1. Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor talhowiriny@yahoo.com

2. Application of samples ► Samples must be applied to thin layers with extreme care and minimal disturbance of the adsorbent layer. Normally samples are applied via capillary tube or micropipette so that the emerging drop just touches the surface of the plate with the appliance tip remaining just above the sorbent layer. A hole in the sorbent causes an obstruction to even solvent flow, with distortion of the moving spots culminating in loss of resolution.

3. Application of samples ► Spots should not be nearer than 1cm centre to centre; they should be 2-5mm in diameter and should not be nearer to the edge of the plate than 1.5cm on a 20X20cm plate. A volume of 0.1-0.5 mm ³ should be applied, and if a larger volume of the sample solution is needed to give the required loading it should be applied in portions. TLC is a micro analytical method and the loading should not be more than about 12 µ g per spot on a layer 250 µ m thick, 10 µ g being the optimum amount for most substances.

4. Application of samples ► In semi – preparative work the sample is often applied as a streak along the start line; up about 4mg may be thus be loaded on 20X20 cm plate 250 µ m thick. Mechanical devices can be obtained for use in conjunction with a syringe to give rapid and even streaking. A line can be drawn across the plate with a sharp pencil. The pencil removes a fine line of adsorbent down to the glass. The solvent flow is forced to stop when the front reaches the line, a useful feature which enables conditions to be standardized very easily.

5. Application of samples ► The plate should no, however, be left standing in the tank for a long time after the front has reached the spot line, as diffusion and evaporation may cause spreading of the separated spots. The Edges of the plate should be rubbed clear with finger before spotting to a width of about 0.5cm, to give a sharper edge to the adsorbent layer ► The solvent in which the sample is dissolved for spotting should be as volatile as possible.

6. Application of samples ► In TLC and paper chromatography the results obtained are described by quoting the R f values, An R f value is defined as: ► R f = distance moved by solute/distance moved by solvent front. ► Due to the larger number of variables, however, which can influence the R f value – for instance, minute differences in solvents composition, temperature, size of tank, the sorbent layer and the nature of the mixture-coincidence f R f values, even in more than one solvent system, should never be taken as unequivocal proof of the structure of a component.

7. Application of samples ► An alternative approach which minimizes the differences in R f values due to the influence of the variables enumerated above, is to quote R f relative to a carefully chosen standard which is run on the same plate. This relative value, Rx or Rst should be constant since under any given conditions the relative R f values remain the same. ► Rst = distance to centre f component spot/distance to centre of standard spot. ► As with the R f value, however, coincidence of Rst values should not be taken as absolute proof of identification and for complete structural characterization the component should be eluted from the sorbent layer and spectroanalytical studies, such as infrared ultraviolet, nuclear magnetic resonance ( ¹³ C and ¹ H) and mass spectroscopy carried out to aid identification.