Mobile phase mass spectrometry

C. J. Hogan, E. Folta-Stogniev, B. Ruotolo, J. Fernandez de la Mora, IMS-MS study of native aggregates, from insulin to GroEL. To be submitted to Anal. Chem. 2009 Hogan, C., Ruotolo, B., Robinson, C., Fernandez de la Mora, J. Tandem differential mobility analysis-mass spectrometry of the GroEL complex Structure compaction in the gas phase and inelastic air-protein interaction, Submitted to J. Phys. Chem. B, 2010. 

From a mass spectrometry perspective, the pump must be pulse free, i.e. it must deliver the mobile phase at a constant flow rate. Pulsing of the flow causes the total ion current (TIC) trace (see Chapter 3) - the primary piece of information used for spectral analysis - to show increases in signal intensity when analytes are not being eluted and this makes interpretation more difficult. 

Before considering these in detail, it is necessary to revisit the inherent incompatibilities between mass spectrometry and liqnid chromatography. These are, as discussed previously, that HPLC utilizes a liquid mobile phase, often containing significant amounts of water, flowing typically at 1 mlmin while the mass spectrometer must be maintained under conditions of high vacuum, i.e. around 10- torr (1.333 22 x IQ- Pa). 

The need for a more definitive identification of HPLC eluates than that provided by retention times alone has been discussed previously, as have the incompatibilities between the operating characteristics of liquid chromatography and mass spectrometry. The combination of the two techniques was originally achieved by the physical isolation of fractions as they eluted from an HPLC column, followed by the removal of the mobile phase, usually by evaporation, and transfer of the analyte(s) into the mass spectrometer by using an appropriate probe. 

A modified nucleotide found in RNA sequencing could either be a new nucleotide of unknown chemical structure or it could correspond to an already known modified nucleotide (up to now about 90 different modified nucleotides have been identified in RNA). Keith [124] proposed preparative purifications of major and modified ribonucleotides on cellulose plates, allowing for their further analysis by UV or mass spectrometry. Separation was realized by two-dimensional elution using the following mobile phases (1) isobutyric acid-25% ammonia-water (50 1.1 28.9,... 

Detection in SFC can be achieved in the condensed phase using optical detectors similar to those used in liquid chromatography or in the gas phase using detectors similar to those used in gas chromatography. Spectroscopic detectors, such as mass spectrometry and Fourier transform infrared spectroscopy, are relatively easily interfaced to SFC compared to the problems observed with liquid mobile phases (see Chapter 9). The range of available detectors for SFC is considered one of its strengths. 

Figure 7.31 Negative-ion thermospray mass spectrum of the disulfonated azo dye Direct Red 81 (mobile phase contains 10mmolL ammonium acetate). After Niessen [568]. Reprinted from W. Niessen, in Encyclopedia of Spectroscopy and Spectrometry (J.C. Lindon, ed.), Academic Press, pp. 2353-2360, Copyright (2000), with permission from Elsevier...

Tholey, A., Toll, H., Huber, C.G. (2005). Separation and detection of phosphorylated and nonphosophorylated peptides in hquid chromatography—mass spectrometry using monolithic columns and acidic or alkaline mobile phases. Anal. Chem. 77, 4618 1625. 

Apffel, A., Fischer, S., Goldberg, G., Goodley, P.C., Kuhlmann, F.E. (1995). Enhanced sensitivity for peptide mapping with electrospray liquid chromatography—mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases. J. Chromatogr. A 712, 177-190. 

The instrumental analysis for the identification of UV filters degradation products formed during the fungal treatment process was performed by means of HPLC coupled to tandem mass spectrometry using a hybrid quadrupole-time-of-flight mass spectrometer (HPLC-QqTOF-MS/MS). Chromatographic separation was achieved on a Hibar Purospher STAR HR R-18 ec. (50 mm x 2.0 mm, 5 pm, from Merck). In the optimized method, the mobile phase consisted of a mixture of HPLC grade water and acetonitrile, both with 0.15% formic acid. The injection volume was set to 10 pL and the mobile phase flow-rate to 0.3 mL/min. 

Perhaps the most mechanically complex solution ever developed for uniting HPLC with mass spectrometry was the moving belt interface [54]. The heart of this system was a mechanically driven continuous belt (analogous to an escalator or moving walkway) to which the HPLC eluent was applied. The majority of the mobile phase was evaporated by a heat source (ideally hot enough to vaporize the solvents but not to... 

However, phosphate salts are not volatile. We must constantly remember that mass spectrometry is a gas-phase experiment. Materials to be examined by mass spectrometry must ultimately be made gaseous. Figure 19.14 shows the atmospheric pressure ionization source chamber of a mass spectrometer after infusion of a 20 mM potassium phosphate-containing mobile phase into the instrument for a few hours. The accumulation of phosphate salts on the striker plate is evident. Visual evidence of salt accumulation is also apparent on the back wall of the source chamber, above the striker plate. The overall haziness of the image is not the result of poor photography, but rather due to the coating of dust on the inner walls of the chamber and all surfaces within. 

When the mobile phase is a gas, the technique is called gas chromatography-mass spectrometry (GC-MS). In this case, the mixture is in the gas phase as it moves through the column, and the individual separated mixture components emerge in the gas phase and mix with the gaseous mobile phase, which is usually helium. When the mobile phase is a liquid, or a mixture of liquids, the technique is called liquid chromatography-mass spectrometry (LC-MS). In this case, the mixture is in the liquid phase as it moves through the column and individual separated mixture components emerge dissolved in this liquid phase. 

A novel pigment has been isolated from the petals of Rosa hybrida with complex chromatographic techniques and the structure was elucidated with spectroscopic methods and high resolution fast-atom bombardement mass spectrometry, lH NMR, and FTIR. Anthocyanins were extracted from 7.9 kg of petals of Rosa hybrida cv. M me Violet with 80 per cent aqueous ACN containing 0.1 per cent TFA. The extract was purified in a Sephadex LH-20 column, and the fraction eluted with 80 per cent ACN was further fractionated in a HP-20 column using water, 15, 20 and 30 per cent ACN as mobile phases. The last fraction was lyophilized and separated by preparative RP-HPLC using an ODS column (50 X 5 cm i.d.). Solvents were 0.5 per cent aqueous TFA (A) and water-methanol... 

TLC coupled with mass spectrometry employing desorption electrospray ionization has been used for the separation of synthetic dyes. The chemical structures of dyes included in the investigation are shown in Fig. 3.7. ODS HPTLC plates (10 X 10 cm) were used as the stationary phase the mobile phase consisted of methanol-tetrahydrofuran (60 40, v/v) containing 50-100 mM ammonium acetate for the positive-ion test and of methanol-water (70 30, v/v) for the negative-ion test. Test mixtures for negative- and positive-ion mode detection consisted of methyleneblue, crystal violet, rhodamine 6G... 

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