Chem. 230 – 9/11 Lecture

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Chem. 230 9/11 Lecture
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Announcements I

• HW Set 1 due
• Additional Resources (show students site)
• Application Paper (pass out)

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Announcements II

• First Quiz
• First 30 minutes next Wednesday
• Will cover materials on Simple Extractions (in
  text and covered in lecture)
• Questions will be similar to those given as
  examples in lecture and in homework
• You should be familiar with equations needed, but
  constants will be provided
• Example Quiz Solutions posted

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Advanced Extraction TechniquesSPE Demonstration

• Procedure
• Clean cartridge with removing solvent, then
  sample solvent
• Apply sample strongly retained compounds will
  remain on stationary phase, weakly
  retained/unretained compounds pass through
• Rinse cartridge with sample solvent
• Apply eluting solvent to remove strongly retained
  compounds
• It is possible to increase solvent strength to
  remove compounds in several fractions

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Advanced Extraction TechniquesSPE Demonstration

• Example
• Mixture of methylene blue (organic cation) and I2
  in 90 water 10 methanol
• Could in theory separate using either C18 or
  cation-exchange solid phase
• C18 used in example (and methylene blue is a
  sticky molecule so sticks to many surfaces,
  though should pass through C18 column)

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Advanced Extraction TechniquesSolid Phase
Extraction

• Solid Phase
• Very Similar to HPLC packing particles
• Smaller column
• Larger particles (allowing low pressure elution)
• Some Types
• Silica Based (octadecyl or C18, phenyl,
  aminopropyl, etc.)
• Ion Exchange (normally charged group on polymeric
  solid)
• Others (e.g. graphitic carbon)

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Advanced Extraction TechniquesSolid Phase
Extraction

• Partitioning Strategies
• As before, both efficient phase transfer and good
  selectivity are desired
• To trap less polar compounds in polar solvents,
  hydrophobic stationary phases (also known as
  reversed-phase) are desired. (Example
  pesticides in water)
• To trap more polar compounds in less polar
  solvents, hydrophillic stationary phases (also
  known as normal phase) are desired. (Example
  sugars in acetonitrile, steroids in hexane)
• Trapping of polar compounds in polar solvents (or
  non-polar compounds in non-polar solvents) is
  difficult. Breakthrough often occurs.
• Larger analyte solvent polarity difference
  allows better trapping but is limited by analyte
  solubility.
• To trap ionic compounds (usually in water),
  stationary phases with charged groups opposite in
  charge to analyte ions are used.
• It may be possible to produce several fractions
  by increasing solvent strength or changing pH.

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Advanced Extraction TechniquesSolid Phase
Extraction

• Reversed-Phase Groups
• C18 (most commonly used) best for trapping
  compounds with alkyl groups
• Phenyl good for enhanced retention of aromatic
  compounds
• Stronger solvent is less polar
• Normal-Phase Groups
• Cyano (-CN)
• Amino (-NH2)
• Hydroxy (diol or SiOH)
• Stronger solvent is more polar

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Advanced Extraction TechniquesSolid Phase
Extraction

• Ion Exchange Stationary Phases
• Sulfonate groups common for cation exchange
• Ammonium groups NR3 common for anion exchange
• Trapping occurs in low ionic strength solvents
  release occurs in high ionic strength
• Weak acids/bases need to be trapped in ion form
  but also can be released by pH adjustment

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Advanced Extraction TechniquesSolid Phase
Extraction

• Breakthrough and Release
• When SPE cartridges are used to trap and release
  compounds, losses can occur from incomplete
  trapping (breakthrough) or release of compounds.
• Breakthrough can occur because the partitioning
  equilibrium is not strong enough or due to
  capacity of cartridge is exceeded (sample
  overload)
• Breakthrough can be determined by measuring the
  concentration of solute passed through cartridge
  (either in whole sample or in intervals)
• Release can be determined by secondary rinses of
  SPE cartridge

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Advanced Extraction TechniquesSPE - Questions

• It is desired to trap benzoic acid in an aqueous
  phase on an SPE cartridge and release it to an
  aqueous phase. Is this possible?
• Fish triglyerides are extracted in hexane.
  Describe a way to separate the triglyerides from
  more polar compounds (free fatty acids and
  steroids with OH groups).
• Trapping of trace amounts of phenols in water is
  attempted. To concentrate phenols, large volumes
  of water are used followed by small volumes of
  acetonitrile. What is a concern?
• Some of the phenols in water contain carboxylic
  acids. Suggest a way to trap both carboxylic
  acid-containing phenols and regular phenols while
  releasing them into two fractions for separate
  analysis. The pKa for carboxylic acids are about
  4 and about 10 for phenols.

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Advanced Extraction TechniquesSolid Phase Micro
Extraction (SPME)

• First described in Arthur, C. Pawlisyzn, J.
  Solid phase microextraction with thermal
  desorption using fused silica optical fibers,
  Analytical Chemistry (1990) 62, 2145-2148.
• Can be used for subsequent analysis by GC or
  HPLC, but most common with GC
• Typically, non-exhaustive type sampling (meaning
  only a portion of analyte in sample is trapped).
  Quantitation is based on keeping exposure to
  samples the same (easier with autosampler).
• While quantitation is often difficult,
  sensitivity is enhanced relative to SPE because
  whole trapped sample is injected.

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Advanced Extraction TechniquesSPME Procedure

• The needle pierces the septum to a sample (sample
  can be gas, liquid, or headspace)
• The sheath is removed allowing trapping of
  analytes on fiber
• Stirring helps the transfer
• The sheath goes back and the needle is withdrawn
• The needle pierces the septum to a GC, the sheath
  is withdrawn and the analyte is desorbed by the
  heated GC injector

Fiber
GC Inlet
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Advanced Extraction TechniquesSolid Phase Micro
Extraction (SPME)

• Sample Types (GC analysis)
• Liquid Samples (best when analyte concentrations
  are low)
• Headspace Sampling (avoids fiber fouling)
• Gas Samples
• In Fiber Derivatization (typically applied to
  polar organic compounds which often decompose on
  GC columns)
• Areas of Applications (reviews on these areas)
• Environmental Analysis (VOCs in air, pesticides
  in water, soil/sediment analysis, toxic metals)
• Biological Samples
• Food Analysis
• Natural Products

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Advanced Extraction TechniquesSPME
Advantages/Disadvantages

• Advantages
• Listed as Solvent-less technique (at least
  great reduction in solvent injected into GC)
• Less interference from solvent peak
• Reduced injection of non-volatiles
• Less sample handling ( ability to automate)
• Can chose fibers for good selectivity
• Disadvantages
• More difficult for quantitative results
• Limited lifetime of fibers
• Memory effects (slow desorption from fibers)

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Advanced Extraction TechniquesOther Methods

• Emphasis toward microscale methods
• Liquid-Liquid Microextraction (drop scale liquid
  liquid extraction)
• Use of semi-permeable membranes (discussed in
  text)
• Stir-Bar Sorptive Extraction

1. Stir bar traps analytes
2. Stir bar transferred to GC inlet
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Advanced Extraction TechniquesSome Questions

• A test for decomposition of a milk sample is made
  by measuring small aldehydes (e.g. butyraldehyde)
  by SPME through direct immersion in milk. A
  non-polar fiber is used and analysis is performed
  by GC with a non-polar stationary phase. Which
  of the following are advantages of using the SPME
  method
• removal of interferents (other parts to milk)
• 2 dimensions of separation (on SPME fiber and on
  GC column)
• increase of concentrations by trapping on fiber
• avoiding need for more labor intensive methods
  (e.g. liquid liquid extraction)
• If a fiber sits in a solution long enough, the
  peak area will reach a constant (be independent
  of time). Why is this? Is this exhaustive
  extraction?
• In SPME for HPLC, analytes are desorbed from the
  fiber into solvent that is injected into the HPLC
  column. Should the solvent be stronger or
  weaker than the sample solvent?
• In comparing direct headspace injections with
  SPME headspace injections, later eluting peaks
  (by GC) are larger in SPME. Explain why.

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Chromatographic TheorySimple Separations vs.
Chromatography

• Simple separations generally involve one to
  several process steps that lead to two to several
  fractions.
• Simple separations are limited to coarse
  fractionation of samples.
• Chromatographic separations are generally capable
  of isolating more than 5 compounds.
• Once the number of simple separation steps goes
  over a few (maybe 5 maximum), it becomes a labor
  inefficient way of performing a separation.

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Chromatographic TheorySimple Separations vs.
Chromatography

• Example of separation of two compounds by LLE.
• Compound X has Kp 0.25 and Compound Y has Kp
  4. Extraction of X and Y using n washes with
  extractant phase (equal volumes and saving all
  extractant phase)
• To get efficient transfer of X means transferring
  a fair amount of Y also (poor selectivity)

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Chromatographic TheorySimple Separations vs.
Chromatography

• Continuation of example
• Better selectivity at same efficiency can be made
  by adjusting extract volume and increasing number
  of extractions
• In past example, using Vraf/Vext 2.5/1 with 5
  extractions results in 99 efficient transfer of
  X, while only transferring 38 Y
• Table shows dependence of Y transferred on Kp
  values (assuming Kp(Y) 1/Kp(X)) and 99
  transfer of Y with 3 extractions (volumes
  adjusted to get 99 transfer of X)

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Chromatographic TheorySimple Separations vs.
Chromatography

• Chromatography example
• Even column of poor efficiency can handle much
  more similar compounds
• Example KY/KX 1.25 ( a value)
• If we assume kX 4, and resolution 1.5
  (minimum for baseline), a plate number of 1000
  would be needed (not very high)

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Chromatographic TheorySimple Separations vs.
Chromatography

• Conclusions to example
• Unless order of magnitude differences in Kp
  values, simple separations have limited use (e.g.
  reduction of interfering substance).
• Simple separations are better for coarse
  separations
• Chromatographic separations can handle similar K
  values much better.

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Chromatographic Theory Chromatography vs. Other
Advanced Separation Techniques

• Chromatography is based on analyte partitioning
  between two phases
• Other methods use different mechanism for
  separation of analytes (e.g. electrolytic
  mobility in capillary zone electrophoresis)
• Some areas of overlap (e.g. Capillary
  electrochromatography and size exclusion
  chromatography)

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Chromatographic Theory Phase Definitions

• Mobile Phase (M subscript in later parameters)
• Fluid phase (gas, liquid or supercritical fluid)
  that moves through stationary phase
• Mobile phase defines the major classes of
  chromatography (GC, LC and SFC)
• Stationary Phase (S subscript)
• A non-moving phase (except in MEKC) to which
  compounds partition via absorption or adsorption
• Phase can be liquid (not very stable),
  liquid-like (most common), or solid (common for
  some applications)
• In past was second part of class name (for
  example GLC for gas-liquid chromatography)

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Chromatographic Theory More on Stationary Phases

• Stationary phases come in several arrangements
  in columns or on plates (used in thin layer
  chromatography)
• In columns, open tubular (coated walls), packed
  columns and monoliths are possible means of
  attaching stationary phase
• Packed columns contain packing material with the
  stationary phase either being the surface or
  being a coating on the surface
• Porous packing material is common
• Most common stationary phase is a liquid-like
  material chemically bonded to packing material or
  to wall (in open tubular chromatography).

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Chromatographic Theory More on Stationary Phases
Open Tubular (end on, cross section view)
Packed column (side view) (e.g. Silica in normal
phase HPLC)
Column Wall
Packing Material Stationary phase is outer
surface (although influenced by adsorbed solvents)
Mobile phase
Stationary phase (wall coating)
Bonded phase (liquid-like)
Expanded View
Stationary Phase Chemically bonded to packing
material
Packing Material
Note true representation should include
micropores in sphere
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Chromatographic Theory Definition Section

• Chromatograph instrument
• Chromatogram detection vs. time (vol.) plot

Chromatograph Components
Sample In
Chromatographic Column
Detector
Flow/Pressure Control
Mobile Phase Reservoir
Waste or fraction collection
Injector
Chromatogram
Signal to data recorder
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Chromatographic Theory Definition Section Flow
Volume Relation

• Relationship between volume (used with gravity
  columns) and time (most common with more modern
  instruments)
• V tF
• V volume passing through column part in time t
  at flow rate F
• Also, VR tRF where R refers to retention
  time/volume (time it takes component to go
  through column or volume of solvent needed to
  elute compound)

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Chromatographic Theory Definition Section More
on Volume

• Hold-up volume VM volume occupied by mobile
  phase in column
• Stationary phase volume VS
• Calculation of VM
• VM Vcolumn Vpacking material VS
• VM tMF, where tM time needed for unretained
  compounds to elute from column