Abstract
Using the Waters AutoPurification System, separation methods can be developed on an analytical scale and transferred to preparatory scale on the same system, reducing a laboratory's overall capital investment. Here we illustrate a systematic approach to scale-up using the separation of Kudzu (Pueraria lobata) root extract to move from a 4.6 mm I.D. analytical column separation through 10, 19, and 30 mm I.D. preparatory column separations. The Waters OBD Prep Calculator is featured.
Introduction
Chromatographic separation methods can be developed on anyscale. To minimize the consumption of sample and solvents, thereis a benefit in developing separation methods on a small scaleand transferring them to a larger scale. Taking into account theimportant parameters and applying appropriate scaling factors,in a logical manner, enables users to scale up from analyticalchromatography to larger-scale preparative separations easilyand successfully. In this application note, the analytical-scaleseparation of Kudzu (Pueraria lobata) root extract is used todemonstrate the calculations and techniques used to move froma 4.6 mm I.D. analytical column separation through 10, 19, and30 mm I.D. preparatory column separations.
Kudzu is a climbing, woody or semi-woody, perennial vinewith a tuberous root. The roots of Kudzu contain a number ofpotentially useful isoflavones, including daidzein, daidzin,genistein, genistin, and quercetin. Kudzu is also a unique sourceof the isoflavone puerarin. Kudzu root extracts are thought toreduce alcohol intake and reduce alcohol withdrawal symptoms.Antibacterial, anti-cancer, anti-inflammatory, and antioxidanteffects have also been noted.1
Figure 1. AutoPurification System.
Experimental
Extraction
Kudzu root pieces (20 g) were added to 100 mL of 9:1water/methanol and shaken for one hour, allowed to standovernight, and shaken for one additional hour. This extractwas centrifuged at 3000 RPM for 20 minutes and usedwithout further treatment.
Separations
Chromatographic separations, at all scales, were carried out usingthe Waters AutoPurification System (Figure 1), which consistedof the following components:
| Pump: | Waters 2545 Binary Gradient Module |
| Detectors: | Waters 2998 Photodiode Array, Waters 3100 Mass Detector |
| Injector/collector: | Waters 2767 Sample Manager |
| Column management: | Waters System Fluidics Organizer An initial analytical-scale separation was developed on a Waters SunFire C18, 5 μm, 4.6 x 50 mm Column, using the conditions described below. |
| Column temp.: | Ambient |
| Flow rate: | 1.5 mL/min |
| Mobile phase A: | Water + 0.1% Formic acid |
| Mobile phase B: | Methanol |
| Gradient: | 5% to 70% B over seven minutes |
| Injection vol.: | 20 μL |
| Detection: | UV (200 to 400 nm) and MS Full Scan 150 to 700 m/z |
The resulting chromatogram (Figure 2) showed a number of resolvedcompounds and was considered an acceptable candidate for scale-up.
Figure 2. Analytical separation (4.6 mm I.D.) of Kudzu root extract.
Results and Discussion
Scale-up method
Asystematic approach to scale up will provide the best possibleresult. The ultimate goal is to maintain chromatographicresolution between key components and enable users to betterpredict chromatographic performance between analytical andpreparative chromatography.
There are a number of key factors to consider when approachingthis scale-up process.
Column chemistry
The heart of the separation is the column. Ideally, you shouldchoose column chemistries that are identical. If the analyticaland preparative columns are of different chemistries, it becomesvery difficult to predict the preparative separation based on theanalytical results. Waters offers a wide range of column chemistrychoices available in analytical- and preparative-scale dimensions.As well as the chemistry itself, particle size should also beconsidered. Columns of the same particle size will provide similarresolution of critical pairs at both separation scales. Columnlength also influences the separation efficiency; columns ofidentical length, when scaled, give similar separation power.It is possible to scale to shorter or longer columns, but keep inmind that the separation will change.
Injection volume
To maintain peak shape and loading capacity, the injection volumeneeds to be suitably scaled using the following equation:
where Vol is the injection volume (μL), D is the inner diameter ofthe column (mm), and L is the column length (mm). For example, a20 μL injection on a 4.6 x 50 mm column corresponds to a 341 μLinjection on a 19 x 50 mm preparative column.
Flow rate
To maintain separation quality, the flow rate must be scaled basedon column dimensions. With columns of identical particle size, thefollowing equation is used to geometrically scale flow rate:
where F is flow rate (mL/min) and D is the inner diameter of the column(mm). For example, a 1.5 mL/min flow rate on a 4.6 mm I.D. columnequates to a 25.6 mL/min flow rate on a 19 mm I.D. column.
Gradient scaling
When columns are of identical length, no changes to the gradientprofile are required. If scaling to longer or shorter columns, thegradient segment volume must be maintained to preserve theseparation profile.
The Waters Optimum Bed Density (OBD) Prep Calculator,a free download, (Figure 3) is an easy-to-use tool that aidsin these analytical-to-preparative scaling calculations(www.waters.com/prepcalculator). The Waters OBD Prep Calculatorwas used to convert the analytical separation method to thepreparatory separation methods described in this application note.
Figure 3. Waters OBD Prep Calculator.
Using the Waters OBD Prep Calculator
To calculate injection volume and flow rates, select the mass loadscaling calculation (Figure 4) from the opening screen. Input youranalytical and preparative column dimensions, analytical flowrate, and injection volume and the calculator returns the correctpreparative values.
Figure 4. Waters OBD Prep Calculator mass load scaling calculation.
If your column lengths are identical, you can simply input thepreparative flow rates into your gradient table using the same gradientsegment times as your analytical method. Alternatively, for gradientmethods, choose the basic gradient scalar calculation (Figure 5) fromthe opening screen, select your analytical and preparative columndimensions, input your analytical gradient table, and click the Calculatebutton. The preparative gradient table is automatically calculated andshown on the bottom half of the page. The Waters OBD Prep CalculatorUser Guide gives detailed instructions on use of all calculator functions.
Figure 5. Waters OBD Prep Calculator basic gradient scalar calculation.
Results
To demonstrate the previously described techniques, the analyticalseparation method described in the experimental section was scaled tothree different preparative dimension columns (10.0, 19.0, and 30.0mm I.D.). The scaled flow rates and injection volumes (all calculatedusing the Waters OBD Prep Calculator) are shown in Table 1.
Table 1. Waters OBD Prep Calculator scaled flow rates and injection volumes.
All of the preparative columns are SunFire Prep C18 OBD, 5 μm,50 mm in length, and all of the separations were performed on thesame system as the analytical-scale chromatography. As can beenseen in Figure 6, regardless of the scale, the chromatography(UV TIC) is very similar. When compared to the original 4.6-mmI.D. scale (Figure 2), it can be seen that in terms of resolution andretention time the chromatography is again very similar.
This simple experiment demonstrates that a systematic approach toscale up meets the goal of maintaining chromatographic resolutionbetween key components, and enables users to better predictchromatographic performance between analytical and preparativechromatography. This exercise also demonstrates the uniquecapability of the Waters AutoPurification System, which allowsusers to perform both analytical and preparatory chromatographyon the same system with no performance compromise.
Figure 6. Scaled preparatory separations, 10 mm I.D. (top), 19 mm I.D. (middle), 30 I.D. mm (bottom).
Conclusion
Analytical chromatography can be successfully scaledto preparatory chromatography easily by using asystematic approach.
- The use of identical column chemistry and identical column lengths maintains separation quality.
- Waters’ proprietary Optimum Bed Density (OBD) Column design offers excellent sample loading and column stability in an extensive array of chemistries and configurations.
- The Waters Prep OBD Calculator aids in the scaling calculations.
- Using the Waters AutoPurification System, separation methods can be developed on an analytical scale and transferred to preparatory scale on the same system, reducing a laboratory’s overall capital investment.
- Developing methods on the analytical scale and transferring them to preparatory scale reduces solvent and sample consumption, while reducing waste disposal cost, compared to developing separation methods only at the preparatory scale.
References
- PDR for Herbal Medicines. Thompson Healthcare Inc, Montvale NJ, U.S.A. 2007; 4th Ed.
