1. Chem. 231 – 3/4 Lecture

2. 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

3. Announcements II Set 3 Labs –Hope to have instructions completed by Monday Todays Lecture –SPE Lab Information –SPME Lab Information –Practical Applications of HPLC

4. 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 OH R OCH 3 guaiacols

5. 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

6. 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

7. 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

8. 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 myrcene

9. SPME/GC Lab Information - Methodology GC – Methodology –Requires splitless injection (so cant 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

10. 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.)

11. 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

12. 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

13. 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)

14. 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

15. 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 column pumps time Pressure gradient starts Effect seen dwell time

16. 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

17. 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 valvesOut Stroke open closed open In Stroke

18. 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 Bad check valve leaking

19. 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 POSITION INJECT POSITION

20. Practical Applications of HPLC Sample Injection –Injection Volumes With sample solvent = eluent, volume injected shouldnt 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 L, so use vol < 25 L