On-line hyphenation

Mass spectrometry provides detailed information regarding molecular weights and structures from extremely small quantities of materials. Several types of ionization sources can be employed for the on-line hyphenation of capillary electromigration techniques with MS, which include... 

A useful approach to detection in polymer HPLC presents the on-line hyphenation of different measurement principles. For example, an RI detector combined with a UV photometer produces valuable additional information on the composition of some copolymers. Further progress was brought with the triple detection RI plus LALS plus VISCO [313], which is especially suitable for branched macromolecules and the tetra detection UV plus RI plus LALS plus VISCO, which enables characterization of some complex polymer systems, exhibiting a distribution not only in their molar mass and architecture, but also in their chemical composition such as long chain branched copolymers. 

Instrumental developments (e.g., of sector field instruments with multiple ion collection, introduced in 1992, or the insertion of collision and reaction cells in order to reduce disturbing isobaric interferences), the progress in applications for ultratrace analysis, also in combination with on line hyphenated separation techniques (HPLC, CE), especially routine capability as well as decreasing price and user friendly maintenance mean that sales are increasing by 10 % every year. To improve the analytical performance of ICP mass spectrometers for precise isotope ratio measurements (e.g., for geochronology or for the study of fine isotope variation in nature) powerful instrumentation with high mass dispersion and multiple ion collector systems instead of single ion collection are commercially available on the analytical market. 

In summary, the variety of applications outlined in this section provides evidence for the concept of on-line hyphenation and demonstrates the enormous potential for the solution of structure elucidation problems in all major research areas. 

On-Line Systems Flowing MMLLE systems have been established in different layouts with automation and on-line hyphenation to GC and HPLC analysis. An automated on-line FS-MMLLE-GC system with a loop-type interface compatible with LVI was used for the extraction of pesticides and PAHs in surface waters.86 In another study, pressurized hot water extraction (PH WE) was coupled on-line to a FS-MMLLE-GC-FID system and applied to the analysis of PAHs in soil, where MMLLE was used as a cleanup and concentration step of the PH WE extract prior to final GC analysis.87 In addition, an HF-MMLLE setup was incorporated in PHWE and GC, resulting in an online PHWE-HF-MMLLE-GC system, where the HF membrane module contained 10-100 HFs. The system served for the extraction and analysis of PAHs in soil and sediments ... 

Flowing FS-MMLLE with on-line hyphenation to FtPLC has also been investigated. Sandahl et al. were the first to interface FS-MMLLE with reversed-phase HPLC for the on-line extraction of methyl-thiophanate in natural water, obtaining an LOD of 0.5 pg L-1.89 Also, a parallel FS-SLM and FS-MMLLE design was coupled on-line to reverse-phase HPLC for the extraction of methyl-thiophanate (by MMLLE) and its metabolites (by SLM) in natural water.90 In addition, on-line coupling of FS-MMLLE and normal-phase HPLC has been successfully applied in the determination of vinclozolin (Ee =118 and LOD = 1 pg I. ) in surface water91 and of in-sample ion-paired cationic surfactants (Ee > 250 and LOD = 0.7-5 ug L-1) in river water and wastewater samples.92... 

The on-line hyphenation of CEC and MS has several potentially challenging instrumental aspects which complicate the successful combination of these two techniques. The first arises due to the absence of a CEC column outlet electrolyte reservoir and the need to achieve electrical continuity for the CEC system, and, in the case of ESI, also for the ESI ion source. Another consideration is the requirement to efficiently remove the mobile phase and simultaneously generate gas phase ions from the analyte, which have to be transferred with high efficiency into the vacuum of the mass analyzer. Because of this situation, numerous designs have been advanced which solve to various degrees these related problems and are discussed individually below. 

A rapid and simple MW-assisted digestion method with alkaline solution (TMAH or methanolic KOH solution) was developed for speciation analysis of inorganic Hg and methyl-Hg in biological tissues [41]. Extracts with quantitative recoveries of Hg species after the alkaline dissolution of the sample were directly analyzed by an automated on-line hyphenated system incorporating aqueous HG, cryogenic trapping, GC, and detection by A AS. The proposed method was validated by the analysis of biological CRMs (CRM 463, DORM-1, TORT-1) and one BCR sample from an interlaboratory study (Tuna Fish 2). 

The usual method of elemental speciation - also in human milk samples - is the use of already well-established separation and detection methods, which have to be combined in novel ways and modified according to the relevant speciation problems. Off-line combination and on-line hyphenation of separation techniques... 

The combination of capillary zone electrophoresis or CIEF with inductively coupled plasma (ICP) MS promises a powerful tool for metal speciation. The on-line hyphenation of these techniques resulted in short separation times (10 min) and a subsequent detection step lasting 100 s. Standard mixtures and body fluids such as human milk and serum have been studied [65],... 

Use of an appropriate detector (e.g., not GFAAS for on-line hyphenation (loss of chromatographic resolution), qlCP-MS may provide questionable results in the range m/z 40-80 due to interferences, ESI-MS mostly unsuitable for ionic (labile) species (total loss of species information)... 

Kitayama, T., Janco, M., Ute, K., Niimi, R., Hafada, K., and Berek, D., Analysis of Poly(ethyl methacrylate)s by On-line Hyphenation of Liquid Chromatography at the Critical Adsorption Point and Nuclear Magnetic Resonance Spectroscopy, Anal Chem., 71, 1518, 2000. 

Mincsovics, E., Papai, K., Ludanyi, K., David,A.Z., Budai, M., Antai, L, Klebovich, I. (2008) Fully on-line hyphenation of an experimental OPEC separation unit with diode-array detection and mass-spectrometry (OPLC-DAD-MS) for anaiysis of xanthine compounds. Journal of Planar Chromatography, 21,361-366. 

Cebolla, V., fame, C., Membrado, L., Saviron, M., and Orduna, J. July 2-4, 2014, Separation, quantitative determination, and fatty acid profiling of monoacylgliceraides in fatty-acid methyl esters (FAME) using an on-line, hyphenated teehnique based on AMD-FDIC-ESI-MS, HPTLC 2014, Lyon, France, abstract P-29. 

Michel T, Destandau E, Elfakir C (2011) On-line hyphenation of centrifugal partition chromatography and high pressure liquid chromatography for the fractionation of flavonoids from Hippophae rhamnoides L. berries. J Chromatogr A 1218 6173-6178... 

D.-D. Wang, F. Li, and X.-P. Yan. On-line hyphenation of flow injection, miniaturized capillary electrophoresis and atomic fluorescence spectrometiy for high-throughput speciation analysis. 

FIGURE 5.9 Two-dimensional plot of the on-flow LCCC—NMR separation of blends of PS and PI, separation at critical conditions of PS, samples 1—5 are indicated by numbers. Source Reprinted from P. Sinha, W. Hiller, H. Pasch, Characterisation of blends of polyisoprene and polystyrene by on-line hyphenation of HPLC and 1-H-NMR LC-CC-NMR at critical conditions of both homopolymers. Journal of Separation Science, 33 (2010) 3494—3500. (2010), used with permission from Wiley-VCH (Ref. [96]). 

Kitayama T, Janco M, Ute K, Niimi R, Hatada K, Berek D. Analysis of polyjethyl methacrylatejs by on line hyphenation of liquid chromatography at the critical... 

Stnha P, Hiller W, Pasch H. Characterisation of blends of polyisoprene and polystyrene by on-line hyphenation of HPLC and 1-H-NMR LC-CC-NMR at critical conditions of both homopolymers. J Sep Sci 2010 33 3494-500. 

Nowadays, atomic absorption spectrometry (AAS) systems are comparatively inexpensive element selective detectors, and some of the instruments also show multi(few)-element capability. There are flame (F AAS), cold vapor (CV AAS), hydride-generating (HG AAS), and graphite furnace (GF-AAS) systems. However, the use of AAS-based detectors for on-line speciation analysis is problematic. F AAS is usually not sensitive enough for speciation analysis at "normal" environmental or physiological concentrations and sample intake is high (4—5 ml/min), which complicates on-line hyphenations with LC an auxiliary flow is necessary. CV AAS and HG AAS use selective derivatization for matrix separation and increased sensitivity for the derivatized species. But, the detector response is species dependent and interferences can be a problem. GF AAS requires only a few microliters of sample and provides low detection limits, between 0.1 and 5 gg/1. Matrix interferences are widely eliminated by Zeeman correction and matrix modifiers nevertheless, quantification errors were reported as atomization temperature does not exceed 2900°C. The most critical problem in respect to speciation analysis is the discontinuous measiuement because of the temperature program operation employed, which takes a few minutes. Therefore, GF AAS is unsuitable for on-line hyphenations as chromatograms carmot be monitored with sufficient resolution. 

Mature technique and instrumentation On-line hyphenation Wide applicability (including QC)... 

On-line GPC-MALDI MS On-line hyphenation of MALDI-MS with GPC is not as straightforward because of the incompatibilities associated with the complex sample preparation schemes required for MALDI and the intrinsically pulsed nature of the laser source with a continuous beam of eluent from GPC. Fei and Murray first demonstrated on-line GPC-MALDI-TOF MS by mixing the eluent from the GPC with a matrix solution, and by irradiating the resulting spray with a laser to promote MALDI. (349... 

Off-line GPC-MALDI MS In contrast to on-line hyphenation, off-line hyphenation of MALDI-TOF MS with GPC is more conunonplace. In one offline approach, GPC fractions are collected and then prepared individually for MALDI-TOF MS analysis. Fig. 5 illustrates MALDI-TOF MS spectra of GPC fractions obtained for the same HB-PDMS sample as illustrated in Fig. 1. In Fig. 5 each fractionated polymer spectrum obtained contains a well-defined monodisperse MMD with the expected decrease in average mass with increasing GPC elution time. The combination of GPC with MALDI-TOF MS serves to simplify the complexity of the polydisperse MMD such that higher mass oligomers can be observed. Comparatively oligomers up to 12,000 Da were observed with approximately the same S/N as that observed for the 4,000 Da... 

Sinha, P. Hiller, W. Pasch, H.Characterization ot Blends ot Potyisoprene by On-line Hyphenation of Critical Chromatography and Nuclear Magnetic Resonance Spectroscopy. Polychar-18 Siegen, Germany, 2010. 

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