1. 1 HPLC Lecture 41

2. 2 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.

3. 3 Sample Injection Valves

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7. 7 Columns Columns are almost always made from stainless steel with most common dimensions in the range from 3-25 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  m. Porous packings are mostly silica based with particle diameters from 3-10  m.

8. 8 Chromatographic Column

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10. 10 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.

11. 11 PoPo P From Column To Waste

12. 12 HPLC-UV-Vis Variable wavelength detector - monochromator PMT

13. 13 HPLC-UV-Vis Diode array detector - polychromator

14. 14 Fluorescence Detectors exc. em From Column To Waste

15. 15 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. Source Detector Sample Reference Mirror

16. 16 Bonded Phase Chromatography In this case, the stationary phase is chemically bonded to the solid support which implies the following: 1.No bleeding 2.Can use stationary phases of any chain length from a C1 to C18 which reflects very well on the retention characteristics of the column 3.The solid support surface is not completely covered with stationary phase and residual silanol groups may complicate retention mechanism and results in tailing 4.If damaged, the stationary phase can not be regenerated in situ 5.Usually, bonded phase stationary phases are expensive 6.Most widely used (almost exclusively)

17. 17 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.

18. 18 Reversed-Phase Liquid Chromatography (RPLC) In this technique, the stationary phase is less polar than the mobile phase where a C 3, C 8, or a C 18 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.

19. 19 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.

20. 20 The chain length in RPLC can serve the following: 1.As the chain length of the stationary phase is increased, the retention times of the less polar solutes are increased. 2.Increasing the chain length of the stationary phase controls the sample size where a C 3 stationary phase can be used for separation of a sample size about one half that of a C 8 stationary phase, providing other conditions are the same. 3.theoretically, shorter stationary phases result in better efficiencies due to decreased H S.

21. 21 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.

22. 22 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 .