Simplifying Conversion of HPLC Methods to LC/MS

1. Simplifying Conversion of HPLC Methods to LC/MS John W. Dolan, James L. Merdink, Michael S. Alexander, and Christopher J. Kemper LC Resources Inc. Laboratory Services 3138 NE Rivergate, Bldg. 301C McMinnville, OR 97128 (503)-472-8882 (503-472-4863 (fax) info@LCResources.com

2. The Quadrupole Mass Spectrometer

3. Conversion Steps • LC Mode • convert to MS-compatible mobile phase • LC or LC/MS • scale to MS (adjust column conditions) • LC/MS • confirm success

4. LC/MS Mobile Phase Requirements • Mobile Phase Solvent • volatility • amount of water • ionization issues • Mobile Phase Additives • volatility • concentration • ion suppression • buffering capacity • Buffer / pH Control • formic acid • ammonium formate • ammonium acetate • (trifluoroacetic acid)

5. Check for Sufficient Retention to Avoid t0 Interferences (blank plasma injection) (drug standard)

6. 0.0 0.6 1.2 1.8 2.4 3.0 Time (min) Ion Suppression blank plasma injected into drug infusion drug standards

7. Scaling Isocratic Separations • pressure = f(linear velocity) • linear velocity = f(flow rate, column cross-sectional area) … so for constant pressure, adjust flow rate = f(diameter2) e.g., 4.6 mm column @ 1.0 mL/min, what flow should be used for 2.1 mm column? (4.6 mm / 2.1 mm)2  5 1.0 mL/min / 5 = 200 L/min

8. Adjust Column Diameter 150 x 4.6 mm 1.0 mL/min 1500 psi 150 x 2.1 mm 0.2 mL/min 1500 psi

9. Eliminate Extra-Column Volume 150 x 4.6 mm 1.0 mL/min 1500 psi 150 x 2.1 mm 0.2 mL/min 1500 psi

10. Reduce Column Length 150 x 2.1 mm 0.2 mL/min 1500 psi 50 x 2.1 mm 0.2 mL/min 500 psi

11. Increase Flow Rate 50 x 2.1 mm 0.2 mL/min 500 psi 50 x 2.1 mm 0.6 mL/min 1500 psi

12. Net Isocratic Change 150 x 4.6 mm 1.0 mL/min 1500 psi 50 x 2.1 mm 0.6 mL/min 1500 psi

13. gradient: tG F  Vm S k*  Isocratic vs. Gradient Retention remember: k controls selectivity isocratic: log(k) = log(k0) - S

14. tG F  Vm S Eliminate Gradient Waste 150 x 4.6 mm, 5 m 1.5 mL/min 5-100%B / 50 min k*  7 15-35%B / 10 min (2%/min)

15. tG F  Vm S Shorten Column 150 x 4.6 mm, 5 m 1.5 mL/min 15-35%B / 10 min 30 x 4.6 mm, 3 m 1.5 mL/min 15-35%B / 2 min

16. tG F  Vm S Reduce Column Diameter 30 x 4.6 mm, 3 m 1.5 mL/min 15-35%B / 2 min 30 x 2.1 mm, 3 m 0.3 mL/min 15-35%B / 2 min

17. tG F  Vm S Dwell Volume -- the Column Scaling Killer 150 x 4.6 mm, 5 m 1.0 mL/min 20-70%B / 15 min VD = 1.0 mL 150 x 2.1 mm, 5 m 0.2 mL/min 20-70%B / 15 min VD = 1.0 mL

18. Dwell Volume -- Equivalent to Isocratic Hold 150 x 4.6 mm, 5 m 1.0 mL/min 20-70%B / 15 min VD = 1.0 mL 150 x 2.1 mm, 5 m 0.2 mL/min 20-70%B / 15 min VD = 1.0 mL

19. tG F  Vm S F VD Adjust VD With Flow Rate scales gradient scales dwell time

20. Dwell Volume -- Scale with Flow Rate Change 150 x 4.6 mm, 5 m 1.0 mL/min 20-70%B / 15 min VD = 1.0 mL 150 x 2.1 mm, 5 m 0.2 mL/min 20-70%B / 15 min VD = 0.2 mL

21. Summary • Use a volatile mobile phase • scaling isocratic runs (i.d., length, flow) • keep k* constant when scaling gradients • minimize extra-column effects • beware of dwell volume reprints: www.LCResources.com