Introduction to HPLC. Basic Module in Bioorganic Chemistry January 16, 2007 Johannes Ranke. Introduction to HPLC p.1/17

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1 Introduction to HPLC Basic Module in Bioorganic Chemistry January 16, 2007 Johannes Ranke Introduction to HPLC p.1/17

2 Sources R. Kellner, J.-M. Mermet, M. Otto, H. M. Widmer (Eds.), Analytical Chemistry, Wiley-VCH, Weinheim, 1998 W. J. Lough and I. W. Wainer (Eds.), High Performance Liquid Chromatography, Blackie Academic & Professional, London, 1996 Power Point slides P. Stepnowski/B. Jastorff W. M. A. Niessen, J. van der Greef, Liquid Chromatography Mass Spectrometry, Chromatography Series Volume 58, M. Dekker, New York, 1992 V. R. Meyer, Praxis der Hochleistungs- Flüssigchromatographie, Salle + Sauerländer, Frankfurt a. M., 1992 Introduction to HPLC p.2/17

3 1D Separation methods Electrophoresis Introduction to HPLC p.3/17

4 1D Separation methods Electrophoresis Chromatography Introduction to HPLC p.3/17

5 1D Separation methods Electrophoresis Chromatography Membrane separation Introduction to HPLC p.3/17

6 1D Separation methods Electrophoresis Separation according to Kinetic properties Equilibrium distribution Combinations thereof Chromatography Membrane separation Introduction to HPLC p.3/17

7 Elution techniques A + B Introduction to HPLC p.4/17

8 Elution techniques A B Introduction to HPLC p.4/17

9 Elution techniques A B Introduction to HPLC p.4/17

10 Elution techniques Detector signal A B Time [min] Introduction to HPLC p.4/17

11 Equilibrium based separation t M Introduction to HPLC p.5/17

12 Equilibrium based separation t M t R Introduction to HPLC p.5/17

13 Equilibrium based separation k = n S n M t M t R Introduction to HPLC p.5/17

14 Equilibrium based separation k = n S n M = t S t M t M t R Introduction to HPLC p.5/17

15 Equilibrium based separation k = n S n M = t S t M = t R t M t M t M t R Introduction to HPLC p.5/17

16 Equilibrium based separation k = n S n M = t S t M = t R t M t M k = c S V S c M V M t M t R Introduction to HPLC p.5/17

17 Equilibrium based separation k = n S n M = t S t M = t R t M t M k = c S V S c M V M =K VS V M t M t R Introduction to HPLC p.5/17

18 Peak shapes c P. Stepnowski Introduction to HPLC p.6/17

19 Column performance S 2σ w b =4σ t R t Introduction to HPLC p.7/17

20 Column performance S N=( t R σ )2 2σ w b =4σ t R t Introduction to HPLC p.7/17

21 Column performance S N=( t R σ )2 N=16 ( t R w b ) 2 2σ w b =4σ t R t Introduction to HPLC p.7/17

22 Column performance S N=( t R σ )2 N=16 ( t R w b ) 2 N=8 ln2 ( t R w 0.5 ) 2 2σ w 0.5 w b =4σ t R t Introduction to HPLC p.7/17

23 Column performance N=16 ( t R w b ) 2 Introduction to HPLC p.8/17

24 Column performance N=16 ( t R w b ) 2 N eff =16 ( t R t 0 w b ) 2 Introduction to HPLC p.8/17

25 Column performance N=16 ( t R w b ) 2 N eff =16 ( t R t 0 w b ) 2 H= L N H is the Height Equivalent of a Theoretical Plate (HETP). Introduction to HPLC p.8/17

26 Column performance N=16 ( t R w b ) 2 N eff =16 ( t R t 0 w b ) 2 H= L N H is the Height Equivalent of a Theoretical Plate (HETP). ResolutionR S = t R,1 t R,2 0.5 (w b,1 +w b,2 ) Introduction to HPLC p.8/17

27 The HPLC system c P. Stepnowski Introduction to HPLC p.9/17

28 Standard HPLC system Introduction to HPLC p.10/17

29 Sample injection valve c P. Stepnowski Introduction to HPLC p.11/17

30 Detector properties Sensitivity Introduction to HPLC p.12/17

31 Detector properties Sensitivity Selectivity Introduction to HPLC p.12/17

32 Detector properties Sensitivity Selectivity Linear range Introduction to HPLC p.12/17

33 Detector properties Sensitivity Selectivity Linear range Range of application Introduction to HPLC p.12/17

34 Detector properties Sensitivity Selectivity Linear range Range of application Analyte characterisation Introduction to HPLC p.12/17

35 Detector properties Sensitivity Selectivity Linear range Range of application Analyte characterisation Gradient elution? Introduction to HPLC p.12/17

36 Detector properties Sensitivity Selectivity Linear range Range of application Analyte characterisation Gradient elution? Relative cost Introduction to HPLC p.12/17

37 UV/VIS detector Lough/Wainer 1996, p. 121 S: light source C: flow cell W: wavelength selection RC: reference cell BS: beam splitter D: photodetector M: mirror RD: reference photodetector L: lens Introduction to HPLC p.13/17

38 UV/VIS detector Meyer 1996, p.66 Introduction to HPLC p.14/17

39 UV cutoff wavelengths Solvent Cutoff wavelength (nm) Acetonitrile 190 Water 190 Cyclohexane 195 Methanol 205 Dichloromethane 220 Introduction to HPLC p.15/17

40 The HPLC crash course Preparation of the eluents Introduction to HPLC p.16/17

41 The HPLC crash course Preparation of the eluents Operating the device Introduction to HPLC p.16/17

42 The HPLC crash course Preparation of the eluents Operating the device Determination of dead time and dead volume Introduction to HPLC p.16/17

43 The HPLC crash course Preparation of the eluents Operating the device Determination of dead time and dead volume Separation of additional aromatic compounds Introduction to HPLC p.16/17

44 The HPLC crash course Preparation of the eluents Operating the device Determination of dead time and dead volume Separation of additional aromatic compounds Method development for two compounds Introduction to HPLC p.16/17

45 The HPLC crash course Preparation of the eluents Operating the device Determination of dead time and dead volume Separation of additional aromatic compounds Method development for two compounds Introduction to HPLC p.16/17

46 In the lab Start collection and evaluation of data during the experiment Introduction to HPLC p.17/17

47 In the lab Start collection and evaluation of data during the experiment Always collect information about chemicals Introduction to HPLC p.17/17

48 In the lab Start collection and evaluation of data during the experiment Always collect information about chemicals Use Prof. Jastorff s color coding scheme for discussing molecular interactions Introduction to HPLC p.17/17

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