Analytical and Bioanalytical Chemistry

, Volume 410, Issue 16, pp 3705–3713 | Cite as

Natural deep eutectic solvents as the major mobile phase components in high-performance liquid chromatography—searching for alternatives to organic solvents

  • Adam T. Sutton
  • Karina Fraige
  • Gabriel Mazzi Leme
  • Vanderlan da Silva Bolzani
  • Emily F. Hilder
  • Alberto J. Cavalheiro
  • R. Dario Arrua
  • Cristiano Soleo Funari
Research Paper


Over the past six decades, acetonitrile (ACN) has been the most employed organic modifier in reversed-phase high-performance liquid chromatography (RP-HPLC), followed by methanol (MeOH). However, from the growing environmental awareness that leads to the emergence of “green analytical chemistry,” new research has emerged that includes finding replacements to problematic ACN because of its low sustainability. Deep eutectic solvents (DES) can be produced from an almost infinite possible combinations of compounds, while being a “greener” alternative to organic solvents in HPLC, especially those prepared from natural compounds called natural DES (NADES). In this work, the use of three NADES as the main organic component in RP-HPLC, rather than simply an additive, was explored and compared to the common organic solvents ACN and MeOH but additionally to the greener ethanol for separating two different mixtures of compounds, one demonstrating the elution of compounds with increasing hydrophobicity and the other comparing molecules of different functionality and molar mass. To utilize NADES as an organic modifier and overcome their high viscosity monolithic columns, temperatures at 50 °C and 5% ethanol in the mobile phase were used. NADES are shown to give chromatographic performances in between those observed for ACN and MeOH when eluotropic strength, resolution, and peak capacity were taken into consideration, while being less environmentally impactful as shown by the HPLC-Environmental Assessment Tool (HPLC-EAT) metric. With the development of proper technologies, DES could open a new class of mobile phases increasing the possibilities of new separation selectivities while reducing the environmental impact of HPLC analyses.

Graphical abstract

Natural deep eutectic solvents versus traditional solvents in HPLC


Green analytical chemistry NADES Low transition temperature mixtures Green solvents Natural designer solvents Green chromatography 



A.S. acknowledges the Australian Commonwealth government for an RTP scholarship. We thank Dr João Luiz Bronzel for assistance with chromatography experiments.

Funding information

This work was supported by a FAPESP SPRINT 4th Edition 2015 grant and the Australian Research Council’s Discovery funding scheme (grant no. 16/50009-2 and DP130101471, respectively). V.S.B., A.J.C., C.S.F., and K.F. are supported by the São Paulo Research Foundation (grant no. 013/07600-3 and no. 13/15086-8).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1027_MOESM1_ESM.pdf (720 kb)
ESM 1 (PDF 719 kb)


  1. 1.
    Yuan X, Richter BE, Jiang K, Boniface KJ, Cormier A, Sanders CA, et al. Carbonated water for the separation of carboxylic compounds: a chromatography approach. Green Chem. 2018;20:440–8.CrossRefGoogle Scholar
  2. 2.
    Welch CJ, Nowak T, Joyce LA, Regalado EL. Cocktail chromatography: enabling the migration of HPLC to nonlaboratory environments. ACS Sustain Chem Eng. 2015;3:1000–9.CrossRefGoogle Scholar
  3. 3.
    Funari CS, Carneiro RL, Cavalheiro AJ, Hilder EF. A trade off between separation, detection and sustainability in liquid chromatographic fingerprinting. J Chromatogr A. 2014;1354:34–42.CrossRefPubMedGoogle Scholar
  4. 4.
    Fritz R, Ruth W, Kragl U. Assessment of acetone as an alternative to acetonitrile in peptide analysis by liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2009;23:2139–45.CrossRefPubMedGoogle Scholar
  5. 5.
    Welch CJ, Wu N, Biba M, Hartman R, Brkovic T, Gong X, et al. Greening analytical chromatography. TrAC Trends Anal Chem. 2010;29:667–80.CrossRefGoogle Scholar
  6. 6.
    Płotka J, Tobiszewski M, Sulej AM, Kupska M, Górecki T, Namieśnik J. Green chromatography. J Chromatogr A. 2013;1307:1–20.CrossRefPubMedGoogle Scholar
  7. 7.
    Koel M. Do we need green analytical chemistry? Green Chem. 2016;18:923–31.CrossRefGoogle Scholar
  8. 8.
    Olives AI, González-Ruiz V, Martín MA. Sustainable and eco-friendly alternatives for liquid chromatographic analysis. ACS Sustain Chem Eng. 2017;5:5618–34.CrossRefGoogle Scholar
  9. 9.
    Tobiszewski M, Namieśnik J, Pena-Pereira F. Environmental risk-based ranking of solvents using the combination of a multimedia model and multi-criteria decision analysis. Green Chem. 2017;19:1034–42.CrossRefGoogle Scholar
  10. 10.
    Prat D, Wells A, Hayler J, Sneddon H, McElroy CR, Abou-Shehada S, et al. CHEM21 selection guide of classical- and less classical-solvents. Green Chem. 2015;18:288–96.CrossRefGoogle Scholar
  11. 11.
    Kittell JE Paul P, Arnold D, Neyer D, DeLand P, Rehm J (2008) Micro-scale HPLC generates < 1% of the solvent waste of conventional analytical LC. Paper presented at the the 12th annual green chemistry and engineering conference, Washington DC, USAGoogle Scholar
  12. 12.
    Armenta S, de la Guardia M. Green chromatography for the analysis of foods of animal origin. TrAC Trends Anal Chem. 2016;80:517–30.CrossRefGoogle Scholar
  13. 13.
    Welch CJ, Brkovic T, Schafer W, Gong X. Performance to burn? Re-evaluating the choice of acetonitrile as the platform solvent for analytical HPLC. Green Chem. 2009;11:1232–8.CrossRefGoogle Scholar
  14. 14.
    Gaber Y, Tornvall U, Kumar MA, Ali Amin M, Hatti-Kaul R. HPLC-EAT (Environmental Assessment Tool): a tool for profiling safety, health and environmental impacts of liquid chromatography methods. Green Chem. 2011;13:2021–5.CrossRefGoogle Scholar
  15. 15.
    Tobiszewski M. Metrics for green analytical chemistry. Anal Methods. 2016;8:2993–9.CrossRefGoogle Scholar
  16. 16.
    Funari CS, Carneiro RL, Khandagale MM, Cavalheiro AJ, Hilder EF. Acetone as a greener alternative to acetonitrile in liquid chromatographic fingerprinting. J Sep Sci. 2015;38:1458–65.CrossRefPubMedGoogle Scholar
  17. 17.
    Lesellier E, West C. The many faces of packed column supercritical fluid chromatography—a critical review. J Chromatogr A. 2015;1382:2–46.CrossRefPubMedGoogle Scholar
  18. 18.
    Vera CM, Shock D, Dennis GR, Farrell W, Shalliker RA. Comparing the selectivity and chiral separation of D- and L-fluorenylmethyloxycarbonyl chloride protected amino acids in analytical high performance liquid chromatography and supercritical fluid chromatography; evaluating throughput, economic and environmental impact. J Chromatogr A. 2017;1493:10–8.CrossRefPubMedGoogle Scholar
  19. 19.
    Yang Y. Subcritical water chromatography: a green approach to high-temperature liquid chromatography. J Sep Sci. 2007;30:1131–40.CrossRefPubMedGoogle Scholar
  20. 20.
    Alghoul ZM, Ogden PB, Dorsey JG. Characterization of the polarity of subcritical water. J Chromatogr A. 2017;1486:42–9.CrossRefPubMedGoogle Scholar
  21. 21.
    El-Shaheny RN, El-Maghrabey MH, Belal FF. Micellar liquid chromatography from green analysis perspective. Open Chem. 2015;13:877–92.CrossRefGoogle Scholar
  22. 22.
    Soares B, Passos H, Freire CSR, Coutinho JAP, Silvestre AJD, Freire MG. Ionic liquids in chromatographic and electrophoretic techniques: toward additional improvements in the separation of natural compounds. Green Chem. 2016;18:4582–604.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Han D, Row KH. Recent applications of ionic liquids in separation technology. Molecules. 2010;15:2405–26.CrossRefPubMedGoogle Scholar
  24. 24.
    Dai Y, van Spronsen J, Witkamp GJ, Verpoorte R, Choi YH. Natural deep eutectic solvents as new potential media for green technology. Anal Chim Acta. 2013;766:61–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Francisco M, Van Den Bruinhorst A, Kroon MC. Low-transition-temperature mixtures (LTTMs): a new generation of designer solvents. Angew Chem Int Ed. 2013;52:3074–85.CrossRefGoogle Scholar
  26. 26.
    Espino M, de los Ángeles Fernández M, FJV G, Silva MF. Natural designer solvents for greening analytical chemistry. TrAC Trends Anal Chem. 2016;76:126–36.CrossRefGoogle Scholar
  27. 27.
    Choi YH, van Spronsen J, Dai Y, Verberne M, Hollmann F, Arends IWCE, et al. Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology? Plant Physiol. 2011;156:1701–5.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Mbous YP, Hayyan M, Wong WF, Looi CY, Hashim MA. Unraveling the cytotoxicity and metabolic pathways of binary natural deep eutectic solvent systems. Sci Rep. 2017;7:41257.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Hayyan M, Mbous YP, Looi CY, Wong WF, Hayyan A, Salleh Z, et al. Natural deep eutectic solvents: cytotoxic profile. Springerplus. 2016;5:913.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Dai Y, Witkamp GJ, Verpoorte R, Choi YH. Natural deep eutectic solvents as a new extraction media for phenolic metabolites in carthamus tinctorius L. Anal Chem. 2013;85:6272–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Wei Z, Qi X, Li T, Luo M, Wang W, Zu Y, et al. Application of natural deep eutectic solvents for extraction and determination of phenolics in Cajanus cajan leaves by ultra performance liquid chromatography. Sep Purif Technol. 2015;149:237–44.CrossRefGoogle Scholar
  32. 32.
    Radošević K, Ćurko N, Gaurina Srček V, Cvjetko Bubalo M, Tomašević M, Kovačević Ganić K, et al. Natural deep eutectic solvents as beneficial extractants for enhancement of plant extracts bioactivity. LWT-Food Sci Technol. 2016;73:45–51.CrossRefGoogle Scholar
  33. 33.
    Bakirtzi C, Triantafyllidou K, Makris DP. Novel lactic acid-based natural deep eutectic solvents: efficiency in the ultrasound-assisted extraction of antioxidant polyphenols from common native Greek medicinal plants. J Appl Res Med Aromat Plants. 2016;3:120–7.Google Scholar
  34. 34.
    Bajkacz S, Adamek J. Evaluation of new natural deep eutectic solvents for the extraction of isoflavones from soy products. Talanta. 2017;168:329–35.CrossRefPubMedGoogle Scholar
  35. 35.
    Tan T, Zhang M, Wan Y, Qiu H. Utilization of deep eutectic solvents as novel mobile phase additives for improving the separation of bioactive quaternary alkaloids. Talanta. 2016;149:85–90.CrossRefPubMedGoogle Scholar
  36. 36.
    Li G, Zhu T, Lei Y. Choline chloride-based deep eutectic solvents as additives for optimizing chromatographic behavior of caffeic acid. Korean J Chem Eng. 2015;32:2103–8.CrossRefGoogle Scholar
  37. 37.
    Dai Y, Witkamp GJ, Verpoorte R, Choi YH. Tailoring properties of natural deep eutectic solvents with water to facilitate their applications. Food Chem. 2015;187:14–9.CrossRefPubMedGoogle Scholar
  38. 38.
    De Faria CMQG, Nazaré AC, Petrônio MS, Paracatu LC, Zeraik ML, Regasini LO, et al. Protocatechuic acid alkyl esters: hydrophobicity as a determinant factor for inhibition of NADPH oxidase. Curr Med Chem. 2012;19:4885–93.CrossRefPubMedGoogle Scholar
  39. 39.
    Snyder LR, Kirkland JJ, Dolan JW. Introduction to modern liquid chromatography. 3rd ed. Hoboken: John Wiley & Sons, Inc.; 2009.Google Scholar
  40. 40.
    Neue UD. Theory of peak capacity in gradient elution. J Chromatogr A. 2005;1079:153–61.CrossRefPubMedGoogle Scholar
  41. 41.
    Zhang Q, De Oliveira Vigier K, Royer S, Jérôme F. Deep eutectic solvents: syntheses, properties and applications. Chem Soc Rev. 2012;41:7108–46.CrossRefPubMedGoogle Scholar
  42. 42.
    Prat D, Hayler J, Wells A. A survey of solvent selection guides. Green Chem. 2014;16:4546–51.CrossRefGoogle Scholar
  43. 43.
    Martín-Calero A, Pino V, Ayala JH, González V, Afonso AM. Ionic liquids as mobile phase additives in high-performance liquid chromatography with electrochemical detection: application to the determination of heterocyclic aromatic amines in meat-based infant foods. Talanta. 2009;79:590–7.CrossRefPubMedGoogle Scholar
  44. 44.
    Tobiszewski M, Namieśnik J. Greener organic solvents in analytical chemistry. Curr Opin Green Sustain Chem. 2017;5:1–4.CrossRefGoogle Scholar
  45. 45.
    Tobiszewski M, Marć M, Gałuszka A, Namieśnik J. Green chemistry metrics with special reference to green analytical chemistry. Molecules. 2015;20:10928–46.CrossRefPubMedGoogle Scholar
  46. 46.
    Koller G, Fischer U, Hungerbühler K. Assessing safety, health, and environmental impact early during process development. Ind Eng Chem Res. 2000;39:960–72.CrossRefGoogle Scholar
  47. 47.
    Koller G, Fischer U, Hungerbühler K. Assessment of environment-, health- and safety aspects of fine chemical processes during early design phases. Comput Chem Eng. 1999;23:S63–S6.CrossRefGoogle Scholar
  48. 48.
    Shaaban H, Górecki T. Current trends in green liquid chromatography for the analysis of pharmaceutically active compounds in the environmental water compartments. Talanta. 2015;132:739–52.CrossRefPubMedGoogle Scholar
  49. 49.
    Desire CT, Hilder EF, Arrua RD. Monolithic high-performance liquid chromatography columns. In: Encyclopedia of Analytical Chemistry. Hoboken: John Wiley & Sons, Ltd.; 2017. p. 1–37.
  50. 50.
    Li X, Row KH. Development of deep eutectic solvents applied in extraction and separation. J Sep Sci. 2016;39:3505–20.CrossRefPubMedGoogle Scholar
  51. 51.
    García-Alvarez-Coque MC, Ruiz-Angel MJ, Berthod A, Carda-Broch S. On the use of ionic liquids as mobile phase additives in high-performance liquid chromatography. A review. Anal Chim Acta. 2015;883:1–21.CrossRefPubMedGoogle Scholar
  52. 52.
    Ferreira VG, Leme GM, Cavalheiro AJ, Funari CS. Online extraction coupled to liquid chromatography analysis (OLE-LC): eliminating traditional sample preparation steps in the investigation of solid complex matrices. Anal Chem. 2016;88:8421–7.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Adam T. Sutton
    • 1
  • Karina Fraige
    • 2
  • Gabriel Mazzi Leme
    • 2
  • Vanderlan da Silva Bolzani
    • 2
  • Emily F. Hilder
    • 1
  • Alberto J. Cavalheiro
    • 2
  • R. Dario Arrua
    • 1
  • Cristiano Soleo Funari
    • 3
  1. 1.Future Industries InstituteUniversity of South AustraliaAdelaideAustralia
  2. 2.Institute of ChemistrySão Paulo State University (UNESP)AraraquaraBrazil
  3. 3.Faculty of Agricultural SciencesSão Paulo State University (UNESP)BotucatuBrazil

Personalised recommendations