There are several instrumental parameters that can utilized to provide optimal data acquisition. The descriptions below should be useful when completing the sample submission form to ensure that your analytical experiments are conducted in the best possible manner.
CHOICES in IONIZATION
Electrospray Ionization – ESI
ESI is an atmospheric pressure, soft ionization technique that utilizes high voltages to generate quasi-molecular ions, [M + H]+ or [M – H]-, from non-volatile, polar liquid-phase analytes. The ions generated are then desolvated over a defined region and introduced in the mass spectrometer inlet as distinct gas phase ions. Electrospray ionization can be used on small molecules ranging in molecular weights from 50-2000 Da. Multiple charging of peptides and proteins can be achieved to enable analysis of very large molecular weight species at lower molecular weights, and data can be transformed to enable parent ion molecular weight determination.
Atmospheric Pressure Chemical Ionization – APCI
In APCI, a sample solution flows through a heated tube where it is volatilized and sprayed into a corona discharge to generate a plasma. Inside the plasma, proton transfer reactions occur to generate charged analyte species. APCI produces [M+H]+ ions but the source parameters must be optimized to avoid significant fragmentation. Polar, semi-volatile samples are the best candidates for analysis by APCI. Note that APCI has the ability to ionize less polar compounds than that of ESI.
Atmospheric Pressure Photonionization – APPI
APPI is ideal for non-polar speciation, aromatics, thiophenes, furans, et cetera, of compounds. LC eluent or sample is directly infused into a nebulizing tube to aid in desolvation of the sample. The desolvated ions are then exposed to a Krypton UV lamp, 120 nm photos ~10ev, where ionization can occur through direct ionization or proton-transfer reactions to generate either [M+H]+or M+⋅. The charged species are then swept into the mass spectrometry inlet for mass analysis. APPI is a soft ionization technique but ionization often time requires the selection of a dopant to increase ionization efficiency and sensitivity.
Positive ion electrospray analysis is commonly used to generate positively charged molecular ions through protonation to yield [M+H]+, where M signifies the molecular compound, and H, the additional proton. The m/z value at which one detects a singly charged ion is therefore 1 Dalton, Da, higher in mass than the molecular weight. Basic chemical functionalities that are most frequently ionized in the positive mode include basic nitrogen, pyridinic, furans and inorganic cations.
Negative ion electrospray analysis commonly used to generate negatively charged molecular ions through deprotonation to yield [M-H]-, where M signifies the molecular compound, and H, the lost proton. The m/z value where one would detect a singly charged deprotonated molecular ion is 1 Da lower in mass than the molecular weight. Acidic chemical functionalities that are most frequently ionized include neutral nitrogen, pyrrole, carboxylic acids and inorganic anions.
In special instances, sample adduction may be required to produce efficient ionization. Adduct formation is carried out in the solution phase prior to sample introduction. In negative ion ESI, the ionization of polar, neutral molecules or very weakly acidic species that do not generate stable negative ions through deprotonation often times form adducts with chloride ions. The resultant ion of increased molecular weight, +35 in the case of chloride, but possessing a single negative charge. In positive mode ESI, the addition of a salt, in very small quantities, less than 1mM, can facilitate ionization through the formation of a positively charged adduct with sodium, lithium, or ammonium. The observed positive ions result in a molecular weight increase equivalent to that of the salt ion, +23 for sodium, et cetera, and typically carry a single positive charge.
Ways to Get Your Sample Into the Mass Spectrometer
Sample introduction via direct infusion requires the sample be diluted to its final concentration, on the order of 1-10 µM, before ESI. The sample is placed in a syringe and pumped at a defined flow rate by a syringe pump through transfer tubing directly to the ESI source. This is the simplest sample introduction method and ideal for samples that do not require separation or additional sample preparation before mass analysis.
This type of sample introduction is often times utilized by an autosampler/HPLC system. Samples are prepared in autosampler vials at a given analytical concentration, usually 10x higher than the limit of detection, approximately 10-100 µM. The autosampler selects the sample of interest when cued and injects the sample into the ESI source via transfer tubing that has solvent flowing through it. The bulk solvent is supplied from solvent reservoirs on the HPLC system and pumped into the ESI source through transfer tubing. The sample is mixed with the bulk solvent in the transfer tubing, therefore it is diluted upon injection.
Liquid Chromatrography – LC:
LC is used a separation technique prior to ionization by one of the methods referred to above. Our laboratory most commonly utilizes reverse phase HPLC. A small aliquot of analyte solution is injected onto a non-polar stationary phase column. A flowing mobile phase, typically increased in organic composition as a function of time, is used to elute the analyte compounds of interest.