Microbore HPLC

The pump must provide stable flow rates from between 10 ttlmin and 2 mlmin with the LC-MS requirement dependent upon the interface being used and the diameter of the HPLC column. For example, the electrospray interface, when used with a microbore HPLC column, operates at the bottom end of this range, while with a conventional 4.6 mm column such an interface usually operates towards the top end of the range, as does the atmospheric-pressure chemical ionization (APCI) interface. The flow rate requirements of the different interfaces are discussed in the appropriate section of Chapter 4. 

The use of the lower flow rates employed with microbore HPLC columns or splitting of the eluate from a conventional column will immediately reduce the volume of liquid being presented to the interface and, while not necessarily totally removing the tendency to form droplets, at least is likely to make the situation more manageable. 

This can potentially be overcome by the use of microbore HPLC columns with flow rates which are directly compatible with mass spectrometer operation, although the necessary decrease in injection volume results in little overall gain in the concentration of sample reaching the mass spectrometer. In addition, at the time that the DLI was available, the use of microbore HPLC, which introduces another set of potential problems related to chromatographic performance, was probably as widespread as the use of LC-MS It has been assessed [2] that in around 25% of the reported applications of DLI, microbore HPLC has been utilized. 

The interface is directly compatible with the low flow rates used with microbore HPLC and, because of the narrow chromatographic peaks, good sensitivity can be obtained with small amounts of sample. 

On-line microbore HPLC monitoring of a catalytic hydrogenation process... 

Figure 9 Typical microbore HPLC chromatogram of catalytic hydrogenation process. Column 1 x 250-mm Partisil ODS (Whatman). Mobile phase acetonitrile water tetrahydrofuran, 85 15 2 (v v v). Flow rate 0.2ml/min. Detection UV at 214 nm. Injection 1 pi.

Microbore HPLC-FTIR detection limits are about 10 times lower than analytical-scale HPLC-FTIR detection limits. The lowest reported LC-FTIR detection limits are approximately 100-1000 times higher than the best GC-FTIR detection limits. The main characteristics of flow-cell HPLC-FTIR are summarised in Table 7.44. Because of mobile-phase interferences, flow-cell HPLC-FTIR is considered as a powerful tool only for the specific detection of major components but is otherwise a method of limited potential, and SFE-SFC-FTTR has been proposed as an alternative [391]. 

For microbore HPLC, with a flow of less than lOOpLmin-1, off-line LC-FT1R has been developed using matrix isolation techniques. The solutes are deposited on a moving IR salt window [504] or on a rotating plated disc [486], and are measured afterwards with the aid of a FITR microscope or a reflectance accessory. FTIR detection was first applied to the analysis of microbore HPLC eluent by Teramae and Tanaka [505]. In microbore HPLC-FTIR the amount of mobile phase required for separation is much less than for conventional scale HPLC. This simplifies both flow-cell and mobile-phase elimination interfaces. Flow-cell... 

As in the case in the analysis of food samples, the introduction of relatively inexpensive MS detectors for GC has had a substantial impact on the determination of methylxanthines by GC. For example, in 1990, Benchekroun published a paper in which a GC-MS method for the quantitation of tri-, di-, and monmethylxanthines and uric acid from hepatocyte incubation media was described.55 The method described allows for the measurement of the concentration of 14 methylxanthines and methyluric acid metabolites of methylxanthines. In other studies, GC-MS has also been used. Two examples from the recent literature are studies by Simek and Lartigue-Mattei, respectively.58 57 In the first case, GC-MS using an ion trap detector was used to provide confirmatory data to support a microbore HPLC technique. TMS derivatives of the compounds of interest were formed and separated on a 25 m DB-% column directly coupled to the ion trap detector. In the second example, allopurinol, oxypurinol, hypoxanthine, and xanthine were assayed simultaneously using GC-MS. 

Determination of two esterquats used as substitution products of DTDMAC, such as diethylester dimethylammonium chloride (DEED-MAC) and diesterquaternary (DEQ) (Fig. 4.2.8) in sewage water samples was carried out by the same ion-pair extraction procedure for the analysis of DTDMAC reported elsewhere [103,111] followed by microbore HPLC-ESI-MS analysis [116] and quantification employing commercial blends. 

Fig. 2.18 Raw data from a model GPC spin column/microbore HPLC ESI-MS primary screen of 400 compounds with PKA protein spiked with both staurosporine and olomoucine, known ligands of PKA. (Left) TIC, UV trace at 214 nm, and corresponding mass chromatograms for olomoucine and...

Figure 6.45 Microbore LC-NMR layout. A Microbore HPLC system with a 0.5 mm X 150 mm C18 column is interfaced to a solenoidal microcoil probe. The transfer capfllary is connected to the NMR flow cell with a polyamide resin. Reproduced from [85] with permission. Copyright 1999 American Chemical Society.

Most of the frequently used comprehensive 2D LC systems employ a microbore HPLC column in the first dimension, operated at low flow rate, both under isocratic and gradient conditions. This enables the transfer of fractions of small volume via the multiport valve equipped with two identical... 

The most common method is RP-HPLC. Microbore-HPLC [613] and narrow bore columns [614] are applied with good results in increasing sensitivity. A recent comparison between NP and RP microbore columns confirms the better suitability of RP for this purpose [615], The mobile phase is usually composed of acetonitrile or methanol. 

However, by no means are microbore HPLC columns nonoptimal when sample volumes are limited. Because of the substantially reduced mobile-phase volumes necessary to carry out a given separation, microbore columns are more easily interfaced to many useful detection systems, for example, electron-capture detectors, nitrogen-specific thermionic detectors, and mass spectrometers. 

Since microbore HPLC columns are usually used for continuous flow FAB, the sample volume is usually 1-5 fjl. 

Since FAB (or LSIMS) requires that the analyte be dissolved in a liquid matrix, this ionization technique was easily adapted for infusion of solution-phase samples into the FAB ionization source, in an approach known as continuous-flow FAB. Continuous-flow FAB was connected to microbore HPLC columns for LC/MS applications (Ito et al., 1985). Since this method is limited to microbore HPLC applications at flow rates of <10 pl/min... 

CF, TB, TP Microbore HPLC-UV at 280 nm. Column Whatman Micro-B of ODS-3. Mobile phase H20-MeOH-acetic acid. pg/ml Cocoa Aqueous extraction. 261... 

Recent advances in chromatography have made it possible to employ microbore HPLC for the determination of NOC. Its main advantage is that it uses a very low mobile-phase flow (20-100 /rl/min). This might make the TEA compatible with a reversed-phase system. Massey et al. (73), in fact, have successfully used reversed-phase chromatography for the HPLC-TEA determination of V-nitroso-V, 7V -di methylpiperazinium iodide. A 500-mm X 1-mm microbore ODS column and a mobile phase consisting of 0.1 M ammonium heptane-sulfonate in methanol water (70 30) (flow rate 20 /zl/min) was used for the HPLC separation. In another study, Riihl and Reusch (74) used a microbore Spherisorb 3 SW column for HPLC-TEA determination of volatile V-nitrosamines. The mobile phase was a mixture of 2-propanol and n-hexane (2.5 97.5). Further application of such techniques for the determination of various polar NOC, especially A-nitrosamides, in foods is desirable. 

I. L. Davies, K. D. Bartle, G. E. Andrews and G. T. Williams, Automated chemical class characterization of kerosene and diesel fuels by on-hne coupled microbore HPLC/capil-lary GC , J. Chromatogr. Sci. 26 125-130 (1988). 

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