Chem. 230 – 11/18 Lecture
Announcements I
• Exam 3 Results
– Lower Average (72%)
– Distribution
• New Homework Posted Online (long problems due next
week)
Exam 3
8
Number of Students
7
6
5
4
3
2
1
0
90-96.7
80s
70s
Range
60s
<60
Announcements II
• Special Topics Presentations
– Need to prepare reading material (link to journal or photocopies in
folder) one week before presentations (due today for group going
11/25)
– Besides presentation, will need Homework Problems (I request 4
per group) on day of presentation
11/25
12/2
12/9
Group
Stephanie
& Diana
Topic
MEKC
Brenden &
Leo
Theo &
Chris
Nancy &
Maria
SMB
chromatogr.
SPMEHPLC
Ion-pairing
HPLC
Sam & Luis
Morgan &
Nicole
Dai & Olga
Emily &
Adriana
Dustin &
Rich
Thao &
Addison
Fluid Flow
Fractionation
SF
extraction
Chiral
Separations
Zirconia
in HPLC
2D LC
Zwitterionic
HPLC
Announcements III
• Today’s Lecture
– Quantification
• Methods of Calibration
– Mass Spectrometry
•
•
•
•
•
Applications
Instrumentation
Use as Chromatographic Detector
Interpretation
Other Topics
Quantitation in Chromatography
Calibration Methods
•
External Standard
•
Internal Standard
– most common method
Area
– standards run separately and calibration
curve prepared
– samples run, from peak areas,
concentrations are determined
– best results if unknown concentration comes
out in calibration standard range
– Common for GC with manual injection
(imprecisely known sample volume)
– Useful if slow drift in detector response
– Standard added to sample; calibration and
AX/AS
sample determination based on peak area
ratio
– F = constant where A = area and C = conc.
(X = analyte, S = internal standard)
F
Concentration
AX / AS
C X / CS
Conc. X (constant conc. S)
Quantitation in Chromatography
Calibration Methods
• Standard Addition
Area
– Used when sample matrix affects
response to analytes
– Commonly needed for LC-MS with
complicated samples
Analyte
– Standard is added to sample (usually
Concentration
in multiple increments)
– Needed if slope is affected by matrix
– Concentration is determined by
extrapolation (= |X-intercept|)
• Surrogate Standards
– Used when actual standard is not
available
– Should use structurally similar
compounds as standards
– Will work with some detector types
(FID, RI, ABDs)
standards in water
Concentration
Added
A  mX  b  0
X  b/ m
Quantitation
Additional (Recovery Standards + Questions)
• Recovery Standards
– Principle of use is similar to standard addition
– Standard (same as analyte or related compound)
added to sample, then measured (in addition to direct
measurement of sample)
%recovered 
amount recovered 100 amount total - amount unknown  100

amount expected
amount expected
– Useful for determining losses during extractions,
derivatization, and with matrix effects
Quantitation
Some Questions/Problems
1.
2.
3.
4.
Does increasing the flow rate improve the sensitivity of
a method?
Does the use of standard addition make more sense
when using a selective detector or a universal
detector?
Is a matrix effect more likely with a simple sample or a
complex sample?
Why is the internal standard calibration more common
when using manual injection than injection with an
autosampler?
Quantitation
Some Questions/Problems
5.
A scientist is using GC-FID to quantitate hydrocarbons. The FID is
expected to generate equal peak areas for equal numbers of
carbons (if substances are similar). Determine the concentrations
of compounds X and Y based on the calibration standard (1octanol). X = hydroxycyclohexane and Y = hydroxypentane.
Compound
1-octanol
cC6-OH
cC5-OH
Area
3520
299
1839
Conc. (ug
mL-1)
10.0
?
?
Quantitation
Some More Questions/Problems
6.
A chemist is using HPLC with fluorescence detection. He wants to
see if a compound co-eluting with a peak is quenching
(decreasing) the fluorescence signal. A set of calibration
standards gives a slope of 79 mL μg-1 and an intercept of 3. The
unknown gives a signal of 193 when diluted 4 mL to 5 mL (using
1 mL of water). When 1.0 mL of a 5.0 μg mL-1 standard is added
to 4.0 mL of the unknown, it gives a signal of 265. What is the
concentration of the unknown compound and is a significant
quenching (more than 10% drop in signal) occurring?
Quantitation
Some More Questions/Problems
7.
A chemist is testing an extraction process for removing DDT from
fish fat. 8.0 g of fat is first dissolved in 50 mL of 25% methylene
chloride in hexane. The 50 mL is divided into two 25 mL
portions, one of which is spiked by adding 2.0 mL of 25.0 ng mL-1
DDT. Each portion is run through a phenyl type SPE cartridge
and the trapped DDT is eluted with 5.0 mL 100% methylene
chloride. The methylene chloride is evaporated off, and the
sample is redissolved in 0.5 mL of hexane and injected onto a GC.
The un-spiked sample gives a DDT conc. (in 0.5 mL of hexane) of
63 ng mL-1, while the spiked sample gives a DDT conc. of 148 ng
mL-1. What is the % recovery? What was the original conc. of
DDT in the fat in ppb?
Mass Spectrometry
Overview
•
•
•
•
•
Applications of Mass Spectrometry
Mass Spectrometer Components
GC-MS
LC-MS
Other Applications
Mass Spectrometry
Applications
• Direct Analysis of Samples
– Most common with liquid or solid samples
– Reduces sample preparation
– Main problem: interfering analytes
• Off-line Analysis of Samples
– Samples can be separated through low or high
efficiency separations
– More laborious
• Chromatographic Detectors
– generally most desired type since this allows
resolution of overlapping peaks
Mass Spectrometry
Applications
• Purposes of Mass Spectrometry
– Quantitative Analysis (essentially used as any other
chromatographic detector)
• Advantages:
– selective detector (only compounds giving same ion fragments
will overlap)
– overlapping peaks with same ion fragment can be resolved
(through deconvolution methods)
– semi-universal detector (almost all gases and many solutes in
liquid will ionize)
– very good sensitivity
• Disadvantages
– cost
– requires standards for quantification
Mass Spectrometry
Applications
• Purposes of Mass Spectrometry - continued
– Qualitative Analysis/Confirmation of Identity
• With ionization method giving fragmentation, few compounds will
produce the same fragmentation pattern
• Even for ionization methods that don’t cause fragmentation, the
parent ion mass to charge data gives information about the
compound identity.
• Some degree of elemental determination can be made based on
isotopic abundances (e.g. determination of # of Cl atoms in small
molecules)
• Additional information can be obtained from MS-MS (further
fragmentation of ions) and from high resolution mass spectrometry
(molecular formula) if those options are available.
– Isotopic Analysis
• Mass spectrometry allows analysis of the % of specific isotopes
present in compounds (although this is normally done by dedicated
instruments to get good enough precision for use as source tracers)
• An example of this use is in drug testing to determine if
testosterone is naturally produced or synthetic
Mass Spectrometry
Instrumentation
• Main Components:
–
–
–
–
Ion source
Analyzer
Detector
Data Processor
Mass Spectrometry
Instrumentation
• Ion Sources
– For Gases
• Electron Impact (EI):
+
gas stream
– electrons from heated element
strike molecules
– M + e- => M+* + 2e– M+ is the parent ion
– Because M+* often has excess
energy, it can fragment further,
usually producing a smaller ion
and a radical
– Fragmentation occurs at bonds,
but electronegative elements
tend to keep electrons
M
-
e- e
CH3-Br+*
CH3+ + Br∙
CH3∙ + Br+
Main
fragment
Minor or unobserved
fragment
Mass Spectrometery
Instrumentation
• Ion Sources
– For Gases
• Chemical Ionization (CI):
– Can produce positive or negative ions
– First, a reagent gas reacts with a corona discharge to
produce a reagent ion: CH4 => => CH5+ (more likely
CH4∙H+)
– Then the reagent ion transfers its charge to a molecule:
M + CH5+ => MH+ (one of largest peak has mass to
charge ratio of MW + 1)
– Less fragmentation occurs, so more useful for identifying
the parent ion
Mass Spectrometery
Instrumentation
• Ion Sources
– For Liquids
• Earlier Methods (particle beam and thermospray) suffered from
poorer efficiency and ability to form ions from large molecules
• Electrospray Ionization (ESI):
–
–
–
–
Liquid is nebulized with sheath gas
Nebulizer tip is at high voltage (+ or –), producing charged droplets
As droplets evaporate, charge is concentrated until ions are expelled
Efficient charging of polar/ionic compounds, including very large
compounds
– Almost no fragmentation, but multiple charges possible
– For positive ionization, major peak is often M+1 peak; or for multiply
charged compounds, peak is [M+n]n+ where n = charge on ion
Nebulizing
gas
High voltage
M+
+ +
Liquid
in
+
+ +
Mass Spectrometery
Instrumentation
• Ion Sources
– For Liquids (continued)
• Atmospheric Pressure Chemical Ionization
– Liquid is sprayed as in ESI, but charging is from a corona
needle nearby
- More restricted to smaller sized molecules
• Atmospheric Pressure Photoionization
– UV light causes photoionization of molecules
Mass Spectrometery
Instrumentation
• Ion Sources
– For Solids (common offline method)
• Matrix Assisted Laser
Desorption Ionization
– Sample plus strong absorber
placed on substrate
– solvent removed
– laser focused on sample
– heat causes desorption and
ionization of analytes
M+
Mass Spectrometry
Instrumentation
• Analyzers
– Separates ions based on mass to charge ratio
– All operate at very low pressures (vacuums) to avoid
many ion – ion or ion – molecule collisions
– Analyzers for chromatographic systems must be fast.
(If a peak is 5 s wide, there should be 4 scans/s)
– Most common types (as chromatographic detectors):
• Quadrupole (most common)
• Ion Trap (smaller, MS-MS capability)
• Time of Flight (higher speed for fast separations and can be
used for high resolution applications)
Mass Spectrometry
Instrumentation
• Mass Spectrometer Resolution
– R = M/ΔM where M = mass to charge ratio and is ΔM difference
between neighboring peaks (so that valley is 10% of peak
height).
– Standard resolution needed:
• To be able to tell apart ions of different integral weights (e.g.
(CH3CH2)2NH – MW = 73 vs. CH3CH2CO2H – MW = 74)
– High Resolution MS:
• To be able to determine molecular formulas from “exact” mass
• example: CH3CH2CO2H vs. CHOCO2H; both nominal masses are 74
amu but CHOCO2H weighs slightly less (74.037 vs. 74.000 amu)
because 16O is lighter than 12C + 41H (Note: need to use main
isotope masses to calculate these numbers – not average atomic
weights). Needed resolution = 74/0.037 = 2000
• To separate similar ions requires very high resolution > 104 to 105
• However, to obtain “accurate” mass (error in mass under 5 ppm) is
not quite as hard in terms of resolution but requires internal
standards and clean peaks
Mass Spectrometry
Instrumentation
• Analyzers – how separation works
– Analyzers can act as filters (only passing a specific
m/z at a time) – e.g. in quadrupoles and ion traps,
can give full spectrum in a short time (time of flights),
or can give full information over an acquisition
(Fourier Transform ion cyclotron resonance)
– Control of ion throughput makes sense in ion traps or
in quadrupoles but in time of flight full spectrum
comes (whether desired or not)
Mass Spectrometry
Instrumentation
• Detectors:
M+
I
– Faraday Cup (simple, but not sensitive)
– Electron Multiplier (most common)
– Array Detector (Multichannel Analyzer)
Detection Process:
Anode
Dynodes
Ion strikes anode
Electrons are ejected
Ejected electrons hit
dynodes causing a
cascade of electron
releases
Current of electrons
hitting cathode is
measured
M+
Cathode
e- e
I
Mass Spectrometery
Use with GC
•
•
•
•
MS matches well to capillary GC flow rates
With EI gives good qualitative information
CI used if compound fragments too much
Total Ion and Selective Ion Modes:
– Total Ion Current (TIC) gives full mass spectra at
every point (better for qualitative analysis)
– Selective Ion Monitoring (SIM) only determines signal
at several ions (the fragments of interest) (better for
quantitative analysis because of better sensitivity)
40
20
0
5
7.632
7.723
7.760
7.859
7.940
8.069
8.151
8.349
8.468
8.662
8.969
9.039
9.078
9.125
9.209
9.262
9.472
9.638
9.751
9.859
9.908
10
15
20
25
25.850
24.784
23.834
22.964
22.120
21.249
20.354
19.438
18.502
15.137
15.201
15.258
15.308
15.386
15.655
16.070
16.146
16.206
16.336
16.604
17.009
17.158
17.291
17.556
20
10
10.2
mostly branched alkanes
10.4
10.6
1-Alkene
10.800
10.742
pA
10.908
10.653
10.585
50
10.412
140
10.284
160
10.332
FID1 B, (Y VONNE\08081301.D)
10.237
1.756
1.824
14.714
180
11.263
11.334
11.386
11.435
11.509
11.669
11.733
11.813
11.864
11.975
12.324
12.356
12.404
12.454
12.527
12.809
12.856
12.965
13.277
13.315
13.369
13.417
13.490
13.810 13.764
14.206
14.259
14.314
14.364
14.439
10.742
9.573
8.269
6.764
5.013
100
4.378
4.407
4.519
4.594
4.643
4.784
4.858
5.142
5.289
5.658
6.008
6.162
6.292
6.423
6.537
6.626
6.706
6.865
6.994
7.164
80
3.244
120
10.237
10.284
10.332
10.412
10.585
10.653
10.800
10.908
60
2.143
pA
2.737
2.768
2.809
2.884
2.974
3.120
3.184
3.317
3.369
3.504
1.634
1.707
1.961
2.040
2.087
2.194
2.267
Mass Spectrometery
Use with GC - Example
• Example of examination of co-eluting peaks
• Synthetic diesel sample shows large number of
peaks – mostly alkanes and alkenes
FID1 B, (Y VONNE\08081301.D)
60
40
C12s
30
10.8
Alkane
11
min
min
peak cluster = (mostly) same number carbons
2-Alkenes
Mass Spectrometery
Use with GC – Example – Cont.
• Analysis didn’t match manufacturer’s assessment of 4%
alcohols
• However, alcohols are hard to determine by MS due to loss of
H2O in fragmentation
– CH3(CH2)6OH → CH3(CH2)5CH·+ (MW = 98 – same as expected for
alkene M peak)
• Linear Alcohols found to elute at time of branched C10 alkanes
mass spectrum shows
alkyl chains
Mass Spectrometery
Use with GC – Example – Cont.
• Careful examination of fragmentation shows differences
between right and left sides of peak with right side close to
that of C7 alcohol standard
right
shoulder
1-heptanol
Mass Spectrometery
Use with GC – Example – Cont.
• Ion Extraction allows separation of chromatographic peaks
based on 70 vs 71 fragments
• Could improve by: using CI, using slight difference in column
polarity
• Identification stronger due to water washing fuel
70 (alcohol)
fragment
71 (branched
alkane) fragment
Mass Spectrometery
Use with HPLC
• One disadvantage is the volume of gas developed as
solvent evaporates
• For this reason, HPLC flows must be low (e.g. semimicrobore), or splitters are needed
• With most common ionization (ESI), little fragmentation
occurs, making identification of unknown compounds
harder
• Because of little fragmentation, MS-MS is more common
• In MS-MS, ions leaving mass analyzer are then
fragmented (by collisions with molecules) before
entering a second mass analyzer or re-entering the mass
analyzer
• Also, some compounds are hard to ionize efficiently
Mass Spectrometery
Interpretation
• Fragmentation Analysis
– Focus on possible structure of fragments (low end of spectrum)
or of fragments lost (high end of spectrum)
• Isotopic Analysis
– For elements with more than 1 isotope in abundance
– Average MW not useful, MW of specific isotopes determines
charge
– Formation of M+1, M+2, M+3 ... peaks to predict elements
present
• Determination of Charge
– Important for interpreting MALDI and ESI peaks where multiple
charges are possible
Mass Spectrometry
Isotope Effects
• It also may be possible to distinguish compounds based
on isotopic composition
• Average MW is not useful (except for very large MW
compounds), but abundance of each isotope gives each
element a “fingerprint”
• Compounds in high resolution example will have
different expected M+1/M and M+2/M ratios (which will
NOT require high resolution to see)
• Go over calculations on board for CH3SSCH3
• Main difficulty is accurately determining ratios (plus
effects of contaminants, variation in ratio, etc.)
Mass Spectrometry
Other Topics – Multiple Charges in ESI
(M+n)/n
Dm/z
Ion current
• In ESI analysis of large
molecules, multiple charges
are common due to extra
(+) or missing (-) Hs (or
e.g. Na+)
• The number of charges can
be determined by looking at
distribution of big peaks
• For + ions m/z = (M+n)/n
(most common)
• For – ions m/z = (M–n)/n
m/z
(M+n+1)/(n+1)
Example: m/z peaks =711.2, 569.3,
474.8, 407.1
Dm/z = (M+n)/n – (M+n+1)/(n+1) = (M+n)(n+1)/[n(n+1)] – (Mn+n2+n)/[n(n+1)] =
M/[n(n+1)] = 141.9, (94.5, 67.7)
Do rest on board
Mass Spectrometry
Other Topics – Multiple Charges in ESI
• Another way to find charge on ions is to examine the
gap in m/z between isotope peaks (0 13C vs. 1 13C)
• The +1 mass difference will be ½ if charge is +2 or 1/3
if charge is +3
gap = 405.73 – 405.23 = 0.50
Glycodendrimer core
Glycodendrimer core
Mass Spectrometry
Other Topics - MS-MS
• In LC-ESI-MS, little fragmentation occurs making
determination of unknowns difficult
• In LC-ESI-MS on complicated samples, peak
overlap is common, with interferants with the
same mass possible (e.g. PBDPs)
• In both of above samples, using MS-MS is useful
• This involves multiple passes through mass
analyzers (either separate MSs or reinjection in
ion-trap MS) and is termed MS-MS
• Between travels through MS, ions are collided
with reagent gas to cause fragmentation
Mass Spectrometery
Questions I
1. Which ionization method can be achieved on
solid samples (without changing phase)
2. If one is using GC and concerned about
detecting the “parent” ion of a compound that
can fragment easily, which ionization method
should be used?
3. For a large, polar non-volatile molecule being
separated by HPLC, which ionization method
should be used?
Mass Spectrometery
Interpretation Questions
1. Determine the
identity of the
compound giving
the following
distribution:
m/z
Abundance
(% of biggest)
25
14
26
34
27
100
35
9
62
77
64
24
Mass Spectrometery
Interpretation Questions
2. Determine the
identity of the
compound giving
the following
distribution:
m/z
Abundance
(% of biggest)
29
9.2
50
30.5
51
84.7
77
100
93
16
123
39
Mass Spectrometery
Interpretation Questions
3. From the following
M, M+n ions,
determine the
number of Cs, Brs
and Cls:
m/z
Abundance
(% of biggest)
117
100
118
1.4
119
98
121
31.1
123
3