1. LC-trap and LC-TOF basic plumbing. The two LC systems - on the ion-trap and the ESI-TOF - are more different than they look. The one on the trap has a maximum flow-rate of 20 無/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 無/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 無/minute, 'standard' for flow-rates up to about 200 無/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 ion-trap is always 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 trap 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 無/minute, you should probably inject less than 1 無; if you're on the TOF, with a flow-rate of 200 無/minute, you could probably go as high as 25 - 30 無.

4. Fragile UV flow-cells. The standard UV cell on the TOF can handle pressures up to around 400 bar, but the micro cell on the trap can only handle around 40 bar. So - if you're using the micro UV cell with capillary columns - you should pay continual attention to your analyses because the microspray capillary clogs from time to time 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 limited amounts of sample.

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. The ion trap should be able to routinely detect mid-femtomoles of pure material (on-column) and the TOF somewhat less, so there is usually no good reason to inject more than a few picomoles (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.

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 may get you better data.

8. Trap vs TOF: sensitivity. Sensitivity on the ion-trap suffers from high background or co-eluting peaks. The trap typically holds only 100,000 ions so analytes present at low abundance may be undetectable in the presence of contaminants or co-eluters. In addition, the wider the mass range the lower the sensitivity at the extremes. So the trap should generally be operated with a relatively narrow mass range with your target m/z in the middle (although - for reasons I don't yet understand - the resolution suffers if the mass range is too narrow). The TOF, on the other hand, is insensitive to this, so - except for the additional burden on hard-drive space - you will lose neither sensitivity nor data quality by operating over a wide mass range. (You may, however, have to work a bit harder to pull your data out from the background.)

9. 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 always get maximum sensitivity with the smallest column diameter and 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 無/minute range on the trap with the micro spray offer a nice compromise, since these are easy to work with and typically give excellent results.

10. 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.

11. 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.

12. 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 (e.g., the ubiquitous m/z 391) with nice isotope peaks. If the background signal is weak and noisy, you should deal with it 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. The most common cause of poor signal is low flow or inappropriate spray conditions, although a dirty spray-shield can also be the villain. When really in doubt, check with me!

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. There's a laminated sheet near the instruments with a set of suggested starting points for a variety of sprayer configurations and flow rates. Remember, though, that these are suggestions and guidelines rather than gospel and rules and should be used only as starting points if you need to optimize for a particular analysis. 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).

14. 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.

15. Autosampler inserts. I'll cover things like this in an upcoming set of sample-prep guidelines, but you should generally use glass inserts rather than plastic inserts in the autosamplers in order to minimize plasticizer contaminants. (The supply is low at the moment, but Jung just ordered a new batch.)

16. 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 trap should be 80 psi or slightly above; for the TOF it should be around 100 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, but which is just right for the instruments. When you replace a tank, notify Amy so she can keep us supplied.

17. Remote data analysis. (Not strictly electrospray, but useful.) The beige Dell computer in 56-747A has data analysis software for the QStar, the ESI-TOF, and the ion trap, along with ChemWindows and Microsoft Office. It also has standard ChemStation software, so you can work with your diode-array data as well. The mass spectrometers and the two computers in 56-747A are all on a local network that interfaces with the web (and thus to DNA-net) via high-speed fiber optics.

18. Tune files on the TOF. Don't create a lot of different tuning ('method') files for the ESI-TOF. Stick to ESIautotune1.m since that always contains the most recent calibration.

19. Tune files on the ion-trap. The ion-trap is much more compatible than the TOF with customized tune files since 1. the tuning and calibration parameters are incorporated into the tuning files and 2. the trap can't be optimized over a wide mass range so it's important that the parameters be optimized for a given analysis. There is an important quirk, though, in that you have to be careful to save your trap files along with your ChemStation LC control files, since the trap is considered just another LC detector. So, if - for example - you decide to use someone else's LC method, then open or create a great trap control method but forget to save it with your renamed method, then the next time you use that method it will bring up the original user's trap control file - perhaps to disastrous results. (This is also true of the LC methods themselves. Any changes you make will be discarded if you don't resave the file.)

20. Stabilizing flow on the ion trap. 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 無/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 無/minute and it'll work fine.

21. 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 ion-trap 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 無/minute. But remember the first sentence of this paragraph!