Counter Current Chromatography Questions Dynamic Extractions are Frequently Asked

The following are the most often asked questions for your immediate access

How to change flying leads on DE Spectrum

Can I use my existing LC equipment to run a DE HPCCC machine?

For the Spectrum and Midi HPCCC units, any HPLC or low-pressure LC system can be used providing the pumps are non-pulsing. Furthermore, they must be able to flow at up to 20 ml/min for use with Spectrum and up to 100ml/min for use with Midi.

However, remember that because these units are low pressure you will need to fit a back pressure in the system to make your pumps work. If in doubt check with Dynamic Extractions.)

I do not know which solvent table to use?

Start initially with the Hemwat solvent screen. We find that this is suitable for 70-80% of the purifications we are asked to perform, whether they be small or complex molecules. This can be found on the method development page in technical support section of this website.

Having used this screen to perform a partitioning study then speak to Dynamic Extractions for further screens that can be used.

Where do I begin the method development procedure?

Firstly download the detailed methodology from this website. This will provide you with a systematic approach to develop your purification method.

If after performing this procedure you are still not making the progress you require contact Dynamic Extractions for support.

My target molecule is not eluting in the first three column volumes of mobile phase?

We assume that you are running in classical elution mode (i.e. running mobile phase until your target elutes. Instead of this run in elution extrusion mode and after 3 column volumes of mobile, stop the mobile phase and begin pumping the stationary phase instead. Collect fractions, which will contain both mobile and stationary phases, for off-line analysis.

If your compound is:

• in the first 50% of extruded stationary phase you will need to modify your solvent system by changing the ratio of solvents to decrease the distribution ratio of your compound;
• in the last 50% of extruded stationary phase, you will need to try another run with the opposite phase as the mobile phase.

My target molecule elutes after more than 20 minutes?

If separation resolution is good enough then increase the mobile phase flow rate to make your compound elutes earlier. Otherwise, change the polarity of the stationary phase to reduce the relative attraction of the target molecule to that phase. This means for example if the molecule is very polar and the stationary phase is aqueous, you need to make the mobile phase more polar.

What initial volume of sample do I load onto a particular column?

For analytical column with volume less than 20ml inject 15-20mg of crude sample loaded into sample loop of 2.5% of column volume. For any columns with bigger volume load 0.1g of crude per each 100ml of column volume (100ml Vc) into 5% of Vc sample loop. In other words, for example, for 1L HPCCC machine it will be equal 1g of crude. If the separation resolution is good enough, then increase loading by mass up to 0.5g or 1g per each 100ml Vac and then by volume up to 7.5 or 10% of column volume.

In case of crude extracts from bark or root, the loading typically is about 0.1-0.3g per each 100ml of Vac; for extracts from leaves and stems the loading is about 0.5-0.7g per each 100ml Vac; for synthetic products loading can rich up to 2.5g per 100ml Vac.

Can HPCCC be used for screening of Natural products?

Yes, it has been successfully used for the screening of Natural products [4, 5]. The separation should be run at a slow flow rate (about 0.5 ml/min for analytical coils with volume less than 20ml) for 60 minutes, then the machine should be stopped and extrusion mode applied. Collected fractions can be used for off-line analysis and bioassays to determine the bioactivity.

If you have a question that is not covered, please ask that question through contact us