Mobile phase composition compatibility

Beyond the density changes that can be used to control method modifications in SFC, the mobile phase composition can also be adjusted. Typical LC solvents are the first choice, most likely because of their availability, but also because of their compatibility with analytical detectors. The most common mobile phase modifiers, which have been used, are methanol, acetonitrile and tetrahydrofuran (THF). Additives, defined as solutes added to the mobile phase in addition to the modifier to counteract any specific analyte-column interactions, are frequently included also to overcome the low polarity of the carbon dioxide mobile phase. Amines are among the most common additives. 

In the development and optimization of a comprehensive LCxLC method, many parameters have to be taken in acconnt in order to accomplish snccessfnl separations. First of all, selectivity of the columns used in the two dimensions must be different to get maximum gain in peak capacity of the 2D system. For the experimental setup, column dimensions and stationary phases, particle sizes, mobile-phase compositions, flow rates, and second-dimension injection volumes should be carefully selected. The main challenges are related to the efficient coupling of columns and the preservation of mobile phase/column compatibility. 

The solvent elimination problem became less of a problem with the commercialization of microbore columns. Hayes et al. (54) studied gradient HPLC-MS using microbore columns and a moving-belt interface. The heart of the system was the spray deposition device designed to be compatible with microbore-column flow rates. Nebulization of the eluent was found to be applicable to a variety of mobile-phase compositions and thus was readily compatible with gradient elution. Figure 13 shows a comparison of UV detection with that obtained with the HPLC-MS system. Applications of this system were demonstrated on water from coal gasification processes. 

Samples are injected onto the turbulent-flow column similar to single-column methods. The analytes of interest are retained in the turbulent-flow column while the large macromolecules are eluted to waste. Once the analytes are separated from the matrix, the samples are then eluted into the analytical column. The characteristics of the analytical column determine the peak shape and separation seen at the MS detector. Flow rates which are compatible with the mass spectrometer can then be used and the chromatograms are based on conventional HPLC parameters. The key to dual-column methods is that the retentive properties of the analytical column must be sufficiently stronger than that of the turbulent-flow column the dual-column approach is performed in such a manner so that the mobile-phase composition needed to elute the analyte from the turbulent-flow column does not elute the analyte from the analytical column. The sample is then focused at the head of the analytical column until the mobile-phase conditions are changed to elute the analyte. The choice of columns is critical to the success of dual-column methods. Table 10.2 lists some of the applications of dual-column methods found in the literature. 

Typically, in gradient elution liquid chromatography, electrochemical detection has been difficult due to base-line shifts that result as a consequence of the altered mobile phase composition. However, a unique property of micelles allows for much improved compatibility of gradients (i.e. gradient in terms of micellar concentration or variation of small amount of additive such as pentanol) with electrochemical detectors. This has been demonstrated by the separation and electrochemical detection of phenols using micellar gradient LC (488). A surfactant (apparently non-micellar) gradient elution with electrochemical detection has also been successfully applied for the assay of some thyroid hormones by LC (491). 

The system should be compatible with a wide range of mobile-phase compositions and with the typical flow-rates of conventional LC column. MAGIC is based on aerosol formation in order to readily achieve evaporation of the solvent and minimum band broadening of the chromatographic peaks and to avoid the need of thermal desorption of the analyte molecules. 

The most appropriate way to solve mobile-phase incompatibility problems in LC-MS is to change the mobile-phase composition to LC-MS compatible conditions. This is the approach taken in most cases. Unfortrmately, it is not always possible to do so. In some cases, specialized LC colunrn materials demand a particular mobile-phase composition. This is the case with for instance some chiral colunms, which will only provide adequate enantiomeric separation in a predefined mobile phase. The retention behaviour in high-performance anion-exchange chromatography (HPAEC) is significantly influenced by the cation (sodium or ammonium) in the mobile phase. In these cases, mobile-phase... 

For production-scale processes economic pressure arises and thus a successive optimization of the chromatographic system will pay off by a reduction in operating costs. Therefore, the development of the chromatographic system has to start with the search for a suitable mobile phase with high solubility for the given solute (Section 4.3.1) and a compatible adsorbent. Dependent on the nature of the phase system the separation is optimized by adjusting the mobile phase composition (Sections 4.3.2-4.3.4). 

It has become painfully obvious that most of the excellent approaches and techniques that have been developed for use in liquid chromatography are not applicable to liquid chromatography/mass spectrometry (LC/MS) with atmospheric pressure ionization. Chapter 5 described the reagents and the range of mobile-phase compositions that are compatible with electrospray and atmospheric pressure chemical ionization (APCI), and these are limited to volatile components that do not cause significant ion suppression. Certain problems that are not significant with standard LC separations become difficult to deal with because of the limitations placed on the mobile phase by atmospheric pressure ionization (API) LC/MS. 

Supercritical fluid chromatography is compatible with both HPLC and GC detectors. As a result, optical detectors, flame detectors and spectroscopic detectors can be used. The FID is the most common detector used. However, the mobile phase composition, column type and flow rate must be taken into account when the detector is selected. Some care must also be taken such that the detector components are capable of withstanding the high pressures of SFC. 

Both the position of the emission wavelength envelope and the emission intensity can be affected by the mobile phase composition and even by the presence of contaminants. Table 5.3. Fluorescence detection is compatible with gradient elution unless one or more components of the mobile phase contain a high level of fluorescent impurities. 

Filtration is required to remove any particulate matter that could cause damage to the pump seals and could block the column and narrow tubing if they are not removed. The choice of filter will depend on the level of particulate contamination and the mobile phase composition. An example of this might be the use of a 0.45 pm nylon membrane filter, which is compatible with most mobile phases. Information in relation to filter compatibility for commonly used mobile phase components for RP-HPLC is given in Table 3.3. 

Despite the automated degassing, it is still advisable to filter each of the components of the mobile phase under vacuum before use. Degassing can be performed manually using a combination of vacuum filtration (see Chapter 3 for filter compatibility information), followed by immersion in an ultrasonic bath for a short period of time. Care should be taken when using an ultrasonic bath because of the generation of heat that can alter the mobile phase composition by evaporation of the more volatile organic solvent. 

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