Chem. 231

1
Chem. 231 3/11 Lecture
2
Announcements I

• Set 1 Lab Reports due 3/13
• Homework Set 3 due today
• Quiz 3 (last quiz) today
• Set 3 Labs
• Pass out handout
• Will Start after Spring Break
• Remaining Discussion Schedule (tentative)
• Today (finish practical aspects of GC/HPLC)
• Mar. 18 (remaining topics and discussion of set 3
  labs)

3
Announcements II

• Remaining Discussion Schedule (cont.)
• April 1 (review for final exam)
• April 8 (Final Exam)
• April 29 (Set 3 Lab Presentations)
• Todays Discussion Todays Lecture
• Practical Aspects of GC (parts not covered
  previously)
• Practical Aspects of HPLC

4
Practical Aspects of GCMore on Matching
Sample/Analytes to Equipment

• Injectors other than split/splitless
• gas samples
• can do direct injections with split/splitless,
  but lack sensitivity
• specialized methods (injection valves, trap
  systems)
• water samples
• purge and trap most common
• SPME methods
• wide variety of applications with standard GC
  injector
• limited to moderately volatile compounds in head
  space methods or moderate to low volatility in
  immersion methods

5
Practical Aspects of GCMore on Matching
Sample/Analytes to Equipment

• Columns
• format
• capillary columns generally preferred over packed
  columns (much higher resolution possible)
• exceptions prep separations, specialized
  stationary phases, thermal conductivity detectors
  (hydrocarbon free analytes)
• capillary column dimensions
• smaller diameter gives better resolution but has
  less capacity and is somewhat slower (for a given
  length)
• MS requires small to medium diameters, while FID
  responds to analyte flux (somewhat greater signal
  for larger diameters)
• length governed by needed resolution vs. time for
  analysis
• thinner films give better resolution and lower
  capacity
• thinner films more useful for higher boiling
  point compounds
• 2D formats can give benefits of high resolution
  without the cost of excessive time

6
Practical Aspects of GCMore on Matching
Sample/Analytes to Equipment

• Columns
• stationary phase
• typically want column polarity to match analyte
  polarity
• not required, but advantageous to trap good
  compounds and pass interferences
• exception can occur if at upper end of columns
  temperature limit
• less volatile compounds require more robust
  columns (high temperature limit is usually due to
  column bleed)

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Practical Aspects of GCMore on Matching
Sample/Analytes to Equipment

• Detectors
• Universal (FID, TCD, MS)
• TCD for non-FID detectable gases (e.g. N2O)
• FID allows surrogate standard calibrations is
  very common
• MS is useful as both universal and selective
  detector
• MS selected if qualitative analysis also needed
  on unknown compounds

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Practical Aspects of GCMore on Matching
Sample/Analytes to Equipment

• Detectors cont.
• Selective
• Generally selected for more complex samples
  (although all analytes must be detectable) where
  selectivity is needed
• Most are sensitive (good for trace analysis)
• ECD for electronegative elements (halogens plus
  some oxygen and nitrogen-containing compounds)
• Element specific detectors (e.g. NPD, FPD) for
  specific elements (N, P, and S)

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Practical Aspects of GCQuestions

1. Pyrethroids are natural pesticides (no N or P
   atoms) with relatively high boiling points.
   There are about 15 types. If one is interested
   in measuring pyrethroids in environmental samples
   (moderate complexity and concentrations are low),
   what type of column, injection, and detector
   should be used?
2. A student is using a GC-FID with a 0.25 mm x 30 m
   DB-5 column and split/splitless injector to
   identify relatively volatile reaction products (a
   mixture of esters) in a hexane solvent. With a
   split ratio of 110, the peak shape is not very
   good leading to peak overlap. Sensitivity is not
   an issue. How could the separation be improved?

10
Practical Applications of HPLC

• Column Selection Stationary Phase
• Normal Phase vs. Reversed Phase
• Solute must be soluble in mobile phase (this may
  rule out the most polar compounds from silica
  based normal phase and the most non-polar
  compounds from reversed phase)
• In complex samples, more retention is desired to
  remove analytes from a mass of less retained
  compounds (this makes reversed phase undesirable
  for highly polar compounds and normal phase
  undesirable for non-polar compounds)
• Silica vs. Silica Bonded Phases
• Silica is undesired because it requires long
  stabilization times
• Bonded silica can bleed, particularly outside
  stable pH range (2 to 8)

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Practical Applications of HPLC

• Column Selection Stationary Phase cont.
• Silica Particles vs. Other Backbones
• Silica production (e.g. cost of controlled
  particle sizes) and column performance
  (efficiency, capacity, pressure range, etc.) is
  generally superior to other columns
• Silica often has more limitations in terms of
  useful conditions (temperature and pH) vs. other
  packing material
• Column Selection dimensions
• packing material size
• small for better resolution
• but also results in greater backpressure
• small particles often selected for fast
  separations with short columns
• must minimize other sources of extra column
  broadening

12
Practical Applications of HPLC

• Column Selection dimensions cont.
• Column length
• longer (e.g. 250 for 5 mm particles) for better
  resolution
• but also slower analyses
• small particles often selected for fast
  separations with short columns
• Column diameter
• prep size (gt4.6 mm diameters)
• conventional size (4.6 mm diameter)
• narrow or micro bore (lt 4.6 mm diameter)
• primarily used to increase mass sensitivity (most
  detectors show increase mass sensitivity but
  decreased concentration sensitivity at lower flow
  rates)
• particularly good for some detectors (MS and ECD)
• must minimize extra-column broadening

13
Practical Applications of HPLC

• How to minimize extra column broadening
• Make sure connections are good
• Reduce dispersion in injection
• Minimize connecting tubing lengths and diameters
  and connections
• Gradients help with pre-column dispersion
• Use fast detectors

void volume
good connection
bad connection
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Practical Applications of HPLC

• Detector Selection
• Universal Detectors
• RI most universal but least sensitive
• UV detection large range of detectable
  compounds, but can not use with surrogate
  standards for calibration
• Aerosol based good for non-volatile compounds
• MS not as universal as with GC due to
  ionization difficulties
• Selective Detectors
• ECD applicable to a wide range of compounds,
  very sensitive, but requires high water in
  mobile phase
• Fluorescence good sensitivity and selectivity,
  but to a small range of compounds
• MS also not as selective as GC

15
Practical Aspects of HPLCQuestions

• A chemist has been using a 5 mm, 150 x 4.6 mm C18
  column to separate and detect 5 compounds in a 15
  minute run. He wants to improve the speed and
  switches to a 3 mm, 100 x 4.6 mm C18 column.
• If run at the same flow rate and using the same
  mobile phase composition, should the resolution
  improve (theoretically)?
• How much faster will the run be?
• What could cause an observed decrease in
  resolution?
• If two compounds from a sample are found to
  co-elute with a k value of 5, what can be done to
  improve selectivity? For example guaiacols and
  corresponding syringols typically have small
  separation factors (a values)