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Frequently Asked Questions
Here are some of the frequently asked questions we've received from you regarding our products.
What are some common reasons that columns clog?
Tips: 4 principle sources of column fouling particles:
- HPLC solvents
- Pump seals
- Poorly cleaved fused silica tubing
- Sample
1.) Filter the solvents either before they go into the solvent bottles, or use an in bottle filter (this is the best way). If the problem is the HPLC solvents than you will see column fouling with just solvents running over the column.
2.) Pump seal particles are typically filtered out using an inline filter, this is OK when you have high flow rates and/or are splitting the flow, but can't really be done on a nanoflow system, especially a high pressure one, so there is really nothing that can be done here. If the problem is the particles from the pumps, than you will see column fouling with just solvents running over the column.
3.) If particles are coming from poor cutting of the fused silica, then you can get a SHORTIX cutting tool (it is recommended that you pass the solvent through any newly cleaved tubing before you attach a column to the flow path) this will make sure that any particles get washed out. Fouling from bad cuts of the tubing should only destroy one column.
4.) Perform some kind of clean up on your samples prior to injection. For protein digests it is recommended that they get passed through a Ziptip or some similar device. This filters out any particles and should also absorb any material that will "irreversibly" bind to your column, after the Ziptip we recommend speedvac to remove the organic solvent.
If your sample is concentrated enough, you can simply dilute. Use filter solutions for this, until you are sure the particles aren't coming from them. Fouling from the sample will occur only after an injection.
Remember that once a column fouls, whatever fouled it (including particles) is still in the system. If you just switch out the column, than that leftover material will end up going over the new column and possible fouling it too. So as you are chasing down your problem you will need to be flushing the flowpath (including the valves and syringes) to get rid of any retained garbage.
What is your advice on using a long sample loading time (~15 minutes) at a high flow rate (~1 uL/min), and then switching to a low flow rate of 300nL/minute for the chromatography with a splitless pump?
It is much easier to do the fast load/slow gradient with a splitter on a conventional or microflow pump then on a splitless pump such as the Microtech system. The reason to do this is that it is faster. Say you are loading a 5 ul sample, this takes 5 min at 1 ul/min and almost 17 min at 300 nl/min. The problem with the Microtech system is that the only place for the pressurized solvent to go is out through your column so you build up a big pressure at 1 ul/min and when you switch to 300 nl/min you still have this big pressure so you have to wait until enough solvent has left the end of the column to equilibrate to the pressure required to run at 300 nl/min. Since the pumps are still moving, albeit slower, this can take a long time! Some other splitless pumps such as the Eksigent can apparently handle this mode of operation.
Solution for the Microtech pump:
Two ways you can do the fast load/slow gradient on the Microtech.
One way is to run for a while at 300 nl/min and note the pressure in the pumps then ramp up to 1 ul/min once the pressure has equilibrated set the flow to 0.000 ul/min and measure the time it takes for the pressure to drop to the level required to run at 300 nl/min. So program your gradient method to start at 1 ul/min for 15 minutes, then set the flow to 0 for the time it takes for the pressure to drop to the 300 nl/min level, then set the flow to 300 nl/min and start the gradient at this point. The pressures and times will be column specific so this will have to be done with each column.
The other way to do this is to put a Tee fitting just before your column. One arm of the Tee goes to the column, one arm goes to the injector valve on the Microtech, and the last arm goes to the unused valve on the Microtech. This unused valve gets plumbed so that the line from the tee is either connected to a line going to waste or to a plug. When the valve is connected to the plug all the flow will go over the column so you will have the valve at this position most of the time. When the position of the valve changes, most/all of the flow will go out the waste line. Have this valve set to the plug position and do your loading at 1 ul/min, change the flow to 300 nl/min and then switch the valve to "waste", the built up pressure will rush out the waste line. If you noted the pressure at 300 nl/min you can easily figure out how long to leave the valve on "waste" before switching it back to "plug". This is essentially the way Microtech runs the instrument if you are using a trapping column, where the trapping column would be between the injector valve and the Tee. I would plumb this with 50 um tubing between the tee and the valve and 75 or 100 um tubing between the valve and your waste container. This should allow you to lower the pressure within a reasonable time scale.
The problem with this set up is that you have introduced some dead volume and "unswept" volume in the Tee, this can lead to sample carry over problems and you will definitely need to flush the system in both the "plug" and "waste" positions between runs
I know it should be easier than this, but you need to keep from having to repressurize the pumps. Because the aqueous will repressurize much faster than the organic (especially at 97% aqueous), this will lead to problems with your gradient.
Solution for the Microtech pump:
This could be due to a lot of things. You may be over loading the column and you are seeing some peptides "breaking through". I wouldn't load much more than 1 pmol of peptide mixture. If you are overloading the column you should also see the same peptides more than once in the run (i.e. you will see them at 7.5 min when they overwhelm the column and then again when the material bound to the column elutes.) The other reason could be due to the flow never equilibrating to 300 and this is wreaking havoc on the gradient. I am not sure how the gradient would develop under these conditions.
Dead volume audits
If you see peptide peaks at 7.5 minutes, and if the dead volume in your system is If the dead volume of your system (from the injection valve to the inlet of the mass spec) is 7.5 ul, then the peaks you see are due to break through (assuming 1 ul/min flow). If the dead volume on the Microtech is about 2 ul, at 500 nl/min oneI would see the garbage from the injection appear at 4 minutes after injection. The easy way to do this is to run an injection of 0.1% AcOH in 30% MeOH (can pretty much use any solvent other than what's coming from the HPLC, 100% isopropanol will give a dramatic change and it can also be used to clean the column at the same time. This will cause a big change in the spray that you can monitor with the mass spec. Just take note of the time it takes from injecting the sample to seeing a change in the mass spec, multiply by flow rate and you get dead volume.
I think most problems can be traced to issues with dead volume. On a traditional system a 1 ul dead volume is probably less than 1% of the flow, on a nanoflow system it can be 500%. That is a huge difference.
What are the advantage and disadvantages of a column heater?”
Maintaining a stable column temperature gives reproducible retention times, especially in labs where temperatures fluctuate. High temperatures also allow one to take advantage of improved mass transfer and reduced pressures. It is more typical to use moderate temperatures (40-50C) as higher temperatures are preferred for performing fast-LC or when lowering pressure is required when using smaller particles or other specific applications.
Advantages:
- Reproducible retention times
- Sharper peaks, improved resolution, greater sensitivity.
- Reduced or eliminated carryover (of anayltes in the pores of the stationary phase). Greater heats assists in washing out the pores in the higher organic wash at the end of a run.
- Reduced pressures allow for use of smaller particle sizes with conventional LC systems for even greater resolution and sensitivity performance.
- Greater flow rates if fast-LC methodology is preferred.
Drawbacks:
- In a recent workshop I attended, bubble formation (or degassing during analysis) was not a problem with those using temperatures up to 100C. Of course, stringent degassing techniques are critical for any nanospray operation (sonication under vacuum is a general accepted practice).
- Thermal decomposition of specific labile analytes is a consideration which requires one to examine their chemistry for appropriate methods and temperatures. An easy validation is to perform analysis of the same sample at room temperature and then at elevated temperatures to test for changes in the chromatographic profile.
Thank you for your interest in Phoenix S&T’s “Programmable High Voltage(HV)” options and our column heater. We at Phoenix S&T are committed to making nanospray a more robust technique with improvements in reliability and performance.
How would one (or the spray controller), choose the optimum voltages to get even spray throughout a gradient?
To set up HV programming, I recommend a blank run to be performed with visual observation of the spray adjusting and recording the optimum voltage every n minutes (n is subjective, generally depending on the length of the run and rate of the LC gradient). Once the voltages/times for a method are established, these parameters are programmed into software that controls our 6kV power supply.
Optimum electrospray conditions are dependent on a number of factors, two being the onset voltage and the surface tension of the mobile phase. My experience, working with both high aqueous and high organic mobile phases, is that minor changes of just 100V alter the jet and plume of the spray. Though some spray emitters are more sensitive to changes in mobile phases than others, optimum spray conditions translate to more stable conditions and higher ion efficiency (thus greater sensitivity). In addition, if one uses a conventional LC pump and splits the flow for nanospray, then flow rates will also trend as the mobile phase composition varies; another reason to incorporate programmable HV.
The programmable HV feature is available with our AutoNano System or as a standalone unit. With the AutoNano System, current sensing is employed which can detect spray failure and perform a number of operations to restore spray thus allowing for unattended performance. In addition, the AutoNano has a dual LC column feature which allows for the equilibration of one column while analysis is being performed on a second column. This feature allows for increased throughput as equilibration time is no longer a factor in analysis run times. Finally, the AutoNano also has Direct Infusion capability in which sample is sprayed directly from a well of a CHIP without mobile phase pumping required. This technique is excellent for top-down proteomics, multiple MSn experimentation for structural elucidation, and high-throughput MS identification and quantitation.
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