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Frequently Asked Questions & Answers for SepaFlash Columns
(1)Whether the silica flash cartridge can be used repeatedly or not?
The silica flash columns are disposable and for single use, but with proper handling, the silica cartridges can be reused without sacrificing performance.
In order to be reused, the silica flash column needs to be simply dried by compressed air or flushed with and stored in isopropanol.
(2)Whether the silica cartridge can be eluted by methanol or not?
For normal phase column, it is recommended to use the mobile phase where the ratio of methanol does not exceed 25%.
(3)What is the limit for using polar solvents like DMSO, DMF?
Generally, it is recommended to use the mobile phase where the ratio of the polar solvents does not exceed 5%. The polar solvents include DMSO, DMF, THF, TEA etc.
(4)Compared with silica flash columns, what is the special performance for the alumina flash columns?
The alumina flash columns are an alternative option when the samples are sensitive and prone to degradation on silica gel.
(5)What are the points of attention for using C18 flash columns?
For optimal purification with C18 flash columns, please follow these steps:
① Flush the column with 100% of the strong (organic) solvent for 10 - 20 CVs (column volume), typically methanol or acetonitrile.
② Flush the column with 50% strong + 50% aqueous (if additives are required, include them) for another 3 - 5 CVs.
③ Flush the column with the initial gradient conditions for 3 - 5 CVs.
(6)What are the suitable preservation conditions for C18 flash cartridge?
Proper storage will allow C18 flash columns to be reused:
• Never allow the column to dry out after using.
• Remove all organic modifiers by flushing the column with 80% methanol or acetonitrile in water for 3 - 5 CVs.
• Store the column in the above mentioned flushing solvent with end fittings in place.
(7)Solutions for solid sample loading?
Solid sample loading is a useful technique to load the sample to be purified onto a column, particularly for samples of low-solubility. In this case, iLOK flash cartridge is a very suitable choice.
Generally, the sample is dissolved in a suitable solvent and adsorbed onto a solid adsorbant which could be the same as used in flash columns, including diatomaceous earths or silica or other materials. After removal / evaporation of the residual solvent, the adsorbent is put on top of a partly filled column or into an empty solid loading cartridge. For more detailed information, please refer to the document iLOK-SL Cartridge User guide for more details.
(8)Questions about thermal effect in the pre-equilibrium process for flash columns?
For the large size columns above 220g, the thermal effect is obvious in the process of pre-equilibrium. It is recommended to set the flow rate at 50-60% of the suggested flow rate in pre-equilibrium process to avoid obvious thermal effect.
The thermal effect of mixed solvent is more obvious than single solvent. Take the solvent system cyclohexane/ethyl acetate as an example, it is suggested that use 100% cyclohexane in the pre-equilibrium process. When pre-equilibration is completed, the separation experiment could be performed according to the preset solvent system.
(9)Questions about back pressure in the process of using the flash column?
The back pressure of flash column is related to the particle size of packed material. The packed material with smaller particle size will result in higher back pressure for the flash column. Therefore the flow rate of the mobile phase used in flash chromatography should be lowered accordingly in order to prevent the flash system from stop working.
The back pressure of flash column is also proportional to the length of column. Longer column body will result in higher back pressure for the flash column. Furthermore, the back pressure of flash column is inversely proportional to the ID (internal diameter) of the column body. Finally, the back pressure of flash column is proportional to the viscosity of the mobile phase used in flash chromatography.
(10)What exactly is a column volume for the flash column?
The parameter column volume (CV) is especially useful to determine scale-up factors. Some chemists think the internal volume of the cartridge (or column) without packing material inside is the column volume. However, the volume of an empty column is not the CV. The CV of any column or cartridge is the volume of the space not occupied by the material pre-packed in a column. This volume includes both the interstitial volume (the volume of the space outside the packed particles) and the particle’s own internal porosity (pore volume).
(11)What is the test method of column volume for the flash column?
Column volume is approximately equal to dead volume (VM) when ignoring the additional volume in the tubings connecting the column with the injector and the detector.
Dead time (tM) is the time required for elution of an unretained component.
Dead volume (VM) is the volume of the mobile phase required for elution of an unretained component. Dead volume can be calculated by the following equation:
VM =F0*tM.
Among the above equation, F0 is the flow rate of the mobile phase.
(12)Does functionalized silica dissolve in methanol or any of the other standard organic solvents?
No, end-capped silica is insoluble in any commonly used organic solvent.
(13)Does functionalized silica dissolve in water?
Bare silica will start to dissolve in aqueous solution of pH 9, albeit very slowly. In the solvent with pH lower than 9 the bare silica is stable. If the pH of the solvent exceeds 9, the dissolution will speed up as the pH increasing. For functionalized silica, the workable range of solvent pH is between 2 and 12 since the residual hydroxyl groups are end-capped.
(14)Questions about the connector for large flash columns?
For column size between 4g and 330g, standard Luer connector is used in these flash columns. For column size of 800g, 1600g and 3000g, additional connector adaptors should be used to mount these large flash columns on the flash chromatography system. Please refer to the document Santai Adaptor Kit for 800g, 1600g, 3kg Flash Columns for more details.
(15)What about the compatibility of SepaFlash columns on other flash chromatography systems?
For SepaFlash Standard Series columns, the connectors used are Luer-lock in and Luer-slip out. These columns could be directly mounted on ISCO’s CombiFlash systems.
For SepaFlash HP Series, Bonded Series or iLOK Series columns, the connectors used are Luer-lock in and Luer-lock out. These columns could also be mounted on ISCO’s CombiFlash systems via extra adaptors. For the details of these adaptors, please refer to the document Santai Adaptor Kit for 800g, 1600g, 3kg Flash Columns.
Frequently Asked Questions and Solutions for SepaBean machine
1. In the welcome page of SepaBean App, “Instrument not found” was indicated. What is the possible reason for this?
Possible reasons: The instrument is not powered on or is still in the booting process without indicating "Ready". Or network connection for iPad is error.
Solutions: Power on the instrument and wait for its prompt "Ready". Make sure the iPad network connection is correct. Also make sure that the router is powered on.
2. The main screen on the front panel of the instrument prompts "Network recovery" and the instrument cannot be found.
Possible reasons: The router is not powered on or the router is faulty.
Solution: Check and confirm router status to make sure the iPad can be connected to the current router.
3. What is the approximate volume of tubings and connections inside the instrument?
The total volume of system tubing, connetors and mixing chamber is about 25 mL.
4. What is the approximate delay time between seeing the peak in the chromatogram and collecting the corresponding fraction in the fraction collector?
The total delay volume from the detector to the collection nozzle is 2.5 mL. For example, if the flow rate is 30 mL/min, it will take about 5 seconds for the eluting peak to be collected in the test tube.The delay volume will be automatically deducted by the system during collection.
5. What is the approximate volume delay between seeing the peak in the chromatogram and eluting out the corresponding fraction from the waste outlet? How long does it take?
The delay volume is approximately 7.5 mL. It will take about 8 seconds at the flow rate of 30 mL/min. Deducting 2 seconds for signal processing, the actual delay time is about 6 seconds.
When automatic collection mode is adopted, the system will automatically calculate the signal delay and the user does not need to worry about the sample loss. When fractions are collected in waste collecting mode, it is recommended to deduct the delay time for more precise collection.
6. How to switch between the normal phase separation and reversed phase separation?
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.
7. There are bubbles in the pre-column tubing.
Possible reasons: The solvent filter head is not clean or sticked with impurities. Or there are immiscible solvents in the solvent lines
Solutions: 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.
8. What need to do if bubbles are found inside the column and the column outlet?
Check whether the solvent bottle is lack of related solvent and replenish the solvent.
If the solvent line is full of solvent, please do not worry. Air bubble does not affect the flash separation since it is inevitable during solid sample loading. These bubbles will be gradually drained out during separation procedure.
9. What is the cleaning function in "Pre-separation"? Does it have to be performed?
This cleaning function is designed to clean the system pipeline before separation run. If "post-cleaning" has been performed after the last separation run, this step could be skipped. If it is not performed, it is recommended to do this cleaning step as instructed by the system prompt.
10. Occasionally, solvent is found leaking from the base of the column holder when the column holder lifts up.
Possible reasons: Solvent leakage might be due to the solvent level in the waste bottle is higher than the height of the connector at the base of the column holder.
Solution: Place the waste bottle below the operation platform of the instrument, or quickly move down the column holder after removing the column.
11. Why do we need to equilibrate the column before separation run? To what extent should the column be equilibrated?
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.
The equilibration is done when the column is totally wetted and looks translucent. Usually this can be done in flushing 2 ~ 3 CVs of the mobile phase. During the equilibration process, occasionally we might find that the column cannot be completely wetted. This is a normal phenomenon and will not compromise the separation performance.
12. When touching the button of “install the flash column”, the column holder head does not lifte up automatically. What’s the problem?
Possible reasons: 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.
Solution: 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 (Please refer to Appendix 1 for more details).
13. There is a small amount of leakage at the top of the loading column when solid sample loading is utilized. What’s the problem?
Possible reasons: The Luer connector is not tightened properly or the connector of the solid loading column is worn.
● Check whether the Luer connector is fully tightened. It is recommended to apply a downward force during the tightening process of the connector in order to tighten the connector.
● Replace the worn sample loading column.
14. After the separation started, the SepaBean App prompts alarm information of "Tube rack was not placed". Any reason for this?
Possible reasons:
1. The tube rack is missing.
2. The tube rack is not correctly placed in right position.
3. The tube rack is faulty and cannot be recognized by the instrument.
1. Check if the tube rack is correctly placed in the right position. When this is done, the LCD screen on the tube rack should show a connected symble as shown in the below picture.
2. If the tube rack is faulty, user can choose customized tube rack from the tube rack list in SepaBean App for temporary use (please refer to Appendix 2 for more details). Or contact the local technical support.
15. The instrument prompts alarm inforamtion of high backpressure and cannot run normally. What’s the problem?
Possible reasons:
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.
Remove the flash column from the instrument and lower the system flow rate to check if the back pressure is normal. If the back pressure is still high, please refer to Appendix 3 for more solution details.
16. There are negative signal responce in the flash chromatogram, or the eluting peak in the flash chromatogram is abnormal, e.g. no peak in chromatogram though the sample has UV absorption.
Possible reasons: The flow cell of the detector module is contaminated by the sample which has strong UV absorption. Another possible reason might be due to solvent UV absorption which is a normal phenomenon.
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. Please refer to Appendix 4 for more details.
17. Chromatogram baseline keeps drifting upward when ethyl acetate was employed as the eluting solvent. What’s the problem?
Possible reason: 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.
Solutions: Change the detection wavelength to another one. 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.
18. When collecting eluents from the colletion nozzle, the eluents sprinkle outside the collection tube. What should we do?
Possible reasons: Sample precipitates or previous residual sample accumulates at the collection nozzle.
Solutions: Go to the maintenance page of the instrument and run the collection nozzle cleaning with proper solvents. At the meanwhile use proper solvent to clean the outer part of the collection nozzle.
19. Why does test tube rack turn “white screen”? How to deal with it?
Possible reason: The circuit controlling the LCD screen on the test tube rack is interfered.
Solutions: As shown in the below picture, long press the button with "light up" sign on the LCD screen for about 10 seconds to reset the screen. Afterwhile the LCD screen should return to normal state. If screen reset is not working, please refer to Appendix 2 for temporary emergency solution.
Appendix 1
Emergency solution for column holder abnormalities
When the column holder head cannot be lifted up normally, please follow the emergency alternative methods as listed below:
1. As shown in the below picture, user can install the column on the top of the column holder head. Liquid sample can be injected directly onto the column. Solid sample loading column can be installed on the top of the separation column.
2. Install the column on the auxiliary column holder with the inlet and outlet connected by related connectors and tubing.
Appendix 2
Emergency solution for test tube rack abnormalities
When the test tube rack has a problem of white screen or black screen, or cannot be correctly detected by the instrument, we can choose a specific type of test tube rack for emergency use. The detailed procedure is described as follows.
1. As shown in the below picture, touch the tube rack drop-down list to select the tube rack corresponding to the model currently in use.
2. Take the 18*180mm-36mL test-tube rack as an example, when only one of the test tube racks is inserted, we put it on Position 1 which is located at the right part of the rack tray (as shown in the below picture). Then choose 18*180mm-36mL*1 from the tube rack list. Now the instrument knows that the test tube rack is currently inserted into Positon 1 of the rack tray and will automatically pause the running when the last test tube has finished collecting fractions.
3. When we plan to insert two tube racks of this model, we just need to choose 18*180mm-36mL*2 from the tube rack list. In this case the instrument knows that two test tube racks of this model have been inserted in the rack tray. By default, the fraction collection will start from Rack 1 then goes to Rack 2 when Rack 1 is full of collected fractions. When Rack 2 is also full of collected fractions, the instrument will automatically pause the running until new rack is inserted.
Appendix 3
How to deal with abnormal high back pressure
When the flash column is removed from the instrument, flushing the system with a low flow rate, e.g. 30 mL/min, and the normal back pressure should be within 20 psi. When the back pressure is higher than 50 psi under this opeartion, it indicates that there may be blockage in the system flow path. It is recommended to use a solvent which is easy to dissolve samples to clean the system lines. The recommended flow rate is around 15 mL/min and user should flush the systme lines for about 15 minutes.
Recommended cleaning solvents
If instrument is running normal phase separation for the most time, try to flush the system lines with dichloromethane or ethyl acetate for the first washing step, then replace with methanol or ethanol for the second washing step.
If instrument is running reversed phase separation for the most time, it is recommended to flush the system lines with methanol or acetonitrile for the first washing step, then replace with dichloromethane or ethyl acetate for the second washing step. It should be ensured that the eluting solvent to be used in the following separation is miscible with the washing solvent used in last step.
After cleaning, user should check whether the back pressure is lowered. If the back pressure goes down to around 20 psi then the instrument is recovered to normal status. When the back pressure cannot be lowered to normal range, please contact the local technical support for further help.
Appendix 4
Flow cell inspection and cleaning
When unusual chromatogram is found during the running process, such as no eluting peak for a long time or negative baseline, probably the detector module especially the flow cell should be inspected. Please find detailed steps as follows.
Step 1: Check the spectrum of the lamp
Open the SepaBean App and go to Instrument Maintenance, then check the spectrum of the lamp at the Deuterium/halogen lamp page as shown in the below figure.
As shown in the above figure, the X axis relates to the wavelength and the Y axis relates to the lamp energy intensity. When the lamp is in a good state, the lamp energy intensity should be higher than 30%. In ideal state the lamp energy intensity should be higher than 80% or even more. In case the lamp energy intensity is lower than 30%, it is probably that the flow cell is contaminated or the lamp has reached its lifetime and needs to be replaced with a new one.
Attention: The lamp energy mentioned here is measured when water is used as the solvent in the flow cell. When organic solvents such as n-hexane, petroleum ether, dichloromethane, ethyl acetate, methanol, etc. are used, the measured lamp energy intensity might be lower than 20% since these organic solvents have a relatively strong absorption in the ultraviolet band (below 250 nm). In this case, it is recommended that user could take lamp energy intensity at 300 nm or higher wavelength as the reference, or replace the solvents in the flow cell with water for inspection.
Step 2: Clean the flow cell
According to the characteristics of the sample in last run, choose a solvent with good solubility for the sample to flush the flow cell in low flow rate for long time. 
● Suggested cleaning paratemers:
Cleaning flow rate: 15 mL/min
Cleaning time: 20 min
Inspect the lamp energy intensity again after cleaning the flow cell. If the intensity is still low, then further ultrasonic cleaning for the flow cell is required.
Step 3: Ultrasonic cleaning for the flow cell
1. Disassemble the flow cell from detector module
i. As marked by red circles in the left figure, use a screwdriver to unscrew the four fixing nuts on the side panel of the detector module.
ii. As marked by red circles in the left figure, unscrew the inlet and outlet line connected to the flow cell.
iii. As marked by red circles in the left figure, unscrew the fixing nuts of the flow cell by hand.
2. Check the cleanliness of flow cell lens.
As shown in the above picture, the flow cell lens is contaminated by sample, which will affect the signal response of the detector. In this case, ultrasonic cleaning for the flow cell is required.
3. Ultrasonic cleaning for the flow cell
Use a beaker added with proper solvent and put the flow cell into the beaker. As shown in the below picture, make sure that the entire flow cell body (the stainless steel part) is immersed in the solvent. Ultrasonically clean the flow cell for about 20 minutes then take it out for inspection. Do the ultrasonic cleaning again or change to another solvent for cleaning in case there are still residual contamination on the flow cell lens.
4. Inspection after cleaning
Check the cleanliness of the flow cell lens when ultrasonic cleaning is done. As shown in the below picture, there are no contaminants on the lens and the flow cell could be re-installed and now ready to use.
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