1. Chem. 231 – 3/18 Lecture

2. Announcements Set 2 Lab Reports –Due 4/10 Final Exam – April 8 th Today’s Discussion –Set 2 Labs – Information for “real” samples (flowers for SPME and lignin products for SPE) –Set 3 Labs – Overview and specific information

3. Set 2 Labs - SPME Real Samples –Analysis of flowers may give peaks where unknowns elute plus will give other peaks –GC-FID method may allow identification of unknown compounds (myrcene, limonene, linalool, and geraniol) if: peaks observed at known retention times these peaks are not overlapping peaks actually due to above compounds (not other compounds with same elution time) –We are not trying to get absolute quantification of volatile concentrations, but we can get relative concentrations with GC- FID (peak area should be roughly proportional to mass C conc.) –We have several other standards that can be used for qualitative analysis (  -pinene,  -caryophyllene, nerolidol, and farnesene) that were prepared in 2011

4. Set 2 Labs - SPME Real Samples – Qualitative Analysis –If GC-FID gives complicated chromatograms (e.g. many overlapping peaks), you should: use a slower eluting (lower temperature) temperature program to improve resolution run standards again and then sample at new program –Use of GC-MS will improve identification success, since you also get the mass spectrum I have set up an SPME method that can be modified for your use may want to run standards to get an idea of fragmentation patterns expected for known monoterpenes

5. Set 2 Labs - SPME Real Samples – Qualitative Analysis –Degree of confidence in unknown peaks is never 100% but may be high. Highest confidence occurs for: well shaped isolated peak same (within uncertainty from injection start) retention time as standard same MS pattern as standard (this gives GC-MS a clear advantage over GC-FID) –Can also have some, but lower, confidence if MS is same as predicted (e.g. based on library) but no standard is available or run –Lower confidence if just based on reasonable fragmentation pattern (monoterpene mass spectra tend to be similar)

6. Set 2 Labs - SPME Real Samples – Qualitative Analysis –Types of Compounds Monoterpenes –all are made from isoprene units –all have formula C 10 H 16 ( MW = 136 and three degrees of unsaturation/cyclization) –main differences are in # of double bonds vs. # rings myrcene - acyclic Mass Spectrum: 136 (small) = parent ion 121 (small) = - CH 3 · 93 (big) = - CH(CH 3 ) 2 limonene (1 ring) mass spectrum is similar but 107 peak

7. Set 2 Labs - SPME Real Samples – Qualitative Analysis –Types of compounds – cont. monoterpene alcohols –linalool and geraniol –addition of H 2 O to monoterpene –have MW of 154 other compounds (sesquiterpenes: C 15 H 24, monoterpene alcohol esters and glycosides) myrcene H2OH2O linalool

8. Set 2 Labs - SPME Selective Analysis by GC-MS Demonstration Chromatogram allows mass spectra at all points (if set up to collect data over full range) GC-MS Software Right Click on Peaks using Mouse provides peak – see lavender example First peak shows monoterpene (parent ion of 136)

9. Set 2 Labs - SPME Selective Analysis by GC-MS – cont. First tall peak – appears to be monoterpene alcohol

10. Set 2 Labs - SPME Selective Analysis by GC-MS – cont. Can set GC to only show specific ions (example for 136 and 154) Use: Chromatograms → Extract Ion Chromatograms

11. Set 2 Labs - SPME GC-MS Example – extract ions top is 136 bottom is 154

12. Set 2 Labs - SPE Real Samples –Use syringe filters –Samples may give peaks where unknowns (probably not for phenol or 4-ethyl phenol) elute plus will give other peaks –HPLC method may allow identification of unknown compounds (in same manner as GC-FID) –HPLC-DAD is less useful than GC-MS, but can also use DAD to get spectral information –More conjugation and aldehydes have peaks at longer wavelengths vanillin 4-EtPhenol 3-hydroxy,4-methoxy- cinnamaldehyde

13. Set 2 Labs - SPE Real Samples – Qualitative Analysis –Can set DAD to take spectra at peaks (when editing method) –After collecting data, can then look at spectra once you select that option Put mouse over chromatographic peak and click on spectrum icon

14. Set 2 Labs - SPE Real Samples – Qualitative Analysis –DAD use – cont. Spectrum for Vanillin

15. Set 3 Labs - Derivatization Overview –Both labs involve converting analyte to one that can be better separated and/or detected –These are common methods, but reagents are more toxic than past labs –In GC, reaction of fatty acids to fatty acid methyl esters (FAMEs) results in more volatile, less polar compound –These can be run through the GC with fewer problems –In HPLC, the derivatives make weak to moderately absorbing carbonyl compounds strongly absorbing

16. Set 3 Labs - GC Fatty Acid Analysis –Fatty acids are the main constituents in triglycerides (fats) –The type of fatty acids can be important for health effects (generally, more unsaturated fatty acids are better, saturated fats are worse, and trans unsaturated fats have the most serious health effects –Besides, triglycerides, other fatty acids can exist: free fatty acids, waxes, phospholipids, other molecules

17. Set 3 Labs - GC Fatty Acid – Derivatization –Reaction involves replacing proton with methyl group –Catalyzed by using BF 3 with methanol as other reactant –Sample clean up is needed to remove excess reactants and catalyst (more polar compounds) –Sample clean up uses liquid – liquid extraction

18. Set 3 Labs - GC FAME Analysis –Can use GC-FID or GC-MS –We will switch column on GC-FID to get better separation of C18:0 from C18:1 FAMEs –GC-MS will use DB-5 column (not very selective), but then can use “Extract Ion” to separated C18:0 and C18:1 FAMEs –We have margaric acid (C17 fatty acid) as a recovery standard (you will need to identify another internal standard for quantification)

19. Set 3 Labs - GC Oil Samples –Cooking oil is mostly triglycerides –Analysis of fatty acid composition gives information about how “healthy” specific oil is –First required step is base-catalyzed saponification (release of fatty acids from triglyceride) –We have tristearin (three C18:0 fatty acids attached to glycerol) as a recovery standard (can also use C17). To use properly, you need to divide the oil into two nearly identical replicates (one with and without tristearin added)

20. Set 3 Labs - HPLC Carbonyl Analysis –Formaldehyde and to a lesser extent other carbonyl compounds are common air pollutants –They originate from oxidation of alkanes/alkenes and from direct sources –They also are present in food samples from oxidation of alcohols

21. Set 3 Labs - HPLC Carbonyl Compound – Derivatization –Reaction involves replacing C=O bond with C=N bond in hydrozone using dinitrophenyl hydrozene –Product absorbs strongly (including in visible) –DNPH reactant is toxic and relatively shock sensitive (don’t drop the bottle) –Reaction requires acidic conditions –Safety gear is needed at all times

22. Set 3 Labs - HPLC Carbonyl Compound – Derivatization – cont. –A concern is contamination of the DNPH (aldehydes are common indoor air pollutants and Chem 125 uses carbonyl compounds) –We will need to purify DNPH before use –We have run this lab without an internal standard and without much clean up (filter samples, though), but you can use an internal standard not in the unknown or sample