Partitioning Between Phases

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Partitioning Between Phases
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Extraction Equation (how to wash glassware)
K S2 / S1 Fraction remaining in phase 1
(q) q (V1 / V1KV2)n n of extractions V1
volume of solvent 1 V2 volume of solvent 2
(extraction solvent) Extraction is a power
function
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Extraction Example
K3 (75 extraction) V1 50 mL V2 45 mL (1
large extraction) q (V1 / V1KV2)n q (50 /
50 3(45))1 q 0.27 (27 remains) K3 (75
extraction) V1 50 mL extract 3 times w/ V2 15
mL q (50 / 50 3(15))3 q 0.15 (15
remains)
K9 (90 extraction) V1 50 mL V2 45 mL (1
large extraction) q (V1 / V1KV2)n q (50 /
50 9(45))1 q 0.109 (10.9 remains) K9
(90 extraction) V1 50 mL extract 3 times w/ V2
15 mL q (50 / 50 9(15))3 q 0.019 (1.9
remains)
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Separation using pH
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Column Chromatography
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Really Big Column Chromatography
Often used for pharmaceutical separations
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Types of Chromatography
Traditional packed column GC or HPLC
Open tube GC
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Gel Permeation
Similar to HPLC
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Immobilized Antibody Separation
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Basic Chromatogram Parameters
tm time for unretained compound to travel
through column t r retention time tr
adjusted retention time (t r t m )
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Basic Chromatographic Parameters

• tr 2 / tr 1 relative retention (a gt 1)
• relatively independent of flow rate
• (can help relocate peaks if flow rate changes)
• k tr / t m capacity factor
• (retention factor capacity ratio partition
  ratio)
• indicator of column performance
• Both a and k can be calculated from basic
  chromatogram
• measurements (t m and t r )

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Properties of a Gaussian Shaped Curve
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Resolution of Gaussian Curves
Baseline Resolution
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Band Broadening in Chromatography
(Idealized)
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van Deemter Equation
A Multiple paths (packed columns only) B
Longitudinal Diffusion C Mass Transfer ( phase
equilibration)
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Multiple Paths
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Longitudinal Diffusion
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Mass Transfer
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Flow and Temperature Variation
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Peak Asymmetry in Gas Chromatography
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Typical GC Schematic
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Cross-sectional View of GC Column
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Typical Fused Silica Column GC
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Common Stationary Phase Configurations
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Features of Common Stationary Phase Configurations

• WCOT has best resolution but smallest sample
  capacity
• SCOT has higher sample capacity but poorer
  resolution
• PLOT is an alternative to packed columns for
  certain types of analysis (e.g., atmospheric
  gasses)

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PLOT GC Column
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Headspace Analysis of Beer
B.P.
21 65 78 77 82 116 130 130 142 167 220
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Column Diameter and Resolution
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Column Length and Resolution
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Column Polarity and Retention Time
polar
non-polar
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Isothermal Programming
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Temperature Programming and Resolution
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Carrier Gas and Plate Height
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Injection Port
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Injection Modes
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Typical Injection Applications

• Split - default, conc. samples (gt0.1), sample
  size of 1 uL or less, 0.2-2 of sample on column
• Splitless dilute samples (lt0.01), 80 of
  sample on column
• On Column easily decomposed samples, 100 of
  sample on column

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Thermal Conductivity Detector
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Flame Ionization Detector
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Electron Capture Detector
Column
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SPME (solid phase micro extraction)
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GC Detectors

• 4 Detectors (TCD, FID, ECD, MS)
• Relative Sensitivity (TCDlt FIDlt ECDMS)
• Analyte Selectivity
• TCD MS- Universal
• FID-Combustible HCs
• ECD-Halogenated HCs
• How They Work (one sentence description)

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Method Development

• Goal of Analysis
• Qualitative, Quantitative, Precision, etc.
• Sample Preparation
• Extraction, Derivatization,
• Detector
• Column
• Stationary Phase (thickness polarity)
• Column Diameter, Length
• Injection Method

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Microdialysis Probe (concentric tube design)
Diffusion drives transport of analyte across
membrane
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Microdialysis Sample Chromatogram
(metabolite)
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Typical HPLC System (ca. 1990)
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HPLC Column Cross-section
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Van Deemter Plot (smaller particles have smaller
A C terms)
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Particle Size and Column Performance
(300 psi) (1300 psi) (10,000 psi) (34,000 psi)
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Protection of Siloxane Bonds With Bulky Alkyl
Groups
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Monolithic Silica Rods Increased Flow Rates with
Excellent Resolution
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Mobile Phase Considerations

• Eluent Strength
• Strong mobile phase compositions make solute
  elute faster.
• Normal vs. Reversed Phase Systems
• Normal Phase
• polar stationary phase non-polar mobile phase
• polar mobile phases have higher eluent strength
• Reversed Phase
• non-polar stationary phase polar mobile phase
• non-polar mobile phases have higher eluent
  strength

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Injection Considerations
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HPLC Detectors

• UV/VIS (diode array)
• LOD 0.1-1 ng Gradient
• Evaporative Light Scattering
• LOD 0.1-1 ng Gradient Large Dynamic Range
• Electrochemical
• LOD 0.01-1 ng Isocratic
• Conductivity
• Refractive Index
• MS

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Optical Flow Cell
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UV/VIS
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UV/Vis Chromatogram
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UV Spectrum (for compound ID)
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Refractive Index Detection
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Evaporative Light Scattering Detection
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Electrochemical Detector Signal Output
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Organic Phase Equivalency Nomograph