HPLC

1. HPLC

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY • High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques for identification, quantification and purification of mixtures of organic compounds. • In HPLC, as in all chromatographic methods, components of a mixture are partitioned between an adsorbent (the stationary phase) and a solvent (the mobile phase). • The stationary phase is made up of very small particles contained in a steel column. Due to the small particle size (3-5 um), pressure is required to force the mobile phase through the stationary phase. • There are a wide variety of stationary phases available for HPLC. In this lab we will use a normal phase(Silica gel), although reverse phase (silica gel in which a 18 carbon hydrocarbon is covalently bound to the surface of the silica) columns are currently one of the most commonly used HPLC stationary phases.

5. Four main chromatographic techniques that use a liquid mobile phase are covered under the broad high performance liquid chromatographic technique. These include: • Partition Chromatography • Liquid-Solid Chromatography • Ion-Exchange Chromatography • Size Exclusion (Gel Permeation) Chromatography The first of the abovementioned chromatographic technique is most widely used. Generally, HPLC uses very high pressures (up to 4000 psi) and very small particle size (down to 3 mm).

6. Column efficiency in Liquid Chromatography We have seen earlier that several factors affect efficiency in chromatographic techniques including: • Particle size • Flow rate • Thickness of stationary phase • Mobile phase viscosity • Diffusion of solute in mobile and stationary phases • How well a column is packed • Sample size (mg sample/g packing)

8. Extra-Column Band Broadening We have discussed three reasons for intra column band broadening including: • Multiple paths effects • Longitudinal diffusion • mass transfer in stationary and mobile phases However, there are other sources of band broadening unrelated to column materials and occur outside the column. These include : • Fittings dead volume • Tubing length and diameter • Detector volume • Injection volume

9. Instruments for Liquid Chromatography Pumps Three types of pumps are known: • Reciprocating pumps • Displacement Pumps • Pneumatic pumps • Reciprocating pumps are by far the most widely used and practically 100% of the pumps used in commercial HPLC equipment are of the reciprocating type.

10. Reciprocating pumps In reciprocating pumps, a motor driven reciprocating piston controls the flow of mobile phase with the help of two ball check valves that opens and closes with the piston movement. The flow is thus not continuous and damping of flow is necessary. This is accomplished using pulse dampers which are a long coiled capillary tube.

13. Displacement pumps Displacement pumps, on the other hand, is composed of a one directional motor driven plunger that pushes the mobile phase present in a syringe like chamber. The volume of displacement pumps is limited which lacks convenience. A constant flow rate is usually obtained with syringe like pumps.

14. Sample Injection Valves

18. Columns Columns are almost always made from stainless steel with most common dimensions in the range from -25 cm long and about 4.6 mm internal diameter. Pellicular or porous packing materials are usually used. Pellicular packings are nonporous glass or polymer beads ranging from 30 to 40 mm. Porous packings are mostly silica based with particle diameters from 3-10 mm.

19. Chromatographic Column

21. Detectors Detector can be classified as bulk or property detectors. Bulk detectors respond to a bulk property of the mobile phase, like refractive index, dielectric constant, conductivity, etc. which is modified in presence of a solute. On the other hand, solute property detectors respond to a property of the solute like its UV-Vis absorption, fluorescence, chemiluminescence, etc. that is not possessed by the mobile phase.

22. From Column Po P To Waste

23. HPLC-UV-Vis Variable wavelength detector - monochromator PMT

24. HPLC-UV-Vis Diode array detector - polychromator

25. From Column lexc. To Waste lem Fluorescence Detectors

26. Sample Source Mirror Detector Reference Refractive Index Detectors, RI These are very good detectors that responds to changes in the refractive index of the mobile phase in presence of an analyte.

27. Bonded Phase Chromatography In this case, the stationary phase is chemically bonded to the solid support which implies the following: • No bleeding • Can use stationary phases of any chain length from a C1 to C18 which reflects very well on the retention characteristics of the column • The solid support surface is not completely covered with stationary phase and residual silanol groups may complicate retention mechanism and results in tailing • If damaged, the stationary phase can not be regenerated in situ • Usually, bonded phase stationary phases are expensive • Most widely used (almost exclusively)

28. Types of Bonded Phase Chromatography Two main techniques are usually used with bonded phase chromatography: Normal Phase Chromatography (NPC) In this technique, the stationary phase is more polar than the mobile phase where a hydroxyl, amino, or cyano terminated stationary phase is used while the mobile phase is a nonpolar solvent like n-hexane. In this type of chromatography, the least polar compound is eluted first while the more polar compound will be retained more.

29. Reversed-Phase Liquid Chromatography (RPLC) In this technique, the stationary phase is less polar than the mobile phase where a C3, C8, or a C18 chain length stationary phase is used while the mobile phase is a polar solvent like methanol, acetonitrile, etc or mixtures with water. In this type of chromatography, the more polar compound is eluted first while the less polar compound will be retained more. RPLC is the most common technique in liquid chromatography and several versions and techniques had emerged from RPLC with some modification of mobile phase.

30. The separation mechanism in RPLC is relatively simple where increasing percentage of organic modifier in mobile phase decreases retention time of less polar solutes, as the polarity of the mobile phase decreases. In contrast, increasing the polarity of the mobile phase increases the retention time of less polar solutes and decreases the retention time of the more polar solutes. The separation mechanism in BPC is simplified by assuming that the chemically bonded stationary phase as if it were a physically retained liquid.

31. The chain length in RPLC can serve the following: • As the chain length of the stationary phase is increased, the retention times of the less polar solutes are increased. • Increasing the chain length of the stationary phase controls the sample size where a C3 stationary phase can be used for separation of a sample size about one half that of a C8 stationary phase, providing other conditions are the same. • theoretically, shorter stationary phases result in better efficiencies due to decreased HS.

32. It should also be observed that the most common solid support in BPC is silica. The Si-O-Si (siloxane bonds) are not stable outside the pH range from 3-8. Therefore, the pH of mobile phases must not exceed this limit, otherwise hydrolysis of silica particles and release of stationary phase will take place which results in deterioration of the packing material and hence the column.

33. 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 a. 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 a.

34. 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 = fAP'A + fBP'B Where, P'AB is the polarity index of the mobile phase containing fA and fB are volume fractions of solvents A and B while P'A and P’B are the polarity indices of pure solvents A and B.

35. 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.

36. A solute having a retention time of 31.3 min is separated using a column with tM = 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 = fAP'A + fBP'B Values of P' can be obtained from tables of polarity indices

38. 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 = fAP'A + fBP'B 6.5 = x*5.1 + (1-x)*10.2 x = 0.73 % Methanol = 0.73*100 = 73% % Water = 100-71 = 27%

39. 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 a 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).

40. 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.

41. 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.

42. 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.

43. The total volume of a column packed with a porous polymer is equal to: Vt = Vg + Vi +Vo Where, Vg = Volume of the solid matrix V0 = Void Volume (Volume in the system outside the porous beads) Vi = Internal Volume (Volume of solvent inside the pores) The following cases can be identified: a. For large molecules (larger than the pore size), full exclusion takes place and the elution volume needed to elute such high molecular weight compounds is: Ve = Vo Where, Ve is the elution volume

44. b. For molecules of small size (smaller than the pore size) the elution volume is: Ve = Vi + Vo c. For molecules of intermediate size (these will transfer to some extent depending on their size and pore size distribution, K, inside the solvent in the pores), the elution volume is: Ve = Vo + KVi K = (Ve - Vo)/Vi = CS/CM In other words: K = 0 for molecules too large to enter the pores K = 1 for molecules that can enter the pores unhindered 1> K >0 for solutes of intermediate size

45. Exclusion Limit Permeation Limit Log MW Vo Vi VR

46. 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

47. 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.