic procedure, narrower particle size distribution, and less active surface results in more efficient and reproducible chromatographic separation compared to a different carbon column from another vendor. The Supel™ Carbon LC column is an ideal choice for polar compounds that are difficult to retain using typical RP columns.