Chem. 231 – 2/11 Lecture

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

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

Simple Extractions Calculations 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

Simple Extractions Calculations 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

Simple Extractions Calculations 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

Simple Extractions Calculations 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

Simple Extractions Tests 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

Low Performance Chromatography Lower 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

Low Performance Chromatography Thin 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)

Low Performance Chromatography Thin 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

Quantitative Chromatography Integration 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

Quantitative Chromatography Integration 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

Quantitative Chromatography Integration 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

Quantitative Chromatography Integration 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

Quantitative Chromatography Integration 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