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Optimization & Monitoring

Optimization & Monitoring. How to achieve optimal conditions?. 1) Use knowledge about chromatography to select conditions 2) Optimize your system by doing experiments 3) After optimization, monitor the system so you are sure the conditions stay optimal. How to achieve optimal conditions?.

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Optimization & Monitoring

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  1. Optimization & Monitoring

  2. How to achieve optimal conditions? 1) Use knowledge about chromatography to select conditions 2) Optimize your system by doing experiments 3) After optimization, monitor the system so you are sure the conditions stay optimal

  3. How to achieve optimal conditions? 1) Use knowledge about chromatography to select conditions 2) Optimize your system by doing experiments 3) After optimization, monitor the system so you are sure the conditions stay optimal

  4. How to avoid chromatographic overlaps Detector signal 0 1 2 3 4 5 6 7 8 9 10 min

  5. How to avoid chromatographic overlaps Rules of thumb (general): If the compounds differ in type or number of functional groups: try to change selectivity If the compounds are isomers: increase the efficiency In complex chromatograms: increase the efficiency

  6. tR(B) – tR(A) Rs = (wb(A) + wb(B)) 1 2 How to avoid chromatographic overlaps B Peak resolution Peak overlap A B B A A Increase efficiency (decrease peak width) Change selectivity (increased tR)

  7. How to change selectivity in LC Change solvent composition Change type of stationary phase B A Detector signal 0 1 2 3 4 5 6 7 8 9 10 min

  8. How to change selectivity in LC Change solvent composition Change type of stationary phase Selectivity issues depend on the applications. Understanding selectivity requires a good understanding of how the analytes interacts with both the stationary phase and the mobile phase  A good understanding of chemistry is required. B A Detector signal 0 1 2 3 4 5 6 7 8 9 10 min

  9. How to achieve high column efficiency in LC B A Detector signal 0 1 2 3 4 5 6 7 8 9 10 min

  10. Solvent flow Solvent flow How to achieve high column efficiency in LC Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity)

  11. N 4 α–1 α k(B) 1+k(B) Rs = Solvent flow Solvent flow B u H = A + + C∙u How to achieve high column efficiency in LC Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity) Van Deemter equation Purnell equation

  12. N 4 α–1 α k(B) 1+k(B) Rs = B u H = A + + C∙u How to achieve high column efficiency in LC Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity) Van Deemter equation Purnell equation

  13. N 4 α–1 α k(B) 1+k(B) Rs = Solvent flow Solvent flow B u H = A + + C∙u How to achieve high column efficiency in LC Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity) Van Deemter equation Purnell equation

  14. N 4 α–1 α k(B) 1+k(B) Rs = Solvent flow Solvent flow B u H = A + + C∙u How to achieve high column efficiency in LC Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity) Van Deemter equation Purnell equation

  15. N 4 α–1 α k(B) 1+k(B) Rs = Solvent flow Solvent flow B u H = A + + C∙u How to achieve high column efficiency in LC Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity) Van Deemter equation Purnell equation

  16. N 4 α–1 α k(B) 1+k(B) Rs = Solvent flow Solvent flow B u H = A + + C∙u How to achieve high column efficiency in LC Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity) Van Deemter equation Purnell equation

  17. N 4 α–1 α k(B) 1+k(B) Rs = Solvent flow Solvent flow B u H = A + + C∙u How to achieve high column efficiency in LC Increased time Pressure limitations Increase column length Decrease particle diameter Uniform particles (Lower solvent strength) (Optimal mobile phase velocity) Pressure limitations Always good Increased time Van Deemter equation Purnell equation

  18. Solvent flow Solvent flow How to achieve high column efficiency in LC • HPLC: • Typical particle diameters of 2-5 m. • Pressures up to 400 bar. • UHPLC (Ultra high performance LC) • Particle diameters < 2 m. • Pressures above 400 bar. • Monodisperse particles (all same size).

  19. N 4 α–1 α k(B) 1+k(B) Rs = How to increase efficiency in gas chromatography Increase column length Decrease column diameter Decrease film thickness (Lower temperature) (Optimal carrier gas velocity) Low efficiency per meter High efficiency per meter Golay equation Purnell equation B u H = + C∙u

  20. N 4 α–1 α k(B) 1+k(B) Rs = How to increase efficiency in gas chromatography Increase column length Decrease column diameter Decrease film thickness (Lower temperature) (Optimal carrier gas velocity) Low efficiency per meter High efficiency per meter Golay equation Purnell equation B u H = + C∙u

  21. N 4 α–1 α k(B) 1+k(B) Rs = How to increase efficiency in gas chromatography Increase column length Decrease column diameter Decrease film thickness (Lower temperature) (Optimal carrier gas velocity) Low efficiency per meter 2 High efficiency per meter Golay equation Purnell equation B u H = + C∙u

  22. N 4 α–1 α k(B) 1+k(B) Rs = How to increase efficiency in gas chromatography Increase column length Decrease column diameter Decrease film thickness (Lower temperature) (Optimal carrier gas velocity) Low efficiency per meter High efficiency per meter Golay equation Purnell equation B u H = + C∙u

  23. N 4 α–1 α k(B) 1+k(B) Rs = Low efficiency per meter High efficiency per meter Purnell equation How to increase efficiency in gas chromatography Increase column length Decrease column diameter Decrease film thickness (Lower temperature) (Optimal carrier gas velocity) Golay equation B u H = + C∙u

  24. N 4 α–1 α k(B) 1+k(B) Rs = Low efficiency per meter High efficiency per meter Purnell equation How to increase efficiency in gas chromatography Increased time Pressure limitations Increase column length Decrease column diameter Decrease film thickness (Lower temperature) (Optimal carrier gas velocity) Pressure limitations Decreased sample capacity Decreased sample capacity Increased time Golay equation B u H = + C∙u

  25. How to increase efficiency in gas chromatography Data for apolar column (kanalyte = 5) Phase ratio, β = 250 High efficiency per meter Low efficiency per meter

  26. How to increase efficiency in gas chromatography Data for apolar column (kanalyte = 5) 10 m column  119 800 plates 25 m column  115 750 plates Phase ratio, β = 250 High efficiency per meter Low efficiency per meter

  27. How to change selectivity in GC Change type of stationary phase (the mobile phase has no selectivity) The mobile phase has no selectivity and there are a limited number of stationary phase types. Understanding and predicting selectivity in GC is therefore much simpler than in LC (Good chemists do LC, poor chemists do GC) B A Detector signal 0 1 2 3 4 5 6 7 8 9 10 min

  28. Time/resolution tradeoff In chromatography there is usually a tradeoff between time and resolution (or efficiency) We want as short runs as possible (time=money) but with sufficient resolution Acceptable resolution Acceptable resolution And half the time

  29. Time/resolution tradeoff • In chromatography there is usually a tradeoff between time and resolution (or efficiency) • We want as short runs as possible (time=money) but with sufficient resolution • If we want to sacrifice chromatographic efficiency to save time there are several options: • Reduce the column length • Use higher than optimal mobile phase velocity • Increase mobile phase strength (LC) or temperture (GC) • Use steeper gradients of solvent strength or temperature

  30. Time/resolution tradeoff Which of the two options that is the best choice will depend on the conditions used and the importance of the C term relative to the A and B terms. The penalty for using higher than optimal velocity depends on the steepness of the curve (C-term)

  31. Time/resolution tradeoff Which of the two options that is the best choice will depend on the conditions used and the importance of the C term relative to the A and B terms. The penalty for using higher than optimal velocity depends on the steepness of the curve (C-term) The general advice for GC is to always use optimal velocity and reduce the column length. That is also cheaper since there will be less carrier gas consumption and since shorter columns are cheaper than long columns.

  32. Time/resolution tradeoff Which of the two options that is the best choice will depend on the conditions used and the importance of the C term relative to the A and B terms. The penalty for using higher than optimal velocity depends on the steepness of the curve (C-term) The general advice for GC is to always use optimal velocity and reduce the column length. That is also cheaper since there will be less carrier gas consumption and since shorter columns are cheaper than long columns. However, if the column is used for several methods with different requirements for efficiency, it is usually more convenient to use higher than optimum flow.

  33. Time/resolution tradeoff Which of the two options that is the best choice will depend on the conditions used and the importance of the C term relative to the A and B terms. The penalty for using higher than optimal velocity depends on the steepness of the curve (C-term) In LC the C term is low with small particles <5 μm and the mobile phase flow will usually be higher than optimum. In LC, pressure limitations of the pump may be more important for the conditions chosen than the C term in the van Deemter equation.

  34. How to achieve optimal conditions? 1) Use knowledge about chromatography to select conditions 2) Optimize your system by doing experiments 3) After optimization, monitor the system so you are sure the conditions stay optimal

  35. How to achieve optimal conditions? Why do we have to do experiments when we have so much theory and so many nice equations to tell us which conditions that are optimal?

  36. How to achieve optimal conditions? Why do we have to do experiments when we have so much theory and so many nice equations to tell us which conditions that are optimal? 1) Because theory will not always fit reality 2) Because we will not have good enough knowledge about the system we are working with

  37. How to achieve optimal conditions? • Why do we have to do experiments when we have so much theory and so many nice equations to tell us which conditions that are optimal? • 1) Because theory will not always fit reality • 2) Because we will not have good enough knowledge about the system we are working with • We will not know exact column dimensions, exact particle diameters, exact diffusion coefficients, exact flow rates and contribution to extra-column effects from the chromatographic system.

  38. How to achieve optimal conditions? • Linear flow rates, pressures and volumes • In GC, flow rate is adjusted from the head pressure, column dimensions are critical • In LC a constant volume is delivered, but the volume of the column that is occupied by the stationary phase is often difficult to estimate.

  39. Optimization Best performance Low performance

  40. Optimization Best performance Using chromatographic knowledge and making clever choices (and having enough money to buy the right equipent) Low performance

  41. Best performance Low performance Optimization Experimenting and modelling Using chromatographic knowledge and making clever choices (and having enough money to buy the right equipent)

  42. Optimization  You have to do experiments But how?

  43. Optimization  You have to do experiments But how? There are many ways to optimize chromatographic separations. But the general methodology is to do experimental design and calculate response surfaces

  44. Optimization  You have to do experiments But how? Experimental design is a systematic way of setting up your experiments

  45. Optimization Response surfaces A response surface tells you how the response (that you want to optimize) varies with different parameters

  46. Optimization Response surfaces Measured response variable Varied parameter 1 Varied parameter 2

  47. Optimization Response surfaces Surface plot Measured response variable Contour plot Varied parameter 1 Varied parameter 2

  48. Optimization Response surfaces Measured response variable Maximum Varied parameter 1 Varied parameter 2

  49. y Measured response variable x1 x2 Varied parameter 1 Varied parameter 2 Optimization Finding the response surface

  50. Optimization Finding the response surface means that you have to solve an eqation explaining how the response, y, varies as function of the x-variables, the interactions between the variables and usually also higher order (squared) terms of the main variables The regression coefficients, b, are found for instance by multivariate regression y = b0 + b1x1 + b2x2 + b12x1x2 + b11x12 + b22x22

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