SepaBean™ machine

Column equilibration can protect the column from being damaged by exothermic effect when solvent quickly flushing through the column. While dry silica pre-packed in the column being contacted by the solvent for first time during separation run, a lot of heat might be released especially when the solvent flushes in a high flow rate. This heat might cause the column body to deform and thus solvent leakage from the column. In some cases, this heat might also damage heat sensitive sample.

It maybe caused by the lack of lubricating oil at the rotating shaft of the pump.

The total volume of system tubing, connetors and mixing chamber is about 25 mL.

The flow cell of the detector module is contaminated by the sample which has strong UV absorption. Or it might be due to solvent UV absorption which is a normal phenomenon. Please do the following operation:

1. Remove the flash column and flush the system tubing with strongly polar solvent then followed by weakly polar solvent.

2. Solvent UV absorption problem: e.g. while n-hexane and dichloromethane (DCM) are employed as the eluting solvent, as the proportion of DCM increases, the baseline of chromatogram may continue to be below zero on Y-axis since the absorption of DCM at 254 nm is lower than that of n-hexane. In case of this phenomenon happens, we can handle it by clicking the “Zero” button on the separation running page in SepaBean App.

3.The flow cell of the detector module is heavily contaminated and needs to be cleaned ultrasonically.

It might be due to that the connectors on the column holder head as well as on the base part are swelled by solvent so that the connectors are stuck.

User can manually lift up the column holder head by using a little bit force. When the column holder head is lifted up to a certain height, the column holder head should be able to be moved by touching the buttons on it. If the column holder head cannot be lifted up manually, user should contact the local technical support.

Emergency alternative method: User can install the column on the top of the column holder head instead. Liquid sample can be injected directly onto the column. Solid sample loading column can be installed on the top of the separation column.

1. Low energy of light source;

2. The circulation pool is polluted; Intuitively, there is no spectral peak or the spectral peak is small in the separation , The energy spectra shows a value of less than 25%.

Please flush the tube with an appropriate solvent at 10ml/min for 30min and observe the energy spectrum.If there is no change in the spectrum,it seems low energy of light source,please replace the deuterium lamp; If the spectrum changed, the circulation pool is polluted，please continue to clean with appropriate solvent.

Please check the tube and connector regularly.

The detection wavelength is set at the wavlength lower than 245 nm since ethyl acetate has strong absorption at the detection range lower than 245nm. The baseline drifting will be most dominant when ethyl acetate is used as eluting solvent and we choose 220 nm as the detection wavelength.

Please change the detection wavelength. It is recommended to choose 254nm as the detection wavelength. If 220 nm is the only wavelength suitable for the sample detection, user should collect the eluent with carefully judgement and excessive solvent might be collected in this case.

Clean the solvent filter head completely to remove any impurities. Use ethanol or isopropanol to flush the system completely to avoid immiscible solvent problems.

To clean the solvent filter head, disassemble the filter from the filter head and clean it with a small brush. Then wash the filter with ethanol and blow-dry it. Re-assemble the filter head for future use.

Either switch from normal phase separation to reversed phase separation or vice versa, ethanol or isopropanol should be used as the transition solvent to completely flush out any immiscible solvents in the tubing.

It is suggested to set the flow rate at 40 mL/min to flush the solvent lines and all the internal tubings.

Please reposition the bottom of the column holder after Loosen the screw.

1. The system flow rate is too high for the current flash column.

2. Sample has poor solubility and precipitates from the mobile phase, thus resulting tubing blockage.

3. Other reason causes tubing blockage.