1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

2. 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.

3. http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

5. http://www.labhut.com/education/flash/introduction07.php TLC vs High Performance Liquid Chromatography (HPLC) HPLC Optimization

6. HPLC – Optimizing Separation Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

7. Schematic Presentation of a Chromatogram

8. HPLC - Resolution Resolution (R S ) of a column provides a quantitative measure of its ability to separate two analytesResolution (R S ) of a column provides a quantitative measure of its ability to separate two analytes R s =  Z /1/2(W A +W B ) R s =

9. HPLC - Resolution RsRs Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

10. HPLC - Resolution Capacity Factor (k’): Also called retention factor. Is a measure for the position of a sample peak in the chromatogram. k’ = (t R1 -t o )/t o specific for a given compound and constant under constant conditions A function of column and mobile phase chemistry Primarily applicable under isocratic conditions In general, a change in the k’ of one peak will move all peaks in the same direction. Selectivity Factor (  ): Also called separation or selectivity coefficient is defined as  = k 2 ’/k 1 ’ = (t R2 -t o ) / (t R1 -t o ) A function of column and mobile phase chemistry Primarily applicable under isocratic conditions Changes in selectivity will affect different compounds in different ways. Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

11. HPLC – Capacity Factor

12. HPLC – Selectivity Factor

13. HPLC - Resolution Theoretical Plates (N): The number of theoretical plates characterizes the quality or efficiency of a column. (N = 16 (t R /W) 2 ) N = 5.54 [(t R ) / w 1/2 ] 2 (N = 16 (t R /W) 2 ) Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

14. Phenomenex catalog, 1999

15. HPLC - Resolution Theoretical Plates (N): The number of theoretical plates characterizes the quality or efficiency of a column. N = 5.54 [(t R ) / w 1/2 ] 2 (N = 16 (t R /W) 2 ) (N = 16 (t R /W) 2 ) Plate Height (H): The height equivalent to a theoretical plate (HEPT = H) H = L / N Resolution (Rs) depends on the number of theoretical plates: R s = Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

16. Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

17. HPLC - General Elution Problem Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

18. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (TLC vs Normal Phase and Reverse Phase HPLC)

19. Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998 Reverse Phase HPLC

20. Normal Phase vs. Reverse Phase HPLC Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

21. RP-HPLC – Stationary Phase Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

22. RP-HPLC – Mobile Phase vs k’ Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

23. Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998 RP-HPLC – Mobile Phase (k’,  )

24. RP-HPLC – Mobile Phase (  ) Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

25. RP-HPLC - Example Alltech Chromatography Sourcebook, 2004-04 catalog

26. RP-HPLC - Optimization Alltech Chromatography Sourcebook, 2004-04 catalog

27. RP-HPLC – Gradient Elution Alltech Chromatography Sourcebook, 2004-04 catalog

30. HPLC – Resolution vs Column Efficiency (N, H) van Deemter Equation H = A + B/u +(C s + C m )u H = L / N Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

31. HPLC - Column Efficiency Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

32. van Deemter Equation H = A + B/u +Cu HPLC - Column Efficiency Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

33. HPLC - Column Efficiency H = A + B/u + Cu A = 2 d p  depends on particle size distribution, the narrower the distribution the smaller the  depends on particle size distribution, the narrower the distribution the smaller the 2.d p = particle size 3.Independent of mobile phase flow rate 4.Also known as eddy diffusion Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

34. HPLC - Column Efficiency particle size Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

35. HPLC Column Efficiency Longitudinal Diffusion (B) H = A + B/u + Cu B/u = 2  D M /u B/u = 2  D M /u  = constant depending on quality of packing 2.D M is the mobile phase diffusion coefficient 3.Inversely related to mobile phase flow rate

36. HPLC Column Efficiency Mass Transfer HPLC Column Efficiency Mass Transfer (C s + C m ) H = A + B/u + (C s + C m )u C S = f S (k’)d f 2 / D S C M = f M (k’)d p 2 / D M D M is the mobile phase diffusion coefficientD M is the mobile phase diffusion coefficient D S is the stationary phase diffusion coefficientD S is the stationary phase diffusion coefficient d f is film thicknessd f is film thickness d p is particle sized p is particle size Directly related to mobile phase flow rateDirectly related to mobile phase flow rate Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

37. RP-HPLC – Variables Alltech Chromatography Sourcebook, 2004-04 catalog

38. 1.35 min. Ibuprofen 7.11 min. Caffeine 1.48 min. Aspirin 2.82 min Acetaminophen AnalgesicRetention Time Acetaminophen2.82 Aspirin1.48 Caffeine7.11 Ibuprofen1.35 Gradient = 0 min: 100% EtOAC (+ 0.2% HOAc) 3 min: 100% EtOAC (+ 0.2% HOAc) 5 min: 15% MeOH, 85% % EtOAc (+ 0.2% HOAc) 8 min: 15% MeOH, 85% % EtOAc (+ 0.2% HOAc) 10 min: 100% EtOAC (+ 0.2% HOAc) SiO 2 Flow Rate = 1 mL/min UV detector set at 240 nm HPLC OF ANALGESICS - UV Detection Standard Analgesics

39. Question The peak areas of aspirin and acetaminophen are very different, even though they are present in equal amounts (250mg/tablet) in Excedrin ES. Caffeine is present at ~ ¼ the concentration of aspirin (65 mg/tablet vs. 250 mg/tablet), but it’s peak area is greater than the peak area of aspirin. WHY? UV Absorbance of analgesics vs UV setting of detector Area % Aspirin 19.5% Acetaminophen 50.0% Caffeine 20.5% Excedrin ES 250 mg aspirin 250 mg acetaminophen 65 mg caffeine HPLC OF ANALGESICS - UV Detection

40. Detector set at 240 nm Detector set at 254 nm Detector set at 280 nm UV Max Aspirin 225, 296 nm Acetaminophen 248 nm Caffeine 272 nm Area % Aspirin 19.5% Acetaminophen 50.0% Caffeine 20.5% Area % Aspirin 7.3% Acetaminophen 81.9% Caffeine 10.8% Area % Aspirin 24.8% Acetaminophen 39.3% Caffeine 35.9% HPLC: Peak Area vs Detector setting

41. Figure 2. HPLC (SiO2) of crude tumeric extract. Gradient 0-2 min, 4% EtOAc/Hexane; 2-9 min 4 to 80% EtOAc; 9-11 min, 80% EtOAc/hexane; 11-13 min, 80 to 4% EtOAc/hex, 13-15 min, 4% EtOAc /hexane. (A)Detector set at 420 nm. (B)Detector set at 254 nm. (C)Detector set at 254 nm (0-3.5 min), 420 nm 3.5-15 min. (A) (B) (C) HPLC – UV Detection