Chem. 231

1
Chem. 231 3/4 Lecture
2
Announcements I

• Set 1 Lab Reports
• Both Lab Reports due 3/13
• Only one of the lab reports needs to be detailed,
  the other can be a summary
• Homework Set 3 due Wednesday
• Quiz 3 (last quiz) next Monday
• Set 2 Labs (two need to do one of them)
• SPE/HPLC lab
• SPME/GC lab
• More instructions coming

3
Announcements II

• Set 3 Labs
• Hope to have instructions completed by Monday
• Todays Lecture
• SPE Lab Information
• SPME Lab Information
• Practical Applications of HPLC

4
SPE/HPLC LabInformation

• Goal Determine concentration of unknown phenol
  in aqueous solution
• Background
• Phenols are possible soil contaminants (e.g.
  pentachlorophenol and nitrophenols) and can
  contaminate water supplies
• Phenols are also in some beverages (e.g.
  vanillin, other guaiacols in mostly aqueous
  environment)
• Solid Phase allows trapping of moderately
  non-polar compounds needed for sensitive analysis

OH
guaiacols
R
OCH3
5
SPE/HPLC LabInformation Methodology

• Overview
• Unknowns will need to be trapped on the SPE
  cartridge, then eluted (with smaller volume) to
  concentrate samples
• Analysis by HPLC (will need to develop method and
  determine elution order as in set 1 lab)
• SPE Procedure
• Clean SPE cartridges (first with removal eluent,
  then with sample solvent)
• Flow rate must be slow enough for transfer
• Apply sample to SPE cartridges

6
SPE/HPLC LabInformation Methodology

• SPE Procedure cont.
• Wash sample with sample solvent (removes polar
  contaminants from pores)
• Remove analytes with stronger solvent
• If removing into volumetric flask, need to fill
  to line (more quantitative method)
• Unknown Analysis
• Will want to use SPE method on unknown to
  determine unknown compounds

7
SPE/HPLC LabInformation Methodology

• SPE Efficiency Testing
• To determine if unknown compound losses (in
  passing through SPE cartridge or in removal from
  SPE cartridge) are significant
• Done by keeping and analyzing multiple fractions

8
SPME/GC LabInformation

• Goal
• Determine the concentration of two flower scent
  monoterpene unknowns in a sample
• Background
• Many of the scent compounds in flowers are
  monoterpenes (consist of two isoprene units)
• Traditional analysis would involve extraction and
  liquid injection
• Alternative of SPME is solvent free

myrcene
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SPME/GC LabInformation - Methodology

• GC Methodology
• Requires splitless injection (so cant use Buck
  GCs)
• We will be using an HP6890 and an Agilent 7890
  GC-MS (this will also give qualitative analysis)
• These have software control closer to the Agilent
  HPLCs
• See 2/4 lecture, slide 16 for procedure using SPME

10
SPME/GC LabInformation - Methodology

• GC Methodology cont.
• SPME equipment is fragile
• SHEATH ALWAYS ON WHEN GOING IN OUR OUT OF SEPTA
  OR DAMAGE OCCURS
• NO CONTACT WITH DICHLOROMETHANE
• SPME fibers have limited lifetimes under normal
  use and may need replacing
• Besides what is shown in past slide, SPME must be
  pre- and/or post- heated (can use injector for
  this if conditions are o.k.)

11
SPME/GC LabInformation - Methodology

• GC Methodology cont.
• Quantitative Analysis by SPME/GC works best when
• SPME conditions are kept as consistent as
  possible (same exposure conditions in vial and
  same desorption conditions in GC inlet)
• An internal standard can help control some
  variables so that if the transfer of two
  compounds is consistent, good results will occur

12
Practical Applications of HPLC

• Review of Instrument Components

Chromatograph Components
Chromatographic Column
Detector
Flow Control
Mobile Phase Reservoir
Injector
Waste or fraction collection
Chromatogram
Signal to data recorder
13
Practical Applications of HPLC

• Mobile Phase Selection
• HPLC class will determine main mobile phase
  requirements
• mostly non-polar solvent for normal phase
• polar solvents for reversed phase
• water plus ion exchanger for ion chromatography
• Other Major Factors
• desired retention (adjusting polarity of solvent
  in HPLC to get desired retention or eluent ionic
  strength in IC)
• need for pH adjustment (to reduce or promote ion
  formation)

14
Practical Applications of HPLC

• Mobile Phase Selection Cont.
• Additional Considerations
• Selectivity (different solvents will have
  different solvent analyte interactions best to
  choose solvent that emphasizes analyte
  differences)
• Solvent viscosity (low viscosity means smaller
  back pressure for given flow rate)
• Solvent miscibility
• Sample solubility
• Detector limitations (e.g. wavelength cut-offs
  for UV detection)
• Compatibility with column packing and tubing

15
Practical Applications of HPLC
To column

• Solvent Selection
• Single Pump
• Manual Solvent Selection
• manual valve selection or replacement of mobile
  phase
• not capable of gradient runs
• Low Pressure Mixing
• controlled valve for selection of eluents (4
  possible with our Agilent system)
• fast proportional opening of valves allows good
  control for gradients
• Dual Pump Systems
• each pump runs independently
• better gradient control and retention time
  stability vs. low pressure mixing
• also smaller dwell time (difference in time
  between switching valves and effects at columns)

Mixing chamber
pump
To column
pumps
gradient starts
Pressure
dwell time
Effect seen
time
16
Practical Applications of HPLC

• Pump Requirements
• High pressures
• Small packing material diameters require high
  pressures (up to 300-400 bar) so that one can
  obtain reasonable flow rates
• Newest packing material diameters (sub 5 mm)
  require even higher pressures or shorter column
  lengths) in UPLC which can go to 1000 bar
• Pumps require specific components capable of
  withstanding such high pressures (e.g. use of
  sapphire piston rods)
• Stable Flow rates
• Usually involve feedback control

17
Practical Applications of HPLC

• Pump how they work
• Most pumps use two piston heads 180º out of phase
  to reduce pressure fluctuations
• Solvents go into and out of piston heads through
  one-way check valves
• Exit check valve closes on in stroke and
  entrance check valve closes on out stroke

In Stroke
Check valves
Out Stroke
open
closed
closed
open
pistons
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Practical Applications of HPLC

• Pump Failures
• Common for check valves to fail
• This results in a drop in pressure during one of
  the cycles resulting in an alternating pressure

Bad check valve leaking
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Practical Applications of HPLC
6 port valve

• Injection Valves
• Fixed loop injector is by far the most common
  type
• small loop volume with manual injection
• with autosamplers, it is common to use partial
  filling (e.g. fill 10 mL of a 250 mL loop)
• For large sample injections, the loop can be
  replaced with a trap
• Specialized injectors
• used for injecting very small samples in
  microbore columns
• SPME injectors

From pump
To column
Waste
sample in
LOAD POSITION
INJECT POSITION
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Practical Applications of HPLC

• Sample Injection
• Injection Volumes
• With sample solvent eluent, volume injected
  shouldnt contribute significantly to extra
  column broadening (rule of thumb injection volume
  lt 0.25(early eluting peak volume))
• Sample Solvent
• Ideal solvent is solvent weaker than eluent (or
  initial eluent in a gradient)
• Then can use larger injection volume since
  analytes will trap on column
• Stronger solvents can result in distorted peak
  shapes or require even smaller injection volumes

width 0.084 min. (or 5.1 s) _at_ flow 1.2
mL/min or peak volume 100 mL, so use vol lt 25 mL