Transcript Oct. 28 Lecture Notes

Chem. 230 – 10/28 Lecture
Announcements I
• HW Set #3 – due today (short answer problems have
been posted)
• Next Exam: Topics + format (still can bring 3” x 5”
notecard)
– Gas Chromatography
– Supercritical Fluid Chromatography
– HPLC (everything except detection)
• Return Application Abstracts
– missing a few
– most looked good; a few seemed to be focused on
technology rather than application to a particular problem
(check to see if you need to improve on your abstract)
Announcements II
• Today’s Lecture
– HPLC
• covered so far: classification, packing material geometry
and composition, gradient elution, size exclusion
chromatography, ion exchange chromatography
• Instrumentation (mobile phase selection and delivery,
injection, column dimensions, detection)
• Aerosol-Based Detectors (in more detail)
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
• Mobile Phase Selection
– See slide 18 of lecture for factors influencing selection
of mobile phase
– Solvents must meet purity requirements (for column
and detector functions)
– Solvent selectivity issue is important because:
• Changing solvent affects retention for different analytes
differently
• HPLC is less efficient than GC so often more likely to have
overlapping peaks
• Changes in pH also are important for acidic/basic compounds
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
Less retention
OH
H3C
CH3
O
O
O
More
retention
– RP-HPLC Separation of
syringols from guaiacols
– Difference is in 2nd MeOH
group
– Water/Acetonitrile eluents
produce poor
syringol/guaiacol
separation factors
– Water/Methanol works
better (although greater
retention with MeOH of
syringol is counter intuitive)
OH
R
Syringols
Guaiacols
HPLC-UV
HPLC Sample
Sample11(MeOH/0.1%TFA)
(ACN/0.1%TFA)
350
350
300
300
250
250
200
200
150
150
100
100
5050
00
-50
-50
00
55
CH3
R
Absorbance
Absorbance
• Example of solvent
changes to affect
selectivity:
1010
Time
(minutes)
Time
(minutes)
1515
acetovanillone
acetosyringone
acetosyringone
acetovanillone
cinnamic
cinnamicacid
acid
isoeugenol
isoeugenol
syringic
syringicacid
acid
20 20
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
• Optimization of Mobile
Phase Composition
– Separation should be
perfomed on three different
water/organic systems
– Then additional
separations can be carried
out using 3 component
mobile phases
– Patterns in retention can be
used to optimize mobile
phase composition
Acetonitrile
(40% in water)
20% ACN, 25%
MeOH, water
Methanol (50%
in water)
THF (30% in
water)
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
• Mobile Phase Selection – pH
Buffering
– In reversed-phase HPLC,
solute generally must be nonionized to be retained
– pH is adjusted by adding
buffer in water/organic
modifier
– pH at pKa means retention
factor about half of nonionized acid retention factor
– In ion-exchange
chromatography, pH should
be in range needed to
produce ions
– In ion-pairing RP-HPLC, an
ion-pairing reagent is added
O
O
OH
O
retained
-
unretained
+
NHNH
2 3
O
-
O
S
O
CH3
pair reagent = pentane
Benzyl amine Ion
(conj.
sulfonic acid (sodium salt)
acid pKa = 9.35)
Non-ionized only at
high pH
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
• Solvent Flow
– HPLC requires high pressures and thus
specific pumps
– The solvent also needs low levels of dissolved
gases for pumps to function (through solvent
degassing)
– For the simplest “dedicated” HPLC, a single
solvent reservoir and pump is needed
– For gradients and/or more method
development work, switching between
different solvents is needed
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
• Pumps
– Most pumps use two piston
heads 180º out of phase to
reduce pressure
fluctuations
– Solvents go into and out of
piston heads through oneway “check valves”
– Exit check valve closes on
“in” stroke and entrance
check valve closes on “out”
stroke
Check valves
In
Stroke
Out
Stroke
closed
open
closed
open
pistons
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
16000
Signal (uV)
14000
12000
10000
8000
6000
4000
2000
0
-2000
7
7.5
8
8.5
9
9.5
10
Time (min)
200
100
Signal (uV)
• Example of pump with
non-functioning check
valves
• Fluctuation in
pressure and signal
can occur
• Changes to retention
time also will occur
0
-100
-200
-300
8
8.2
8.4
8.6
Time (min)
Bad check valve leaking
8.8
9
9.2
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
• Solvent Flow (for gradient/greater flexibility
operations)
– Dual Pumps (high pressure mixing)
– Low Pressure Mixing (stream “open” in proportion to
fraction)
To column
To column
pump
pumps
Mixing chamber
Liquid Chromatography
Instrumentation – Injection
• Fixed Loop Injectors (see GC slides for diagram)
– Used in almost all cases
– For some injectors, partial filling of loop is possible (Vinj < Vloop),
but then filling precision must be good
– Special injection valves needed for small injections (< 1 to 5 μL)
– Small injections often needed for microbore columns
• Other Injectors
– Traps replace loops (can be used if sample is in weak solvent)
– SPME (not as common as for GC but with solvent removing
trapped compounds)
– SPME requires special injector
Liquid Chromatography
Instrumentation – Injection
• Sample Matrix
– Best chromatography solvent – should be weaker than mobile
phase, particularly for larger volume injections
– Remember, weaker solvent allows on-column concentrating
– With traps, sample must have weaker solvent, but must be
pulled off with significantly stronger solvent so pulled off in
narrow injection plug
– Other concern can be solvent miscibility and solute solubility
(example: in reversed phase HPLC, water is a good solvent, but
many compounds such as aromatic compounds have limited
solubility in weakest solvents)
Liquid Chromatography
Instrumentation – Columns
• Column dimensions
– Length: balance between flow,
pressure and efficiency
– Diameter:
• Choice depends on separation
purpose
• Preparative for isolation of
larger quantities
• Microbore usually results in
smaller mass detection limits
but greater concentration
detection limits (good when
limited sample)
• Special care is needed using
microbore with sample
injection, pump stability, and
extra-column broadening
(tubing diameter and fitting
connections)
Type
Diameter
(mm)
Typical
Flow Rate
(mL/min)
Preparative >7.8
>3
Analytical
4.6
1
Microbore
<1
< 0.05
Liquid Chromatography
Instrumentation – Columns
• Column dimensions
– Equation for extra-column broadening:
2
2
2
Wtot2  Wcol
 Winj2  Wtubing
 Wdet
ector
– Extra-Column broadening is more of a
problem when using 1) low H columns,
2) early eluting peaks (where Wcol is
small)
– Demonstration of Extra-Column
Broadening on Narrowest Bore Columns
– note: W can have time or volume
dimensions, but hard to get very small
volume W for tubing, and some
detection
Liquid Chromatography
Some Questions
1.
2.
3.
4.
5.
6.
7.
A student is running a RP-HPLC separation using methanol and
water. The selectivity (a value) is not good. He decides to
switch to ethanol in water. Is this a good decision?
A chemist is planning on purchasing an HPLC instrument for
developing isocratic analysis methods. Is there an advantage to
being able to select multiple solvents?
In order to decrease H in a column, which column or packing
material dimension should be changed? and in which direction?
Why would one want to go to a microbore HPLC system?
Why is the decrease in H observed often less than predicted when
using smaller diameter packing material or small diameter
columns?
If injecting large volumes of a sample containing trace levels of
benzoic acid in water for a reversed phase separation, will it make
any difference what the pH of the sample is?
In anion exchange chromatography, what type of sample would
allow on-column trapping? What type of samples would give
broad peaks if using large injection volumes?
Liquid Chromatography
Instrumentation – Detectors
• Some Generalizations
– Relative to GC, HPLC detectors perform poorly and cost more
• Universal Type
–
–
–
–
UV absorption (also considered selective)
Refractive Index
Aerosol-based detectors (will cover later)
Conductivity (for ion chromatography)
• Selective Type
– Fluorescence
– Electrochemical
• Hyphenated Detectors
– Photodiode Array Detector (type of UV detector)
– Mass Spectrometer
Liquid Chromatography
Instrumentation – Detectors
• UV Absorption Detectors
– The most common type of detector
– Principle: absorption of ultraviolet (or visible) light
– Follows Beer’s Law: A = -log(I/Io) = εbC
•
•
•
•
Light beam
I = intensity of light (Io for blank)
ε = molar absorptivity (constant)
b = path length
C = concentration
b
Cell
– Best results for 0.001 < A < 1
– Fast response – sensitivity trade off in path length
(can select cell volumes)
Liquid Chromatography
Instrumentation – Detectors
• UV Absorption Detectors
– Sensitivity to Compounds (ε values)
• Best for compounds with conjugated double bonds,
aromatic groups or strongly absorbing functional groups
(e.g. R-NO2, R-I, R-Br)
• Poor response for compounds with few or weakly
absorbing functional groups (worst for R-CN, R-NH2, R-F;
poor for R-OR’, R-OH, R-COOH, R-COOR’)
– Solvents:
• Requires use of solvents that absorb poorly in UV
Liquid Chromatography
Instrumentation – Detectors
• UV Absorption Detectors
solvent
– Wavelength Selection:
• Must choose λ > solvent
cut-offs
• Most compounds absorb
strongly at short
wavelengths but many
also absorb less
moderately at longer
wavelengths
• More sensitivity at shorter
wavelengths (provided
little mobile phase
absorption)
• More selectivity at longer
wavelengths
analyte
180
220
260
Wavelength (nm)
Sensitive λ
More selective λ
Liquid Chromatography
Instrumentation – Detectors
• UV Absorption Detectors
– General Properties
•
•
•
•
Reasonably good (but variable) sensitivity
Good linearity, reproducibility
Good stability (but baseline drift and warm up time)
Poor as a universal detector
– Types:
• Fixed wavelength (absorption at single wavelength)
• Variable wavelength (can select one wavelength using
monochromator)
• Photodiode array (can measure at multiple wavelengths
simultaneously) – these give some qualitative information
and allow more peak overlap
Liquid Chromatography
Instrumentation – Detectors
• Application of UV Detection to Weak
Absorbers
– Use short wavelengths (method must be
selective; not always effective)
– Derivatize compounds to add strong absorber
(common for amino acids, carbohydrates)
– Use indirect UV absorption (absorber added to
eluent, analytes displace eluent and give
negative peak)
Liquid Chromatography
Instrumentation – Detectors
• Refractive Index Detectors
– Principle:
• liquids with different refractive index will diffract light
differently
• Composition will determine refractive index
• Any compound with a refractive index different than the
solvent’s is detectable
– Advantage:
• Most universal detector (can detect weakly absorbing
compounds)
– Disadvantages:
• Gradients are not possible
• Requires thermal stability
• Generally not very sensitive
Liquid Chromatography
Instrumentation – Ion Exchange Chromatography
• Types of Instruments:
– Single column
– With analytical plus suppressor columns
• Detection in Single Column Instruments
– Other detection methods (fairly common)
– Conductivity detection
• Conductivity Detector
From HPLC column
– Resistance measured (AC circuit)
– Conductivity = 1/(resistance)
– Ions in solution create conductance
– Conductivity depends on ion
concentration and size
Conductivity
cell
Electronics
Liquid Chromatography
Instrumentation – IC
• Difficulties with single column
instruments:
– Both analytes and ion exchanger conduct
electricity
– High concentration of ion exchanger means
high background conductance and difficulties
in detecting small concentrations of analytes
– Often large ions used as exchanger (such as
potassium hydrogen phthalate for anion
exchange)
Liquid Chromatography
Instrumentation – IC
• Suppressor Columns
– The purpose of the suppressor is to convert the ion exchanger to
mostly non-ionic compounds
– Example below with sodium bicarbonate eluent (Na+HCO3- is the
ion exchanger) in anion exchange
– In the suppressor column, Na+ is replaced with H+
– This converts conductive Na+HCO3- to non-conductive H2CO3
– NaCl is converted to HCl (still conductive)
– Na2HPO4 is converted to H+H2PO4- (less conductive)
– This reduces the baseline and increases sensitivity
Na+HCO3Separation column
Suppressor Column
To Conductivity detector
Na+2HPO42-
Na+Cl-
H+Cl-
H+ In; Na+ out
Liquid Chromatography
Instrumentation – Detectors
• Electrochemical Detectors
– Principle:
• Redox reactions occur at electrodes
following column
• Potential cycle used to periodically
oxidize/reduce analytes at electrode
• Current depends on concentration
of analyte being reduced or oxidized
(similar to A in UV detector)
• Electrode potential determines
classes of compounds that are
detectable (similar to λ in UV
detector)
From column
Analyte
electrode
Reference
electrode
Voltage supply/
electrometer
Liquid Chromatography
Instrumentation – Detectors
• Electrochemical Detector
– Advantages:
• Very sensitive (limits of detection under 1 pg possible)
• Adjustable selectivity
• Wide range of compounds can be detected (including UV
inactive compounds)
• Advantageous for microbore
– Disadvantages
• Electrode fouling
• Variable analyte response
• Requires ions to “complete circuit”
– Array Detectors:
• Can have multiple electrodes in detector (set to different
potentials)
Liquid Chromatography
Instrumentation – Detectors
• Fluorescence Detectors
– Detection Principle:
• Light promotes molecules to
excited electronic state
• Excited molecules transition from
lowest excited state back to the
ground state and emit light in the
process
M + hν → M*
M* → M*’ (lower vibrational level)
M*’ → M + hν’
Light Source
– Equipment:
• High intensity light source
• Filters or monochromators to
select wavelengths (before and
after cell)
• Sensitive light detector
Filter or
monochromator
Light
detector
Liquid Chromatography
Instrumentation – Detectors
• Fluorescence Detectors
– Advantages:
• Greater sensitivity possible (for molecules with high fluorescence
efficiencies) because easy to detect small signal against zero background
(see below)
• Much greater selectivity because few molecules fluoresce, particularly at
selected wavelengths
– Disadvantages:
• Limited to relatively few molecules (although derivatization is also possible)
Absorption of light
95% transparent
(equiv. to A = 0.022)
Emission of light
Weak light in black
background
Liquid Chromatography
Detector Questions
1.
2.
3.
A compound has an absorptivity of 493 M-1 cm-1 at 210
nm and 32 M-1 cm-1 at 280 nm. Why would one even
consider setting the wavelength to 280 nm?
Describe one way to use a UV detector for detecting
weakly absorbing organic compounds.
Describe how you could use a photodiode array
detector to determine if the odd shaped peak below is
from one or multiple compounds.
A (254 nm)
Time
Liquid Chromatography
More Questions
1.
2.
3.
4.
Why is electrochemical detection difficult to use with
non-bonded silica HPLC?
When weakly absorbing compounds are derivatized, it
is more common to use fluorescent derivatizing
agents. Why is this?
What is the advantage of using suppression in ion
chromatography?
Why is suppressed ion chromatography not so useful
for weak acid anions vs. strong acid anions?
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?