Chromatography General Principle of Sample Separation

Questions: 1. Explain briefly the general principle of sample separation by chromatography. 2. Give the three equations required to calculate Rf-values, the CV and the CV. 3. An unknown sample has been applied to 4 different TLC plates. Each TLC plate has been run in a different mobile phase. The stationary phase of the TLC plates is made of unmodified silica gel. Complete the table using the TLC plates above. (10 points) A B C D Rf Rf Rf - - CV CV CV - - CV CV CV 4. Consider the results obtained in the table under task 3. Summarise your observations and discuss these in terms of the quality of the sample separation. (10 points) 5. Consider the system used in TLC A;Ethanol/CH2Cl2 1:6. a) Which TLC will show a larger change in the Rf values, a change to a Ethanol/CH2Cl2 1:7 system or Ethanol/CH2Cl2 2:6? b) Why? Explain briefly. Answers: 1. Liquid chromatography isolates molecules in liquid mobile phase by applying a solid stationary phase. Thin layer chromatography is said to be a chromatographic technique applied to divide non-volatile mixtures. After a sample is applied on plate, a mixture of solvent or solvent is put together on the plate by capillary action (Qin et al., 2015). Diverse compounds within the sample mixture pass through at various rates because of differences in the attraction to stationary phase and due to solubility differences within the solvent (Waksmundzka-Hajnos, Sherma and Kowalska, 2008). By altering the solvent or applying mixture, the separation of the substances can be changed. Compound separation is depending upon the competition of mobile and solute phase for attaching position on stationary phase (Lin, 2013). For example if silica gel is applied as stationary phase, this is considered as polar. If it is compared between two polar compounds then the more polar substance has stronger int eraction with silica and hence more competent to drive out the mobile phase from binding places. The less polar component moves top of the plate. 2. The equation required to calculate Rf values: The equation required to calculate the CV: CV=r2h The equation required to calculate the CV: 3. A B C D Rf ac/ad ac/ad ad/ae ac/ad Rf ab/ad ab/ad ac/ae ab/ad Rf ab/ae - ab/ae aa/ad CV (ap)2.ac (ap)2.ac (ap)2.ad (ap)2.ac CV (ap)2.ab (ap)2.ab (ap)2.ac (ap)2.ab CV - - (ap)2.ab (ap)2.aa CV Change in (ap)2.ac/1unit/ (ap)2.ac Change in (ap)2.ac/1unit/ (ap)2.ac Change in (ap)2.ad/1unit/ (ap)2.ad Change in (ap)2.ac/1unit/ (ap)2.ac CV Change in (ap)2.ab/1unit/ (ap)2.ab Change in (ap)2.ab/1unit/ (ap)2.ab Change in (ap)2.ac/1unit/ (ap)2.ac Change in (ap)2.ab/1unit/ (ap)2.ab CV Change in (ap)2.ab/1unit/ (ap)2.ab Change in (ap)2.aa/1unit/ (ap)2.aa 4. An unknown sample has been applied to 4diffrent TLC plates. Each TLC plate has been run in a different mobile phase. The stationary phase of the TLC plates is made of unmodified silica gel. The table has enquired for the Rf values of the unknown sample 1 2 and 3. Rf means the retention value. To make the chromatography technique more scientific than just an interpretation, Rf value can be applied. A particular sample will travel same distance along stationary phase by specific solvent when other conditions are kept constant. Every unknown sample has specific retention value for specific solvent and the concentration of the solvent. In case of plate A, B, C and D four hypothetical points are measured. These are: a, b, c and d; where the unknown samples are pointed as 1, 2 and 3. Therefore, to measure the Rf of plate A, sample 1 and 2 in plate A, hypothetical data is applied. In case of plate B to measure the Rf of sample 1 and 2 hypothetical data is applied, thus continued to plate number. Then next to calculate the column volume of 1, 2 and 3 of 4 plates, hypothetical formula is applied, where the column volume is calculated by using the formula of r2h. Hence, for individual points (samples) in individual plate the respective data is entered. 5. a) A change to a Ethanol/CH2Cl2 1:7 system will show a larger change in the retention values. b) This is because the eluent ratio of ethanol and dichrolomethane is greater in 1:7 systems, where the system contains 1part of ethanol and 7parts of dichrolomethane. Rather in case of Ethanol/CH2Cl2 2:6 systems, it contains 1part of ethanol and 3parts of dichrolomethane (Komsta, 2007). Higher and lower retention value is depending on the polarity of the compound. The more polar compound has strong contact with silica gel and hence more potent to dispel the liquid phase resulting in lesser retention value and less polar substance move top of the plate and results in higher retention value (Rogatsky, 2012). Therefore a change to an Ethanol/CH2Cl2 1:7 systems will show a larger change in the retention values. References Komsta,  . (2007). Prediction of the retention in thin layer chromatography screening systems by atomic contributions.Analytica Chimica Acta, 593(2), pp.224-237. Lin, S. (2013). Wonderlab Comic Thin Layer Chromatography.ChemViews. Qin, Y., Qi, N., Tang, Y., He, J., Li, X., Gu, F. and Zou, S. (2015). Isolation and identification of a high molecular weight protein in sow milk.animal, pp.1-8. Rogatsky, E. (2012). Application of Fused-Core Particle Column in Two Dimensional Reversed Phase - Reversed Phase LC/MS Analysis of Biological Samples. Impact of Extra-Column Volume.J Chromat Separation Techniq, 03(08). Waksmundzka-Hajnos, M., Sherma, J. and Kowalska, T. (2008).Thin layer chromatography in phytochemistry. Boca Raton: CRC Press.