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

Varian 940-LC Application note

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

Factor = r2(prep)/r2(analytical) where r = radius 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

Scale up factors are applied to the flow rate and the injection volume 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

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.

This is achieved in 2 ways: 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!!!

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

Table 1. Sample composition Sample 1 composition for analytical injection Uracil 40 mg/L Acetophenone 60 mg/L Methyl benzoate 60 mg/L Toluene 3g/L Naphthalene 500 mg/L Sample 2 composition for preparative injection 10 x Sample 1 Solvent HPLC grade acetonitrile

Table 2. Chromatographic Conditions Analytical conditions Preparative. conditions Column Pursuit™ XRs C18, 5 μm, 4.6 mm ID x 150 mm length Pursuit XRs C18, 5 μm, 21.2 mm ID x 150 mm length Flow rate 1.0ml/min 21.2ml/min Sample injection volume 20 µL 420µL Solvent 60:40 Acetonitrile/H2O Collection mode NA Threshold Trigger parameter 40mAu Tube Size 50mL

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

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.

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

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