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

Dennis HoobinVarian Australia Pty Ltd

679 Springvale Rd Mulgrave VIC Australia

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Varian 940-LC Application note

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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

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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

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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

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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.

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•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!!!

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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

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Sample composition

Table 1. Sample compositionSample 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

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Conditions

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 60:40 Acetonitrile/H2O

Collection mode NA Threshold

Trigger parameter NA 40mAu

Tube Size NA 50mL

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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

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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.

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Fraction collection log

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

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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

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