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

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 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 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


• 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.)


• 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


• 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)


• 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


• 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


• 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


• 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


• 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


• 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


• 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

Documents

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