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Listed in this appendix are common LC/MS questions from students and customers and my answers. This list is not exhaustive. One of the most common questions that I did not include was: “Why won’t my system start up?” I would ask the person on the phone if the system was plugged in. After the explosion on the other end settled down, I would say: “Sir, sometimes janitors unplug lines so they can plug in their polishers. Would you please check to see if it is plugged in?” Often, after about a minute or so, I would hear a quiet, embarrassed click as the phone was hung up.

1. Why do I need to use helium gas on a liquid chromatograph?
   Helium might be used for two reasons. Low-pressure-mixing-valve gradient systems suffer from bubbles being pulled out of solution and stalling the pump head unless air is flushed out of the solvents by helium purging. Sometimes the solvent reservoirs are pressurized with helium gas to aid in smooth solvent flow. Helium or nitrogen may also be used as the nebulizer gas in an atmospheric-pressure ionization interface to remove solvent, volatile buffers, and aid in ionizing compounds in the LC effluent.
   
   
2. Do I need a gradient system, and if so, why?
   Gradient systems let you control flow rate and solvent/buffer changes to improve chromatographic separations. They can be used to sharpen separations and to speed column reequilibration. A four-solvent gradient system is useful for methods development when equipped with methanol, acetonitrile, ammonium acetate buffer, and formic acid solution. But many quality control laboratories prefer to use inexpensive isocratic systems that run a constant-composition premixed mobile phase for rapid separations.
   
   
3. Do I need an autosampler?
   Autosamplers and robotic workstations provide reproducible injections and allow automation of the chromatographic separation, but add significant cost to a system. Many university laboratories prefer to substitute graduate students to do the job.
   
   
4. Why does my LC system keep shutting itself off?
   HPLC pumps are equipped with an overpressure setting to protect fragile columns. Perhaps your settings are too low, or your column frits may be plugged, providing too much backpressure. If the pressure settings are correct, you may have to clean the line from the injector to the column or the column frit. The most common cause of plugs in lines or frits is material not filtered from samples or mobile phase. Buffers precipitate when you switch between incompatible solvents. Washing buffers out with water before moving to a new organic mobile phase will help prevent this problem in the future.
   
   
5. What kind of sample preparation do I need to do before I inject?
   That depends on what you are analyzing. As much of the interfering compound(s) needs to be removed as possible: proteins precipitated, lipids extracted, cells and particulates filtered or removed. So me samples need to be concentrated to aid in detecting trace amounts in dilute samples. Check the literature for your particular compound, use traditional procedures for compound purification, and look into the possibility of using SPE columns for precolumn purification and concentration.
   
   

1. Which is better: ion spray, electrospray, or nanospray?
   Each interface has its use in LC/MS. Ion spray works best with neutral and nonpolar compounds. Electrospray is preferable with ionized or polar materials such as proteins. Nanospray is electrospray optimized to give the best production of ions for polar compounds from microflow columns, especially when only trace amounts of sample are available.
   
   
2. What is that liquid running off on the floor?
   The MS interface is designed to ionize only a tiny portion of the available sample. Part of the LC mobile phase is vaporized, but much of it is left behind and can be diverted to a secondary detector. At 1 to 2 mL/min, this represents a large volume of solvent that must be diverted to some type of reservoir for disposal or mopped up off the floor.