Introduction to RP-HPLC

Basics of RP-HPLC

1. RP-HPLC – BASICS S.Karthikumar ., M. Sc .,M.Phil., M.Tech .,( Ph.D ) Assistant Professor , Department of Biotechnology Kamaraj College of Engineering and Technology Virudhunagar-626001, Tamilnadu , INDIA [email protected] 1

2. HPLC Normal Phase HPLC Reverse Phase HPLC [email protected] 2

3. 3 Invention of Chromatography by M.Tswett Ether CaCO 3 Chlorophyll Chromato graphy Colors [email protected]

4. High Pressure Liquid Chromatography High Performance Liquid Chromatography [email protected] 4

5. Advantages of High Performance Liquid Chromatography High separation capacity, Superior quantitative capability and reproducibility Moderate analytical conditions Unlike GC, the sample does not need to be vaporized. Generally high sensitivity Low sample consumption Easy preparative separation and purification of samples [email protected] 5

6. Fields in Which High Performance Liquid Chromatography Is Used Biogenic substances Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids, amines, etc. Medical products Drugs, antibiotics, etc. Food products Vitamins, food additives, sugars, organic acids, amino acids, etc. Environmental samples Inorganic ions Hazardous organic substances, etc. Organic industrial products Synthetic polymers, additives, surfactants, etc. [email protected] 6

7. Interaction Between Solutes, Stationary Phase, and Mobile Phase Differences in the interactions between the solutes and stationary and mobile phases enable separation. Solute Stationary phase Mobile phase Degree of adsorption, solubility, ionicity, etc. [email protected] 7

8. 8 Stationary Phase and Mobile Phase Used in Normal Phase Mode Stationary Phase Silica gel: -Si- OH Cyano type: -Si-CH 2 CH 2 CH 2 CN Amino type: -Si-CH 2 CH 2 CH 2 NH 2 Diol type: -Si-CH 2 CH 2 CH 2 OCH( OH )-CH 2 OH Mobile Phase Basic solvents: Aliphatic hydrocarbons, aromatic hydrocarbons, etc. Additional solvents: Alcohols, ethers, etc. [email protected]

9. 9 Relationship between Hydrogen Bonding and Retention Time in Normal Phase Mode OH HO SiOH SiOH Strong Weak Steric hindrance Very weak ? Polar Functional Groups -COOH Carboxyl groups -NH 2 Amino groups -OH Hydroxyl groups [email protected]

10. Reversed Phase Chromatography Stationary phase: Low polarity Octadecyl group-bonded silical gel (ODS) Mobile phase: High polarity Water, methanol, acetonitrile Salt is sometimes added. Stationary phase Mobile phase Normal phase High polarity (hydrophilic) Low polarity (hydrophobic) Reversed phase Low polarity (hydrophobic) High polarity (hydrophilic) Nonpolar Functional Groups -(CH 2 ) n CH 3 Alkyl groups -C 6 H 5 Phenyl groups [email protected] 10

11. Separation Column for Reversed Phase Chromatography C 18 (ODS) type C 8 ( octyl ) type C 4 (butyl) type Phenyl type TMS type Cyano type Si -O-Si C 18 (ODS) CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 [email protected] 11

12. 12 Hydrophobic Interaction H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O Network of hydrogen bonds H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O Nonpolar solute If a nonpolar substance is added... …the network is broken and... H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O Nonpolar solute Nonpolar stationary phase …the nonpolar substance is pushed to a nonpolar location. [email protected]

13. Effect of Chain Length of Stationary Phase C 18 (ODS) Strong C 8 C 4 Medium Weak [email protected] 13

14. Relationship Between Retention Time and Polarity C 18 (ODS) CH 3 Strong Weak OH [email protected] 14

15. Comparison of Normal Phase and Reversed Phase Normal Phase Effective for separation of structural isomers Offers separation selectivity not available with reversed phase Stabilizes slowly and is prone to fluctuations in retention time Eluents are expensive Reversed Phase Wide range of applications Effective for separation of homologs Stationary phase has long service life Stabilizes quickly Eluents are inexpensive and easy to use [email protected] 15

16. HPLC - Hardware Solvent Delivery System, Degasser, Sample Injection Unit, Column Oven [email protected] 16

17. Pump Sample injection unit (injector) Column Column oven (thermostatic column chamber) Detector Eluent (mobile phase) Drain Data processor Degasser Flow Channel Diagram for High Performance Liquid Chromatograph [email protected] 17

18. 18 Solvent Delivery Pump Performance Requirements Capacity to withstand high load pressures. Flow rate does not fluctuate. Solvent replacement is easy. The flow rate setting range is wide and the flow rate is accurate. [email protected]

19. 19 Solvent Delivery Pump: Schematic Diagram of Plunger Pump Motor and cam Plunger Plunger seal Check valves Pump head 10 -100µL [email protected]

20. Pump – Mixing & Flow Rate System Isocratic system Constant eluent composition Gradient system Varying eluent composition HPGE (High Pressure Gradient) LPGE (Low Pressure Gradient) [email protected] 20

21. Difference in the Chromatogram In isocratic mode Long analysis time!! Poor separation!! CH 3 OH / H 2 O = 6 / 4 CH 3 OH / H 2 O = 8 / 2 If the eluent composition is changed gradually during analysis... Gradient system [email protected] 21

22. Problems caused by dissolved air in the eluent Unstable delivery by pump More noise and large baseline drift in detector cell In order to avoid these problems, the eluent must be degassed. [email protected] 22

23. Degasser Heated stirring is used to reduce the dissolved air in the solution in the mobile phase bottle to the saturated solubility level. [email protected] 23

24. Mixing, Filtration, and Offline Degassing of the Eluent Decompression by aspirator Ultrasonic cleaning unit Decompression by aspirator Membrane filter with pore size of approx. 0.45 µ m [email protected] 24

25. Online Degasser Gas-liquid separation membrane method Helium purge method Helium cylinder To draft To pump Eluent container Regulator Drain valve To pump Eluent container Polymeric film tube Vacuum chamber [email protected] 25

26. Preparing pH Buffer Solution Use a weak acid with a p K a value close to the desired pH value. Example: Preparing a buffer solution for a pH value of around 4.8.  Use acetic acid, which has a p K a value of 4.8. Make the concentrations of HA and A - roughly equal.  Mix an acid with its salt. Example: Mix acetic acid and sodium acetate so that they have the same molar concentration. [email protected] 26

27. Buffer Solutions Used for HPLC Eluent Requirements High buffering power at prescribed pH. Does not adversely affect detection. Does not damage column or equipment. Inexpensive. Commonly Used Acids Phosphoric acid p K a 2.1, 7.2, 12.3 Acetic acid p K a 4.8 Citric acid p K a 3.1, 4.8, 6.4 Concentration If only to adjust pH, 10 mmol/L is sufficient. [email protected] 27

28. pH of Eluent and Retention of Ionic Solutes COOH COO H pH of eluent Acidic Alkaline + Increased hydrophobicity Increased hydrophilicity High RT Low RT [email protected] 28

29. Replacement of Eluent Mutually insoluble solvents must not be exchanged directly. Aqueous solutions containing salt and organic solvents must not be exchanged directly. Water Hexane 2-Propanol Buffer solution Water-soluble organic solvent Water [email protected] 29

30. Sample Injection Unit (Injector ) INJECT position LOAD position From pump To column From pump To column [email protected] 30

31. Column Oven Air circulation heating type Block heating type A luminum block heater Insulated column jacket type Water bath [email protected] 31

32. HPLC Detectors UV-VIS absorbance detector Photodiode array-type UV-VIS absorbance detector Fluorescence detector Refractive index detector Evaporative light scattering detector Electrical conductivity detector Electrochemical detector Mass spectrometer [email protected] 32

33. UV-VIS Absorbance Detector A = e · C · l = – log ( E out / E in ) l C : Concentration ( A : absorbance, E : absorption coefficient) Detection cell E in E out A C [email protected] 33

34. Comparison of Detectors Note: The above table indicates general characteristics. There are exceptions. Selectivity Sensitivity Possibility of Gradient System Absorbance Light-absorbing substances ng Possible Fluorescence Fluorescent substances pg Possible Differential refractive index None µ g Impossible Evaporative light scattering Nonvolatile substances µ g Possible Electrical conductivity Ionic substances ng Partially possible Electrochemical Oxidizing / reducing substances pg Partially possible [email protected] 34

35. Chromatogram t R t Intensity of detector signal Time Peak t R : Retention time h A t : Non-retention time A : Peak area h : Peak height [email protected] 35

36. Retention Factor, k t R t Strength of detector signal Time t R : Retention time t : Non-retention time [email protected] 36

37. Theoretical Plate Number, N W W 1/2 H 1/2 H 2 . 2 1 R R / R W t W 2 2 2  54 5 16 · = = = Area H t t N [email protected] 37

38. [email protected] 38

39. THANK YOU [email protected] 39