Chem. 231 – 3/18 Lecture

Announcements• Set 2 Lab Reports

– Due 4/10• Final Exam – April 8th

• 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

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

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

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)

Set 2 Labs - SPME• Real Samples –

Qualitative Analysis– Types of Compounds

• Monoterpenes– all are made from isoprene

units– all have formula C10H16

( MW = 136 and three degrees of unsaturation/cyclization)

– main differences are in # of double bonds vs. # rings

CH3

CH3

CH2

CH2

myrcene - acyclic

Mass Spectrum:136 (small) = parent ion121 (small) = - CH3·93 (big) = - CH(CH3)2

limonene (1 ring)mass spectrum is similar but 107 peak

CH3

CH3CH2

Set 2 Labs - SPME• Real Samples –

Qualitative Analysis– Types of compounds –

cont.• monoterpene alcohols

– linalool and geraniol– addition of H2O to

monoterpene– have MW of 154

• other compounds (sesquiterpenes: C15H24, monoterpene alcohol esters and glycosides)

CH3

CH3

CH2

CH2

myrcene

H2O

OHCH3

CH3

CH3

CH2

linalool

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)

Set 2 Labs - SPME• Selective Analysis by GC-MS – cont.

• First tall peak – appears to be monoterpene alcohol

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

Set 2 Labs - SPME• GC-MS Example – extract ions

top is 136

bottom is 154

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 wavelengthsO

CH3

O

OH

vanillin

CH3

OH

4-EtPhenol

CH3O

O

OH

3-hydroxy,4-methoxy-cinnamaldehyde

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

Set 2 Labs - SPE• Real Samples – Qualitative Analysis

– DAD use – cont.

Spectrum for Vanillin

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

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

Set 3 Labs - GC• Fatty Acid – Derivatization

– Reaction involves replacing proton with methyl group

– Catalyzed by using BF3 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

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)

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)

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

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

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