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 GCFID (peak area should be roughly proportional to mass C conc.)
– We have several other standards that can be used for qualitative
analysis (a-pinene, b-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
H3C
CH2
H3C
– 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
limonene (1 ring)
mass spectrum is similar
but 107 peak
H2C
myrcene - acyclic
CH3
Mass Spectrum:
136 (small) = parent ion
121 (small) = - CH3·
93 (big) = - CH(CH3)2
H3C
CH2
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)
H2O
H3C
H3C
CH3
CH2
OH
H3C
H3C
H2C
H2C
linalool
myrcene
Set 2 Labs - SPME
• Selective Analysis by GC-MS
GC-MS Software
• Demonstration
• Chromatogram allows mass
spectra at all points (if set up
to collect data over full range)
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
wavelengths
O
H3C
O
OH
OH
OH
O
CH3
O
CH3
vanillin
4-EtPhenol
3-hydroxy,4-methoxycinnamaldehyde
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