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 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

5. 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] = = 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 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

7. 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

8. 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

9. 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

10. 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)

11. 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

12. 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

13. 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

14. 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

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

16. 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