On-line HPLC analysis

Figure 1 Goals of temporary vs. permanent on-line HPLC analysis.

Stalmach A, Mullen W, Nagai C, Crozier A. On-line HPLC analysis of the antioxidant activity of phenolic compounds in brewed, paper-filtered coffee. Brazilian Journal of Plant Physiology. 2006 18(1) 253-262. 

W. Golkiewicz, C. E. Werkhoven-Goewie, U. A. Th Brinkman, R. W. Frei, H. Colin and G. Guiochon, Use of pyrocarbon sorbents for trace enrichment of polar compounds from aqueous samples with on-line HPLC analysis , J. Chromatogr. Sci. 21 27-33 (1981). 

The most popular method is automated Edman chemistry (mn on a sequencer), a method which removes one amino acid at a time from the N-terminus, resulting in the sequential liberation of phenylthiohydantoin (PTH) amino acids, which are identified by on-line HPLC analysis. Edman sequanators are completely automated high-sensitivity instrument systems that can routinely detect and identify as little as 0.2 pmol to 1 pmol of amino acid in a given cycle and carry out more than 20 cycles with 1 to 5 pmol of protein. Most peptides of length 3-30 amino acids can be sequenced completely. Although amino... 

As we have seen in this book, three developments appear to have provided the necessary momentum, and all three are still works in progress . The first was the development of TOF instruments, which could accomodate electrospray ionization by using orthogonal extraction or combining ion trapping with the TOF analyzer, which led to the possibility of carrying out on-line HPLC analysis. Considerable improvements are required to enable ESI-TOF instruments to be commercially competitive with existing quadrupole (and triple quadrupole) ESI mass spectrometers, but this is sure to be a major focus of development by both University laboratories and instrument manufacturers. 

Mondello et al. (54) have developed some applications of on-line HPLC-HRGC and HPLC-HRGC/MS in the analysis of citrus essential oils. In particular, they used LC-GC to determine the enantiomeric ratios of monoterpene alcohols in lemon, mandarin, bitter orange and sweet orange oils. LC-GC/MS was used to study the composition of the most common citrus peel, citrus leaf (petitgrain) and flower (neroli) oils. The oils were separated into two fractions, i.e. mono- and sesquiterpene... 

Besides on-line UV analysis, off-line HPLC analysis was performed after about 48 h dark reaction [72, 74]. In this period, the photoinduced reaction proceeds by radical paths in the dark. 

The Gilson Aspec automatic sample preparation system is a fully automated system for solid-phase extraction on disposable columns and online HPLC analysis. The Aspec system offers total automation and total control of the entire sample preparation process including clean-up and concentration. In addition, Aspec can automatically inject prepared samples into on-line HPLC systems. 

E. J. Benjamin and D. L. Conley, On-line HPLC method for clean-up and analysis of hydrocortisone and sulconazole nitrate in a cream, Int. J. Pharm., 75 205 (1983). 

Godejohann, M., Preiss, A., Miigge, C. and Wii-nsch, G, Application of on-line HPLC- H NMR to environmental samples analysis of ground-water near former ammunition plants, Anal. Chem., 69, 3832-3837 (1997). 

The direct coupling of liquid chromatography with proton NMR has been attempted numerous times. Early experiments of coupled HPLC- H-NMR were conducted in a stop-flow mode or with very low flow rates [193-195]. This was necessary to accumulate a sufficient number of spectra per sample volume in order to improve the signal-to-noise ratio. Other problems associated with the implementation of on-line HPLC-NMR have included the need for deuterated solvents. However, with the exception of deuterium oxide, the use of deuterated eluents is too expensive for routine analysis. Therefore, proton-containing solvents, such as acetonitrile or methanol, must be used. To get rid of the solvent signals in the spectra, the proton NMR signals of the solvents have to be suppressed. 

The analysis of fatty alcohol ethoxylate based surfactants by on-line HPLC- H-NMR has been described by Schlotterbeck et al. [211]. Using a reversed stationary phase and ACN/deuterium oxide as the eluent, surfactant mixtures were... 

Fig. 39. Contour plot of chemical shift vs. retention time and chemigram of the on-line HPLC-NMR analysis of a technical poly(ethylene oxide) (from [210] with permission)...

D. Louden, A. Handley, S. Taylor, I. Sinclair, E. Lenz, and I. D. Wilson, High temperature reversed-phase HPLC using deuterium oxide as a mobile phase for the separation of model pharmaceuticals with multiple on-line spectroscopic analysis (UV, IR, IH-NMR and MS), Analyst 126 (2001), 1625-1629. 

K. Mock, J. Firth, J.S. Cottrell, Application of on-line HPLC-FAB MS to the analysis of enzymatic digests of ribonuclease B, Org. Mass Spectrom., 24 (1989) 591. 

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