I have an HPLC column with an i.d. of 4.6 mm and a flux of 400 microL/min which in principle requires the ESI source to have between 60 and 100 arbitrary units of sheath gas. I intend to do non-targeted metabolomics.
However, the equipment only achieves 64 arbitrary units of sheath gas, ruling out the possibility of optimizing that parameter. Is it relevant that eventual optimization that is no longer possible, and therefore should I get a new column with lower i.d. (and lower sheath gas requirement)? Or it is not important?
If the sheath gas flow is not high enough, you will lose sensitivity, perhaps massively. You therefore need to make sure that the sheath gas is at least sufficient.
Run your mobile phase through your column isocratically for 30 mins or so at its maximum % aqueous and examine the source. Most sources have a window which you can view the spray, and if this shows signs of instability, condensation or fogging of the window, this suggests that your gas flow is not high enough. You may also be able to plot the chamber current, which should be stable in isocratic conditions. Try running at 40, 50 and 60 units. If you have no signs of fogging or instability at 40 / 50 units, you will be fine at 60. If there are signs of an insufficient gas flow a 50 units, then 60 may not be high enough and you should attempt to improve matters.
Why are you limited to 64 units? Is this the capacity of your gas generator, or has one of the internal compressors failed or is leaking? If the capacity is insufficient, you could try adding bottled gas to boost the gas flow, but may need clever plumbing to make it work.
How much you can do depends on how advanced your method development is.
1 Drop to a smaller column diameter to give a lower flow. If you already have a draft method, this may need work to get the gradient right due to the gradient delay volume
2 If you have good MS sensitivity you could split the eluent post column, effectively reducing the mobile flow rate but using the same HPLC conditions
3 Reduce the flow rate and slow everything down. You should not need to adjust the gradient too much and the retention times will remain proportional to the existing/draft method.
4 Optimise some of the other source parameters to try to improve the desolvation. You may be able to increase the needle voltage or gas temperature or the positioning of the needle. The gains are likely to be small, but may be enough to make a difference
5 Adjust the mobile phase composition so that the target analytes elute at a higher organic percentage, diverting the higher aqueous flow to waste. You may be able to do this by adjusting the gradient rate or the aqueous modifiers.
6 Add a volatile solvent post-column to assist desolvation. Although the flow would increase, this may be offset by the easier vaporisation
You will need to at least investigate (especially if this is part of a thesis) whether you have a problem, and you may find that a combination of these actions gives you the best results
Richard
Only way to answer is do it experimentally. The 4.6 mm is old technology that requires higher flow rate and needs higher gas flow and temperature to dry the droplets. I would recommend to use a smaller diameter to increase peak height and you can use lower flow rate to dry mobile phase quickly with lower gas flow / temperature. Since you may get better sensitivity, you can inject less to reduce matrix suppression. You can use halo-type column instead of fully porous particle to speed up the analysis and get better peak shape.
If the sheath gas flow is not high enough, you will lose sensitivity, perhaps massively. You therefore need to make sure that the sheath gas is at least sufficient.
Run your mobile phase through your column isocratically for 30 mins or so at its maximum % aqueous and examine the source. Most sources have a window which you can view the spray, and if this shows signs of instability, condensation or fogging of the window, this suggests that your gas flow is not high enough. You may also be able to plot the chamber current, which should be stable in isocratic conditions. Try running at 40, 50 and 60 units. If you have no signs of fogging or instability at 40 / 50 units, you will be fine at 60. If there are signs of an insufficient gas flow a 50 units, then 60 may not be high enough and you should attempt to improve matters.
Why are you limited to 64 units? Is this the capacity of your gas generator, or has one of the internal compressors failed or is leaking? If the capacity is insufficient, you could try adding bottled gas to boost the gas flow, but may need clever plumbing to make it work.
How much you can do depends on how advanced your method development is.
1 Drop to a smaller column diameter to give a lower flow. If you already have a draft method, this may need work to get the gradient right due to the gradient delay volume
2 If you have good MS sensitivity you could split the eluent post column, effectively reducing the mobile flow rate but using the same HPLC conditions
3 Reduce the flow rate and slow everything down. You should not need to adjust the gradient too much and the retention times will remain proportional to the existing/draft method.
4 Optimise some of the other source parameters to try to improve the desolvation. You may be able to increase the needle voltage or gas temperature or the positioning of the needle. The gains are likely to be small, but may be enough to make a difference
5 Adjust the mobile phase composition so that the target analytes elute at a higher organic percentage, diverting the higher aqueous flow to waste. You may be able to do this by adjusting the gradient rate or the aqueous modifiers.
6 Add a volatile solvent post-column to assist desolvation. Although the flow would increase, this may be offset by the easier vaporisation
You will need to at least investigate (especially if this is part of a thesis) whether you have a problem, and you may find that a combination of these actions gives you the best results
Richard
Definitively, buy a new column (smaller i.d., I mean): it's a much more sensitive, cost effective and durable alternative to a long, painful and probably worthless investigation (unless that's a crucial point in your work!)
Thank you all for your answers!
Richard, I am limited to 64 units due to the capacity of the gas generator. As you suggested, I have run at 40, 50 and 60 units and indeed have not observed any fogging or condensation. However, as you all suggested, I am going to try first to exchange the column to one with a smaller i.d..
Thanks again
It definitely impacts your sensitivity since in part the sensitivity in ESI is dependent upon the desolvation of the ions. Most MS systems are limited to somewhere between 0.4 and 0.5 mL/minute; with the 4.6 column you are below the optimum flow range and pushing the upper limit of your MS. If you go to a 2.1 your optimum flow is 0.25 mL/minute; it is a great place to operate as far as your MS is concerned. If you change your column you can then optimize your gas flow for your specific analysis.
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