Determining Chloride and Sugars in Food Samples Using HPLC

Accurate quantification of sugars in saline solution is now possible using a new high performance liquid chromatography (HPLC) method. The Column spoke to Abdelrahman Saleh Zaky from the School of Biosciences at the University of Nottingham (Nottingham, UK) about this method.

Q. Your group has established an HPLC method for the determination of chloride in the presence of sugars in food. What led your group to begin this study?

A: The main objective of this project was to establish the use of seawater as an alternative source of water for the fermentation industry, particularly for the production of bioethanol. Therefore, our fermentation media will contain high amounts of salt, mostly NaCl. In addition, the media will contain high amounts of sugar, especially glucose, (up to 20% w/v) because sugar is the main substrate for bioethanol production. However, obtaining an accurate quantification of sugars in samples containing high concentrations of NaCl using the existing high performance liquid chromatography (HPLC) methods has proven difficult because of the similar retention time for Cl ions and sugar molecules, especially glucose and sucrose (1). Hence, there was a requirement to develop a suitable analytical method for sugars and alcohol derived from samples that contain high concentration of salts. HPLC is the preferred method for sugar quantification according to the guidelines of the Association of Official Analytical Chemists (2). Therefore, our goal was to investigate a new HPLC method that can accurately determine the concentrations of sugars in samples containing salts.

Q. What is novel about your research and what were your main findings?

A: The results from our investigation revealed that a ion-exchange ligand-exchange column, which was designed for sugar and organic acid determination, could also separate the salt ions in the seawater, and as a result sugar quantification of samples from seawater-based media could be accurately obtained. Later, we decided to investigate the ability of this method for the determination of NaCl. Our investigations validated the use of this method for the accurate quantification of Cl ions. The method was successfully applied to a variety of food samples for salt determination and the results were comparable with three other methods (3). This was the first report of the possibility of measuring chloride ions using the organic acid column and reflective index (RI) detector. This method also reported for the first time the possibility of measuring glucose and sucrose in samples containing high concentrations of NaCl using HPLC. The method also allows the simultaneous measurement of chloride salts and sugars in a variety of samples (see Figure 1).

Figure 1: HPLC chromatograms from five mixed solutions of different chemical components.

Q. What advantages does your method offer over existing methods?

A: Classical titration methods, Mohr (4) and Volhard (modified) (5) use silver nitrate (AgNO₃) for the determination of NaCl. Silver nitrate is considered a very toxic and corrosive compound even at very low concentrations (6). In addition, those methods are associated with several limitations such as: a) they are time consuming, b) the results are sensitive to the pH and the presence of heavy metals in the sample, c) they can have false end points, d) they are difficult to automate, and e) the safe disposal of silver compounds after testing. Hence, the chloride analyzer has been suggested as an alternate method. This is a rapid test but still requires AgNO₃ to operate (7).

Our method, on the other hand, does not use any hazardous chemicals and can measure several components including salts, sugars, organic acids, and alcohols in one analysis using a single sample preparation. This saves time and effort and provides accurate results.   

The standard methods for Na⁺ or Cl^- determination, such as flame photometer or ion chromatography, can provide an accurate quantification for salt but they cannot measure the other components of the sample and therefore extra time and cost is needed for the determination of the other components.

Q. What were the biggest challenges involved in the establishment of this method?

A: The chemical structure of salts (which are inorganic substances) are totally different to the chemical structure of sugars (which are organic substances). Finding a method that could quantify both of them in a single analysis was difficult. After finding the suitable method, we had to do many tests to validate our findings.

Q. Can this method be applied to other foods aside from those analyzed in your research?

A: The method was successfully applied to a variety of solid and liquid food samples including milk, tomato juice, different types of cheeses, sauces, and soft drinks. Therefore, this method can, theoretically, be applied to other samples that contain salt at a concentration above 0.2 g/L, including those samples which contain sugars. However, applying the method to other products may need to be investigated on a case by case basis because food products contain a complex mixture of chemical components that may interfere with the salt analysis by this method.

Q. What other areas research are you involved with?

A: I am a microbiologist and I have extensive experience in food microbiology, especially the microbiological analysis of baker’s yeast (8). I am also interested in all sectors of industrial biotechnology especially research on how to minimize the negative impact of industrial biotechnology on the environment. I am currently focusing on the establishment of the marine fermentation concept. Marine fermentation is an approach where seawater, marine biomass, and marine microorganisms are used in the fermentation process for the production of value-added compounds such as bioethanol. The main purpose of marine fermentation is to reduce pressure on the use of freshwater and arable land, allowing these resources to be dedicated to the production of food and feeds (9–12).

References

1. A. Sims, Journal of Agricultural and Food Chemistry 43(2), 377–380 (1995)

2. AOAC, Methods of Analysis for Nutrition Labelling. 1993, Arlington, VA, USA: Association of Official Analytical Chemists.

3. A.S. Zaky et al., Journal of Food Composition and Analysis 56, 25–33 (2017).

4. H.W. Doughty, Journal of the American Chemical Society46(12), 2707–2709 (1924).

5. O. Schales and S.S. Schales, Journal of Biological Chemistry140(3), 879–884 (1941).

6. C.-M. Zhao and W.-X. Wang, Environmental Toxicology and Chemistry30(4), 885–892 (2011).

7. M.E. Johnson and N.F. Olson, Journal of Dairy Science68(4), 1020–1024 (1985).

8. N.F. Nasr, A.S. Zaky, and Z.Y. Daw, Journal of Pure and Applied Microbiology 4(2), 455–462 (2010).

9. M.A. Kyyaly, A.S. Zaky, and C.D. Powell, New Biotechnology 33(Supplement), S71 (2016).

10. A.S. Zaky, G. Tucker, and C. Du, New Biotechnology33(Supplement), S52–S53 (2016).

11. A.S. Zaky and C. Du, New Biotechnology31(Supplement), S132 (2014).

12.  A.S. Zaky et al., Journal of Microbiology and Biotechnology26(11), 1891–1907 (2016).

Abdelrahman Saleh Zaky is an academic staff member (on sabbatical) at the Department of Agricultural Microbiology at Cairo University, Egypt. Currently, he is attending the University of Nottingham as an associate researcher at the School of Biosciences. He received his B.Sc. degree in biotechnology and M.Sc. degree in food microbiology from Cairo University in Egypt. He conducted his Ph.D. jointly between the University of Nottingham and the University of Huddersfield in the United Kingdom. His PhD project aimed to reduce the water footprint of bioethanol by establishing a new fermentation strategy termed Marine Fermentation, in which seawater is substituted for freshwater for the production of bioethanol. Dr. Zaky has previously worked in the areas of microbial isolation, identification, and fermentation; biomass hydrolysis and analysis; food microbiology, safety and analysis; and bioenergy and industrial biotechnology. He also has experience in academic teaching through participating and leading several modules on microbiology, industrial biotechnology, and chemical engineering at universities in Egypt, India, and the UK. In addition, he has presented his research in more than 20 international conferences around the world since 2010. He is an active member of many scientific societies, editor of two journals, and reviewer in several journals.    

E-mail: Abdelrahman.Zaky@agr.cu.edu.eg

Websites: https://www.linkedin.com/in/aszaky/ https://www.researchgate.net/profile/Abdelrahman_Zaky3