Transcript Chem. 230 – 11/4 Lecture Announcements I • • • • Exam 1 today No Class Next Tuesday 11/18 and 11/25 on remaining topics Special Topics Presentations – Sign.

Chem. 230 – 11/4 Lecture
Announcements I
•
•
•
•
Exam 1 today
No Class Next Tuesday
11/18 and 11/25 on remaining topics
Special Topics Presentations
– Sign up for Presentation
– Dates:
• 11/25 (one group – if desired, not required)
• 12/2 (4 or 5 groups – also have exam 4)
• 12/9 (5 or 6 groups)
– Need to prepare reading material (link to journal or
photocopies in folder) one week before presentations
Announcements II
• Today’s Lecture
– HPLC
• Aerosol-Based Detectors (in more detail)
– Quantification
•
•
•
•
•
Performance Measures
Sensitivity Considerations
LOD and LOQ Calculations
Data Smoothing and Integration
Methods of Calibration
– Mass Spectrometry (Introduction and instruments)
Aerosol-Based Detectors for
HPLC
Example Advanced Method
Presentation
Aerosol-Based Detectors for HPLC
Outline
• Introduction to Technology
• Theory Including Three Types of
Detectors
• Advantages and Disadvantages of ABDs
• Some Applications
• Conclusions
• References
Aerosol-Based Detectors for HPLC
Introduction
• Limitations of Conventional Detectors
– UV Absorption Detectors:
• Not very universal
• Poor sensitivity for many classes of compounds
(carbohydrates, fats, amino acids, dicarboxylic acids, etc.)
– Refractive Index Detectors:
• Low and somewhat variable sensitivity
• Not gradient compatible
– Mass Spectrometer Detectors:
• Not all compounds ionize readily
• Expensive, large, expensive to operate
Aerosol-Based Detectors for HPLC
Introduction
– Effluent from column is
nebulized producing spray
of solvent and solute
– Spray droplets are heated
in an oven, evaporating
solvent gas and producing
aerosol particles from
solute
– Aerosol passes to an
aerosol detector to produce
a signal
Nebulizer
N2(g)
HPLC Column
• Processes in AerosolBased Detectors:
Oven
Aerosol Detector
droplet
particle
Spray
Chamber
Aerosol-Based Detectors for HPLC
Introduction
• Mobile Phase Requirements
– Solvent must be volatile (and cause little
column bleed)
• Analyte Requirements
– Works best if analyte is non-volatile
– Semi-volatile compounds give reduced
response
Aerosol-Based Detectors for HPLC
Theory
C
d p  d d 
 p
1/3




where: dd, dp are drop and
particle diameters, C is mass
concentration, and ρp is
particle density
Size Distributions
1 mg mL-1 solute
0.9
0.8
0.7
number (dn/dlogd)
• Nebulization produces a
distribution of drop sizes
• Solvent viscosity and surface
tension can affect distribution
of droplet sizes
• Evaporation shifts this to
distribution of particle sizes
based on:
0.6
0.5
0.4
Particles
Droplets
0.3
0.2
0.1
0
1.E-03
1.E-02
1.E-01
1.E+00
diameter (mm)
1.E+01
1.E+02
Aerosol-Based Detectors for HPLC
Theory
• Types of Aerosol-Based Detectors
– Depends on method of detecting aerosol particles
– Evaporative Light Scattering Detection (ELSD)
(Charlesworth, J. M. Anal. Chem. 1978, 50, 1414)
– Condensation Nucleation Light Scattering Detection
(CNLSD) (Allen, L. B.; Koropchak, J. A. Anal. Chem.
1993, 65, 841)
– Charged Aerosol Detector (CAD)/Aerosol Charge
Detector (Dixon, R. W.; Peterson, D. S. Anal. Chem.,
2002, 74, 2930)
Aerosol-Based Detectors for HPLC
Theory
• ELSD principles
– Detection by lightscattering by particles
– Efficient detection when dp
~ λ; less efficient at other
sizes
– Non-linear response results
– At low concentrations, dp <
λ so sensitivity is poor
(detection limits of around
0.1 to 1 μg mL-1)
Expanded Region
concentration
Aerosol-Based Detectors for HPLC
Theory
condensor
– Detection principle also uses particle lightscattering but overcomes poor detection of
small particles by growing small particles
to bigger particles by condensation of
vapor on to particles
– This technology is very sensitive (a single 3
nm particle can be detected)
– This can translate to very low detection
limits (~10 ppb or ~50 pg) under optimal
conditions
– Commercialized recently
Particles In
Butanol
• Condesation Nucleation Light
Scattering Detection
To light-scattering
detector
Aerosol-Based Detectors for HPLC
Theory
•
Charged Aerosol Detection
– Particles charged as aerosol jet collides with ion-rich jet from corona discharge
(commercial version)
– Charged particles are collected on a filter with charge passed to electrometer
(current measured)
– In another version, particles are charged as they pass near a corona discharge
region
– Sensitivity has equalled CNLSD (at least at standard HPLC flows)
– Large response range and linearity at lower concentrations
Aerosol In
To
Electrometer
Gamache et al., LCGC North America (2005).
Corona
Discharge Wire
Ion Filter
(negatively
charged rod)
Aerosol
Filter
Aerosol-Based Detectors for HPLC
Advantages and Disadvantages
• Advantages:
– Better performing universal detectors than refractive index
detectors
– Universal response for non-volatile analytes
– CNLSD and CAD sensitivity is similar to typical UV sensitivity
• Disadvantages:
– Requires analytes of low-volatility, volatile mobile phases
– CNLSD and CAD are often limited by solvent purity and column
bleed
– Non-linear calibration often is needed
– Cost is higher than UV Detectors
Aerosol-Based Detectors for HPLC
Some Applications
• Food
– ELSD has been used extensively to characterize carbohydrates and
lipids.
– Methodology requires no derivatizations and allows analysis of whole
lipids (as opposed to just fatty acids)
• Polymers (with SEC)
– Useful for polymers without chromophores
• Pharmaceutical Industry
– ABDs are useful for assessing contaminants in pharmaceutical products
• Biotechnology and Environmental Samples
– Greater potential with CNLSD and CAD for analyzing low concentration
samples (some carbohydrate examples)
• Analysis of Cations, Anions and Neutrals
– Use in combination with zwitterionic stationary phase allows
simultaneous detection of three categories in single run
Aerosol-Based Detectors for HPLC
Triglyceride Example
•
•
•
•
•
•
By Lísa et al (J. Chromatogr. A,
1176 (2007) 135-142).
Homogenous trigylcerides shown
above without (left) and with
“gradient compensation” (right)
Gradient compensation allows
response to remain proportional to
area with a gradient
An alternative is to use a 2
dimensional calibration
(Hutchinson et al., J. Chromatogr.
A, 1217 (2010) 7418-7427)
Gradient compensation uses 2
additional pumps pumping eluent
after the column to produce a
constant eluent composition
Plant oil samples shown below
Aerosol-Based Detectors for HPLC
Paclitaxel Example
•
•
•
•
•
•
By Sun et al. (J. Chromatogr. A,
1177 (2008) 87-91).
Looked at impurities in paclitaxel
(a anti-cancer natural product
from Pacific yew tree) using UV
and CAD
Shown in upper figure (standards
– highest and stressed paclitaxel –
lower)
Paclitaxel impurity response
shown to be uniform by CAD but
not by UV detection
Pharmaceutical impurity analysis
used for determining acceptable
pharmaceuticals
If no standards available, CAD
provides better estimation of
impurity levels
Aerosol-Based Detectors for HPLC
Smoke Tracer Example
• My work (published in Dixon
and Baltzell and Ward et al. –
see my research webpage)
• Detected levoglucosan and
related monosaccharide
anhydrides
• These are thermal breakdown
products from cellulose and
hemicellulose
• It was possible to use the
levoglucosan concentrations to
estimate the total particulate
matter (2.5) derived from
woodsmoke
OH
H
R
HO
H
H
OH
H
OH
O
O
OH
H
HO
H
O
O HO
OH H
H
H
H
H
O
R
OH H
cellulose
H
O
HO
H
HO
H
H
O
levoglucosan
OH H
Chico Winter Air Sample
mannosan
levoglucosan
Aerosol-Based Detectors for HPLC
Glycan Profiling
Frog Egg example
ADC1 A, ADC1 CHANNEL A (NOAH\050409000002.D)
26.420
14.150
200
12.983
10.428
mV
175
150
125
25
7.5
10
12.5
20
22.5
25
28.048
25.328
25.615
25.765
23.846
24.172
24.450
24.776
24.819
24.894
23.297
21.434
19.577
19.607
17.813
17.5
18.551
17.261
15.942
15
16.565
16.632
15.251
14.736
50
12.167
75
12.585
100
27.094
27.451
26.242
11.596
•
Peptide backbone
10.980
•
•
•
8.531
8.856
9.134
9.326
9.604
9.962
•
6.722
•
My more recent work (with Thomas
Peavy, Biological Sciences) also
preliminary work done by Ignaki et al.
Glycans (glycoprotein
oligosaccharides) are difficult to
quantify
Glycans are post-translational
modifications and composition can
depend on host organism/cells
Profiles change in cancer cells
Standards are unavailable or expensive
Currently running surrogate standards
to prepare multi-dimensional
calibration (depending on mass
concentration and retention time)
Test standards show errors of ~0 to
25%
7.306
7.617
7.791
•
oligosaccharides
min
Aerosol-Based Detectors for HPLC
Conclusions
• ABDs have been replacing RID as a
universal detector (at least for non-volatile
compounds)
• ABDs can be used without exact standards
for quantification (much as an FID is used
in GC)
• Biggest limitations are volatility/nonvolatility requirements, cost, and linearity
Aerosol-Based Detectors for HPLC
References
•
ELSD
–
–
–
•
CNLSD
–
–
•
Text (p. 247-248)
Charlesworth, J. M., Evaporative analyzer as a mass detector for liquid
chromatography, Anal. Chem., 50, 1978, 1414-1420.
Review: Koropchak et al., Fundamental Aspects of Aerosol-Based LightScattering Detectors for Separations, Adv. Chromatogr. 40, 2000, 275.
Allen, L. B. and J. A. Koropchak, Condensation nucleation light scattering: A
new approach to development of high-sensitivity, universal detectors for
separations, Anal. Chem., 65, 1993, 841-844.
Same review listed for ELSD
CAD
–
–
Dixon, R. W. and D. S. Peterson, Development and testing of a detection
method for liquid chromatography based on aerosol charging, Anal. Chem., 74,
2002, 2930-2937.
Gamache, P.H., R.S. McCarthy, S.M. Freeto, D.J. Asa, M.J. Woodcock, K.
Laws, and R.O. Cole, HPLC analysis of nonvolatile analytes using charged
aerosol detection, LCGC North America, 23, 150, 152, 154, 156, 158, 160-161,
2005.
Aerosol-Based Detectors for HPLC
References
•
For Applications: (See my faculty web page for CAD references)
–
Foods:
•
•
•
–
Asa, D., Carbohydrate and oligosaccharide analysis with a universal HPLC detector, Am. Laboratory,
38, 16, 18, 2006.
Moreau, R. A.. The analysis of lipids via HPLC with a charged aerosol detector, Lipids, 41, 727-734,
2006.
Lísa, M., F. Lynen, M. Holčapek, and P. Sandra, Quantitation of triacylglycerols from plant oils using
charged aerosol detection with gradient compensation
Pharmaceuticals:
•
•
Loughlin, J., H. Phan, M. Wan, S. Guo, K. May and B. Lin, Evaluation of charged aerosol detection
(CAD) as a complementary technique for high-throughput LC-MS-UV-ELSD analysis of drug discovery
screening libraries, Am. Laboratory, 39, 24-27, 2007.
Sun, P., X. Wang, L. Alquier, C. A. Maryanoff, Determination of relative response factors of impurities
in paclitaxel with high performance liquid chromatography equipped with ultraviolet and charged
aerosol detectors, J. Chromatogr., A, 1177, 87-91, 2008.
–
Biotechnology:
–
Atmospheric Aerosols:
•
•
Inagaki, S., J.Z. Min, and T. Toyo’oka, Direct detection method of oligosaccharides by highperformance liquid chromatography with charged aerosol detection, Biomed. Chromatgr., 21, 338342, 2007.
Dixon, R. W. and G. Baltzell, Determination of levoglucosan in atmospheric aerosols using high
performance liquid chromatography with aerosol charge detection, J. Chromatogr. A, 1109, 214-221,
2006.
Aerosol-Based Detectors for HPLC
Questions
1.
2.
3.
4.
5.
For a complicated sample with several analytes present at
moderate concentrations (around 50 μg mL-1), is it advantageous
to use an ELSD (vs. a UV Detector) 1) if the compounds are weak
absorbers, 2) if the compounds are strong absorbers?
What instrument components will ELSD and CNLSD have in
common that are not present in CAD?
ABDs can not detect volatile analytes. How should weakly
absorbing volatile compounds be determined?
With a single calibration standard (over different concentrations),
is it possible to estimate concentrations of unknown compounds
(e.g. for compounds without any standards)? and under what
conditions?
Protein concentration can be estimated by looking at absorption
from aromatic amino acids? Why might using an ABD be a better
way of quantifying unknown proteins?
Quantitation in Chromatography
Overview
•
•
•
•
•
•
Performance Measures
Detector Response
Levels of Detection and Quantification
Data Smoothing
Integration
Calibration Methods
Quantitation in Chromatography
Performance Measures
• Precision
– How reproducible a measurement is
• Accuracy
– How close measured concentration is to true value
• Sensitivity
– The ability to measure small concentrations or amounts of
analyte
• Selectivity
– Can be an issue in quantification when overlapping/interfering
peaks occur
• % Recovery
– % of analyte added to sample that is measured in sample
Quantitation in Chromatography
Detector Response
• Concentration Type vs. Mass Flow Type
– In concentration type, signal depends on analyte in sample cell;
so generally flow independent
– In mass flow type, signal depends on mass transport to detector
(e.g. in FID without compounds entering flame, no signal will
result)
– Note: for some mass flow (HPLC-ABDs and HPLC-MS) transport
efficiency depends on liquid flow so signal is not directly
proportional to flow rate
Concentration Detector
Mass Flow Detector
flow on
flow off
Time
flow off flow on
Time
Quantitation in Chromatography
Detector Response
• Concentration Type - examples:
– PID (GC)
– UV-Vis (HPLC)
– Fluorescence (HPLC)
• Mass Flow Type - examples:
– FID (GC)
– NPD (GC)
Quantitation in Chromatography
Detector Response
• Detector Signal
– Depends on concentration of analyte or mass of
analyte reaching detector
– Most (but not all) detectors give linear response over
portion of detectable range
• Detector Noise
– Present in all detectors
– High and low frequency types
• Ability to Detect Small Quantities Depends on
Signal (Peak Height) to Noise Ratios
Quantitation in Chromatography
Levels of Detection and Quantification
• Noise can have high and low frequency parts
• Ways of defining noise
– peak to peak (roughly 5σ)
– standard deviation (more accurate way)
peak to peak noise
• Signal = peak height
high frequency
component
low frequency component
Quantitation in Chromatography
Levels of Detection and Quantification
• Limit of Detection (LOD):
– minimum detectable signal can be defined as S/Npeak-to-peak = 2
or S/σ = 3.3
– minimum detectable concentration = concentration needed to
get S/Npeak-to-peak = 2 or S/σ = 3.3
– Calculate as 2N/m where m = slope in peak height vs. conc.
calibration plot
– Minimum detectable quantity = (minimum detectable
conc.)(injection volume)
• Limit of Quantification (LOQ):
– Calculated in similar fashion as LOD
– Lowest concentration to give an “reasonable” conc. (e.g. can be
“auto-integrated” using software)
– Typically 5∙LOD
Quantitation in Chromatography
Data Smoothing
•
Data should be digitized with a frequency ~20/peak width
High frequency noise (where fnoise >> fsignal) can be removed by filtering
– see example below
– note: overfiltering results in reduction of signal and loss of resolution
– overfiltering result also can occur if detector response is too slow (or cell volume
is too large
Difficult to remove noise with frequency similar to or lower than peaks
550
500
response
•
•
450
Raw Data
Filtered Data
Excess Filtering
400
350
300
15
17
19
time
21
23
Quantitation in Chromatography
Integration
• Integration of peak
should give:
– peak height
– peak area
– peak width (often just peak
area/peak height)
• Difficulty comes from
determining if a peak is a
peak (or just noise), and
when to “start” the peak
and “end” the peak.
• Can use “auto
integration” or “manual
integration”
we want to pick
up this peak
but not these
noise spikes
Quantitation in Chromatography
Integration
• Other issues in
integration (besides
noise peaks)
– start and ends to
peaks
– how to split
overlapping peaks
Quantitation in Chromatography
Integration
• Peak Height vs. Peak Area
– Reasons for using peak area
• peak area is independent of retention time
(assuming linear response), while the peak height
will decrease with an increase in retention time
• peak area is independent of peak width, while the
peak height will decrease if the column is
overloaded (non-linear response)
– Reasons for using peak height
• Integration errors tend to be smaller if samples are
close to the detection limits
Quantitation in Chromatography
LOD/LOQ example
• Determine the LODs and LOQ for the
following example. Determine it for the
4.6 min peak if the concentration is 0.4 ng
μL-1. Use the 3.3 and 2N LOD defintions.
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
amountrecovered 100 amounttotal - amountunknown 100

amountexpected
amountexpected
– 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 Spectrometery
Overview
•
•
•
•
•
Applications of Mass Spectrometry
Mass Spectrometer Components
GC-MS
LC-MS
Other Applications
Mass Spectrometery
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 Spectrometery
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 Spectrometery
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)
• An example of this use is in drug testing to determine if
testosterone is naturally produced or synthetic
Mass Spectrometery
Instrumentation
• Main Components:
– Ion source (more details on subsequent slides)
– Analyzer (more details on subsequent slides)
– Detector: most common is electron multiplier
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