An Inquiry-Based Experiment Exploring the Theory Behind HPLC Quantification

Shaeen Chetty¹, Su-Ané Greyling¹ and Letitia Pillay^1,

¹Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Johannesburg, South Africa

Cite this paper:
Shaeen Chetty, Su-Ané Greyling and Letitia Pillay. An Inquiry-Based Experiment Exploring the Theory Behind HPLC Quantification. World Journal of Chemical Education. 2024; 12(4):81-86. doi: 10.12691/wjce-12-4-2

Abstract

High-performance liquid chromatography (HPLC) is one of the most used analytical techniques for the identification and quantification of organic molecules in a variety of samples. In this simple laboratory experiment, quantification parameters and the factors affecting them were investigated using organic acids in plant material. Molar absorptivity (ε) and its implications on limit of detection (LoD) and limit of quantification (LoQ) were examined. Students prepared leaf samples by extraction and quantified the malic and fumaric acid concentrations based on calibration curves of a set of standards. Using the data the LoD, LoQ and ε were calculated and the implications of their values tested through a series of structured assessment questions. The learning goals include sample preparation, the application of theoretical calculations on method development, and calculating the organic acid concentrations. The experiment is suitable for senior undergraduate and early postgraduate students in analytical and environmental chemistry courses.

Keywords:
HPLC organic acids molar absorptivity LoD LoQ

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References:

[1]	O. Coskun, “Separation techniques: chromatography,” North. Clin. Istanb., vol. 3, no. 2, Art. no. 2, 2016.
[2]	C. Horváth, High-performance liquid chromatography: advances and perspectives, vol. 2. Elsevier, 2013.
[3]	T. J. Wenzel, P. B. Forbes, B. C. Galarreta, B. A. Patel, M. Vogel, and D. K. Wong, “Incorporation of analytical chemistry in the undergraduate curriculum: examples from different regions of the world,” Anal. Bioanal. Chem., pp. 1–10, 2024.
[4]	J. F. van Staden, “Analytical chemistry at the University of Pretoria,” Fresenius J. Anal. Chem., vol. 357, pp. 221–223, 1997.
[5]	S. P. Perone, J. Pesek, C. Stone, and P. Englert, “Transforming Traditional Sophomore Quant into a Course on Modern Analytical Science,” J. Chem. Educ., vol. 75, no. 11, p. 1444, Nov. 1998.
[6]	M. J. Hengel, J. W. Wong, Z. C. Redman, C. Rering, and K. L. Williams, “Analysis of Pesticides in Plant Foods by QuEChERS and Gas Chromatography–Mass Spectrometry: An Undergraduate Laboratory Experiment,” J. Chem. Educ., vol. 97, no. 1, pp. 226–233, Jan. 2020.
[7]	P. Gaurav, S. Kushwaha, H. Tiwari, and K. K. Chandrul, “Review on Application of HPLC and UPLC.,” Int. J. Pharm. Life Sci., vol. 15, no. 3, Art. no. 3, 2024.
[8]	G. Chawla and K. K. Chaudhary, “A review of HPLC technique covering its pharmaceutical, environmental, forensic, clinical and other applications,” Int J Pharm Chem Anal, vol. 6, no. 2, Art. no. 2, 2019.
[9]	C. Burgess, Valid analytical methods and procedures. Royal Society of Chemistry, 2000.
[10]	J. Fabian and H. Hartmann, Light absorption of organic colorants: theoretical treatment and empirical rules, vol. 12. Springer Science & Business Media, 2013.
[11]	D. A. Skoog, D. M. West, F. J. Holler, and S. R. Crouch, Fundamentals of Analytical Chemistry, Tenth edition. Boston, MA: Cengage, 2022.
[12]	F. J. Linnig, E. J. Parks, and J. E. Stewart, “Some changes in double-bond structure during the vulcanization of natural rubber,” J. Res. Natl. Bur. Stand. Sect. Phys. Chem., vol. 68A, no. 5, p. 499, Sep. 1964.
[13]	M. Locatelli, G. Carlucci, S. Genovese, M. Curini, and F. Epifano, “Use of HPLC in the Determination of the Molar Absorptivity of 4′-Geranyloxyferulic Acid and Boropinic Acid,” Chromatographia, vol. 73, no. 9, pp. 889–896, May 2011.
[14]	M. Pelillo, M. E. Cuvelier, B. Biguzzi, T. Gallina Toschi, C. Berset, and G. Lercker, “Calculation of the molar absorptivity of polyphenols by using liquid chromatography with diode array detection: the case of carnosic acid,” J. Chromatogr. A, vol. 1023, no. 2, pp. 225–229, Jan. 2004.
[15]	H. Quitmann, R. Fan, and P. Czermak, “Acidic organic compounds in beverage, food, and feed production,” Biotechnol. Food Feed Addit., pp. 91–141, 2014.
[16]	N. Violeta, I. Trandafir, and M. E. Ionica, “HPLC organic acid analysis in different citrus juices under reversed phase conditions,” Not. Bot. Horti Agrobot. Cluj-Napoca, vol. 38, no. 1, Art. no. 1, 2010.
[17]	I. Mato, S. Suárez-Luque, and J. F. Huidobro, “A review of the analytical methods to determine organic acids in grape juices and wines,” Food Res. Int., vol. 38, no. 10, Art. no. 10, 2005.
[18]	A. Robles, M. Fabjanowicz, T. Chmiel, and J. Płotka-Wasylka, “Determination and identification of organic acids in wine samples. Problems and challenges,” TrAC Trends Anal. Chem., vol. 120, p. 115630, 2019.
[19]	S. F. Palme, A. H. Johnson, and G. R. Wyllie, “Color, Candy, and Chromatography: A Combined TLC and HPLC Activity for the General Chemistry Laboratory,” J. Chem. Educ., vol. 101, no. 1, Art. no. 1, 2023.
[20]	R. E. Leacock, J. J. Stankus, and J. M. Davis, “Simultaneous determination of caffeine and vitamin B6 in energy drinks by high-performance liquid chromatography (HPLC),” J. Chem. Educ., vol. 88, no. 2, Art. no. 2, 2011.
[21]	S. C. Purcell et al., “Extraction and antibacterial properties of thyme leaf extracts: authentic practice of green chemistry,” J. Chem. Educ., vol. 93, no. 8, Art. no. 8, 2016.
[22]	D. Arias-Carmona, A. Romero-Rodriguez, and M. Vazquez-Oderiz, “Determination of Organic Acids in Brassica Rapa L. Leaves (Turnip Greens and Turnip Tops) Regulated by the Protected Geographical Indication"Grelos De Galicia",” J. Food Nutr. Res., vol. 2, no. 11, pp. 786–791, Oct. 2014.
[23]	F. Trombete et al., “Determination of Aflatoxins in Wheat and Wheat by-products Intended for Human Consumption, Marketed in Rio de Janeiro, Brazil,” J. Food Nutr. Res., vol. 2, no. 10, pp. 671–674, Sep. 2014.
[24]	A. Shafaei, H. Abbiss, S. Coutts, M. Bannister, J. Bruemmer, and M. C. Boyce, “Tocopherols in seeds and nuts: QuEChERS extraction, HPLC separation, and fluorescence detection,” J. Chem. Educ., vol. 99, no. 5, Art. no. 5, 2022.
[25]	D. C. Stone, “Teaching Chromatography Using Virtual Laboratory Exercises,” J. Chem. Educ., vol. 84, no. 9, p. 1488, Sep. 2007.
[26]	M. C. Boyce and K. Singh, “Student Learning and Evaluation in Analytical Chemistry Using a Problem-Oriented Approach and Portfolio Assessment,” J. Chem. Educ., vol. 85, no. 12, p. 1633, Dec. 2008.
[27]	M. L. Kovarik et al., “Survey of the Undergraduate Analytical Chemistry Curriculum,” J. Chem. Educ., vol. 99, no. 6, pp. 2317–2326, Jun. 2022.
[28]	M. K. Seery, H. Y. Agustian, and X. Zhang, “A Framework for Learning in the Chemistry Laboratory,” Isr. J. Chem., vol. 59, no. 6–7, pp. 546–553, 2019.
[29]	G. M. Novak, “Just-in-time teaching,” New Dir. Teach. Learn., vol. 2011, no. 128, pp. 63–73, 2011.
[30]	A. Udvari-Solner, “Collaborative Learning,” in Encyclopedia of the Sciences of Learning, N. M. Seel, Ed., Boston, MA: Springer US, 2012, pp. 631–634.
[31]	I. Alzahrani and J. Woollard, “The Role of the Constructivist Learning Theory and Collaborative Learning Environment on Wiki Classroom, and the Relationship between Them,” in International Conference for e-Learning & Distance Education 2013, 2013. [Online]. Available: https:// www.learntechlib.org/ p/54801.
[32]	R. H. Chen, “Fostering students’ workplace communicative competence and collaborative mindset through an inquiry-based learning design,” Educ. Sci., vol. 11, no. 1, Art. no. 1, 2021.
[33]	I. Warsah, R. Morganna, M. Uyun, M. Afandi, and H. Hamengkubuwono, “The impact of collaborative learning on learners’ critical thinking skills,” Int. J. Instr., vol. 14, no. 2, Art. no. 2, 2021.
[34]	J.-M. G. Rodriguez, K. H. Hunter, L. J. Scharlott, and N. M. Becker, “A Review of Research on Process Oriented Guided Inquiry Learning: Implications for Research and Practice,” J. Chem. Educ., vol. 97, no. 10, pp. 3506–3520, Oct. 2020.