"Ask Kamel"
Students and others ask me questions almost daily. Although I know some of the questions at the present time, I am going to include them as they come along. These questions come from all levels. Some from students who never even heard about mass spectrometry. Some are somewhat informed, although via regular classes. Others are fairly informed, but they are just missing some of the more specific pieces of the puzzle. A good number of those latest questions make me think and search for answers. At the present time, I am going to lay out those questions as they occur or as they are given to me.
Q: I have a sample, and I want to do mass spec. What should I do?
A: Just for short, my answer will be: what kind of compound(s) are you expecting? Is it a mixture? Do you know some of the chemical and physical properties of your sample, or maybe how you obtained this sample? If your sample contains organic, non thermolabile, then we should try EI and/or CI.
Q. Should I submit my sample or request training?
A. That depends. In general, all GCMS work must be done by the student. Staff will provide training and help with the development and implementation of "special projects". LCMS projects should be done by the student. Extensive training and assistance will be provided. "Pure samples" just requiring a nominal mass scan or elemental formula confirmation can be submitted. Students submitting samples do have certain responsibilities, however, related to proper sample submission and aspects of data analysis and interpretation.
Q: How high do you heat the sample when doing mass spec?
A: Obviously, we are talking solids prob,e EI or CI. The maximum probe temperature is about 400 degrees Celsius. Solids-probe EI/CI is not routinely done in our lab anymore. The technique is still available by special request on the GCT mass spectrometer. Pure samples previously analyzed using solids-probe EI/CI methods are now analyzed primarily using solution-based atmospheric pression ionization methods, including ESI (electrospray) or APCI (atmospheric pressure chemical ionization).
Q: I have a sample. I was told by a colleague to do CI, but I don't know why. Why not EI?
A: In practice, we generally use EI prior to using CI. These two techniques are complementary and not competitive. Let me be clear: When doing EI, we are hoping to observe the molecular ion peak. If we don't, well then, perhaps it is decomposed in the gas phase due to the excess internal energy of the ion. In this case, CI with CH4 or NH3 gases is warranted. In the positive ion mode CH4, we are usually looking at MH+, while for NH3, we are expecting MH+ and /or MNH4+. So to answer your question, EI and CI are complementary, and EI is usually used before CI, especially for unknowns.
Q: My sample does not seem to go through the GC. What is your suggestion?
A: Well, if you are doing everything right, then perhaps your sample contains thermolabile compounds. If this is the case, we have to try to do ESI. Perhaps try a direct infusion or loop injection first. If we see some relevant peaks, then we can consider doing online HPLC/ESIMS. Another technique to consider is APCI. If your sample works with the APCI, online LC/APCI will maybe be better. I will tell you why when we get there.
Q: I want to do ESI, but my sample is not soluble in anything. What do you suggest?
A: First, solubility, even if it's poor, is required for doing ESI. Samples may be poorly soluble, but nonetheless, some of the molecules are trapped in the solvent. So, let's try. Solubility is relative... Samples do need to be centrifuged and filtered to remove all undissolved compounds, especially when solubility is poor. Also, the miscibility of the solvent with the mobile phase has to be considered. We do not want your sample "crashing" out of solution in the mobile phases. That can lead to instrument clogging.
Q: I heard from some sources that for ESI, we have to use only a methanol/water mixture. But my sample is not soluble in that mixture, but very soluble in methylene chloride. What should I do:
A: That is not true. Any solvent can be used successfully with ESI. The reason you have got that info is that in the early days of ESI, people were using it mainly for peptides and proteins, and for those types of samples, a mixture of methanol/water/1% acetic acid is recommended for positive ion mode ESI. The sample must be soluble to be analyzed by ESI, and the miscibility of the solvent with the mobile phase must be considered.
Q: What kind of samples is ESI used for?
A: ESI covers a large spectrum of samples. From low molecular weight polar compounds to high molecular weight biopolymers such as proteins.
Q: I have gotten the ESI data, but there is a peak at 23 mass units higher than what MW is!!
A: Obviously, we are talking positive ion mode; well, that is probably a sodium adduct. Sometimes, potassium adducts are observed too at 39 mass units higher. In fact, in some case,s we spike the sample with some sodium to promote the formation of MNa+.
Q: But my sample doesn't contain sodium. How did it get there?
A: First, sodium, in small quantities, is everywhere, including in your glass sample vial. Consider using plastic vials or inserts. Sometimes, even when we use deionized water or high-grade solvents, we may still see the sodium adduct. If the affinity for the sodium is high, then if there is any out there, it will be in play.
Q: I want to analyze a compound that is present as a salt. Will ESI do?
A: Yes. In general, only the positive ion portion is observed, or if doing negative ion mode, only the counter ion (-) will be observed. For example, if you have a sodium salt compound, negative ion mode ESI will yield [M-Na]-
Q: I want to analyze a protein with ESI. What is the concentration needed?
A: Of course, it depends on the nature of the protein and other factors. Just to tell you a good range: micromolar to less than millimolar solution will be fair.