Microbore column, interface

LC-PB-MS is especially suited to NPLC systems. RPLC-PB-MS is limited to low-MW (<500 Da) additives. For higher masses, LC-API-MS (combined with tandem MS and the development of a specific mass library) is necessary. Coupling of LC via the particle-beam interface to QMS, QITMS and magnetic-sector instruments has been reported. In spite of the compatibility of PB-MS with conventional-size LC, microbore column (i.d. 1-2 mm) LC-PB-MS has also been developed. A well-optimised PB interface can provide a detection limit in the ng range for a full scan mode, and may be improved to pg for SIM analyses. 

LC-NMR hyphenation consists of a liquid chromatograph (autosampler, pump, column and oven) and a classical HPLC detector. The flow of the detector is brought via an interface to the flow-cell NMR probe. Using commercial NMR flow-cells with volumes between 40 and 180 p,L, in connection with microbore columns or packed capillaries, complete spectra have been provided from 1 nmol of sample. These micro-cells allow expensive deuterated solvents to be used, and thus eliminate solvent interference without excessive cost. The HPLC eluent can be split in order to allow simultaneous MS detection. 

Maximum flow-rates compatible with DLI interfaces are in the range of 50 to 100 pL/min. Microbore columns (<1.0mm i.d. column) operating at 5 to lOOpL/min are ideally suited for DLI LC/MS. A flow splitter is required to couple conventional LC with a DLI interface so that only a fraction of the total eluent is introduced into the mass spectrometer. Splitting the flow outside the mass spectrometer results in loss of sensitivity, an undesirable consequence. Henion and co-workers have reported an approach in which the splitter is incorporated into the desolvation chamber of the mass spectrometer. The removal of solvent is achieved by diverting the vapor generated by the solvent without loss of sample and, therefore, sensitivity. Excess pressure inside the mass spectrometer is therefore avoided while higher flow-rates can be accommodated. 

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. 

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. 

Tomlinson and Caruso [28] also performed the speciation of Cr(III) and (VI) using a Dionex AS-11 anion-exchange microbore column and 6 mM 2,6-PDCA-8.6 mill lithium hydroxide mobile phase. A thermospray source was used as the interface between LC and ICP-MS. Absolute limits of detection were at the pg level for both species using this instrument assembly. 

For LC/MS the main problem is the large amount of mobile liquid phase that must be removed to get the effluent reduced to the high vacuum of the MS. Microbore columns are desirable for this reason.22 The three most popular devices have been summarized by Majors23 direct liquid interface (DLI), moving belt transport, and thermospray.24 The thermospray device consists of a small bore capillary tube that is heated to produce a stable, high-velocity jet consisting mostly of vapor with a small amount of mist. It not only provides an interface to the MS, it also causes the ionization of analytes necessary for the MS. Some think it may find more widespread use as a transport device. 

A recent modification of the atmospheric pressure ionization technique involving a special low dead volume interface was described by Thomson etal. [27]. They employed packed microbore columns (170 p, 320 p, and 500 p I. D. with lengths ranging from 5 to 15 cm) in conjunction with a low-volume, wall-coated capillary column as an interface. The total ion current chromatogram of the tryptic digest sample of about 1 picomole of human growth hormone is shown in figure 29. The column was packed with an octadecyl bonded phase... 

An interface for use with microbore columns in conjunction with FTIR was described by Johnson and Taylor [33] and with their system... 

For HPLC-FTIR, GPC-FTIR, or SFC-FTIR, the design of the interface is more challenging since the mobile phases used for these chromatographic systems normally have strong infrared absorbencies thus, it is important to remove the mobile phase prior to measuring the spectrum. For the interface between the two systems flow-cells or mobile-phase elimination techniques may be used. Some recent developments point toward the elimination of mobile-phase techniques. A microbore column can help to reduce the mobile-phase volume in the system. ... 

With respect to packing geometry and colunm efficiency, microbore colunms are equivalent to conventional colunms, except with respect to the internal diameter. Since most electrospray (ESI) interfaces are optimized for operation with flow-rates between 50 and 200 pl/min, the use of 1-3-mm-ID microbore columns is advantageous, because no post-column solvent splitting is required. 

The Finnigan chemical ionization source was modified by the addition of two cartridge heaters. Eluate entered the source through a heated 1/2" probe and excess solvent was removed by a mechanical vacuum pump connected directly opposite the eluate entrance. The interface consisted of 1 meter deactivated silica capillary tubing (ID, 60 OD, 0.008"), led from the outlet of microbore column and threaded through the probe. The probe design has been previously described (26). For all analyses, the probe was operated at 240 C and the source at 250 C. The analyzer pressure was 10 b torr. 

Direct Interfacing of LC-FT-IR Is performed via a flow cell technique. To avoid the Interferences from mobile phase absorbance In the mld-lR region, a compatible (transparent) mobile phase should be used. A "Buffer Memory" technique for Indirect combinations of microbore column and FT-IR was also reported by Jlnno et. al. The buffer memory technique was reported to have advantages over conventional direct LC-FT-IR In achieving a continuous chromatogram and In preventing mobile phase Interference. 

The flame-based detector was reported to accept in excess of 20 ui/mln of 10-25Z aqueous methanol without extinction of the flame Optimum response was obtained at flow rates below 5 iii/min. Compatible solvent systems were aqueous methanol (up to 50%), acetone and ethanol (up to 40%) The minimum detectable quantity (at 5 times noise) measured for the FPD was 2 pg P. The dual-flame TSD can also be directly Interfaced with mlcrocaplllary packed columns The TSD was reported to be compatible with 75 to 100% aqueous methanol The utilization of microbore column LC-TSD for the analyls of nitrogen, phosphorous, and halogen containing compounds is particularly Important in studies of biomolecules, and drugs and their metabolites in physiological fluids ... 

These tendencies are Illustrated with an example from microbore column LC/MS. "Figure 13" describes the LC/MS interface used and gives a schematic view of the interface principle. "Figure 14" represents the mass spectrum of an analyzed sample, involving a compound with a molecular weight of 530. 

Despite the numerous advantages the instrumental demands of microcolumn LC are considerable, and these demands are further accentuated as the requirements vary from one column type to another. A consequence of the reduced flow rates is that the detector flow-cell volume should be reduced to <10nl for OTCs, 0.1 pi for packed microcapillaries and 1 pi for microbore columns. An additional demand of the detector is that it should have a rapid response, <0.5 s. Development of suitable detectors is paramount if the potential of micro-LC is to be realised. Study of detector systems has focused in two areas firstly, the miniaturisation of ultraviolet, fluorescence and electrochemical systems, using in the former two systems LASERS as excitation sources and ultraviolet fibre optic and on-line cells to reduce band broadening and increase sensitivity [123,124] secondly, the direct interfacing with systems which previously required transport and/or concentration of the eluant. Interfacing of HPLC with mass spectroscopy has been undertaken by Barefoot et al. [125] and Lisek et al. [126] and flame systems (FPD and TSD) have been reviewed by Kientz et al. [127]. Jinno has reviewed the interfacing of micro-LC with ICP [128]. 

Micronization in the field of column hardware and packing materials led to the development of microbore and short columns the latter allow separation times sometimes to be shortened by a factor of 10 (83, 385). The advent of microbore columns created a whole new methodology in HPLC with detection limits lowered into the femto-mole range (224, 260, 291). Special micro-metering pumps give pulseless flow rates down to pl/min, and allow direct interfacing of the LC apparatus to a mass or infrared spectrometer. They also open the field of fused silica columns for HPLC use, and it might be not too optimistic to expect theoretical plate numbers of >500000 per LC column for the near future. 

In contrast to flowcell interfaces, solvent-elimination approaches lead to spectra free of solvent interferences. Various sampling techniques are possible the sample can be deposited on a flat ZnSe plate, on a smooth metallic substrate or a thin layer of powdered alkali halide salt, whereas the spectrum can be taken in conventional transmission, external-reflection or diffuse-reflection arrangement. In one of the first applications a synthetic mixture of three quinones was separated on a microbore column packed with silica, using a mobile phase of 5% methanol in supercritical carbon dioxide. The peaks were collected on a plate on which a layer of KCl powder was deposited, and then spectra were measured by a diffuse-reflection accessory. Test measurements on acenaphthenequi-none (AQ) showed later that conventional transmission spectra of samples on flat infrared-transmitting windows give the best compromise between high sensitivity, correct relative band intensities and adherence to the Lambert-Beer law. 

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. 

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