Sample Preparation and Submission Guidelines
Mass spectrometry, like all chemical analytical methods, works best when performed within a narrow range of analytical parameters. Operating outside of this range normally results in severe degradation of performance. It is therefore vitally important that you prepare samples for mass spectral analysis in the correct manner for the particular analytical method you have selected.
Listed below are sample preparation guidelines for each of the techniques employed at JHU chemistry, along with links to downloadable (in .pdf format) sample submission forms for the VG-70S high resolution mass spectral service. If you have further questions regarding sample preparation, please contact the facility for advice before submitting your sample.
We normally request ~0.5 mg of sample for FAB and direct probe EI /CI analyses (though we can often make do with less when only a small amount of sample exists – please seek advice PRIOR to submission). This may seem like a lot of material, but these techniques are not as sensitive as other techniques such as GCMS or Electrospray.
EACH individual sample must be submitted with a correctly completed sample submission form indicating the service required. Samples should be purified and dried prior to submission, and submitted for analysis as crystals or as an oil in glass screw-top 1DRAM vials (or other alternative containers where appropriate – please contact the MS facility for advice prior to submission) that are CLEARLY labeled with the investigators Name, Research Group and a systematic sample ID (please, no complete chemical names). Do not submit solutions. Sample vials must be securely attached to the completed submission form. Loose sample vials will NOT be accepted (the exception being for samples that require cold storage prior to analysis – please deposit these samples in the refrigerator in Rm. B14 and mark the submission form for these samples “In Fridge” ). Samples should be deposited in the appropriate trays in room B14, which is normally open from 9-5, Monday -Friday. There is one tray for EI and CI samples and one for FAB samples. Blank submission forms are also located in Rm. B14 or may be downloaded from this link as a pdf file. Samples without completed submission forms, forms which are incomplete or incorrect, or are submitted in inappropriate containers will NOT be accepted for analysis. Radioactive, Highly Toxic or other “Dangerous” samples requiring specialist handling will NOT be accepted.
Samples for EI or CI analyses are normally introduced using a heated direct insertion probe and so it is important that the vaporization point of these samples is noted on the submission form in order to obtain the correct sample volatization rate to achieve good quality data. Samples for direct probe EI or CI should volatilize at temperatures below 350C under high vacuum conditions. Samples for FAB analyses are dissolved in an appropriate solvent and mixed with a matrix (usually m-nitrobenzyl alcohol) deposited on a target. Please specify an appropriate solvent to dissolve your sample (NOTwater) that is miscible with the matrix. Common solvents employed are Methanol, Ethyl Acetate, Chloroform and dichloromethane. However, if you require the use of a specialist solvent and/or matrix, please indicate this. Failure to specify a suitable solvent may result in delayed analysis.
Most samples for the ESI instrument are introduced via syringe pump. They must be purified and dissolved in an appropriate carrier mixture. Carriers are normally semi-polar solvents such as MeOH or CH3CN, along with 0.1 – 1% volatile modifier. Organic carriers are often mixed 1:1 with deionized water, but the exact ratio of the organic / aqueous content can be varied, though it should NOT contain >80% water. Organic solvents should be HPLC grade. Preferred modifiers include Formic Acid, Acetic Acid, Ammonium Acetate and Ammonium Formate. TFA should NOT be used and TEA and TEAA should only be used if no other alternatives are available. Non-volatile modifiers such as phosphates, borates, or similar salts should NOT be used as they cause spray problems. Acidic modifiers are typically used when obtaining positive ion spectra, and typically result in the formation of protonated species. Basic modifiers should be used when obtaining negative ion spectra. Sample concentration should be around 1 micromolar (although it is recommended that you prepare a dilution series of 10, 1 and 0.1 uM of your analyte in solution) and the entire solution should be filtered prior to introduction to remove any insoluble material which would otherwise block the sample introduction line. 1-2 mL of solution is normally sufficient for analysis.
Samples should be prepared by dissolution in non- or moderately polar volatile solvents such as methanol, acetone, pentane, hexane, etc. Do NOT use water. Sample concentration should be prepared with the aim to achieve a column loading of approximately 10ng, which should yield reasonable mass spectra. E.g. a concentration of approximately 0.1mg/mL would be appropriate for samples injected with a split ratio of 10:1 and a 1uL injection volume. Alternatively, a 1uL splitless injection of 10ug/mL will also yield a 10ng column loading. Samples should be prepared in glass 1.5mL GC autosampler vials. Where only small volumes of sample solution exist, vial inserts can be used allowing samples to be analysed from as little as 20uL of solution. It is important that you ensure your sample components are volatile at temperatures of less than 300 degC, otherwise they will remain on the column and contaminate it.
The scientific literature contains many different methods of preparing samples for MALDI mass spectra and you will have to chose the method that is best for your particular samples. A guide to Maldi matrix selection and sample preparation for this instrument can be found here. However, in general, you need to mix and deposit your sample on the target plate in a 1:10 sample:matrix ratio. Typical matrices used are a-cyano-4-hydroxycinnamic acid and sinapinnic acid. A quick method of MALDI sample preparation is to prepare a solution mixture containing 1uM (1pmol/uL) of sample and 10uM (10pmol/uL) of matrix. 0.5uL of the mixture is deposited on the target and allowed to dry. Several repeat depositions can yield improved spectra. Allowable sample concentrations can range from 0.1uM to 50uM, with matrix concentration scaled accordingly.
Each user should have their own MALDI target which can be obtained from the Chemistry department stockroom. Existing users of the ABI Voyager DE-STR instrument located at the Johns Hopkins Medical School MS Facility can utilize their existing target plates with the Autoflex by using the provided adapter.
A sample identification grid corresponding to the standard Bruker 384 spot MTP target plates can be downloaded here.