Non-volatile buffer

Multiple hyphenation ( hypemation ) provides comprehensive spectroscopic information from a single separation. The first doubly hyphenated HPLC-NMR-MS appeared in 1995 [661], and its value is now accepted meanwhile fully integrated on-line LC-NMR-MS and MSn systems (QMS, QTTMS) are commercially available. On-line LC-NMR-MS coupling is by no means trivial. For example, the sensitivity of NMR is limited, while MS is incompatible with non-volatile buffers. The... 

Somsen, G. W, Mol, R., and de Jong, G. J. (2003). On-line micellar electrokinetic chromatography-mass spectrometry feasibility of direct introduction of non-volatile buffer and surfactant into the electrospray interface. /. Chromatogr. A 1000, 953—961. 

Ammonium formate and phosphate buffers CEC-ESI-MS for the analysis of leucine enkephalin and substance E, which are respectively singly and triply charged peptides. The good mass spectra obtained for the peptides in both the volatile and non-volatile buffers (Figures 17 and 18) indicate the non-crystallization of the non-volatile buffer which is further diluted by the sheath liquid. 

The sensitivities obtained with the volatile buffer (ammonium formate) and non-volatile buffer (sodium borate) in CEC-ESI-MS were assessed by comparing the spectra obtained in the two modes using leucine enkephalin and substance P that carry charges of -Fl and -1-3, respectively. 

This observation widens the potential applications of this monolithic chemistry in that the high selectivities obtained with non-volatile buffers do not have to be sacrificed to obtain good ESI spectra. 

Spin Column Media Advantages and Disadvantages, Volatile vs Non-volatile Buffers... 

Non-volatile buffers such as phosphates, borates, perchlorates and phosphoric acid should be avoided at all costs because of high background ion current, source contamination and blockages, and in the case of perchlorates, explosions. Figure 6.4 shows the mass spectrum of typical background when using phosphoric acid in the eluent. If the solvent system for a particular analysis does not assist the electrospray process, it is possible to enhance ionisation by postcolumn addition of a suitable volatile buffer. 

General rules can be given to choose between ESI and APCl ESI is preferred for compounds which are ionic or very polar or thermo labile, or with masses higher than 1000, whilst APCl is preferred for compounds of lower molecular mass that are not very polar [264], The use of non-volatile buffers is usually avoided when performing ESI or APCl sources [265] and the analyst must make sure he uses eluents compatible with the stainless steel parts of the mass spectrometer and avoids inorganic salt buildup. 

The CLND is limited, of course, to mobile phases that do not contain nitrogen. Acetonitrile and amine modifiers, commonly used in HPLC, are therefore precluded. In addition, the CLND is not readily amenable to non-volatile buffers in the mobile phase. However, it is still possible to determine RRF values for samples run under these non-CLND-compatible HPLC conditions. In such cases, a two-step process is used. First, a CLND-compatible mobile phase (e.g., methanol/water/trifluoroacetic acid) is used to separate the compounds of interest and determine RRF values under those conditions (RRF ). Separately, the UV peak areas obtained using both the CLND-compatible and non-compatible HPLC conditions are compared by analyzing a common sample by both sets of HPLC conditions (apart from the CLND). The peaks of interest must, of course, be tracked to avoid misassignment (e.g., through UV spectra comparison). The relative response factor (RRF ) obtained for the CLND-compatible method can then be used to determine the relative response factor (RRF2)... 

The selection of an appropriate mobile phase is another aspect of concern. Most often the commonly used LC mobile phase is not compatible with thermospray LC/MS, for instance owing to the use of non-volatile buffers can be left out or replaced by volatile ones. In other cases the buffers are present for retention time reproducibility, which mostly is not very important for identification. In other applications however a correspondence between UV or fluorescence peaks and MS identification is obligatory, which makes mobile phase changes unattractive. In this respect it is often overlooked that LC-UV and LC/MS give different responses as a result of different detection principles. For not too complex samples a UV photo-diode array detector can be used to link up the chromatographic peaks obtained under different mobile phase conditions. To cut short, despite many successes also many potential problems are met in LC/MS to which tailor-made creative solutions are needed. 

Gradients formed by increasing ionic strength (constant pH) are preferable to gradients formed by changing the pH. Volatile buffers, such as formates and acetates of pyridine and ammonium are usually used because they can be easily removed from the eluted fractions. However, the use of non-ionic hydrophobic resins and the silica bonded alkyl phases offer a convenient alternative to remove also non-volatile buffers. 

The main issue with atmospheric pressure sources is efficient sampling of the molecular ion species which must be transferred from normal atmospheric pres-siue to a vacuum chamber. This is effected by passing the ion stream through a pin hole and a series of focusing lenses into a two-stage pumped separator which removes the bulk of the neutral molecules. However, the nature of this transfer process makes the inlet prone to contamination, and the use of inorganic and non-volatile buffers in the HPLC separation will rapidly block these systems. Volatile buffers, such as ammonium formate, ammonium acetate and small concentrations of trifluoroacetic acid are best suited for LC/MS procedures. 

Carbamates. A fast, sensitive and selective method for the concentration and analysis of 9 N-methylcarbamate pesticides was reported by Volmer et al. [507]. Three different SPME fibres combined with short-column ESI-LC-MS(-i-) and MS/MS were applied. The detection limits observed were 0.3-1.9 pg Signal intensities increasing by a factor of 2-7 were observed [508] using non-volatile buffers in the separation process prior to ESI-MS. After EC removal of the non-volatile buffers was essential. The results obtained by ESI and APCI-LC-MS and MS/MS for the analysis of the eight N-methylcarbamate pesticides and their degradation products were compared with results obtain with the application of TSP or PBI (cf. 15.3.3.1 TSP, carbamates) [108]. ESI-LC-MS and TSP-LC-MS were used for quantitative determination of 10 different carbamate pesticides which showed a broad variety in polarity. ESI-SIM detection limits were typically 10-60 pg which was 10-150 times better than using TSP-MS (cf. 15.3.3.1 TSP, carbamates) [509]. Interfacing a commercial ESI source to an ITMS allowed the determination of carbamates as well as triazines and azo dyes. Identification could be performed either by IT-MS/MS or by ESI-CID [424]. 

One advantage of ELSD is that a wide range of solvents can be used, including acetone and chloroform which are not useful with UV detection. One drawback is that the solvent must be significantly more volatile than the analytes thus the use of non-volatile buffers should be strictly avoided. Only high-quality HPLC solvents with minimum particulates should be used. 

Incompatibility with some of the non-volatile buffers and other mobile phase additives. Hence, phosphates, ion pairing agents, and amine modifiers are replaced by ammonium acetate, ammonium formate, etc. 

Excessive heating of the layer occurs when the ionic strength is too high. The concentration of non-volatile buffers then rises, due to evaporation this further increases the ionic strength and the heatiag in a vicious spiral. Unequal evaporation of cation and anion components of a volatile buffer can lead to changes in pH, e. g., with triethylamine (B. P. 89° C) and acetic acid (B. P. 118.1° C). Working with either lower current or more dilute buffer is then advisable. Table 12 contains a compilation of available buffer solutions for thin-layer electrophoresis. 

---