Chem. 230 10

1. Chem. 230 � 10/05 Lecture Updated to include info on Q2 and GC

2. Announcements Homework � Long Problems due today Additional Problems and Solutions for Quiz 2 will be posted What we are covering today: Intermolecular Forces (Section 4.1) Method Optimization (Chapter 5) GC: not on Quiz

3. Announcements Quiz 2 � Next Tues. Topics When chromatography is needed Phases (plus how stationary is connected to columns) Instrument Components Definitions (k, Kc, V, t, F, u, a, RS, N, H, L, wb, s) What affects k Efficiency What causes odd shaped peaks van Deemter Equation and broadening Extra-column broadening Intermolecular forces Optimization

4. Announcements Quiz 2 - cont. Format � similar to 1st Quiz Equations Given: (2.5, k = (tR � tM)/tM, 2.12, 2.19, 2.23, 2.27, 3.4, and RS equation below)

5. Fused-Core Particles � Effects on van Deemter Curve Which term of the van Deemter Curve is decreased by going from fully porous to partially porous (superficially porous) particles?

6. 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)

7. Chromatographic TheoryIntermolecular Forces � Types of Interactions Modeling interactions Somewhat of a one-dimensional model can be made by assigning a single value related to polarity for analytes, stationary phases, and mobile phases (See section 4.3) These models neglect some interactions however (e.g. effects of whether an analyte can hydrogen bond with a solvent)

8. Chromatographic TheoryIntermolecular Forces � Some Questions For which molecules (to the right) will London Dispersion forces be larger? How does going from DB-1 (100% methyl stationary phase) to DB-17 (50% methyl � 50% phenyl) in GC affect elution of fatty acid methyl esters? (e.g. C16 vs. C18 vs. C18:1)

9. Chromatographic TheoryIntermolecular Forces � Some Questions Silica has many SiOH groups on the surface (pKa ~2). What interactions will occur with the analyte phenol, C6H5OH, if the eluent is a mixture of hexane and 2-propanol? Sugars are often separated on amino columns. A sugar that has a carboxylic acid group in place of an OH group will have extremely large retention times (at least at neutral pH values). What does this say about the state of the amino groups?

10. Chromatographic TheoryOptimization - Overview How does �method development� work? Goal of method development is to select and improve a chromatographic method to meet the purposes of the application Specific samples and analytes will dictate many of the requirements (e.g. how many analytes are being analyzed for and in what concentration?, what other compounds will be present?) Coarse method selection (e.g. GC vs HPLC and selection of column type and detectors) is often based on past work or can be based on initial assessment showing problems (e.g. 20 compounds all with k between 0.2 and 2.0 with no easy way to increase k) Optimization then involves making equipment work as well as possible (or limiting equipment changes)

11. Chromatographic TheoryOptimization � Some trade offs Flow rate at minimum H vs. higher flow rates (covered with van Deemter Equation) � low flow rate not always desired because of time required and sometimes smaller S/N Maximum flow rate often based on column/instrument damage � this can set flow rate Trade-offs in reducing H In packed columns, going to small particle sizes results in greater back-pressure (harder to keep high flow) In GC, small column and film diameters means less capacity and can require longer analysis times Trade-offs in lengthening column (N = L/H) Longer times due to more column (often not proportional since backpressure at same flow rate will be higher)

12. Chromatographic TheoryOptimization � Improved Resolution Through Increased Column Length Example: Compounds X and Y are separated on a 100 mm column. tM = 2 min, tX = 8 min, tY = 9 min, wX = 1 min, wY = 1.13 min, so RS = 0.94. Also, N = 1024 and H = 100 mm/1024 = 0.097 mm Let�s increase L to 200 mm. Now, all times are doubled: tM = 4 min, tX = 16 min, tY = 18 min. So DtR (or d) now = 2 min. Before considering widths, we must realize that N = L/H (where H is a constant for given packing material). N200 mm = 2*N100 mm. Now, N = 16(tR/w)2 so w = (16tR2/N)0.5 w200 mm/w100 mm = (tR200 mm/tR100 mm)*(N100 mm/N200 mm)0.5 w200 mm/w100 mm = (2)*(0.5)0.5 = 21-0.5 = (2)0.5 w200 mm = 1.41w100 mm RS = 2/1.5 = 1.33 Or RS 200/RS 100 = d/wave = (d200/d100)*(w100/w200)= (L200/L100)*(L100/L200)0.5 So RS is proportional to (L)0.5

13. Chromatographic TheoryOptimization � Resolution Equation Increasing column length is usually the least desired way to improve resolution (because required time increases and signal to noise ratio decreases) Alternatively, k values can be increased (use lower T in GC or weaker solvents in HPLC); or a values can be increased (use different solvents in HPLC or column with better selectivity) but effect on RS is more complicated

14. Chromatographic TheoryOptimization � Resolution Equation How to improve resolution Increase N (increase column length, use more efficient column) Increase a (use more selective column or mobile phase) Increase k values (increase retention) Which way works best? Increase in k is easiest (but best if k is initially small) Increase in a is best, but often hardest Often, changes in k lead to small, but unpredictable, changes in a also

15. Chromatographic TheoryGraphical Representation

16. Chromatographic TheoryOptimization � Back to 1st Example Compounds X and Y are separated on a 100 mm column. tM = 2 min, tX = 8 min, tY = 9 min, wX = 1 min, wY = 1.13 min, so RS = 0.94. Also, N = 1024, kY = 4.5 and a = 1.13. What change is needed in N, k, and a to get RS = 1.5? N (RS)2/(RS)1 = (N2/N1)0.5, N2 = 2607 (e.g. a 250 mm column) k In this case, it is not possible to increase k enough to get RS = 1.5 (assuming a and N are not changing significantly with change in T or solvent strength). RS = K[kY/(1 + kY)] (where K represents the N and a parts of the equation). For RS = 0.94 and kY = 4.5, K = 1.15. The maximum ratio of kY to 1 + kY is 1, so the maximum RS = 1.15 a (RS)2/(RS)1 = (a1/a2)(a2 - 1)/(a1 - 1) 1.60 = 1.13/0.13[(a2 � 1)/a2]a2 = 1.23

17. Chromatographic TheoryOptimization � 2nd Example tM = 1 min, tX = 2 min, wX = 0.1 min, tY = 2.1 min, wY = 0.105 so: RS = 0.98, a = 1.1 With small initial k values, increasing k helps more After k > 5, only minor increases in resolution possible

18. Chromatographic TheoryOptimization � Some Questions Indicate how the chromatograms could be improved?

19. Chromatographic TheoryOptimization � Some Questions Why is it usually more difficult to improve the separation factor (a) when there are a larger number of analytes/contaminants? Both using a longer column or using a column of smaller H will improve resolutions? Which method will lead to a better chromatogram? Why? RS = 0.93 and kB = 2.7. What is the maximum RS value just by changing kB?

20. Chromatographic TheoryOptimization � Some Questions Why is it usually more difficult to improve the separation factor (a) when there are a larger number of analytes/contaminants? Both using a longer column or using a column of smaller H will improve resolutions? Which method will lead to a better chromatogram? Why? RS = 0.93 and kB = 2.7. What is the maximum RS value just by changing kB?

21. Gas ChromatographyOverview of Topics Comparison of mobile phases (Chapter 6) History, analyte � stationary phase interaction (Section 7.1) Instrumentation (Section 7.2, 7.3) Stationary phase (Section 7.4) Temperature issues (Section 7.6

22. Gas ChromatographyComparison of Mobile Phases Two key differences between GC and LC: No analyte � mobile phase interaction in GC Temperature is routinely changed (and always controlled) in GC Effects of gases (vs. liquids) Much higher diffusivity (larger B term of van Deemter equation but very small CM term) Greater viscosity of liquids (cause of high backpressure) Much lower density (capacity of column is a big issue with liquid samples) Gases are compressible

23. Gas ChromatographyCompressibility of Gases The volume flow rate will not be a constant along a column because as the pressure drops, the volume increases There are various ways to calculate average flow rates which we will not go into

24. Gas ChromatographyAdvantages vs. HPLC Main practical advantage comes from high N values (although H is usually larger) achieved with open tubular columns. Another advantage comes from being able to use quite long columns (60 m vs. 250 mm for HPLC) because backpressure is not a major issue Other advantages have to do with instrument cost and better detectors Main disadvantage is for analysis of non-volatile compounds

25. Gas ChromatographyDevelopment and Theory Initially, GC was developed to improve upon fractional distillations In fractional distillations, the liquid at each plate is a mixture of analytes In gas chromatography analytes are present, but stationary phase interactions are dominant and analyte X and Y generally don�t interact

26. Gas ChromatographyDevelopment and Theory Types of Columns Packed Columns Older type of column Both solid and liquid stationary phase Best column for preparatory GC and for use with thermal conductivity detectors Sometimes used for very specific applications Open Tubular Columns More modern columns Much better analytical performance (large N values) Most common in wall coated format (WCOT) Variety of diameters (0.25 to 0.53 mm most common) allow high resolution vs. easier injection Stationary phases are mainly bonded of varying amounts of polarity

27. Gas ChromatographyDevelopment and Theory Retention of Compounds KC value depends on: Volatility Polarity of analyte vs. polarity of stationary phase Measure of volatility Best measure is vapor pressure at temperature Boiling point temperature is used more frequently Depends on molecule�s size and polarity Polarity in separations Polar stationary phases increase retention of polar compounds vs. non-polar compounds

28. Gas ChromatographyDevelopment and Theory Application of GC Gas samples Somewhat different equipment (injector and oven range) is needed vs. liquid samples Liquid samples Compounds must be volatile (plus small amounts of non-volatile interferences) Compounds must be stable at GC temperatures Separations are better for less polar compounds Less volatile compound elution may be limited by maximum temperature stationary phase can handle.

29. Gas ChromatographyDevelopment and Theory Application of GC Extension to non-volatile, thermally labile compounds Derivatization: example � fatty acids are highly polar and do not produce narrow peak with non-polar columns, but they can be reacted to produce fatty acid methyl ester (same reaction used to produce biodiesel) that are volatile and stable Pyrolysis GC: non-volatile samples are heated and breakdown products are measured by GC. This give information about compound�s �building blocks�

30. Gas ChromatographyStationary Phase Selection of stationary phase affects k and a values Main concerns of stationary phase are: polarity, functional groups, maximum operating temperature, and column bleed (loss of stationary phase) More polar columns suffer from lower maximum temperatures and greater column bleed

31. Gas Chromatography Will pick up next time with instrumentation for GC