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Chromatography is the most widely used technique for the separation of mixtures. It was used at the beginning of the 20th century by M. S. Tswett to separate pigments from plants. The name comes from Greek chroma, “color”, and graphein, “to write”. In chromatography, a mixture of analytes which is going to be separated is injected on a column where one phase flows and passes over another one which is immobile or stationary. The mixture of analytes passes through the column with the mobile phase. Analytes generally have different interactions with stationary and mobile phases resulting in different migration rates. The components of the mixture which have longer interaction with the stationary phase are retarded and the components which have poor interaction are moved faster by the mobile phase. The physical states of the mobile and stationary phases are one of the ways to classify different types of chromatography techniques. If the mobile phase is a gas, it is called gas chromatography (GC) and if it is a liquid, it is called liquid chromatography (LC). Thin layer chromatography (TLC) and HPLC are one of the most common examples of the last group; the first one uses an open column and the other one is on a layer. If the stationary phase is a solid and the mobile phase is a liquid, it is called liquid-solid chromatography (LSC) and if both are liquid, it is called liquid-liquid chromatography (LLC). In these different types of chromatography we could identify other groups according to phenomena inside the system. [1] Generally, a separation process takes place inside a column where the stationary phase is and where the mobile phase moves through.

As it was said before, HPLC consists of a liquid mobile phase passing through a column where the stationary phase is attacked, which generally is formed of particles a few microns in diameter. The components of the equipment are: solvent reservoirs, pumps, injector, column and detector. According to the polarities of the phases, the technique can be classified into reversed and normal phase. In reversed phase liquid chromatography (RPLC), the stationary phase is less polar than the mobile phase. In normal phase liquid chromatography (NPLC), the stationary phase is more polar than the mobile phase [1]. Since there is a difference among the migration rates of different analytes if there is a difference between the interaction of different analytes and the phases, separation can be achieved. The detector is set at the end of the column and its response is related to analyte concentrations. This device monitors the presence of analytes; their responses are shown in a graphic called chromatogram, in Figure 1 [2]. Every analyte is represented by a peak at the time when they leave the column; faster analytes appear at the beginning of the chromatograms and slower ones appear at the end of them.

Figure 1. Chormatogram

    Figure 1: Chromatogram.

Micro-High Performance Liquid Chromatography

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Chromatographic methods permanently tend to increase the speed of determinations, reduce solvent consumption and work with small sample volumes. One option is the use of UHPLC (Ultra Performance Liquid Chromatography -High Performance), miniaturization of equipment, the use of new stationary phases (Core -Shell or fused- core) or monolithic columns.

Systems of miniaturized HPLC
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The miniaturization of liquid chromatography systems was because of its great benefits, including the small sample volume required for analysis and use low solvent flows. The first steps were taken by Horváth et al in 1967 [3, 4] who filled columns of 0.5-1.0 mm internal diameter with particulate materials. The classification of the different systems is based primarily on the size of the columns used, as well as on flow rate:

table 1

   Table 1: Classification of HPLC systems according to the scale used [5].

The main disadvantage of these columns is that they operate at very high pressures. This problem was solved by the development of monolithic stationary phases, which allow to achieve similar efficiencies with low pressures and highly efficient separations in a significantly shorter analysis time. These advantages have contributed to the development of monolithic columns to be used in micro-HPLC and electrochromatography, with very promising results [6]. These changes led to the commercialization of equipment of micro and nano - HPLC. It is also possible to adapt conventional HPLC equipment by: I) microflow split valve, which reduces and regulates the flow rate of the mobile phase; II) micro- injection, whose volume is determined by capillary injection loop or “loop” on the order of nanoliters and III) UV detector with nanocells or online detection (“on-column”).

Figure 1: Diagram of a possible adaptation of a conventional HPLC to microscale

    Figure 2: Diagram of a possible adaptation of a conventional HPLC to microscale.

Sample pretreatment

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The main aims of a sample pretreatment are: to eliminate interferences from a sample matrix; to preconcentrate some analytes which are in very low concentration so that they will be detected and quantifiable, and to derivatizate them to allow their detection. This step precedes the injection in the HPLC system. Some sample pretreatment methods are: liquid-liquid extraction (LLE), solid-phase extraction (SPE), evaporation, distillation, solid-liquid extraction, soxhlet extraction, microwave-assited extraction, etc. In LLE, extraction is carried out using two immiscible liquids, one of which is an organic solvent, while the other is an aqueous solution. Analytes of interest partition between those two phases in which they have different solubility values. The main objective is to retain the analytes in one or another phase, generally in the organic phase. After that, the organic solvent is usually evaporated, and this allows us to concentrate the analytes. If we are going to use the sample in an HPLC system, the organic solvent has to be acceptable for it. The relationship between the analyte concentration in the organic phase, [A]o, over the analyte concentration in the aqueous phase, [A]aq, is called partition coefficient, k.

Ecuation partition

The larger the value of k, the greater the analyte concentration in the organic phase. Microextration is another form of LLE, and in this case a micro volume of organic solvent is used. According to solvent densities, the method needs different arrangements. When the organic solvent is denser than water, conical tubes are needed. In the opposite case, tubes with narrow necks are needed. This technique has worse recovery values but better extraction efficiency values because of the low organic phase volume. Another environment friendly alternative to organic solvents is ionic liquids. These substances are replacing organic solvents due to their unique properties such as low volatility, chemical and thermal stability, and good solubility for both organic and inorganic molecules [7].

Chiral chromatography

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Chirality is a geometric property related to asymmetry. A chiral molecule lacks an internal plane of symmetry and has a molecule which is its non-superimposable mirror image. Those molecules are called enantiomers and a mixture of them is known as a racemic mixture. They have very similar physical properties, so their separation is very difficult.

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    Figure 3: Example of enantiomeric molecules and its comparison with the hands. 

Separating chiral molecules is important in organic, environmental, biological, pharmaceutical and analytical chemistry fields. Many techniques are used to analyze chiral organic molecules, such as Chiroptical Methods (Polarimetry, Optical Rotation Dispersion, Circular dicroism, Vibrational Optical Activity, etc); Nuclear Magnetic Resonance (NMR), and Chromatography.

Chiral Chromatography uses a chiral selector, a molecule with chiral centers. The most common chiral selectors, especially in gas chromatography, are cyclodextrins. They are macromolecules made of glucose molecules bonded together in a cone form. According to the number of sugar members, the names of the cyclodextrin are: alpha for six members, beta for seven and gamma for eight. Native cyclodextrins are solid and have poor solubility. This is the reason why they have to be dissolved in a proper solvent or derivatized in order to be used as stationary phases in Gas Chromatography. The most common solvents are polysiloxanes and they are also used to prepare cyclodextrin derivative solutions.

     Figure 4: Chemical structure of the three main types of cyclodextrins (Wikipedia)


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[1] Introducción a la HPLC. Oscar Alberto Quattrochi, Sara Abelaira de Andrizzi, Raúl Felipe Laba. Editorial.
[2] Fundamentos de Química Analítica. Skoog, West, Holler. Editorial Reverté, S. A., 1997. ISBN 84-291-7556-3.
[3] Horváth, C.; Preis, B. A.; Lipsky, S. R. (1967) Analytical Chemistry, 39, 1422.
[4] Horváth, C.; Lipsky, S. R. (1969) Analytical Chemistry, 41, 227.
[5] Smith, N. W., Legido-quigley, C., Marlin, N. D., &Melin, V. (1967). Capillary Liquid Chromatography Capillary and Micro ‐ High Performance Liquid Chromatography, 1–22.
[6] Legido-quigley, C., Marlin, N. D., Melin, V., Manz, A., & Smith, N. W. (2003). 917–944.
[7] Practical HPLC Method Development. Lloyd R. Snyder, Joseph J. Kirkland, Joseph L. Glaich. Editorial: John Wiley & Sons, 2012. ISBN 1118591518, 9781118591512