Chem. 231 – 3/4 Lecture
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
Announcements II
• Set 3 Labs
– Hope to have instructions completed by
Monday
• Today’s Lecture
– SPE Lab Information
– SPME Lab Information
– Practical Applications of HPLC
SPE/HPLC Lab
Information
• 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
R
OH
OCH3
guaiacols
SPE/HPLC Lab
Information – 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
SPE/HPLC Lab
Information – 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
SPE/HPLC Lab
Information – 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
SPME/GC Lab
Information
• 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
H C
3
CH2
H3C
myrcene
H2C
SPME/GC Lab
Information - Methodology
• GC – Methodology
– Requires splitless injection (so can’t 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
SPME/GC Lab
Information - 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.)
SPME/GC Lab
Information - 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
Practical Applications of HPLC
• Review of Instrument Components
Chromatograph Components
Chromatographic
Column
Flow Control
Mobile Phase
Reservoir
Injector
Signal to data
recorder
Detector
Waste or fraction
collection
Chromatogram
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)
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
Practical Applications of HPLC
To column
• Solvent Selection
– Single Pump
• Manual Solvent Selection
Mixing chamber
pump
– 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)
gradient starts
Pressure
dwell time
time
Effect seen
To column
pumps
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
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 oneway “check valves”
– Exit check valve closes on
“in” stroke and entrance
check valve closes on “out”
stroke
Check valves
In
Stroke
Out
Stroke
closed
open
closed
open
pistons
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
200
Signal (uV)
100
0
-100
-200
-300
8
8.2
8.4
8.6
Time (min)
Bad check valve leaking
8.8
9
9.2
Practical Applications of HPLC
• Injection Valves
6 port valve
– 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
Practical Applications of HPLC
• Sample Injection
– Injection Volumes
• With sample solvent = eluent, volume injected shouldn’t contribute
significantly to extra column broadening (rule of thumb injection volume <
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) @ flow = 1.2 mL/min
or peak volume = 100 mL, so use vol < 25 mL
6.129
DAD1 D, Sig=230,16 Ref =400,25 (HERRERA\02201303.D)
mAU
800
0
1
2
4
5
6
6.748
6.864
7.568
7.134
5.412
4.338
3
4.559
4.666
1.357
200
2.272
1.585
400
5.708
5.839
6.031
5.579
6.382
600
9
8
7
min