1 simple scale-up on a 940-lc analytical to preparative hplc dennis hoobin varian australia pty ltd...
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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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