Basics of liquid chromatography: Thin-layer chromatography (TLC) Column liquid chromatography (CLC) Teresa Kowalska Institute of Chemistry University of Silesia 9, Szkolna street 40-006 Katowice, Poland E-mail: teresa.kowalska@us.edu.pl http://chromtheory.us.edu.pl/

Outline of presentation Separation sciences; the notion Chromatography; the IUPAC definition Classification of chromatographic techniques Classification criteria Different classifications Analytical and preparative chromatography mode Analytical chromatography mode Preparative chromatography mode Planar chromatography Column chromatography Basic mechanisms of separation in liquid chromatography Adsorption Partition Parameters of retention Parameters of resolution Applications of liquid chromatography – general view Applications of TLC Applications of CLC Conclusions

Separation sciences Separation sciences are the sciences focusing on the theory of the techniques targeting the separation of the mixtures of different chemical species. Examples of the separation techniques: Distillation Cristallization Extraction Separation with use of membranes Electrochemical techniques Chromatography etc.

Chromatography – the IUPAC definition Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary, while the other moves in a definite direction. Chromatography – the IUPAC definition

Hence, the first message to remember from the IUPAC definition From this definition it simply comes out that each chromatographic system is composed of the following two elements: stationary = immobile phase, mobile phase, and of course, of a mixture of substances to be separated.

Classification of chromatographic techniques Classification criteria Geometrical Physical Chemical

Geometrical criterion In this case, the criterion is the geometrical shape of stationary phase. If (solid) stationary phase is thin and flat (i.e., it looks like a sheet of paper), then the chromatographic mode is planar chromatography. Planar chromatography additionally sub-divides into: paper chromatography thin-layer chromatography

Geometrical criterion (continued) If stationary phase assumes a cylindrical shape and is encapsulated in a tubular container, then the chromatographic mode is column chromatography.

Physical criterion liquid gas solid Chromatography mode Mobile phase liquid-liquid chromatogr. (partition chromatography) liquid-gaseous chromatogr. liquid gas solid-liquid chromatogr. (adsorption chromatography) solid-gaseous chromatogr. solid Chromatography mode Mobile phase Stationary phase This classification is based on physical parameter, i.e., on the condensation degree of stationary phase and mobile phase.

Physico-chemical mechanism Chemical criterion This classification is based on physico-chemical mechanism of distribution of the analyte between the stationary and mobile phase. This classification is still open, as novel chromatographic modes are still invented and added to the list. Electrophoresis Isoelectric focusing Physical adsorption + electrophoresis Electrochromatography Mixed mechanisms Different mixed modes Isotachophoresis (Bio)receptor coupling mechanism (Bio)affinity chromatography Molecular sieves Size-exclusion chromatogr. Coulomb forces + molecular sieves + physical adsorption Ion-exclusion chromatography Coulomb forces + physical adsorption Ion-pair chromatography Chemisorption Ion-exchange chromatography The Nernst liquid-liquid partition Partition chromatography Physical adsorption Adsorption chromatography Physico-chemical mechanism Technique

Stationary phases in liquid chromatography Paper chromatography In paper chromatography, stationary phase is chromatographic paper (cellulose).

Stationary phases in liquid chromatography Thin-layer chromatography (TLC) Octadecyl silica (ODS; C18; RP-18) Octyl silica (C8; RP-8) Dimethyl silica (C2; RP-2) 3-Cyanopropyl silica (CN) 3-Aminopropyl silica (NH2) Diol silica Phenyl silica etc. Silica gel (silica; SiO2) Aluminium oxide (alumina; Al2O3) Florisil (MgO·SiO2) Microcrystalline cellulose powder Polyamide 6 (Nylon 6; ε-aminopolycaprolactame) Second generation stationary phases First generation stationary phases

Stationary phases in liquid chromatography (continued) High-performance liquid chromatography (HPLC) Octadecyl silica (ODS; C18; RP-18) Octyl silica (C8; RP-8) Dimethyl silica (C2; RP-2) 3-Cyanopropyl silica (CN) 3-Aminopropyl silica (NH2) Diol silica Phenyl silica etc. Second generation stationary phases Non-pressurized column liquid chromatography (CLC) Silica gel (silica; SiO2) Aluminium oxide (alumina; Al2O3) First generation stationary phases

water + over 80 organic solvents Mobile phases in liquid chromatography (both planar and column chromatography mode) water + over 80 organic solvents Basic demands posed on solvents used as mobile phase components: They should not be toxic; They should not be explosive; They should not be volatile; They should not be viscous; They should be easily miscible with other solvents used as mobile phase components; They should be chemically inactive (they should not react with components of a separated mixture; They should be the low-molecular-weight compounds; etc.

Analytical and preparative chromatography mode Each isotherm of distribution of an analyte between the stationary and mobile phase of the chromatographic system can be divided into the linear and non-linear part. linear part favorable: convex unfavorable: concave non-linear part Linear part of isotherm: analytical chromatography mode Non-linear part of isotherm: preparative chromatography mode

Simple isotherms of analyte distribution between stationary and mobile phase Isotherms are mathematical models which illustrate the course of the distribution. The Langmuir isotherm (the simplest model of the convex isotherm) The anti-Langmuir isotherm (the simplest model of the concave isotherm) adsorbed monolayer adsorbed multilayer _ where qi and qs - analyte concentration on stationary phase and maximum analyte concentration on stationary phase, respectively, ci – analyte concentration in mobile phase, and K – the isotherm constant. qi = qs 1 + K ci K ci 1 – K ci where qi and qs - analyte concentration on stationary phase and maximum analyte concentration on stationary phase, respectively, ci – analyte concentration in mobile phase, and K – the isotherm constant.

Analytical chromatography mode Main targets: Identification of each separated chemical species (qualitative analysis); Separation of a mixture of substances to individual chemical species; Quantification of each separated chemical species (quantitative analysis). Planar chromatography Column liquid chromatography Calibration curve In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration.

Preparative (and process) chromatography mode Main targets: The laboratory isolation of selected chemical species from reaction mixture on preparative scale; The industrial isolation of selected chemical species from reaction mixture on technological scale. E.g., chemical laboratory of organic synthesis Technological establishment (e.g., pharmaceutical orbiotechnological industry

Planar chromatography Basic notions chromatographic chamber mobile phase chromatographic plate Most often, the analysis is carried out in vertical chromatographic chambers. Horizontal chromatographic chambers (known as sandwich chambers) are also used. Mobile phase migrates upward, owing to capillary action. This upward migration mode is called the ascending mode. In the course of mobile phase migration, a mixture of compounds undergoes separation to individual chemical species. This process is called the development of the chromatogram. The plate with separated chemical species is called the chromatogram. Planar chromatogram Vertical chromatographic chamber Horizontal (sandwich) chromatographic chambers

Visualization and acquisition of planar chromatograms (a) Destructive Spraying with conc. H2SO4 and heating Organic compounds become fully dehydrated and carbonized. As a result, black or dark brown spots of the separated zones are visualized on the white background. (b) Non-destructive (i) Physical visualization in iodine vapours Iodine easily evaporates. If we place the developed chromatogram in a tightly closed chamber with a vessel filled with iodine crystals, iodine vapours will better adsorb on separated chromatographic zones than on chromatographic plate. Hence, the brown spots will appear of the pale yellow background. The process is reversible. Chromatogram visualized in iodine vapours

Visualization and acquisition of planar chromatograms (continued) (ii) Physical visualization by staining separated zones with dyeing reagents Video imaging and acquisition of chromatograms in a digital form

Visualization and acquisition of planar chromatograms (continued) Densitometric scanning of the chromatograms in UV-Vis light Densitometer Densitogram Chromatogram Transformation of planar chromatograms into a digital signal, which enables quantitative analysis

Hyphenated technique: TLC-MS (thin-layer chromatography with mass spectrometric detection) The separated chromatographic bands can be eluted directly to mass spectrometer. This can be obtained with use of the TLC-MS interface (which is a specially designed device to elute separated chromatographic bands and to introduce them directly to mass spectromter). TLC-MS interface

Hyphenated technique: TLC-MS (thin-layer chromatography with mass spectrometric detection) Practical example: „Fingerprinting” of botanical material (a) (b) 1 2 3 4 5 (a) The densitogram, (b) the videoscan, and (1)-(5) the mass spectra of the five separated chromatographic bands derived from the essential oil sample of Salvia hians

Column liquid chromatography (CLC) Basic notions Non-pressurized column liquid chromatography (an omnipresent tool in laboratories of organic synthesis, etc.) Preparative lab-scale column Mobile phase moves down the chromatographic column under the inluence of gravitation. Separated fractions can be automatically collected with use of an automatic fraction collector. Small-scale sample preparative isolation / purification applications Automatic fraction collector

Column liquid chromatography (CLC) (continued) Basic notions Slightly pressurized column liquid chromatography, known as FLASH CHROMATOGRAPHY (FC) (an advanced lab scale tool for rapid isolation of compounds) FC preparative lab-scale column Illustration of preparative separation Automatic fraction collector Mobile phase moves down the chromatographic column under he influence of mechanical force (the pump)

Column liquid chromatography (CLC) (continued) Basic notions Macroscale preparative chromatography Chromatographic column systems used in biotechnology (e.g., for macro-scale isolation and purification of proteins)

Column liquid chromatography (CLC) (continued) Basic notions High-performance liquid chromatography (HPLC) Chromatographic systems used for micro-scale separation, identification, and quantification of the compound mixture (micro-scale analytical mode) A flow scheme for HPLC system The HPLC system The HPLC column

Detection systems applied in CLC All detection systems applied in CLC are based on advanced physical principles. Below, a list is given of the most widely employed physical principles used in the CLC detection systems: light refraction; UV/Vis absorption spectroscopy; IR absorption spectroscopy; 1H NMR spectroscopy; mass spectrometry; fluorometry; Rayleigh light scattering. Coupling of an advanced HPLC separation technique with the physically advanced (mostly spectroscopic) detection techniques results in the so-called hyphenated (i.e., hybrid) analytical techniques.

Main application areas for chromatography Analytical mode Classical analytical chemistry Life sciences analysis (in the first instance, pharmaceutical and biomedical analysis) Environmental analysis Geochemical analysis Extraterrestrial analysis Preparative and technological mode Pharmaceutical industry Biotechnology