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. 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

5. 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

6. 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. 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!!!

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. 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

10. 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

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