Post on 11-Dec-2015
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 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
OHR
OCH3
guaiacols
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
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
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
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
CH3
CH3
CH2
CH2myrcene
SPME/GC LabInformation - 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 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.)
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
Practical Applications of HPLC
• Review of Instrument Components
Chromatograph Components
Mobile Phase Reservoir
Flow Control
Injector
Chromatographic Column Detector
Waste or fraction collection
Signal to data recorder
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
• 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)
To column
Mixing chamber pump
To columnpumps
time
Pressure
gradient starts
Effect seendwell time
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 m) 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 one-way “check valves”
– Exit check valve closes on “in” stroke and entrance check valve closes on “out” stroke
pistons
Check valves Out Stroke
open
closed
closed
open
In Stroke
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
-300
-200
-100
0
100
200
8 8.2 8.4 8.6 8.8 9 9.2
Time (min)
Sig
nal
(u
V)
Bad check valve leaking
Practical Applications of HPLC
• 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 L of a 250 L 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
sample in
Waste
6 port valve
From pump
To column
LOAD POSITIONINJECT 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
min1 2 3 4 5 6 789
mAU
0
200
400
600
800
DAD1 D, Sig=230,16 Ref =400,25 (HERRERA\02201303.D)
1.3
57
1.5
85
2.2
72
4.3
38
4.5
59 4
.666
5.4
12 5
.579
5.7
08 5
.839
6.0
31 6
.129
6.3
82
6.7
48 6
.864
7.1
34 7
.568
width = 0.084 min. (or 5.1 s) @ flow = 1.2 mL/min
or peak volume = 100 L, so use vol < 25 L