1. 1 HPLC Lecture 42

2. 2 Mobile Phase Selection in Partition Chromatography Optimization of the mobile phase composition and polarity is vital for obtaining good separations. The optimization of the sedparation process involves optimization of N, k', and . Changing the mobile phase composition can well control k' and a as well as indirectly improving N. Initially, k' is usually adjusted in the range from 2-5 and if the required resolution is not obtained, one can look at conditions that may change .

3. 3 Effect of Solvent Strength on k' Selection of a suitable mobile phase polarity is very important for successful separations. The polarity of the different solvents can be derived from Snyder's polarity index where: P' AB =  A P' A +  B P' B Where, P' AB is the polarity index of the mobile phase containing  A and  B are volume fractions of solvents A and B while P' A and P’ B are the polarity indices of pure solvents A and B.

4. 4 The retention factor is also related to mobile phase polarity by the relation: log k' 2 /k' 1 = (P' 2 – P' 1 )/2 for RPLC, and: log k' 2 /k' 1 = (P' 1 – P' 2 )/2 for NPC Where, P’ 1 and P' 2 are the initial and final polarity indices of the mobile phase that will result in bringing the value of the retention factor from k' 1 to k' 2.

5. 5 A solute having a retention time of 31.3 min is separated using a column with t M = 0.48 min and mobile phase composition of 30%methanol, 70% water. Find k', and the mobile phase composition that can bring k' to 5. Solution: K' = (31.3-0.48)/0.48 = 64 P' AB =  A P' A +  B P' B Values of P' can be obtained from tables of polarity indices

6. 6

7. 7 P' AB = 0.30*5.1 + 0.70*10.2 = 8.7 log k' 2 /k' 1 = (P' 2 – P' 1 )/2 log 5/64 = (P' 2 – 8.7)/2 P' 2 = 6.5 P' AB =  A P' A +  B P' B 6.5 = x*5.1 + (1-x)*10.2 x = 0.73 % Methanol = 0.73*100 = 73% % Water = 100-71 = 27%

8. 8 Therefore, the mobile phase composition that will result in k' = 5 is 73% methanol and 27% water. However, if k' was judged suitable but still the two peaks overlap, one should look at optimizing  while keeping k' constant. This can be done by changing the chemical nature of the mobile phase, rather than its polarity (i.e. by changing the nature of the organic modifier say for example tetrahydrofuran or dioxane instead of methanol).

9. 9 Size Exclusion Chromatography (SEC) This technique is also called gel permeation (GPC) or gel filtration (GFC) chromatography. It is used for the separation of high molecular weight species (polymers, enzymes, proteins, etc.). No interactions of solutes with the packing material take place and retention is a function of molecular size. The packing material is porous and is characterized by certain range of pore size. Large molecular weight species are retained less since they do not enter the pores while species which have dimensions smaller than the pores will be retained more since they travel through the pores.

10. 10 The separation takes place in a chromatographic column filled with beads of a rigid porous material (also called a gel). The technique can also be used for polymer molecular-weight determinations where highly crosslinked porous polystyrene is the preferred packing materials. The pores in these gels are of the same range as the dimensions of analytes. A sample of a dilute polymer solution is introduced into the mobile phase. As the dissolved polymer molecules flow past the porous beads, they can diffuse into the internal pore structure of the gel to an extent depending on their size and the pore size distribution of the gel.

11. 11 Larger molecules can enter only a small fraction, if at all, of the internal portion of the gel, or are completely excluded; smaller polymer molecules penetrate a larger fraction of the interior pores of the gel. The different molecular species are eluted from the column in order of their molecular size. A specific column or set of columns (with gels of different pore sizes) is calibrated empirically to give such a relationship relating retention to log molecular weight. For convenience, commercially available narrow-distribution polystyrenes (anionic form) are often used as standards.

12. 12 ViVi VoVo VRVR Exclusion Limit Permeation Limit Log MW

13. 13 The permeation limit indicates molecular weight below which all solutes have the same retention time and thus will elute together as a single peak. The exclusion limit indicates the molecular weight of solutes above which all solutes having a molecular weight greater than the exclusion limit will elute at the same retention time as a single peak. A specific column is practically usable for separation of solutes with molecular weights within the molecular weight window between the exclusion and permeation limits

14. 14 A series of commercially available polymerized polystyrenes (anionic form) is used as standards for calibration. The elution volume corresponding to a peak in the chromatogram is related to the molecular weight of a particular polystyrene. After assigning molecular weight of each component to its elution volume, a plot of log (MW) versus elution volume can be constructed. A straight line should result and this is known as the calibration curve.