LC mobile phase

For LC, temperature is not as important as in GC because volatility is not important. The columns are usually metal, and they are operated at or near ambient temperatures, so the temperature-controlled oven used for GC is unnecessary. An LC mobile phase is a solvent such as water, methanol, or acetonitrile, and, if only a single solvent is used for analysis, the chromatography is said to be isocratic. Alternatively, mixtures of solvents can be employed. In fact, chromatography may start with one single solvent or mixture of solvents and gradually change to a different mix of solvents as analysis proceeds (gradient elution). 

Paper filters are available to filter LC mobile phases, but nylon filters are used more often. Why is that ... 

Miniaturizing the column i.d. is of great benefit to the sensitivity of ESl-MS, which behaves as a concentration-sensitive detection principle, because the concentration of equally abundant components in the LC mobile phase is proportional to the square of the column internal diameter. Column diameters from 150 to 15 jm with flow rates 20-200nL improve detection limits of peptides 1-2 orders magnitude over microliter flow rates. Several references referred to in other sections of this chapter discuss the use of LC-ESI MS to characterize separation products. and a sample chromatogram from Ito and coworkers. is seen in Figure 3.8. Table 3.4 provides additional and references that have used this technique. 

Solvent restriction An LC mobile phase is a liquid of variable composition, and sample components are led to the detector together with a given solvent combination. Separation and ionization of analytes often depend on different and, sometimes, antagonist mobile-phase components. 

Solvent-elimination approaches include evaporative spray deposition onto infrared-transparent surfaces (141) or reflective surfaces and powders (142, 143). Other approaches include partial evaporation of the mobile phase before spray deposition (144, 145), and continuous liquid-liquid extraction systems that transfer solutes from LC mobile phases to solvents possessing an infrared window (146). Spray systems include both pneumatic and ultrasonic nozzles (147). 

Unfortunately, not much experimental work has been carried out on the combination of supercritical fluid extraction and liquid chromatography systems (43, 44). One of the reasons for this arises from the difficulties in achieving compatibility between the extraction solvent and the LC mobile phase. Baseline perturbations have been... 

Minimal consumption of organic solvents (elution with LC mobile phase)... 

Another aspect affecting the solvent choice in the case of aH NMR measurements is that deuterated solvents are the most convenient option.53 In principle, the deuterated version of regular LC mobile phases could be used, as deuteration has been shown to have only a minor effect on the partition,52 and they were applied in the studied outlined above. 

In general, method development follows a logical progression, but changes made to one part of the system may have repercussions elsewhere. For example, a change in LC mobile phase composition may affect ionization in addition to LC separation. 

The nature of the stationary phase can be incorporated in this nomenclature. The convention hereby is to denote the character of the stationary phase by inserting one or two letters in the middle. Hence, LSC and LLC are both forms of liquid chromatography (LC mobile phase is a liquid), while the stationary phase is a solid (S) and a liquid (L), respectively. 

The liquid from an LC is compatible with normal IR sampling and is less of a problem. However, LC mobile phases may not be transparent in the IR, and water is a particularly difficult solvent to handle. For volatile organic solvents, evaporation is possible, and the remaining nonvolatile analytes can be deposited in KBr and pressed into pellets. Further discussion can be found in reference 20. 

In a similar fashion, Snyder64 has attempted to characterize LC mobile phases. Using Rohrschneider s data, he has calculated partition coefficients corrected for dispersion interactions and molecular weight effects. As originally defined in Chapter 1, the partition coefficient K is... 

Likewise, the luminescence properties of many analytes can be altered in the presenoe of surfactant aggregates (4,7.,8.). Consequently, addition of micelle-forming surfactants (present either in the LC mobile phase or added post-column) can improve the sensitivity of fluorimetric LC detectors (49,482). Micellar spray reagents have been utilized to enhance the fluorescence densitometric detection of dansylamino acids or polycyclic aromatic hydrocarbons (483). The effect was observed for TLC performed on cellulose or polyamide stationary phases with the micellar spray reagent being either CTAC, SB-12, or NaC (483). More recently, use of nonionic Triton X-100 has been found to improve the HPLC detection of morphine by fluorescence determination after post-column derivatization (486) as well as improve the N-chlorination procedure for the detection of amines, amides, and related compounds on thin-layer chromatograms (488). 

More importantly, the use of heavy metal anionic micellar media has been shown to allow for observation of analytically useful room-temperature liquid phosphorescence (RTLP) (7.484.487). There are several examples in which phosphorescence has been employed as a LC detector with the required micellar assembly being present as part of the LC mobile phase (482) or added post column (485). More recently, metal ions have been determined in a coacervate scum by utilizing the micellar-stabilized RTLP approach (498). Thus, the future should see further development in RTLP detection of metal ions in separation science applications. 

To achieve this whilst maintaining chromatographic integrity through the LC/MS interface was likened by Arpino 6 to an improbable love affair between a goldfish, symbolising the liquid LC mobile phase, and a parrot, symbolising the gas phase in MS (Fig. 5.1). 

In developing on-line LC-MS, three fundamental compatibility problems had to be solved, viz., the amount of solvent eluting from the LC column, the composition of the LC mobile phase, and the nature of the analytes. Interfaces have been... 

J.J. Zhao, A.Y. Yang, J.D. Rogers, Effects of LC mobile phase buffer contents on the ionization and fragmentation of analytes in LC-ESI-MS-MS determination, J. Mass Spectrom., 37 (2002) 421. 

Figure 10.8 H/D exchange to facihtate metabolite identification. Prodnct-ion MS-MS spectra of an oxidized metabohte (stmcture from [15]) obtained with (bottom spectrum) and withont (top) D2O in the LC mobile phase. Reprinted from [59] with permission (02001, John Wiley Sons, Ltd.) and modified.

Two other groups [16-17] demonstrated that similar good results could be obtained without the on-line SPE step. Holt et al. [16] performed an off-hne liquid-liquid extraction (LLE) of the basified supernatant with 1-chlorobutane, evaporation to dryness and reconstitution of the sample in LC mobile phase, while Keevil et al. [17] directly injected the supernatant of the protein precipitation step onto the 33x3.0-nun-ID cyano colunm. 

The nebulizer is normally interfaced directly to the LC column. It combines the eluent with a stream of gas to produce an aerosol. Much of the theoretical and practical basis of nebulization comes from atomic spectroscopy. The average droplet diameter and uniformity of the aerosol are the most important factors for ELSD sensitivity and reproducibility. As larger solute particles scatter light more intensely, an aerosol with large droplets and a narrow droplet size distribution leads to the most precise and sensitive detection. A good nebulizer should produce a uniform aerosol of large droplets with narrow droplet size distribution. The droplets cannot be too large, however otherwise, the solvent in a droplet will not be completely vaporized and errors in detection will occur. The nebulizer properties that can be adjusted to obtain the desired droplet properties are, primarily, the gas flow rate and the LC mobile phase flow rate. ... 

Experimentally, the major problem experienced in the early development of LC interfaces was the fact that the worst scenario centered around an aqueous reversed-phase LC mobile phase at the flow rate of 1 mL/min would... 

Eluent delivery was provided by two model 510 high-pressure pumps coupled with automated gradient controller model 680 (Waters Chromatography Division, Millipore, MA, USA) and a model 7125 infection valve with a 20-(il loop from Rheodyne (Cotati, CA, USA) Stainless-steel columns (30 x 0.40 cm I.D.) packed from Tracer Analltica (Barcelona, Spain) with 10 un particle diameter Spherisorb ODS-2 (Merck, Darmstadt, FRG) were used. Four different LC mobile phase compositions were tested acetonitrile-water... 

The purpose of the last step is to keep the final sample solvent of similar composition to the LC mobile phase to maintain the sulfonylurea LC peak shape. Standards were prepared at the 0.1 - 2.0 ig/mL range in 50% acetonitrile/water. 

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. 

A previous purification of wine sample can be performed by reverse-phase SPE. A volume of 5 mL of wine is added of 15 mL of water and passed through a C18 cartridge previously activated by passage of methanol and water. After washing the cartridge with 6 mL of 0.3% formic acid aqueous solution and 4mL of water, anthocyanin compounds are recovered with 5 mL of methanol. The solution is dried and the residue re-dissolved in the LC mobile phase (Kosir et al., 2004). 

Molecular model of acetonitrile. Acetonitrile (CH3C=N) is a widely used organic solvent. Its use as an LC mobile phase stems from its being more polar than methanol but less polar than water. 

Certain types of traditional LC mobile phase additives should be avoided due to nonvolatility and ion suppression effects. Mobile-phase related ion suppression will not depend on the analyte proximity to the solvent front, or capacity factor. These additives include detergents surfactants ion pairing agents inorganic acids such as sulfuric, phosphoric, hydrochloric, and sulfonic acids nonvolatile salts such as phosphates, citrates, and carbonates strong bases and quaternary amines. Complete suppression of ionization as well as interferences in both positive and negative ion mode will occur when these agents are utilized. 

Table 1 summarizes the commonly used and recommended LC mobile-phase components as well as those that should not be used. 

It is widely recognized that sensitivity in either ESI or APCI is improved as the percentage of organic modifier is increased, due to the improved facility for desolvation. Adequate reverse-phase retention (/< = 1 to 5) is therefore important when LC-MS is performed. Temesi and Law studied the effect of LC mobile-phase composition on ESI response for a series of 35 compounds [18]. In their investigation, the signal response for positive-ion ESI was on average 10-20% higher in MeOH than ACN. 

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