Chem. 231 – 2/11 Lecture

1. Chem. 231 – 2/11 Lecture

2. Announcements I • Return Homework Set 1 • Quiz 1 Today (15 min.) • New Homework Set (Set 2) • Website Update • Homework 1 Solutions • Adding demonstration for HW2

3. Announcements II • Today’s Topics: • Finish Extractions • Quantitative calculations • How to determine if method is working and how to improve methods • Low Performance Chromatography • Lower pressure chromatography • Thin layer chromatography • Quantitative Chromatography • Starting early • Focus today on integrating chromatograms

4. Simple ExtractionsCalculations for Liquid – Liquid Extraction • For liquid-liquid extractions, partitioning between two layers can be calculated: • for unreactive compounds, Kp = [X]raffinate/[X]extractant • k = nraf/nextr = Kp(Vraf/Vext) • Q = fraction transferred to extractant phase • Q = 1/(1+k) • For weak acids/bases, we need to consider that X can exist in two forms in aqueous layer (HA or A- for weak acids and B or BH+ for weak bases) raffinate = original sample layer

5. Simple ExtractionsCalculations for Liquid – Liquid Extraction • Sample Calculation for Butyric acid CH3(CH2)2CO2H with KOW = 5.75 and Ka = 4.82. • Assuming an octanol raffinate phase, lets calculate fraction extracted to an aqueous phase as a function of pH assuming 20 mL aqueous phase and 10 mL octanol • KD = [HA]total aq/[HA]Octanol and Kp = 5.75 • Ka = [H+][A-]/[HA] = 10-4.82 = 1.51 x 10-5 • Since Kp = [HA]Oct/[HA]aq and KD = [HA]Oct/([A-] + [HA])aq, KD/Kp = [HA]aq/([A-] + [HA]) = a = nonionized fraction • a = [HA]/([A-] + [HA]) = [HA]/(Ka[HA]/[H+]+ [HA]) = [H+]/(Ka+ [H+]) = f(pKa, pH) [note: different equation for weak bases] • KD = Kpa • k = Kpa(Voct/Vaq) • Q = 1/(1 + k) • Go to Excel Demonstration

6. Simple ExtractionsCalculations for Liquid – Liquid Extraction • For best separation, want high Q value for one compound and low Q value for other compound • Go to 3-Mepyridine, 2-naphthaleneamine separation

7. Simple ExtractionsCalculations for other methods (SPE) • Not Quantitative (too many variables) • Can Make Predictions about Relative Retention • Example: want to know if 2-chlorophenol will be retained on SPE cartridge • If phenol has smaller KOW and has near 100% retention, 2-chlorophenol should also be retained

8. Simple ExtractionsTests • Numerous losses are possible: • inefficient retention • inefficient sample transfers • inefficient removal of trapped compounds • Best strategy is to test recovery (and for each step if recovery is low) • Small sample sizes and concentrations are challenging

9. Low Performance ChromatographyLower Pressure Chromatography • Purpose of Separation • Typically used for preparative chromatography • Commonly used when simple extractions don’t have sufficient resolution (e.g. removal of desired product from reactants and distinctly different side reaction products) • Equipment • packed columns (usually normal phase) • injection system or manual placement of sample • flow provided by low pressure pump, gravity or gas pressure (Flash) • means for fraction collection more common than means for detection • Advantages/Disadvantages • better separation than simple methods and lower cost than HPLC • limited resolution is main disadvantage

10. Low Performance ChromatographyThin Layer Chromatography • Purpose of Separation • Typically used for fast qualitative analysis • Can be used for initial method optimization or preparative separations • Equipment • plate (typically normal phase) • method to “spot” sample • solvent/container • visual detection or fluorescent detection common spots placed at base Chamber (solvent in base)

11. Low Performance ChromatographyThin Layer Chromatography • Advantages • relatively fast (especially considering start up time) • low cost • simple detection • can run multiple samples in parallel • whole sample seen (whether doesn’t migrate or moves with solvent) • Disadvantages • not very quantitative • limited sample size • limited resolution • not as reproducible

12. Quantitative ChromatographyIntegration Concerns • Goals of Integration: • pick up peaks, but avoid noise • pick up small peaks that overlap with big peaks • give realistic separations of overlapping peaks • avoid bias in peak integrations Both Peaks and Noise Picked Up No Noise Picked Up, but peaks missed

13. Quantitative ChromatographyIntegration Concerns • How to process collected chromatograms • Shown for Agilent 1100 (Chemstation) • Use Correct Window View → Data Analysis • Select your file • File → Load Signal use “offline” version

14. Quantitative ChromatographyIntegration Concerns • Picking up peaks vs. noise • Choose channel of interest and then go to Integration Events to set “Slope Sensitivity” (may need to change peak width, peak area reject or peak height reject) • Slope Sensitivity Set to 1 • Slope Sensitivity Set to 15

15. Quantitative ChromatographyIntegration Concerns • Picking up peaks vs. noise - more • May need to zoom in to “see” small peaks • Slope Sensitivity too low • Improved Settings • Last peak can be picked up, but at the expense of poor integration; better to just use manual integration Area on diagonal indicates manual integration

16. Quantitative ChromatographyIntegration Concerns • Other problems • large solvent (especially GC) or unretained peaks • overlapping peaks Relatively good “auto” integration settings however, some small peaks may have area overestimated “Best” baseline between pink and black lines