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Preparative columns
For applications in the field of preparative chromatography, MZ-Analysentechnik GmbH supplies ready-packed HPLC columns with almost the entire range of chromatographic materials in dimensions from 8.0 to 50.0 mm ID (semi-prep - prep). In the manufacture of our in-house preparative columns, we use a specially optimised packing procedure for each packing material and each pre-packed preparative column is then checked to ensure that it meets our strict quality requirements.
Technical Data
How do I choose the right preparative column?
In principle, the stationary phase should already be defined. Normally, experiments are carried out with analytical columns and these methods are then scaled up to the preparative scale. It is important to ensure that the particle size ideally remains the same. If a larger particle size is used for the preparative column, it is advisable to maintain the ratio of particle size to column length. In addition, the mobile phase and the solvent of the sample should also be identical.
Therefore, when selecting the correct preparative column, the question of which dimension is required should be asked. As maximum loadability is desired in preparative chromatography, it is advisable to first optimise the analytical method and achieve the highest possible resolution. Once the highest resolution has been achieved, the loadability can be analysed:
- A sample with a high concentration is prepared and the injection volume is increased.
- Several samples with different concentrations are prepared and the injection volume remains constant
This should maximise the sample quantity so that the peaks to be separated can still be separated with sufficient purity. This maximum loadability varies from column to column and is highly dependent on the individual separation problem and resolution. This loadability must therefore be determined empirically for each separation. The resulting poor peak shapes or overloading of the detector can be ignored during such preparative preparation.
The maximum loading capacity of the analytical column has been determined - what happens now?
Once the maximum load (defined amount of analyte in a defined injection volume) has been determined, you should consider how much of the analyte should be separated in one run. This injection volume can then be used to calculate the appropriate internal diameter using the following formula. If the particle size of the analytical and preparative column is identical and therefore the length remains the same, the length term becomes one and can be neglected.
Further adjustments
With the appropriate inner diameter of the preparative column, the remaining parameters such as the flow velocity and possibly the gradient time can also be calculated.
Adjusting the flow velocity
Adjustment of the gradient
Different systems are often used for an analytical and preparative method. This also results in different dwell and dead times (dwell volume and dead volume). To compensate for this, short isocratic steps can be incorporated at the start of the gradient. The following equation applies to gradients that are transferred from one system to another:
tD,Analy Dwell time for the analytical system
tI,Analy Time of the isocratic step at the beginning of the gradient for the analysed system system
tc,Analy Dead time for the analytical system
tD,Prep Dwell time for the preparative system
tI,Prep Time of the isocratic step at the beginning of the gradient for the prep. system system
tc,Prep Dead time for the preparative system
Compensation of different dwell volumes of both systems
In order to compensate for different residence volumes between an analytical and preparative system, short isocratic steps can be incorporated before the actual gradient. The time of such a step can be calculated using the following formula, whereby the residence volumes as well as the column dead volume must be known for both systems.
tI,Prep Time of the isocratic step at the beginning of the gradient for the prep. system. system
tI,Analy Time of the isocratic step at the beginning of the gradient for the analy. system system
FPrep Flow rate for the preparative system
FAnaly Flow rate for the analytical system
vC,Prep column dead volume for the preparative system
vD,Prep Dwell volume for the preparative system
vC,Analy column dead volume for the analytical system
vD,Analy Residual volume for the analytical system
With all these calculations, you can scale up your analytical method to a preparative method. In this process you can also use our HPLC method transfer tool can also support you in this process. We are also happy to help you with the calculations. Get in touch with us! At MZ-Analysentechnik GmbH, you can also obtain any material as a preparative column to ensure seamless scaling from analytical to preparative methods.
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Technical support
MZ Tips for the Preparative HPLC (EN)
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