Chem. 230 9

1. Chem. 230 � 9/28 Lecture Updated 9/28 (see slides 3 and 6) + added more slides

2. Announcements Quiz 1 Results Solutions have been posted See class distribution Grade cut-offs have not been set, will depend somewhat on class scores Based on Quiz 1, I might expect: 87% for A-, 75% for B-, and 62% for C-

3. Announcements What we are covering today: More Definitions: Last part plus questions Band broadening theory (Section 3.2) Intermolecular Forces (Section 4.1) HW 2 Long Problems Due Next Week

4. Chromatographic Theory Definitions - Peak Capacity Peak Capacity is the theoretical maximum number of peaks that can be separated with RS = 1.0 within a given time period. We won�t cover calculation, but for example, about 2X more peaks could be possible between 5 and 13 min. Peak capacity 2.3 to 20 min. would be ~27 peaks. Greater peak capacity is typical with temperature/gradient programs (like in example).

5. Chromatographic Theory Definitions - Separation Factor Separation Factor = a = ratio of distribution constants a = KB/KA = kB/kA = (t�R)B/(t�R)A Where (tR)B > (tR)A so that a > 1 Smaller a (closer to 1) means more difficult separation In example chromatogram, (1st 2 peaks) a = (5.77 � 2.37)/(4.96 � 2.37) = 1.31

6. Chromatographic Theory Definitions - Overview The �good� part of chromatography is separation, which results from differences in KC values giving rise to a > 1 The �bad� part of chromatography is band broadening or dispersion, leading to decreased efficiency (and also reducing sensitivity) The �ugly� part of chromatography is non-Gaussian peak shapes (leads to additional band broadening plus need for new equations)

7. Chromatographic TheoryQuestions on Definitions When is chromatographic separation needed vs. only simple separations? An analyte interacts with a stationary phase via adsorption. The stationary phase is most likely: a) Liquid b) Liquid-like c) Solid What are the required two phases in chromatography called? What are advantages and disadvantages with the three common stationary phases (liquid, liquid-like, and solid)?

8. Chromatographic TheoryQuestions on Definitions List two ways in which a stationary phase is �attached� to a column? 6. What column component is present in packed columns but not open-tubular columns? 7. In HPLC, typical packing material consist of �m diameter spherical particles. Even though tightly packing the spheres should lead to > 50% of the column being sphere volume, the ratio of VM/Column Volume > 0.5. Explain this.

9. Chromatographic TheoryQuestions on Definitions List 3 main components of chromatographs. 9. A chemist perform trial runs on a 4.6 mm diameter column with a flow rate of 1.4 mL/min. She then wants to scale up to a 15 mm diameter column (to isolate large quantities of compounds) of same length. What should be the flow rate to keep u (mobile phase velocity) constant? A chemist purchases a new open tubular GC column that is identical to the old GC column except for having a greater film thickness of stationary phase. Which parameters will be affected: KC, k, tM, tR(component X), �, a.

10. Chromatographic TheoryQuestions on Definitions What �easy� change can be made to increase KC in GC? In HPLC? A GC is operated close to the maximum column temperature and for a desired analyte, k = 10. Is this good? 13. In reversed-phase HPLC, the mobile phase is 90% H2O, 10% ACN and k = 10, is this good? Column A is 100 mm long with H = 0.024 mm. Column B is 250 mm long with H = 0.090 mm. Which column will give more efficient separations (under conditions for determining H)?

11. Chromatographic TheoryQuestions on Definitions Given the two chromatograms to the right: Which column shows a larger N value? Which shows better resolution (1st 2 pks top chromatogram)? Which shows better selectivity (larger a; 1st 2 pks on top)? Should be able to calculate k, N, RS, and a

12. Chromatographic TheoryRate Theory We have covered parameters measuring column efficiency, but not yet what factors influence efficiency In order to improve column efficiency, we must understand what causes band broadening (or dispersion) van Deemter Theory where H = Plate Height u = linear velocity and A, B, and C are �constants�

13. Chromatographic TheoryRate Theory

14. Chromatographic TheoryRate Theory How is u determined? u = L/tM u = F/A* (A* = effective cross-sectional area) �Constant� Terms A term: This is due to eddy diffusion Eddy diffusion results from multiple paths

15. Chromatographic TheoryRate Theory A Term Independent of u Smaller A term for: a) small particles, b) spherical particles, or c) no particles (best) Small particles (trend in HPLC) results in greater pressure drop and lower flow rates

16. Chromatographic TheoryRate Theory B Term � Molecular Diffusion Molecular diffusion is caused by random motions of molecules Larger for smaller molecules Much larger for gases Dispersion increases with time spent in mobile phase Slower flow means more time in mobile phase

17. Chromatographic TheoryRate Theory C term � Mass transfer to and within the stationary phase Analyte molecules in stationary phase are not moving and get left behind The greater u, the more dispersion occurs Less dispersion for smaller particles and thinner films of stationary phase

18. Chromatographic TheoryRate Theory More generalities Often run at u values greater than minimum H (saves on time; reduces s � so better S/N even if resolution decreases) For open tubular GC, A term is minimal, C term minimized by using smaller column diameters and stationary phase films For packed columns, A and C terms are minimized by using small particle sizes

19. Chromatographic TheoryRate Theory Some Questions: What are advantages and disadvantages of running chromatographs at high flow rates? Why is GC usually operated closer to the minimum H value than HPLC? Which term is nearly negligible in open tubular GC? How can H be decreased in HPLC? In open tubular GC?

20. Chromatographic TheoryEffects of Intermolecular Forces Phases in which intermolecular forces are important: solid surfaces, liquids, liquid-like layers, supercritical fluids (weaker) In ideal gases, there are no intermolecular forces (mostly valid in GC) Intermolecular forces affect: Adsorption (partitioning to surface) Phase partitioning Non-Gausian Peak Shapes

21. Chromatographic TheoryIntermolecular Forces � Asymmetric Peaks More than one possible cause (e.g. extra-column dispersion) One common cause is sample or analyte overloading of column Analyte loading shown ? More common with solid stationary phase More common with open tubular GC; less common with HPLC

22. Chromatographic TheoryIntermolecular Forces � Asymmetric Peaks Most common for solid stationary phase and GC because Less stationary phase (vs. liquid) GC behavior somewhat like distillations At low concentrations, column �sites� mostly not occupied by analyte As conc. increase, % sites occupied by analyte increases, causing change in analyte � stationary phase interaction

23. Chromatographic TheoryIntermolecular Forces � Asymmetric Peaks As concentration increase, interactions go from analyte � active site to analyte � analyte If interaction is Langmuir type (weak analyte � analyte vs. strong analyte � active site), tailing occurs (blocking of active sites causes additional analyte to elute early) If interaction is anti-Langmuir type (stronger analyte � analyte interactions), fronting occurs (additional analyte sticks longer)

24. Chromatographic TheoryIntermolecular Forces � Asymmetric Peaks If tailing is caused by saturation of stationary phase, changing amount of analyte injected will change amount of tailing

25. Chromatographic TheoryIntermolecular Forces � Odd Peak Shapes Other Reasons for Odd Peak Shapes Large volume injections Example: 1.0 mL/min. + 0.1 mL injection Injection plug time = 0.1 min = 6 s (so no peaks narrower than 6 s unless on-column trapping is used) Injections at high temp./in strong solvents

26. Chromatographic TheoryIntermolecular Forces � Odd Peak Shapes Analyte exists in multiple forms Example: maltotetraose (glu[1?4]glu[1?4]glu) Has 3 forms (a, �, or aldehyde on right glu) a and � forms migrate at different rates At low T, interconversion is slow relative to tR. At high T, interconversion is faster Extra-column broadening/turbulent flow Multiple types of stationary phase

27. Skipped MaterialExtra-Column Broadening Extra-Column broadening is contributions to the peak width from any part of the chromatograph except in the column (e.g. in the injector, in connecting tubing, or in the detector). Extra-column broadening is usually independent of tR and affects peak widths (see equation) and/or peak shapes Minimization of extra-column broadening is most important for 1) early eluting compounds, 2) when using microbore columns, and 3) when using higher efficiency columns

28. Chromatographic TheoryIntermolecular Forces � Odd Peak Shapes Some Questions: In an HPLC analysis of an analyte present at 0.14 mg injected, a tailing peak is observed. How can it be determined if this is caused by sample overloading? Which technique is more susceptible to column overloading, reversed phase HPLC (C18 stationary phase) or normal phase HPLC? If reducing the concentration of analyte injected has little effect on peak shape, what other tests could be performed to determine the cause of tailing peaks? What does a fronting peak say about analyte � analyte and analyte � stationary phase interactions?

29. Chromatographic TheoryIntermolecular Forces � Types of Interactions Knowledge of analyte � stationary phase and analyte � mobile phase interactions can allow prediction of retention of compounds Interactions by decreasing strength: Ion � ion interactions Strong interaction for ions of opposite charge Important in ion exchange chromatography + in use of ion pairs Stronger interactions for more charged ions Ion � dipole interactions Allows stability of ions in polar solvents Partly responsible for polar analytes to stick on charged surfaces (e.g. carbohydrates on cation exchange columns) Dipole strength depends on atom electronegativity and molecule geometry

30. Chromatographic TheoryIntermolecular Forces � Types of Interactions Interactions by decreasing strength � continued (non-ionic interactions = van der Waal interactions) Hydrogen bonding Occurs between H attached to O or N atoms and electronegative elements (primarily O or N) on other molecules Important in solvent choice in HPLC (methanol vs. acetonitrile) and in retention with polar stationary phases Normally not good in GC (often compounds capable of hydrogen bonding show tailing) Dipole � dipole interactions Important in molecule interactions in polar stationary phases and in analyte � mobile phase interactions (reversed phase HPLC)

31. Chromatographic TheoryIntermolecular Forces � Types of Interactions Interactions by decreasing strength � continued (non-ionic interactions = van der Waal interactions) London dispersion forces Present in all molecules Most important intermolecular interaction for non-polar compounds Based on molecule �polarizability� Larger, more electron-rich molecules are more polarizable Important in analyte interactions with non-polar stationary phases and analyte � mobile phase interactions (normal phase HPLC)