Journal of Chromatography A, 1378 (2015) 32–36 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma Solvent minimization in two-dimensional liquid chromatography Krisztián Horváth a,∗ , Annamária Sepsey b , Péter Hajós a a Department of Analytical Chemistry, University of Pannonia, Egyetem utca 10, H-8200 Veszprém, Hungary b MTA–PTE Molecular Interactions in Separation Science Research Group, Ifjúság útja 6, H-7624 Pécs, Hungary a r t i c l e i n f o Article history: Received 2 September 2014 Received in revised form 10 November 2014 Accepted 1 December 2014 Available online 9 December 2014 Keywords: Two-dimensional liquid chromatography Optimization Eluent consumption Green chromatography Separation power Solvent reduction a b s t r a c t An algorithm was developed for the minimization of consumption of organic solvent in comprehensive two-dimensional liquid chromatography (2DLC). It was shown that one can reach higher peak capacities only by using more eluent. The equilibration volume of the second dimension, however, did not affect the solvent consumption significantly. Calculations confirmed that the same target peak capacity could be achieved by consuming significantly different volume of organic modifier depending on the number of fractions analyzed in the second dimension suggesting that 2D separations can be optimized for eluent consumption. It was shown that minimization of eluent usage requires the use of small and high efficient columns in the second dimension. A simple equation was derived for the calculation of the optimal number of collected fractions from the first dimension that allowed the minimization of eluent usage, cost and environmental impact of comprehensive 2DLC separations. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Determination of organic compounds in various matrices is usually carried out by the means of liquid chromatography. Chro- matographic procedures are used in wide area of research, medical, industrial, food and environmental analysis [1]. As a result, very large number of chromatographic analyses performed worldwide. In some cases, solvent employed as mobile phase can be more toxic than the species being determined. As a result, side effects of chromatographic methods can generate greater environmental and human impact than the problem analyzed [2]. Considering that it is not uncommon for a single pharmaceutical company to have more than 1000 HPLC instruments and that a single liquid chromatog- raphy can potentially generate 1–1.5 L of liquid waste daily [3], the environmental impact of solvents should be taken into account during method development. The idea of green analytical chemistry and green chromatogra- phy has been introduced at the end of the last century [4] based on the 12 well-known principles of green chemistry [5]. The concept of green analytical chromatography can be summarized in the three “R”s: Reduce, Replace, Recycle [3]. For years, the green approach alone could not gain a solid ground in the practice of HPLC. Fortun- ately, reduction of eluent consumption in liquid chromatographic ∗ Corresponding author. Tel.: +36 88624485. E-mail address: raksi@almos.uni-pannon.hu (K. Horváth). separations has gained attention due to the worldwide acetonitrile shortage happened last decade and the increasing cost of waste disposal. Nowadays, the interest in green analytical techniques is growing. Books were published [6–8], special issues of journals and reviews were dedicated to green analytical chemistry [9–12] and green chromatography [3,13,14] recently. In bioanalysis, high-performance liquid chromatography (HPLC) is often called to resolve highly complex samples containing hundreds or even thousands of components in a very wide range of concentrations. Despite the continuous progress in column and instrument technologies, these separations exceed the possibilities of conventional chromatographic methods [15]. Two-dimensional liquid chromatography (2DLC) is being developed to improve the separation power of chromatography [16–18]. In these systems, the separation of solutes is implemented through two columns sequen- tially. Small-volume aliquots from the first column are collected and analyzed by the second column. Successful implementation of 2DLC requires the separation mechanism in the two dimen- sions be orthogonal. Two separations are said to be orthogonal if there is no relationship between the retention data of the differ- ent components of the sample in the two separations [19]. Another parameter that significantly affects the separation power of 2DLC systems is the number of fractions collected from the first dimen- sion. This problem was studied thoroughly by different theoretical approaches [20–22] and statistical methods [23] as well. The stud- ies showed that undersampling the first dimension reduces the effective peak capacity, i.e. gives an effective peak capacity that is http://dx.doi.org/10.1016/j.chroma.2014.12.001 0021-9673/© 2014 Elsevier B.V. All rights reserved.