Off-line hyphenation

Z. Bie, Y. Chen, H. Li, R. Wu and Z. Liu, Off-line hyphenation of boronate affinity monolith-based extraction with matrix-assisted laser desorp-tion/ionization time-of-flight mass speetrometry for effieient analysis of glycoproteins/glycopeptides, AnaZ. Chim. Acta, 2014, 834,1-8. 

Orinak, A., Orinakova, R., Slesarova, L., and Gazdova, V. (1997). Off-line hyphenation of thin layer chromatography with atomic absorption spectrometry in the study of stability of zinc complexes. J. Planar Chromatogr.—Mod. TLC 10 44-47. 

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... 

Trace analysis is particularly attractive for SFE-HPLC since quantitative transfer of all analytes extracted to the chromatographic system becomes possible. At present, on-line SFE-HPLC appears to be feasible for qualitative analysis only quantitation is difficult due to possible pump and detector precision problems. Sample size restrictions also appear to be another significant barrier to using on-line SFE-HPLC for quantitative analysis of real samples. On-line SFE-HPLC has therefore not proven to be a very popular hyphenated sample preparatory/separation technique. Although online SFE-HPLC has not been quantitatively feasible, SFE is quite useful for quantitative determination of those analytes that must be analysed by off-line HPLC, and should not be ruled out when considering sample preparatory techniques. In most cases, all of the disadvantages mentioned with the on-line technique (Table 7.15) are eliminated. On- and off-line SFE-HPLC were reviewed [24,128]. 

Figure 7.16 Schematic representation of off-line SFC-FTIR. After deposition of the eluites on to a moving ZnSe substrate the window is moved to the focus of a stand-alone FTIR microscope, where the spectmm of each spot is measured with the plate stationary. After Griffiths et al. [374]. Reprinted from P.R. Griffiths et al., in Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction (K. Jinno, ed.), pp. 83-101, Copyright (1992), with permission from Elsevier...

FTIR in multiply hyphenated systems may be either off-line (with on-line collection of peaks) [666,667] or directly on-line [668,669]. Off-line techniques may be essential for minor components in a mixture, where long analysis times are required for FT-based techniques (NMR, IR), or where careful optimisation of the response is needed. In an early study a prototype configuration comprised SEC, a triple quadrupole mass spectrometer, off-line evaporative FTIR with splitting after UV detection see Scheme 7.12c [667]. Off-line IR spectroscopy (LC Transform ) provides good-quality spectra with no interferences from the mobile phase and the potential for very high sensitivity. Advanced approaches consist of an HPLC system incorporating a UV diode array, FTIR (using an ATR flow-cell to obtain on-flow IR spectra), NMR and ToF-MS. 

Applications Ideally, multiply hyphenated systems should be assembled rapidly in response to real need. Access to these means is restricted to a few laboratories only. Multiple LC hyphenations have been used to analyse test mixtures of polymer additives see Table 7.74. The relative ease with which SEC-UV using CDCI3 as a solvent can be coupled to on-line 1II NMR and an in series off-line FUR (Scheme 7.12b), has been shown for a mixture of polymer additives (BHT, Irganox 1076, DIOP) [666]. Figure 7.35 shows representative spectra for on-flow NMR and MS and off-line FTIR of 2,6-di-f-butyl-4-methoxyphenol. 

Linking TLC with a tandem instrument differs from combining GC or LC with an appropriate spectrometer. Hyphenation of planar chromatographic techniques represents a niche application compared to HPLC-based methods. Due to the nature of the development process in TLC, the combination is often considered as an off-line in situ procedure rather than a truly hyphenated system. True in-line TLC tandem systems are not actually possible, as the TLC separation must be developed before the spots can be monitored. It follows that all TLC tandem instruments operate as either fraction collectors or off-line monitoring devices. Various elaborate plate extraction procedures have been developed. In all cases, TLC serves as a cleanup method. 

Integration of sample preparation and chromatography by on-line coupling aims at reduction of analysis time. It is apparent from Section 7.1 that these hyphenated techniques are not yet contributing heavily to the overall efficiency of polymer/additive analysis in industry. On-line SFE-SFC requires considerable method development, and MAE-HPLC is off-line. Enhancement of sensitivity for trace analysis requires appropriate sample preparation and preconcentration schemes, as well as improved detection systems. 

Nanospray is a miniaturized version of electrospray. In the original setup of Wilm and Mann (8) it is utilized as an off-line technique using disposable, finely drawn (1 -gm tip), metallized glass capillaries to infuse samples at 10-30 nL/min flow rates. This allows more than 50 min analysis time with just a 1-pT sample. Due to the formation of much smaller droplets and the more effective ionization, there is often no need for LC separation, since the separation is accomplished in m/z or by MS/MS. However, limited reproducibility with respect to quantification and a more complex sample preparation can be seen as drawbacks. An on-line version for hyphenation with capillary and nano-LC as well as CE (slightly modified) is now commercially available. 

The elution character of the FFF techniques allows for it to be used in combination with other methods for further on-line or off-line characterization of the analytes (see Figure 12.1). FFF can be hyphenated with selective detection systems like mass spectrometry, multiangle laser scattering and can be combined with different separation techniques in multidimensional modes. In Figure 12.3, the trend in the number of published papers is reported. 

To avoid interferences of isobaric atomic ions of different elements and polyatomic ions at the same nominal mass, off line separation of matrix elements or analyte separation can be applied and/or enrichment and hyphenated techniques such as HPLC- or CE-ICP-MS. Analytical procedures have been developed in the author s laboratory for the determination of spallation nuclides in an irradiated tantalum target using HPLC and CE coupled on line to ICP-MS after dissolution and separation of the tantalum matrix.12,17 The mass spectra of rare earth elements (REEs)... 

Shortly afterwards, this work was extended by the incorporation of a mass spectrometer into the system, thus enabling on-line NMR and MS data to be obtained with on-line collection of the eluent for off-line FT-IR spectroscopy [22]. The incorporation of the mass spectrometer required the addition of a small proportion of ammonium acetate, dissolved in methanol, to the deuterated chloroform used as the eluent in order to promote the ionisation of the analytes. The inclusion of methanol and ammonium acetate to the solvent obviously introduced new signals into the NMR spectra, and in addition resulted in the loss of exchangeable protons from the analytes which had been observable when chloroform alone was used as the solvent. This work demonstrated the feasibility of multiple hyphenation ( hypernation ) but the off-line nature of the FT-IR data acquisition, with the inevitable delay inherent in offline analysis, represents a slight disadvantage. In addition, volatile components may well be lost as the solvent is evaporated. This can be a problem that, together with analyte instability, is exacerbated with such interfaces when reversed-phase eluents are used since these require heating in order to ensure removal of the solvent. 

Two-dimensional H NMR experiments acquired in stop-flow mode represent to some extent a link between on-flow LC-NMR screening and the detailed structural elucidation of isolated compounds employing two-dimensional NMR techniques in conventional off-line probe-heads. As the LC-NMR-MS hyphenation offers the possibility of triggering the stopping of the LC pump by the MS signal, this technique is particularly well suited for the reliable detection of compounds showing only weak UV absorbances. 

In addition to the ruberosides A, B and G discussed in some detail here, the application of LC-NMR-MS hyphenation has resulted so far in the targeted isolation and off-line structural elucidation of a total of seven new asterosapo-nins. In a single chromatogram of an asterosaponin fraction, up to 17 individual constituents could be characterised (see Table 5.1.1). 

There are several review papers on SLM extraction that touch upon off-line or on-line SLM hyphenation to analytical instruments as well as the fields of SLM applications.6772 The following discussion will focus mainly on other aspects of SLM, such as transport mechanisms, selectivity, and equilibrium sampling with SLM. 

The conclusions that can be drawn from the work presented in this chapter emphasize the miniaturization trends currently being pursued in modern analyte extraction techniques. The trends are focused mainly on miniaturization of the core idea of the extraction techniques utilized for analyte extraction from water samples. This has in turn promoted automation and hyphenation of extraction principles with on-line or off-line connection to analytical instruments. 

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... 

Methods for Speciation Analysis in Human Milk Typically Using Hyphenated Techniques and Off-Line Combinations... 

As indicated previously (see Section 1.2) pyrolysis must be associated with an analytical technique in order to provide information on a sample. Several common analytical techniques such as GC, GC/MS or GC/FTIR have been utilized either hyphenated with pyrolysis or off-line and were described previously. Less frequently, techniques such as HPLC, preparative LC, TLC, SFE/SFC, or NMR also have been used for the analysis of pyrolysates. These types of techniques are commonly applied off-line. They are used mainly for obtaining information on that part of the pyrolysate that is difficult to transfer directly to an analytical system such as a GC or for the analysis of materials associated with the char. However, the analysis of the non-volatile part of pyrolysates is frequently neglected, although this leads to an incomplete picture regarding the chemical composition of pyrolysates. 

The analysis of samples in biological matrices is extremely powerful compared with drawbacks such as narrow concentration range for quantitative measurements and long time required for spot detection. Digital autoradiography can be used in hyphenated off-line and... 

---