FAST LC columns

With the trend toward using Fast LC and small-bore columns (3-and 2-mm i.d.) in pharmaceutical analysis, increasing demand is placed on reducing the system dispersion or IBW. Figure 15 shows the deleterious effect of IBW on the performance of a Fast LC column showing that... 

In dynamic FAB, this solution is the eluant flowing from an LC column i.e., the target area is covered by a flowing liquid (dynamic) rather than a static one, as is usually the case where FAB is used to examine single substances. The fast atoms or ions from the gun carry considerable momentum, and when they crash into the surface of the liquid some of this momentum is transferred to molecules in the liquid, which splash back out, rather like the result of throwing a stone into a pond (Figure 13.2). This is a very simplistic view of a complex process that also turns the ejected particles into ions (see Chapter 4 for more information on FAB/LSIMS ionization). 

By passing a continuous flow of solvent (admixed with a matrix material) from an LC column to a target area on the end of a probe tip and then bombarding the target with fast atoms or ions, secondary positive or negative ions are ejected from the surface of the liquid. These ions are then extracted into the analyzer of a mass spectrometer for measurement of a mass spectrum. As mixture components emerge from the LC column, their mass spectra are obtained. 

The LC/TOF instmment was designed specifically for use with the effluent flowing from LC columns, but it can be used also with static solutions. The initial problem with either of these inlets revolves around how to remove the solvent without affecting the substrate (solute) dissolved in it. Without this step, upon ionization, the large excess of ionized solvent molecules would make it difficult if not impossible to observe ions due only to the substrate. Combined inlet/ionization systems are ideal for this purpose. For example, dynamic fast-atom bombardment (FAB), plas-maspray, thermospray, atmospheric-pressure chemical ionization (APCI), and electrospray (ES)... 

High flow-rates (up to 2mLmin-1) fast LC-MS for short columns... 

The current status of chromatography is shown in Table 10.25. Since reducing separation time is a major issue, there is a pronounced trend toward shorter columns filled with small particles. The current trends for lower flow (micro- and nano-LC) columns, and great strides to achieve (ultra-) fast chromatographic... 

HPLC with microchip electrophoresis. Capillary RPLC was used as the first dimension, and chip CE as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet end of LC column into the cross-channel on a specially designed chip. Laser-induced fluorescence was used for detecting the FITC-labeled peptides of a BSA digest. The capillary HPLC effluents were continuously delivered every 20 s to the chip for CE separation. 

When a fast LC system is connected to a detector, care must be taken to ensure that the detector is well suited for the expected flow ranges and peak widths. Most manufacturers, especially those offering dedicated systems for sub-2-micron particle columns, offer efficient UV detectors. Flow rate is usually not an issue for UV and other flow-through cell-based detection systems. However, flow rate can become limiting for dead-end detectors that alter the column effluent, mainly by eliminating mobile phases such as ELSD, CAD, CLND, and mass spectrometers. 

Such columns are excellent filters and require more sample preparation to ensure the removal of all solids. To benefit from the full power of LC optimization, the detectors must be optimized as well. Data rates and duty times must match the narrower peaks in very fast (and well resolving) separations. Careful consideration and optimization of all instrument components and software can produce significant cycle time improvements of fast LC separations and further increase throughput. An important aspect of cycle time improvement is parallelization of components of individual analyses. 

For these reasons, smaller-particle columns are particularly well suited for fast LC and high-throughput screening applications. 

Recently, Chu et al. reported an ultra-fast LC/MS method for analysis of cytochrome P450 3A4 and 2D6 inhibition assaysd Testosterone and dextromethorphan were used as the specific substrates for CYP3A4 and CYP 2D6, respectively. LC/MS analyses were performed on a Sciex API 3000 mass spectrometer equipped with a Shimadzu LC-lOAdvp pump and a PE 200 autosampler. A Phenomenex Luna CIS (4.6x30 mm) column was used along with very steep gradients. Each sample analysis was completed in 0.5 min. 

In work concerning the directed evolution of enantioselective enzymes, there was a need for fast and efficient ways to determine the enantiomeric purity of chiral alcohols, which can be produced enzymatically either by reduction of prochiral ketones (e.g., 26) using reductases or by kinetic resolution of rac-acetates (e.g., 19) by lipases (111). In both systems, the CD approach is theoretically possible. In the former case, an LC column would have to separate the educt 26 from the product (A)/(J )-20, whereas in the latter, (5)/(J )-20 would have to be separated from (S)/(R)-19. 

Dams et al. [19] determined seven different opium alkaloids and derivatives in seized heroin using fast LC-MS analysis. Analytes were separated in 5 min on a monolithic silica column with a gradient elution system and an optimized flow of 5 mL/min. Detection was carried out using a sonic spray ion source [20] a modified ESI source were ionization is achieved using a nebulizer gas at sonic speed instead of applying an electrical field. 

Beside column dimension the size of stationary phase particles is a matter of recent progress. More traditional columns are packed with 3.0-5 pm particles enabling satisfying resolution and reasonable column back pressure of solvent suitable to be processed by conventional HPLC pumps. In contrast, sub 2-pm particles (e.g. 1.7 and 1.8 pm) as applied in rapid or fast LC or ultra high-performance LC (UHPLC) allow better resolved separations in shorter run times. Column back pressure (>12,000 psi) is remarkably high demanding more robust solvent pumps. 

Figure 15. Repetitive chromatograms from drug analyzer—therapeutic drugs in serum (l) theophylline, 10v.g/mL (2) internal standard ((2-hydroxytheophylline), 30 fug/mL column FAST LC-8, 4.6 X 150 mm (5p), 3.5 mL/min, 2000 psi (19)...

In LC practice, we do observe an increase in the sensitivity for columns packed with small (3 pm) particles ( FAST LC or High speed LC columns) in comparison to conventional columns (5 pm or 10 pm particles). 

Fast LC. Some very short columns that are only 3 to 5 cm long and are packed with very small particles (3 xm) have become popular. Sometimes referred to as 3 x 3 columns, they are less costly and give good separations with minimum consumption of mobile phase. The term fast or high-speed LC has been applied to their use at high flow rates around 4 mL/min. Figure 9.26 compares a conventional column with a 3 x 3 column for the separation of six explosives. In general, separations that need 4,000 or fewer plates can be separated with these columns in a minimum of time with conventional detectors. The instrument requirements are more severe, and special detectors may be needed some of the practical aspects of fast LC have been described by van der Wal.46... 

The disadvantage is the instrumental requirement to keep all dead volumes and detector volumes sufficiently small to prevent extracolumn zone broadening. It is also possible that special pumps, sample valves, and detectors may be required. The same requirements apply to the short columns used in fast LC. 

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