Column eluate

Figure 10.4 shows a schematic representation of the multidimensional GC-IRMS System developed by Nitz et al. (27). The performance of this system is demonstrated with an application from the field of flavour analysis. A Siemens SiChromat 2-8 double-oven gas chromatograph equipped with two FIDs, a live-T switching device and two capillary columns was coupled on-line with a triple-collector (masses 44,45 and 46) isotope ratio mass spectrometer via a high efficiency combustion furnace. The column eluate could be directed either to FID3 or to the MS by means of a modified Deans switching system . 

The major advance in the way in which column eluate is deposited on the belt was the introduction of spray deposition devices to replace the original method which was simply to drop liquid onto the belt via a capillary tube connected directly to the outlet of the HPLC column. These devices, based on the gas-assisted nebulizer [5], have high deposition efficiencies, transfer of sample can approach 100% with mobile phases containing up to 90% water, and give constant sample deposition with little band broadening. 

The maximum flow rate that can be accommodated while still allowing the mass spectrometer to operate is in the range of 10-20 tilmin" Typical flow rates used in conventional HPLC separations are between 500 and 1000 tilmin and therefore only between 1 and 4% of the column eluate, and therefore ana-lyte(s), enter the mass spectrometer source. The sensitivity, or more accurately the lack of sensitivity, of the DLl interface is one of its major limitations. 

Two forms of interface have been commercially developed [7] which allow analytes in a flowing liquid stream - it has to be pointed out, not necessarily from an HPLC system (see below) - to be ionized by using FAB. These are essentially identical except for that part where the HPLC eluate is bombarded with the heavy-atom/ion beam. Both of these interfaces consist of a probe in the centre of which is a capillary which takes the flowing HPLC eluate. In the continuous-flow FAB interface (Figure 4.3), the column eluate emerges from the end of the capillary and spreads over the probe tip, while in the frit-FAB interface the capillary terminates in a porous frit onto which the atom/ion beam is directed. 

Figure 5.31 LC-electrospray-MS-MS spectrum of the column eluate at around 22 min in the analysis of the peptide mixture from the tryptic digest of glycoprotein TIME-EA4 from silkworm diapause eggs. Reprinted from Bioorg. Med. Chem., 10, Kurahashi, T., Miyazaki, A., Murakami, Y., Suwan, S., Franz, T., Isobe, M., Tani, M. and Kai, H., Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS , 1703-1710, Copyright (2002), with permission from Elsevier Science.

F. 6 Schematic representation of HPLC-HPTLC coupling by means of the OSP-2 system (Merck) for post-column enrichment of the column eluate fractions. 

Fig. 7 Linomat C (Camag) for on-line transfer of column eluate fractions to TLC/HPTLC plates (A) and application scheme (B).

The silica gel column eluates (Module Cl or C2) are injected, if necessary with the addition of an internal standard, into a gas chromatograph followed by ECD or NPD. The determinations can be performed with different gas chromatographs and fused-silica capillary columns. 

Precondition a Cig (EC) SPE column (l-g/6-mL) with methanol (5mL) followed by another 5 mL of acetonitrile-water (3 7, v/v). Transfer the sample on to the column and allow it to percolate through the column under vacuum, discarding the column eluate. Wash the column with 5 mL of acetonitrile-water (3 7, v/v). Dry the column under high vacuum for 15 min and wash it with hexane (5 mL). Elute the azoxystrobin from the column with 5 mL of ethyl acetate-dichloromethane (11 9, v/v), and evaporate the eluate to dryness under a stream of air in a heating block at 50 °C. Dissolve the sample in 1 mL of acetonitrile-water (1 1, v/v) and filter the solution through a 0.45-p.m syringe filter, transferring the filtrate to an autosampler vial ready for LC/MS/MS analysis. 

Silica gel column cleanup. Clean up the sample with a 15-g silica gel column as described for ginned cottonseed. Evaporate the column eluate just to dryness using rotary evaporation under reduced pressure in a <40 °C water-bath. 

Transfer the sample to the column with a Pasteur pipet and drain the solvent to the top of the sodium sulfate layer. Rinse the round-bottom flask three times with 3-mL portions of hexane-ethyl acetate (10 1, v/v), adding these rinses sequentially to the column and draining the solvent to the top of the sodium sulfate layer before the next addition. Discard the accumulated eluate and place a 100-mL round-bottom flask under the column. Elute the residues with 28 mL of hexane-ethyl acetate (10 1, v/v). Evaporate the column eluate just to dryness by rotary evaporation under reduced pressure in a <40 °C water-bath. Reconstitute the sample in 2.0 mL of toluene for analysis (Section 6.2). 

Vidrine, D. W., Use of subtractive techniques in interpreting on-line FTIR spectra of HPLC column eluate, ]. Chromatogr. Sci., 17, 477, 1979. 

In reduced-flow LC-MS systems, the solvent flow into the spectrometer is reduced to a level where the pumping system can cope. Essentially, three such systems have been developed direct-liquid-introduction (DLI), flowing FAB [531] and electrospray [532]. An alternative approach to belt transport interfacing is to deliver the column eluate directly into the MS source and use Cl techniques. Methods based on this principle are called direct-liquid-injection systems, which are comprised of capillary flow restrictors, diaphragms,... 

Roets and Vanderhaeghe also examined neomycin by chromatography on Dowex 1X2 but these authors monitored the column eluate conductometrically. Gillet et al applied a similar procedure to the examination of various samples of neomycin to ascertain the ratio of neomycin B C. 

An automated colorimetric assay for the quantitation of the separate components of neomycin (B, C and neamine) has also been reported- - . The method utilizes a separation of the components on a column of carbon and Kieslguhr G (4 1). The column eluate is reacted with nin-hydrin to determine the amounts of neomycin B, C and neamine. 

The ninhydrin reaction requires heat and therefore the stream of column eluate plus ninhydrin reagent must pass through a coil of narrow-bore tubing (approximate diameter 1 mm) held in a 100°C heating bath. It is important to ensure that the flow is not restricted because excessive heating may cause the ninhydrin to precipitate in these micro-bore tubes, resulting in complete stoppage of the analyser. 

When all of the material absorbing at 418 nm (associated with the cytochrome P-448 fractions) was eluted from the DEAE-cellulose column (which in some experiments required more than 1 liter of Buffer II), elution was continued with a linear KC1 gradient (0-0.5 M) in Buffer II, as shown in Fig. 5. A different form(s) of cytochrome P-450 (fractions 130-155), having maximal absorption near 451 nm in the carbon monoxide ligated and reduced form (Fig. 6), was obtained although only 2- to 3-fold purification, relative to microsomes, was achieved. This form of cytochrome P-450 was extensively contaminated with epoxide hydratase activity. However, by combining fractions 130-150 (Fig. 5), it was possible to obtain cytochrome P-451 essentially free of cytochrome b5. The relative content of cytochrome P-448 and cytochrome P-451 Tn the DEAE-column eluates ranged from 1 1.1 to 1 1.6 in several different experiments. 

Partial Purification. The cell-free suspension, before and after protease treatment, was subjected to gel-filtration chromatography on Sephadex G-75 according to the procedure described in our earlier report (li). Peak II of the sephadex column eluate will be referred to as flavoprotein preparation (Fig. 1). 

The refractive index detector operates by comparing the refractive index of the mobile phase prior to the column with the refractive index of the column eluate. This detector responds to nearly all solutes but it is highly temperature-sensitive (Skoog et al., 1998). This type ofdetector can be used for sugars and fatty acids. 

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