Trace analysis mobile phase

Multidimensional HPLC offers very high separation power when compared to monodimensional LC analysis. Thus, it can be applied to the analysis of very complex mixtures. Applications of on-line MD-HPLC have been developed, using various techniques such as heart-cut, on-column concentration or trace enrichment applications in which liquid phases on both columns are miscible and compatible are frequently reported, but the on-line coupling of columns with incompatible mobile phases have also been studied. 

Figure 12.18 LC-SFC analysis of mono- and di-laurates of poly (ethylene glycol) ( = 10) in a surfactant sample (a) normal phase HPLC trace (b) chromatogram obtained without prior fractionation (c) chromatogram of fraction 1 (FI) (d) chromatogram of fraction 2 (F2). LC conditions column (20 cm X 0.25 cm i.d.) packed with Shimpak diol mobile phase, w-hexane/methylene chloride/ethanol (75/25/1) flow rate, 4 p.L/min UV detection at 220 nm. SFC conditions fused-silica capillary column (15 m X 0.1 mm i.d.) with OV-17 (0.25 p.m film thickness) Pressure-programmed at a rate of 10 atm/min from 80 atm to 150 atm, and then at arate of 5 atm/min FID detection. Reprinted with permission from Ref. (23).

Figure 12.22 SFC-GC analysis of aromatic fraction of a gasoline fuel, (a) SFC trace (b) GC ttace of the aromatic cut. SFC conditions four columns (4.6 mm i.d.) in series (silica, silver-loaded silica, cation-exchange silica, amino-silica) 50 °C 2850 psi CO2 mobile phase at 2.5 niL/min FID detection. GC conditions methyl silicone column (50 m X 0.2 mm i.d.) injector split ratio, 80 1 injector temperature, 250 °C earner gas helium temperature programmed, — 50 °C (8 min) to 320 °C at a rate of 5 °C/min FID detection. Reprinted from Journal of Liquid Chromatography, 5, P. A. Peaden and M. L. Lee, Supercritical fluid chromatography methods and principles , pp. 179-221, 1987, by courtesy of Marcel Dekker Inc.

From a mass spectrometry perspective, the pump must be pulse free, i.e. it must deliver the mobile phase at a constant flow rate. Pulsing of the flow causes the total ion current (TIC) trace (see Chapter 3) - the primary piece of information used for spectral analysis - to show increases in signal intensity when analytes are not being eluted and this makes interpretation more difficult. 

Reaction detectors are a convenient means of performing online postcolumn derivatization in HPLC. The derivative reaction is performed after the separation of the sample by the column and prior to detection in a continuous reactor. The mobile phase flow is not interrupted during the analysis and reaction, although it may be augmented by the addition of a secondary solvent to aid the reaction or to conform to the requirements of the detector. Reaction detectors are finding increasing application for the analysis of trace components in complex matrices where both high detection sensitivity and selectivity are needed. Many suitable reaction techniques have been published for this purpose [641-650]. 

Fig. 2.55. Gradient reversed-phase HPLC analysis of flavonoids in white onions (a) and celery (b). ODS column of 150 X 3.9mm i.d particle size 5pm. Mobile phase 20min gradient of 15-35 per cent acetonitrile in water adjusted to pH 2.5 with TFA. Fowrate lml/min. Upper and lower traces represent samples before and after hydrolysis, respectively. Detection wavelength 365 nm. IS = internal standard Qc = quercetin Ap = apigenin Lt = luteolin. Reprinted with permission from A. Crozier et al. [159],...

Fig. 3.38.The IUPAC names of Sudan azo dyes are as follows Sudan 1 = 1— [(2,4-dimethylphenyl)azo]-2-naphtalenol Sudan II = l-(phenylazo)-2-naphtol Sudan III = l-(4-phenylazophenylazo)-2-naphtol Sudan IV = o-tolyazo-o-tolyazo-beta-naphtol and Disperse Orange 13 = 4-[4-(phenylazo)-l-naphtylazo]-phenol. Azo dyes were separated in an ODS column (250 x 2.1 mm i.d. particle size 5 /xm) at 35°C. The isocratic mobile phase consisted of 0.1 per cent formic acid in methanol-0.1 per cent formic acid in water (97 3, v/v). The flow rate was 200 /xl/min. MS conditions were nebulizing and desolvation gas were nitrogen at the flow rates of 50 and 5551/h, respectively electrospray voltage, 3.0 kV cone voltage 25 V source temperature, 110°C desolvation temperature, 110°C. Azo dyes were extracted from the samples by homogenizing 1 g of sample with 10 ml of acetone, then the suspension was centrifuged and an aliquot of 3 ml of supernatant was mixed with 1 ml of deionized water, filtered and used for analysis. LC-ESI-MS/Ms SRM traces of standards and spiked samples are listed in Fig. 3.39. It was found that the detection and quantitation limits depended on both the chemical structure of the dye and the character of the accompanying matrix. LOD and LOQ values in chilli tomato sauce...

Fig. 3.58. LC-MS-MS analysis of an effluent sample taken through an analytical method involving SPE. LC conditions mobile phase acetonitrile-0.01 g/100 ml ammonium acetate in water (90 10, v/v) flow rate 0.30 ml/min sample volume 20 pi. The left-hand traces show the MS-MS ion chromatograms, while the corresponding right-hand spectra show the presence of the daughter ion indicative of each dye. RT = retention time. Reprinted with permission from W. F. Smyth et al. [128].

LC/MS-APCI in the positive ion mode was used for trace analysis of TATP [20]. Chromatographic separation was carried out with a C18 (150 X 2.0 mm, 3 (Xm particle size) column, with a mobile phase of methanol—water (70 30) with 5 mM ammonium acetate buffer, at flow rates between 0.1 and 0.2mL/min. Samples were injected as acetonitrile solutions. 

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