Electrospray helpful hints and answers to FAQs.

Some general information that everyone should have…

1. QQQ and LC-TOF basic plumbing. The two LC systems - on the QQQ and the ESI-TOF - are more different than they look. The one on the triple-quad is usually configured for a maximum flow-rate of 20 μL/minute, and should only be used with columns 1mm ID or less. The LC on the TOF can be used routinely with standard columns (2 - 4 mm ID, but I prefer 2 mm). This can also be used with capillary columns, but a splitter and manual injector are required except for isocratic analyses. The reason for this requirement is that gradients won’t reach the column for a LONG time at the low flow rates (a few μL/minute or even less) used with narrow-bore columns. Another consideration is that there are three flavors of sprayers, ‘micro’ for capillary columns and flow-rates up to about 20 μL/minute, ‘standard’ for flow-rates up to about 200 μL/minute, and ‘standard-with-spacer’ for higher flow-rates. All of these will at least work under all conditions (although the ‘micro’ will develop high back-pressure at the higher flows), but the spray assembly should match your flow rate for optimum performance. (Don’t try changing the sprayer, though, unless I’ve checked you out on the procedure.) The QQQ is usually set up with the micro sprayer, but the configuration on the ESI-TOF is changed frequently, so if you’re having sensitivity issues, check to see which sprayer is being used.

2. How little can I inject? The autosampler on the triple-quad can reliably inject as little as 200 nL, but the one on the TOF is not reproducible at those levels.

3. How much can I inject? In this same vein, injection volumes should be based on the flow rate. If, for example, you’re using a narrow-bore column at a flow-rate of 3 μL/minute, you should probably inject less than 1 μL; if you’re on the TOF, with a flow-rate of 200 μL/minute, you could probably go as high as 25 - 30 μL.

4. Fragile UV flow-cells. The UV cell on the TOF can only handle pressures below 60 bar. So you should pay continual attention to your analyses because the spray capillary can occasionally clog, and this can quickly develop enough back-pressure to destroy the UV cell (replacements cost over $1000.00).

5. Basic buffer guidelines. Use the minimum buffer concentration that will give you adequate chromatography, and use only volatile buffers. Even volatile buffers, e.g., ammonium acetate, can suppress ionization and can thus cause problems if you have low analyte concentrations.

6. Be gentle. Try to never inject crude reaction mixtures (I know it’s tempting), especially if there is a small amount of product in the presence of huge amounts of starting material or - worse - salts. Protein digests should be zip-tipped before injection. The triple-quad should be able to routinely detect low-to- mid-femtomoles of pure material (on-column) and the TOF somewhat less, so there is usually no good reason to inject more than a picomole or so (and some good reasons not to inject more, e.g., the ion source gets dirty faster or the chromatography may deteriorate). Start with a dilute sample and increase the concentration if necessary, rather than vice-versa. Also, see the next section.

7. How accurate is ‘accurate’? If you’re using the ESI-TOF for accurate mass, be sure to check the calibration. I check and calibrate frequently, and the instrument is fairly stable, so the mass accuracy is usually within specifications, i.e., 3 ppm or better. But ‘usually’ does not mean ‘always’, so if mass accuracy is important, you should take the time to check the calibration. In addition, the instrument often calibrates at far better than 3 ppm, so the extra effort will usually get you better data. If you need the best accuracy that the instrument can deliver, you should use the reference sprayer (although there is often enough residual calibrant to use as an internal reference). The detector on this instrument will begin to saturate at ion-counts near or above 10⁷, leading to sometimes-wildly inaccurate masses.

8. Why should I use a small column? Remember that electrospray is concentration-dependent and flow-rate/droplet-size dependent. For a given amount of material, you will get maximum sensitivity with the smallest column diameter, lowest flow-rate and the smallest spray aperture. The downside is that the smaller you go the more technical problems you encounter. Semi-micro or micro columns - 1 mm to 0.35 mm ID with flow rates in the 10 - 3 μL/minute range on the triple-quad with the micro spray offer a nice compromise, since these are easy to work with and typically give excellent results.

9. Will I see anything at all? Always run something familiar before running a real sample. This may seem too cumbersome - especially if you’re in a hurry - but will definitely save you time in the long run.

10. Those dangerous first few minutes. Never leave the instrument during the first 10 minutes or so of an analysis that you’ve just set up. Stay around long enough to make sure that the flow is stable, that the injection completes, and that no leaks develop. Check back frequently to make sure that everything remains ok. If you’re running a long work-list, make sure the first two or three samples run smoothly before leaving the lab. Don’t push ‘Start’ and walk away.

111. Is the spray working? Before starting an analysis, make sure that the mass spectrometer signal is stable and robust. You should always see some background ions with nice isotope peaks. If the background signal is weak and noisy, with no stable peaks, you should deal with this before running a sample. When in doubt, go to a low mass range, e.g., 80 - 600; there are always solvent contaminants in this range that show up well if the instrument is working properly. The most common cause of poor signal is low flow (e.g., a leak) or inappropriate spray conditions, although a dirty spray-shield can also be the villain.

13. Optimizing the spray. The four things most often adjusted to optimize the spray itself are 1. nebulizing-gas pressure; 2. drying gas flow; 3. drying gas temperature; and 4. spray voltage. The actual values depend mainly on solvent composition and solvent flow but also to some extent on the analyte. Drying gas temperature can be a problem with low flows and concentrated samples; if it’s too high, the solvent may evaporate too quickly from the spray capillary and precipitate sample, leading to a plugged capillary (these are almost impossible to clean and cost over $100.00 to replace).

13. What should I use to dissolve my sample? The solvent that you use for the autosampler should closely match the initial solvent composition for your gradient. If there is a significant mismatch, you may get bizarre chromatography or - worse - the sample may precipitate out and clog the injector needle or the switching valve.

14. Autosampler inserts. You should generally use glass inserts rather than plastic inserts in the autosamplers in order to minimize plasticizer contaminants, and always use Agilent vials. Never put tape labels on the vials since this can prevent them from going completely into the tray; this can halt the injection sequence.

15. Liquid nitrogen. Always keep an eye on the supply of liquid nitrogen. The indicators on the tanks are notoriously unreliable, so you may have to shake the dewar to see how heavy it is. The nitrogen pressure for the ion triple-quad should be 80 psi or slightly above; for the TOF it should be around 90 psi. The high-pressure setting on the regulator is often confusing, since it usually reads just above the peg. Most people are familiar with gas cylinders, which often have pressures above 2000 psi when fresh. The liquid nitrogen dewars in the lab, however, are set for about 240 psi as head pressure, which barely moves the needle on a standard regulator, but which is just right for the instruments. When you replace a tank, notify Marcia so she can keep us supplied.

16. Remote data analysis. (Not strictly electrospray, but useful.) The new Dell computer in 56-747A has data analysis software for the QStar, the ESI-TOF, and the triple-quad, along with ChemDraw and Microsoft Office. The electrospray mass spectrometers and two of the computers in 56-747A are all on a local network; the Spectrum Mill server is on the web but - for security reasons - is not connected to the local network.

17. Tune files on the QQQ. The QQQ is more compatible than the TOF with customized tune files since the analytical requirements vary widely depending on the acquisition mode.

18. Stabilizing flow on the triple-quad. The Agilent capillary pumping systems need a fair amount of backpressure to stabilize. So - if you try to run the pumps at low flows (1 - 5 μL/minute) without a column, they’ll overshoot the setpoint, oscillate wildly, and then shut down. If you need to troubleshoot without a column, set the flow to 20 μL/minute and it’ll work fine.

19. How much background should I see? This depends on lots of things, e.g., solvent composition, solvent quality, spray parameters, and scan range. A typical/reasonable background on the QQQ base-peak chromatogram would be ion counts in the low-to-mid 10⁵ range with a semi-capillary column (0.35 mm - 1 mm), but this could go below 1 x 10⁴ with nanospray. The TOF will give roughly comparable counts (low-to-mid 10⁵ in the total ion chromatogram) with a fairly clean solvent at 200 μL/minute. But remember the first sentence of this paragraph!