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Chem. 231

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Title: 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)

7
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

8
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)

9
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)

11
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
14
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)
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