Chem. 231

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Chem. 231 2/11 Lecture
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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

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Announcements II

• Todays 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

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Simple ExtractionsCalculations for Liquid
Liquid Extraction

• For liquid-liquid extractions, partitioning
  between two layers can be calculated
• for unreactive compounds, Kp Xraffinate/Xext
  ractant
• k nraf/nextr Kp(Vraf/Vext)
• Q fraction transferred to extractant phase
• Q 1/(1k)
• 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
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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 HAtotal aq/HAOctanol and Kp 5.75
• Ka HA-/HA 10-4.82 1.51 x 10-5
• Since Kp HAOct/HAaq and KD HAOct/(A-
  HA)aq,
• KD/Kp HAaq/(A- HA) a nonionized
  fraction
• a HA/(A- HA) HA/(KaHA/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

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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

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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

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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

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Low Performance ChromatographyLower Pressure
Chromatography

• Purpose of Separation
• Typically used for preparative chromatography
• Commonly used when simple extractions dont 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

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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)
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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 doesnt migrate or
  moves with solvent)
• Disadvantages
• not very quantitative
• limited sample size
• limited resolution
• not as reproducible

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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
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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
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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

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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
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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