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Frequently Asked Questions Below are answers to the most commonly asked questions regarding mass spectrometry here at the JHU chemistry department. This list is by no means exhaustive and you should contact the facility manager for further information should you need it. How much sample do I need to submit? – Please see the relevant section of the sample preparation guidelines page. What is the procedure for booking instrument time or submitting samples for High resolution analyses? – To book time on one of the self-service instruments (GC-MS/MaldiToF/ESI-IonTrap), you must first have completed a training and instructional session on the instrument with the facility manager and filled out an instrument authorization request. You will then be entered into the on-line instrument scheduling and billing system. You MUST NOT use these instruments without having completed a training session on the relevant instrument. Instrument time is reserved using the online scheduler. When you come to use the instrument, you should logon to the system at the kiosk provided in the lab. After using the instrument, ensure that you logout of the system before you depart. If you cannot make your appointment, please cancel your reservation time before it commences so that others may use the instrument. When making a reservation, please ensure that you allow enough time to complete your work AND to clean up the instrument. For more detailed instructions on how to use the online scheduler, please contact the facility manager. When submitting samples for the VG70S high resolution service, please complete a mass spectrum request form for each sample, indicating the service required. These forms can be found in Remsen B.14 or can be downloaded and printed out from this link. Please complete the form as fully as possible, the more information that you provide about your samples, the better (and the faster the analysis will be completed). In particular, please ensure that you provide contact details, a systematic sample ID (NOT the full chemical name), a valid billing code, suggested solvents for dissolving samples and an estimate of the vaporization temperature (for EI/CI samples). Samples submitted without this information may be returned unanalyzed. Please note any special sample storage and handling conditions. Attach the sample (in a clearly labeled sample vial) to the form using sticky tape and deposit in the correct tray in located in Remsen B14 (there is one for FAB samples and one for EI/CI analyses). Samples that are thermally sensitive should be placed in the refrigerator rather than being attached to the sample form, but the form should be clearly labeled that the sample is stored in the fridge. Please DO NOT deposit large blocks of samples at a single time (i.e. accumulated samples at the end of the year) as this slows up the sample queue. Samples will be analyzed as quickly as possible on a first-come, first-served basis and results will be returned to your mail box (Chemistry users) or to the specified mailing address (external users). How long does it take to get results? – We aim to return results from samples submitted to the high resolution MS service to you within one working week. Often results are returned faster than this. However, this can vary depending upon the sample load, instrument problems and maintenance needs, vacation schedules, etc. I requested an exact mass measurement, but I only received a nominal mass spectrum. Why wasn’t the exact mass done? - We usually run a nominal mass, full mass range scan on all samples first. If the nominal mass spectrum does not show the expected molecular ion, we cannot do an exact mass. What is the difference between chemical mass and exact mass? – For most chemical calculations, chemists normally employ the statistically average atomic masses listed in the periodic table. These chemical atomic masses represent a weighted average of all of the naturally occurring isotopes of an element. Because the mass spectrometer will separate molecules with different isotopic compositions, these average atomic masses are not correct for mass spectrometry of small molecules (under 2000 Da) where you have at least unit mass resolution. In this instance, you must use the exact masses of the individual isotopes. For example, carbon has a chemical atomic mass of 12.0110, but the correct value to use for small molecule mass spectrometry would be the mass of carbon-12, which is 12.0000. As another example, the chemical atomic mass of chlorine is 35.4527, but the correct value to use for small molecule mass spectrometry is the mass of chlorine-35, which is 34.9688. On the other hand, for mass spectrometry of large molecules (such as large peptides or proteins) by MALDI-TOF, the individual isotopic peaks are not usually resolved. In this case, the chemical or average atomic masses are the better values to use. What kinds of adducts are commonly observed in mass spectrometry? - By CI with methane, it is common to observe an MH+ ion, but many compounds also exhibit a significant (M-H)+ ion. By CI with methane you will also sometimes observe much smaller (M+C2H5)+ and (M+C3H5)+ adduct ions. For FAB, you usually detect an (MH)+ molecular ion and some fragments. You will also observe fragment and adduct ions from the matrix. At JHU, we normally employ 3-nitrobenzyl-alcohol as the matrix (m-NBA) which forms significant ions at m/z = 136, 154, 289, 307 and 460. For Electrospray MS in the positive mode, it is typical to detect (M+H)+ and relevant fragment ions, whilst with the negative ion mode, (M-H)+ molecular ions are normally observed. It is also common to observe clusters and other adduct ions as well. Molecules tend to cluster when run by electrospray, particularly if the sample concentration is high. Larger molecules that readily take on multiple charges, such as proteins and oligonucleotides, commonly appears as multiple groups of ions with increasing charge. What MS method should I choose? – This all depends on what results you are trying to achieve. If you require accurate mass analyses, here at JHU chemistry, such analyses require the use of the VG70S, which means that the sample must be ionizable by EI, CI or FAB. For sample preparation conditions and instructions, see the sample preparation and guidelines page. Accurate mass analyses are generally only useful for when you want to confirm the molecular formula of a sample, i.e. for publication purposes. They are performed over a narrow mass range, using added reference masses. If you do not know the composition of your sample, it is not usually worth requesting an accurate mass analysis. Most journals will accept an accurate mass analysis as confirmation of a molecular formula if the measured mass agrees with the theoretical mass with a difference of less than 5ppm (some journals will accept 10ppm). Accurate mass measurements at JHU Chemistry are normally performed in the range 100-900 Da. However, if you are require a nominal mass “survey” spectra (i.e. scanning over a large mass range to see which ions are present), then the range of options available is increased and the selected method is dictated by the characteristics of the sample to be analyzed. It is often the case that there is more than method mass spectral analysis available. You need to consider aspects such as how large the molecules is (large molecules may require use of the Maldi-ToF or electrospray instrument), how delicate or thermally sensitive the sample is (delicate samples may decompose when analyzed by use of direct insertion EI and CI MS), etc. Some examples of the types of analysis performed are given below. However, if you have questions regarding the type of analysis to select, please contact the MSF for advice. Stable organic molecules that are easily vaporized under vacuum and mild heating can usually be analyzed using EI. Small molecules that do not produce a molecular ion by EI will often display good adduct ions by CI with methane or by FAB. Volatile mixtures that need to be separated prior to analysis should be analyzed by GC-MS. Electrospray MS is useful for delicate samples that form solution phase ions, particularly those that acquire multiple charges such as proteins, peptides and nucleotides. Compounds containing large numbers of electronegative atoms or groups often run well by negative ion Electrospray. Can you analyze air sensitive samples? – It depends on how air-sensitive the samples are. Contact the facility ahead of sample submission for advice. What are those "extra" peaks we see in an exact mass spectrum? - In order to obtain an accurate exact mass measurement, it is necessary to introduce reference compounds that generate peaks of known mass simultaneously along with the sample. The "extra" peaks are from those from the reference compounds. For FAB, we generally use a 10% mixture of polyethylene glycol (PEG) in with the matrix as the reference. For EI and CI, the reference is generally high boiling Perfluorokerosene (PFK). A list of the most common reference peaks can be found here: PFK and PEG. |