HPLC (High Pressure Liquid Chromatography) for High Performance in Food Analysis

Recently, to separate and detect additives and contaminants, High- Performance Liquid Chromatography (HPLC) is being used increasingly in the analysis of food samples. This method is suitable for compounds with minimal thermal stability since separation and detection occur at or slightly above ambient temperature. The method works on breaking down a complex mixture of samples into individual compounds. This is, in turn, identified and quantified by suitable detectors and data-handling systems. HPLC is a very sensitive analytical technique with its ability to inject large sample amounts (up to 1-2 ml per injection). The HPLC and the non-destructive detection techniques also require fractions to be collected for further study. Modern sample preparation techniques, such as SPE (Solid-Phase Extraction) and SFE (Supercritical Fluid Extraction) allow for high sensitivity HPLC analysis in the ppt (parts per trillion) range. These different detection techniques enable not only highly sensitive, but also highly selective, analysis of compounds in the food samples. Most analyses are nowadays carried out by HPLC on reverse-phase columns. HPLC with UV-vis and fluorescence detector is used for amino acid and protein analyses in food. Considering GC, HPLC, with its selective detectors, together with its ability to connect to a mass spectrometer (MS) for peak identification, makes it the most popular and versatile chromatographic method. It is at ambient temperatures; HPLC detects and separates compounds. For this reason, HPLC has been adopted and recommended by the US Food and Drug Administration (FDA) to analyze thermally labile, non-volatile, and highly polar compounds (Smith and Thakur, 2010).

In a work reported by Song et al. (2019), an HPLC-based detection method was developed for the identification of adulteration in coffee powder with roasted barley, wheat, and rice powders. Numerous chemical indicators, including monosaccharides (glucose, galactose, mannose, rhamnose, xylose, and arabinose), trigonelline, and nicotinic acid, were used. The recovery efficiencies obtained for monosaccharides were 84.1-90.2 percent, for trigonelline 113.6 percent and for 114.9 percent nicotinic acid. The LoD (Limits of Detection) was found to be

  • 0.047-0.070 mmol kg-1 for monosaccharides, for trigonelline 0.209 mg kg4 and 0.117 mg kg 1 for nicotinic acid respectively. Significant differences were noticed in the glucose concentration on comparison of the control coffee sample with samples of coffee adulterated with roasted barley, wheat and rice in the 99:1 (w/w) mixing ratio. It was also noticed that when glucose was used as a chemical index, the limit of discrimination from adulterated coffee samples was found to be 1 per cent (w/w) (P < 0.05).
  • 2.2.1 HPLC with Evaporating Light Scattering Detector (ELSD) for Sugar Analysis

An ELSD (Evaporative Light Scattering Detector) is a universal HPLC detector whose detection principle uses the light scattering phenomenon that occurs from residual particles of non-volatile components. It is done after eliminating the volatile mobile phase with a heat-gas combination for nebulization. LED is the light source used in the ELSD detector. It also uses a photomultiplier so as to measure the changes in the intensity of light in the optical path as the analyte passes through it. One of the advantages here is that the compounds that are usually quantified using a short wavelength of UV detector or a refractive index detector can be identified and measured with more sensitivity and increased stable baseline using ELSD. Valliyodan et al. (2015) used HPLC equipped with an Evaporative Light Dispersing Detector (ELSD) to isolate, classify, and quantify seven sugars in soybean, namely glucose, galactose, fructose, sucrose, melibiose, raffinose, and stachyose. For this, the gradient elution was programmed into two mobile stages. Phase A was pure water, and phase В is mobile, which was a mixture of acetonitrile and acetone 75:25 (v/v).

High Performance Capillary Electrophoresis (HPCE) for Food Analysis

Capillary electrophoresis (CE) is a relatively new and swift separation technique. It is used in the analysis of food routinely. Initially, CE was primarily applied in the analysis of biological macromolecules. This is also used for extracting vitamins, amino acids, chiral medicines, inorganic ions, organic acids, toxins, coloring agents and surfactants. HPCE is a technique to separate mixtures into constituent or distinct compounds. Here the compounds are separated on the basis of variations in their charge to size ratios. In a comparative study between HPLC and HPCE in the analysis of green tea catechins, HPCE offered several advantages with respect to sensitivity, solvent consumption, and time of analysis (Bonoli et al., 2003).

Thus, HPCE is very suitable for the analysis of molecules with a broad range of sizes, charge, and hydrophobicity, such as proteins and peptides in food samples. The other feature of HPCE resides in its ability to manage very low sample amounts, like nanolitre injection volumes that make it ideal for its application with limited sample volumes. Here the electrophoresis is performed in narrow bore, fused silica capillaries.

For example, in the analysis of natural colors, especially of individual pigments, can be carried out only through chromatographic techniques. Solvent extraction of pigments can be done by individual solvents or mixtures of solvents, like acidified alcohol/water mixtures for anthocyanins, ethyl acetate or dichloromethane for carotenoids, etc. Here the detection is carried out in the visible spectrum—diode array detectors to confirm the peak identity. Though the quantification of these pigments can be done through HPLC, using an external standard, the qualitative identification of pigment origin can be done through the powerful tool, like capillary electrophoresis. By this, fruit desserts colored with grapeskin or with black carrot extracts can be easily detected and sauces colored with paprika extracts or p-carotene can be easily distinguished and identified from each other. Due to the heterogeneous nature, caramels and phenolic oxidation products cannot be analyzed as single chemical entities due to their heterogeneous nature, although the presence of byproducts such as hydroxymethylfurfural may be useful for an indirect assessment of caramel addition. Here new techniques, such as capillary zone electrophoresis, hold new promise for the analysis of charged materials, such as the class IV E150(d) caramels (Scotter, 2003).

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