Lab.2. Thin layer chromatography 
                 Key words:  
                 Separation  techniques,  compounds  and  their  physicochemical  properties  (molecular 
                 volume/size,  polarity,  molecular  interactions),  mobile  phase,  stationary  phase,  liquid 
                 chromatography,  thin  layer  chromatography,  column  chromatography,  retardation  factor, 
                 elution,   chromatogram  development,  qualitative              and    quantitative    analysis    with 
                 chromatography techniques, eluotropic series, elution strength.  
                  
                 Literature:  
                 D.A. Skoog, F.J. Holler, T.A. Nieman: Principles of Instrumental Analysis; 637 - 718  
                 Search on www pages “Thin-layer chromatography principles” 
                 For example: MIT Digital Lab Techniques Manual you find on 
                 http://www.youtube.com/watch?v=e99nsCAsJrw&feature=player_detailpage 
                 Basic equipment for modern thin layer chromatography: 
                 www.camag.com/downloads/free/brochures/CAMAG-basic-equipment-08.pdf  
                 other examples: 
                 en.wikipedia.org/wiki/Thin_layer_chromatography 
                 www.chemguide.co.uk/analysis/chromatography/thinlayer.html 
                 www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/th
                 in_layer_chrom.html 
                  
                 Theoretical background 
                 Chromatography  is  the  separation  technique  in  which    separated  solutes  are  distributed 
                 between  two  phases:  stationary  and  mobile.  The  first  phase  can  pose  a  layer  of 
                 sorbent/adsorbent (0.1 to 0.25 mm in thickness) fixed to a carrier plate made of glass, plastic 
                 or aluminum (used in technique named as thin-layer chromatography, TLC) or placed inside 
                 of  a  steel  tube  as  a  column  bed  (used  in  a  technique  named  as  high-performance  liquid 
                 chromatography, HPLC, or generally in column liquid chromatography, LC). The second 
                 phase,  mentioned  above,  constitute  liquid  or  gas  phase.  Various  organic  (e.g.  methanol, 
                 hexane,  acetone)  and  inorganic  (e.g.  water)  solvents  or  their  mixtures  (e.g.  acetone  and 
                                   Lab.2. Thin layer chromatography 
              hexane, methanol and water) can be used as the mobile phases. So each chromatographic 
              system consists of: 
                  a)  stationary phase, 
                  b)  mobile phase, 
                  c)  mixture of components to be separated. 
              A solution of the component mixture is usually introduced into the chromatographic system 
              by injection (in HPLC or classical column chromatography in entrance to the column) or by 
              spotting/application onto start line (in TLC). In column chromatography the mobile phase is 
              pumped through the adsorbent bed or its flow is caused by gravitation as it is demonstrated in 
              Fig 1A. In thin layer chromatography mobile phase is driven into movement by capillary 
              forces (solvent wets adsorbent layer on the chromatographic plate by capillary forces) as it is 
              demonstrated in Fig 1B. Under such circumstances mixture components migrate along the 
              stationary phase (adsorbent) according to the direction of flow of the mobile phase.  
               
                       Mobile  
                       phase 
                                    A                           B 
                       Station
                       ary 
                       phase 
                        
                        
                                                                            Chromatographic 
                                                                            plate 
                         Valve 
                                                                              Chromatographic 
                                                                              chamber 
                                                                               Mobile 
                                                                               phase 
               
               
              Fig.  1.  (A)  Classical  column  chromatography,  (B)  chromatogram  development  in 
              conventional chamber (in cuboid vessel) 
               
              Migration velocities of mixture components are slower from that o the mobile phase. It is 
              because of time, which separated molecules spend in the stationary phase. Arrangement of 
              solute zones on the chromatographic plate after chromatogram development is demonstrated 
              in Fig. 2. 
                                    Lab.2. Thin layer chromatography 
                         
                                                                             Solvent front 
               
               
               
               
                                                                             Start line 
               
              Fig. 2.  Thin layer chromatogram of dyes, 1 and 10 – dye mixture, 2 – 9 single dyes  
               The time the separated molecules spend in the stationary phase depends on their interactions  
              with stationary and mobile phases. It means the mixture components can be separated in the 
              chromatographic system if they demonstrate different migration distances, i.e. if they show 
              different energy of molecular interactions with components of the chromatographic system. 
              Following molecular interactions of solutes with elements of stationary and mobile phases can 
              take place in any chromatographic system: hydrogen bond, dipole – dipole, dipole – induced 
              dipole,  ion – dipole, instantaneous dipole – induced dipole (London dispersion forces),   ion – 
              ion.  
               
              The stationary phase  
                  1.   Silica gel 
              Silica gel  is composed of silicon dioxide (silica). The silicon atoms are bonded via oxygen 
              atoms in a giant covalent structure. However, at the surface of the silica gel -OH groups are 
              attached  to  the  silicon  atoms.  So,  on  the  surface  of  silica  gel  Si-O-H  groups  are  present 
              instead of Si-O-Si ones. This makes silica surface very polar. 
              Fig. 3 shows the model of a small part of the silica surface. 
               
               
               
                                              Lab.2. Thin layer chromatography 
                                                                                                  
                  Fig.3. A simplified model of  silica gel surface  
                   
                  There are also silica based adsorbents, which are non-polar, i.e. chemically modified silica. 
                  Modified  silica  gel  is  formed  by  chemical  reaction  of  its  surface  with  e.g. 
                  trichlorooctadecylsilane or other reagents. Thus the surface polarity decreases and then its 
                  hydrophobicity increases.  
                   
                       2.  Aluminum oxide 
                  Aluminum oxide (Al O ) is another adsorbent, which is often used as stationary phase in 
                                            2   3
                  laboratory practice. TLC aluminum oxide plates usually comprise neutral or basic aluminum 
                  oxide. These kinds of plates provide distinct separation features with regard to a pH range of 
                  the mobile phase used. Under aqueous conditions basic compounds can be well separated 
                  with basic aluminum oxide plates, while neutral compounds can be successfully separated 
                  with neutral aluminum oxide ones.  
                   
                       3.  Cellulose 
                  Cellulose  is  the  next  adsorbent  used  as  a  stationary  phase  in  chromatography  systems, 
                  especially in TLC. Macromolecules consisting of D-glucose units coupled  -glycosidically at 
                  positions 1 and 4 by oxygen atoms stand for this adsorbent. A section of a cellulose chain is 
                  shown in Fig. 4.  
                                                                                                                                 
                  Fig. 4. Fragment of cellulose macromolecule