Natural food components, HPLC

Similarly to the methods used to characterize natural chlorophylls, RP-HPLC has been chosen by several authors to identify the individual components in Cn chlorophyllin preparations and in foods. The same ODS columns, mobile phase and ion pairing or ion suppressing techniques coupled to online photodiode UV-Vis and/or fluorescence detectors have been used. ° ... 

Detection of peptides in HPLC can be achieved by measuring natural absorbance of peptide bonds at 200-220 nm. Unfortunately at these wavelengths a lot of food components and also the solvents used for analysis absorb, demanding an intensive sample pretreatment and clean-up [129]. Peptides with aromatic residues can be detected at 254 nm (phenylalanine, tyrosine, and tryptophan) or 280 nm (tyrosine and tryptophan). Taking advantage of the natural fluorescence shown by some amino acids (tyrosine and tryptophan), detection by fluorescence can also be used for peptides containing these amino acids [106]. 

More recently, enantiomer ratios have been used as evidence of adulteration in natural foods and essential oils. If the enantiomer distribution of achiral component of a natural food does not agree with that of a questionable sample, then adulteration can be suspected. Chiral GC analysis alone may not provide adequate evidence of adulteration, so it is often used in conjunction with other instrumental methods to completely authenticate the source of a natural food. These methods include isotope ratio mass spectrometry (IRMS), which determines an overall 13C/12C ratio (Mosandl, 1995), and site-specific natural isotope fractionation measured by nuclear magnetic resonance spectroscopy (SNIF-NMR), which determines a 2H/ H ratio at different sites in a molecule (Martin et al 1993), which have largely replaced more traditional analytical methods using GC, GC-MS, and HPLC. 

Organic compounds sought include naturally derived materials, such as mycotoxins and off-flavours (produced by rancidification or spoilage), and man-made/industrial chemicals, e.g. pesticides, veterinary drugs, environmental contaminants (such as polychlorodibenzo-p-dioxins, polychlorinated biphenyls, polynuclear aromatic hydrocarbons, etc.) and food tainting compounds (e.g. 2,4,6-trichloro-anisole, the compound responsible for musty cork taint in wine, arising from the inappropriate use of wood preservatives). GC-MS and HPLC-API-MS are widely used for these types of analyses. Desirable food components present at trace levels, such as nutrients, are also determined using these techniques. 

The science of flavor chemistry has concentrated on volatile compounds for two main reasons. First, volatile compounds are primarily responsible for aroma which is often the component of flavor of greatest significance. Second, the techniques developed for the separation and identification of individual components of a complex natural product are much better for volatile compounds than for non-volatile compounds. For example, gas chromatography (GC) is a far more effective method of separation than high pressure liquid chromatography (HPLC), the best method for separation of non-volatile compounds. Therefore, the literature shows much less published information on the presence of non-volatile compounds in foods than it does for volatile compounds. (1-5 ). 

Vanilla extract is used to flavor a wide variety of foods and 1s very expensive. Herrman and StockH (48) developed an HPLC method to determine the major components of vanilla extracts. This method can be used to help differentiate natural vanilla extracts from cheaper substitutes. Shown in Figure 2a 1s a separation of standards of the major components 1n vanilla. Shown in Figure 2b is the chromatogram from a typical vanilla extract. It should be noted in Figure 2b that natural vanilla extracts apparently lack ethyl vanillin, a commonly used flavor substitute. 

Applications of HPLC Of the bioanalytical separation technologies described in this book, arguably HPLC has the widest range of applications, being adopted for the purpose of clinical, environmental, forensic, industrial, pharmaceutical and research analyses. While there are literally thousands of different applications, a few indicators of how HPLC has been used are as follows (i) Clinical quantification of drugs in body fluids (ii) Environmental identification of chemicals in drinking water (iii) Forensic analysis of textile dyes (iv) Industrial stability of compounds in food products (v) Pharmaceutical quality control and shelf-life of a synthetic drug product (vi) Research separation and isolation of components from natural samples from animals and plants. 

GC was the first instrumental technique that allowed the separation of optical isomers [89], This technique offers relatively high peak efficiency compared with HPLC, but the volatility and thermostability requirements of the analyte limit the application of GC for the separation of drug enantiomers. However, GC is used for the enantioseparation of several drugs and many drug intermediates, natural compounds, food and beverage components, essential oils and perfumes. 

The same principle as described above can be used for the estimation of the carotenoid content of extracts of food colorants, pharmaceuticals, foods, biological samples, or chromatographic fractions. This procedure employs calculations used for individual carotenoids of high purity and thus will estimate the total carotenoids present in a food, biological, or natural extract, where a mixture of carotenoids would be expected. Greater accuracy can be obtained as extracts are purified to contain single components. A spectrum scan is not employed in this procedure as the fine structure of a mix of carotenoids can only be identified after HPLC separation and identification. 

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