Title: Chem. 231
1Chem. 231 3/11 Lecture
2Announcements 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)
3Announcements 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
4Practical 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
5Practical 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
6Practical 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)
7Practical 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
8Practical 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)
9Practical Aspects of GCQuestions
- 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? - 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?
10Practical 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)
11Practical 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
12Practical 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
13Practical 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
14Practical 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
15Practical 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)