The extraction and chromatographic analysis of organic acids and inorganic anions found in milk and dairy produce

Jones, Martin (2018) The extraction and chromatographic analysis of organic acids and inorganic anions found in milk and dairy produce. Masters thesis, Waterford Institute of Technology.

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Abstract

Dispersive liquid-liquid microextraction (DLLME) is a ternary extraction system that consists of an extraction solvent, dispersing solvent and an aqueous sample containing the analyte of interest. DLLME is a powerful, miniaturised extraction technique that can increase an analytes concentration and achieve high enrichment factors (EFs) which makes it ideal for trace analysis. DLLME consists of injecting the binary mixture of extraction/dispersing solvent into the sample. This creates a very large surface area of fine extraction droplets. It is these droplets that analytes enrich, almost instantaneously into. Given the speed of the extraction, DLLME is a very attractive procedure that can increase sample throughput whilst lowering solvent consumption, waste and associated costs. Several organic acids were identified as common constituents in milk and dairy produce. Organic acids appear in milk and dairy produce due to natural biochemical processes within the animal, bacterial activity, as preservatives and due to adulteration [1–3]. Levels of organic acids in milk and dairy produce vary drastically due to a number of factors such as: breed of animal, time of year, geography, diet, age, health, stage of lactation and starter culture type [4–9]. Organic acids are important in areas such as flavour studies, cheese ripening, human nutrition, monitoring the health of the animal and monitoring the quality of the product prior to sale to the consumer [10–12]. Milk and dairy produce consists of several components such as proteins, peptides, amino acids, carbohydrates, vitamins, minerals and lipids that require removal prior to analysis. These interferences were removed by mixing two solutions, carrez 1 (zinc acetate) and carrez 2 (potassium hexaferrocyanate) with the sample, followed by centrifugation. This left the sample in an aqueous matrix, which is ideal for DLLME. A number of chromatographic techniques were investigated during the course of the project, those being: high performance liquid chromatography (HPLC), gas chromatography (GC) and capillary zone electrophoresis (CE). The technique most appropriate to the use of DLLME and the separation of organic acids was found to be GC. The optimised GC method consisted of injecting 1 μL of extraction solvent using a 10:1 split ratio then separating on a gradient method in 10 minutes using an Altech AT-100 polyethyleneglycol (PEG) column (15 m x 530 μm i.d. x 1.2 μm) . The method was validated for the analysis of six organic acids. Those acids were acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acid. The method gave retention times of 8.79, 10.06, 10.67, 11.18, 11.68 and 12.49 minutes, respectively and %RSD of: < 0.06%. Peak area was also assessed and gave %RSD of < 0.0%. Coefficients of determination (R2) were all ≥ 0.999. LODs for acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acid were estimated to be: 21.88, 67.25, 8.04, 6.86, 39.38 and 21.68 μg/mL, respectively and LOQs were: 66.32, 203.79, 24.38, 20.82, 119.36 and 65.71 μg/mL, respectively. The optimised extraction consisted of injecting a mixture of 100 μL chloroform (extraction solvent) and 700 μL acetone (dispersing solvent) into a 10-mL sample containing 20% w/v NaCl and pH adjusted to 2.50. This produced EFs up to ~ 45 times more concentrated than in the original sample. The optimised and validated method was then applied to real samples of milk and dairy produce, the following results were obtained: cow’s milk: acetic: (NQ); n-butyric: 10.41 μg/mL. Buttermilk: acetic: (NQ); n-butyric: 14.38 μg/mL; iso-valeric: 12.22 μg/mL; n-valeric: 12.78 μg/mL. Goat’s milk: acetic acid (NQ); iso-butyric: 13.23 μg/mL; n-butyric: 16.46 μg/mL; iso-valeric: 13.12 μg/mL; n-valeric: 12.72 μg/mL. Cottage cheese: acetic acid (NQ); n-butyric: 4.04 μg/g. Brie cheese: acetic acid (NQ); n-butyric: 42.31 μg/g; iso-valeric: 0.39 μg/g; n-valeric: 0.72 μg/g. Probiotic yogurt: acetic acid (NQ); iso-butyric: 6.13 μg/g; n-butyric: 6.90 μg/g; iso-valeric: 5.91 μg/g; n-valeric: 6.06 μg/g. Greek yogurt: acetic acid (NQ); n-butyric: 11.00 μg/g. Due to DLLME’s ease of use, the quickness of the extraction procedure, large EFs, and the ability to carry out an extraction using readily available consumables within any laboratory (centrifuge tube, syringe and syringe needle), DLLME is the ideal extraction procedure for high throughput laboratories that are looking to minimise cost and labour while preserving the quality of results. Further to this, no method could be found at the time of writing that utilised DLLME to extract the highly volatile organic acids mentioned above from milk and dairy produce which lends to the novelty of this body of work.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Chromatographic analysis, anions
Departments or Groups: *NONE OF THESE*
Divisions: School of Science > Department of Chemical and Life Sciences
Depositing User: Derek Langford
Date Deposited: 30 Nov 2018 14:55
Last Modified: 06 Feb 2019 11:59
URI: http://repository.wit.ie/id/eprint/3305

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