Simple Scale-up on a 940-LC Analytical to Preparative HPLC

1. Simple Scale-up on a 940-LC Analytical to Preparative HPLC Dennis Hoobin Varian Australia Pty Ltd 679 Springvale Rd Mulgrave VIC Australia

2. Varian 940-LC Application note

3. Introduction • Preparative chromatography at mg to kg levels has the main aim of producing purified material • After having developed an analytical separation, scaling modifications need to be made such as: • Flow rate • Column size • Sample injection volume • Need to apply a linear scale up factor to produce an effective preparative HPLC separation

4. Scale Up Factors • linear scale-up factor is the ratio of the cross sectional areas of the analytical column and the intended preparative column • The calculation is: • Factor = r2(prep)/r2(analytical) • where r = radius • Scaling up from a 4.6mm ID column to a 21.2mm ID preparative column gives a scaling factor of: • (21.2mm/2)2/(4.6mm/2)2=21.24

5. Scale Up Factor cont. • Scale up factors are applied to the flow rate and the injection volume • Other parameters are kept constant such as: • column length, • packing material, • sample concentration • run time

6. Scale Up Process • In general, to perform the scale-up process: • 1. Optimize the preparative separation on an analytical column. Check sample solubility in mobile phase. • 2. Multiply all of the scaleable elements of the system by the appropriate linear scale-up factor. Do not change any of the ‘absolute’ elements. • 3. Perform the preparative separation.

7. Column overloading allows the user to achieve greater yields than linear scale up would allow • This is achieved in 2 ways: • Increased sample concentration • Increased injection volume • Requires accurate and reproducible detection algorithms for fraction collection to collect the distorted peaks • The 940-LC allows the user to do this!!!

8. Instrumentation • Varian 940-LC Semi-Prep HPLC (part no 00-100898-00) • Binary gradient pumps • 128 position autosampler with 5mL syringe • 445-LC Scale Up Module to allow seamless scalability from Analytical to Semi-preparative applications • Optimised Dual path flowcell • Diode Array detector • 440-LC Fraction collector to allow accurate fraction collection based on Galaxie peak finding algorithms

9. Sample composition • Table 1. Sample composition

10. Conditions • Table 2. Chromatographic Conditions

11. Chromatographic analysis • Figure 1. Upper chromatogram: Pursuit XRs C18 5 μm 4.6 mm x 150 mm, 1 mL/min and 20 μL injection volume. Lower Chromatogram: Pursuit XRs C18 5 μm 21.2 mm x 150 mm, 21 mL/min and 420 μL injection volume Good correlation of retention times between prep and analytical analyses

12. Overloading and Fraction Collecting • A 1 mL injection of sample 2 was made onto the preparative column and this run was fractionated. • Figure 2. 1 mL injection of sample 2 onto a preparative column with a flow rate of 21 mL/min. Color-coded bars indicate the fraction and vertical lines indicate fractionation times.

13. Fraction collection log • Galaxie chromatography software has a colour coded fraction collection log shown below • Allows easy identification of fractions collected

14. Collect and Reinject • Resulting fractions were reanalysed on the 940-LC using a PDA. • Use of spectral libraries were used to gauge peak purity • Note the excellent match of fraction 5 for naphthalene showing high peak purity