liquid chromatography 1 and solid- phase extraction lecture date: april 9 th, 2008
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Liquid Chromatography 1 and Solid-Phase Extraction
Lecture Date: April 9th, 2008
Reading Material
● Skoog, Holler and Crouch: Ch. 28
● Cazes: Ch. 22, 26
● For those using LC in their work, see:L. R. Snyder, J. J. Kirkland, and J. L. Glajch, “Practical HPLC
Method Development”, 2nd Ed., Wiley, 1997.
Basic LC Terminology
● Adsorption chromatography• The stationary phase is an adsorbent (like silica gel or any
other silica-based packing)• The separation is based on repeated adsorption-desorption
steps.
● Normal-phase chromatography• The stationary bed is strongly polar in nature (e.g., silica gel),
and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran).
• Polar samples are retained on the polar surface of the column packing longer than less polar materials.
● Reversed-phase chromatography • The stationary bed is nonpolar (hydrophobic) in nature, • The mobile phase is a polar liquid, such as mixtures of water
and methanol or acetonitrile. • The more nonpolar the material is, the longer it will be retained.
● Size exclusion chromatography (SEC)• column filled with material having precisely controlled pore
sizes, and the sample is simply sieved or filtered according to its solvated molecular size.
• Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the pores of the packing particles and elute later.
• Also called gel permeation chromatography (GCP) although the stationary phase is not restricted to a "gel"
● Ion-exchange chromatography (IC)• the stationary bed has a charged surface of opposite charge
to the sample ions. • Used almost exclusively with ionic or ionizable samples. • The stronger the charge on the sample, the stronger it will be
attracted to the ionic surface and thus, the longer it will take to elute
• The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time
Basic LC Terminology
Analytical Applications of LC
The “branches” of the LC family:Note – this means analyte polarity
Basic Mechanisms used in LC Separations
High Performance Liquid Chromatography (HPLC)
● HPLC utilizes a high-pressure liquid mobile phase (ca. 100-300 bar) to separate the components of a mixture
● These analytes are first dissolved in a solvent, and then forced to flow through a packed small-particle chromatographic column, where the mixture is resolved into its components
● HP = high pressure and high performance
● Resolution depends upon the extent of interaction between the solute components and the stationary phase
Differences between HPLC and “Classical” LC Small ID (2-5 mm), reusable stainless steel columns Column packings with very small (3, 5 and 10 m)
particles and the continual development of new substances to be used as stationary phases
Relatively high inlet pressures and controlled flow of the mobile phase
Precise sample introduction without the need for large samples
Special continuous flow detectors capable of handling small flow rates and detecting very small amounts
Automated standardized instruments Rapid analysis High resolution From now on, LC refers to HPLC
Advantages and Disadvantages of LC
Advantages:• Speed (minutes)• High resolution• Sensitivity• Reproducibility• Accuracy• Automation
Disadvantages:• Cost• Complexity• Low sensitivity for some compounds• Irreversibly adsorbed compounds not detected• Co-elution difficult to detect
More on Reversed-phase (RP) LC
RP is the most widely used mode of HPLC (75%?)
Separates molecules in solution on basis of their hydrophobicity– Non-polar stationary phase
– Polar mobile phase
In practice: non polar functional group bonded to silica– Stationary phase
functional group bonded to silica this corresponds to a volume (Van deemter) Alkyl groups ( C4, C8, C18) retention increases exp. with chain length
Mobile Phases– Polar solvent (water) with addition of less polar solvent (acetonitrile
or methanol)
The Packed Column and the Stationary Phase
Packed LC columns, usually made of stainless steel and carefully filled with material, are the heart of the LC experiment
The stationary phase fills the column – its properties are critical to the separation
Review of Molecular Interactions
The basis of separations (and most of chemistry)…
Name Energy (kcal/mol) Description
Covalent 100-300Hold molecules together, orbital
overlap
Ionic 50-200 Electrostatic attraction
Polar• Hydrogen bonding• Dipole-dipole-stacking
3-10Vary from electrostatic-type interactions (e.g. hydrogen
bonds) to much weaker
Non-Polar• Van der Waals
(dispersion)1-5 Weak, induced dipole
Retention Mechanisms in LC
● HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process
● Hydrophobic interactions are the main ones in reversed-phase separations
● Dipole-dipole (polar) interactions are dominant in normal phase mode.
● Ionic interactions are responsible for the retention in ion-exchange chromatography.
● Retention in LC is competitive: ● Analyte molecules compete with the eluent molecules for the
adsorption sites. So, the stronger analyte molecules interact with the surface, and the weaker the eluent interaction, the longer analyte will be retained on the surface.
Retention Mechanisms in LC
Remember the elution order! Normal-phase vs. reversed-phase LC
Physical Properties of Stationary Phase Particles
HPLC separations are based on the surface interactions, and depends on the types of the adsorption sites (surface chemistry). Modern HPLC adsorbents are the small rigid porous particles with high surface area.
Key parameters:• Particle size: 3 to 10 µm • Particle size distribution: as narrow as possible, usually within 10%
of the mean• Pore size: 70 to 300 Å• Surface area: 50 to 250 m2/g• Bonding phase density (number of adsorption sites per surface
unit): 1 to 5 per 1 nm2
Electron microphotograph of spherical and irregular silica particles. [W.R.Melander, C.Horvath, Reversed-Phase Chromatography, in HPLC Advances and Perspectives, V2, Academic Press, 1980]
The Most Popular Particle: Silica
Macroporous spherical silica particle. [K.K.Unger, Porous silica, Elsevier, 1979]
Different morphology for different applications:
Different chemistry:
Si OH Si OH O
H
H
Si
OH
OH
Free Silanol Adsorbed Water Geminal Silanol
Si
O
SiO
Dehydrated Oxide Siloxane
O
H
O
H
Si
Si
O
Bound and Reactive Silanols
Chemical Modifications to Silica
Silica (or zirconia, or alumina) by itself cannot do the job needed by modern LC users – it must be functionalized and modified to suit the analytical problem
Residual silanols
SiO
SiO
SiO Si
O
SO
Si
Si OSi
OSiOH
SOH
OHOH i
i
OO
Diagram from Crawford Scientific
Functionalized groups
Chemical Modifications to Silica
Groups are usually attached via reaction of an organosilane (which can be pre-polymerized in solution)
Besides attaching groups, it is also possible to polymerize the silica (or the attached group)
Purpose: stability at low pH, more coverage
– High-carbon load
Monomeric phases are more reproducible (easier reactions to control)
– Monomeric phases are also known as “sterically-protected”
Endcapping: fully react the silica surface, remove silanols and their acidity, more coverage
Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
Common LC Stationary Phases
Name Structure Description
SilicaNormal phase, for separating polar, non-ionic
organics
PropylReversed-phase, for hydrophobic interaction
chromatography (proteins, peptides)
C8Reversed-phase, like C18 but less retentive,
used for pharmaceuticals, steroids, nucleotides
C18Reversed-phase, retains non-polar solutes
strongly. When bonded to 300A silica can be used for large proteins and macromolecules
CyanoReversed-phase and normal-phase, more
polar than C18, unique selectivity
AminoReversed-phase, normal-phase, and weak
anion exchange. RP used to separate carbohydrates
Si C3H7
Si C8H17
Si C18H37
Si CH2CH2CH2CN
Si CH2CH2CH2NH2
Si OH
Common LC Stationary Phases
Name Structure Description
PhenylReversed-phase, retains aromatic
molecules. Also used for HIC (proteins)
Diol
Both reversed-phase and normal-phase utility. Used for RP SEC,
also used for NP separations as a more robust alternative to silica
(not ruined by trace water)
NitroNormal-phase, separates aromatic and alkene-containing molecules
Si NO2
Si O
OH
OH
Si CH2CH2CH2
Polar Stationary Phase Interactions
Sorbents Interactions
CN
NH2
2OH
Dipole/Dipole
Hydrogen-Bonding
Hydrogen-Bonding
OH
Si NH
H
SiN
OH
C
OSi
OH
OOH
H
Source: Crawford Scientific.
Ionic Stationary Phase Interactions
Sorbents Interactions
PRS
CBA
SAX
Electrostatic
Electrostatic
Electrostatic
H3+N
SO3-Si
Si
H3+N
O-
O
N+(CH3)3Si
-O3S
Source: Crawford Scientific.
Non-Polar Stationary Phase Interactions
Sorbents Interactions
C8
PH
C2
van der Waals
van der Waals
van der Waals
Si
Si
Si
Source: Crawford Scientific.
A Good Choice of Stationary Phase Depends on the Analyte
NNHH22
NNHH33++
NNHH22
Functionality Analyte Mechanism
Hydrophobic
H-Bonding
Ionic
Non-Polar
Polar
Ion-Exchange
Source: Crawford Scientific.
More Subtle Effects
Shape selectivity (correlates with stationary phase order), temperature, coverage (and the role of bonding chemistry):
Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
More Subtle Effects
The effects of temperature on the order of the stationary phase are often surprising:
Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
Chiral Stationary Phases
Interactions between chiral analytes (enantiomers and molecules with more than 1 chiral center) and chiral stationary phases are also possible
Normal-phase is most common because of binding modes
A. Berthod, “Chiral Recognition Mechanisms”, Anal. Chem. 78, 2093-2099 (2006).
Chiral Stationary Phases
Interactions between chiral analytes and chiral stationary phases are also possible.
Common chiral stationary phases:
Adapted from L. R. Snyder, J. J. Kirkland, and J. L. Glajch, “Practical HPLC Method Development”, 2nd Ed., Wiley, 1997. Pg 545.
Name Chiral Recognition MechanismAnalyte and Mobile Phase
Requirements
Protein basedHydrophobic and electrostatic
interactionsAnalyte must ionize, helpful if it contains an aromatic. RP only.
Cyclodextrin Inclusion complexation, H-bondingPolar and aromatic groups, RP
and NP.
Polymer-based
carbohydrates
Inclusion interactions, attractive interactions
H-bonding donors/acceptors, steric bulk at chiral center, RP and NP.
PirkleH-bonding, interactions, dipole-
dipole interactionsH-bonding donor/acceptors, mostly
NP.
A Chiral LC Separation Example: separation of
naproxen enantiomers Chiral AGP column
– AGP = 1-acid glycoprotein (orosomucoid), 181 amino acid residues and 14 sialic acid residues
Isocratic (no change in mobile phase composition during separation)
Adapted from L. R. Snyder, J. J. Kirkland, and J. L. Glajch, “Practical HPLC Method Development”, 2nd Ed., Wiley, 1997. Pg 545.
O
HO
(S)
O
(S)-naproxen
O
HO
(R)
O
(R)-naproxen
Ion Chromatography (IC) Form of LC, also known as ion-exchange chromatography Basic mechanism is electrostatic exchange:
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
Typical IC Results
Example: an isocratic method for monovalent cations in ammonium nitrate based explosives
Detection limits 50-100 ppb, max working range 40 ppm
Method:– Sample Loop Volume: 50 µL– Columns: IonPac® CS3 Analytical,
IonPac CG3 Guard– Eluent: 25 mM HCl, 0.1 mM DAP•HCl,
4% Acetonitrile– Eluent Flow Rate: 1.0 mL/min– Suppressor: Cation MicroMembrane™– Suppressor (CMMS)– Regenerant: 100 mM
Tetrabutylammonium Hydroxide– Detector: Conductivity, 30 µS full
scale– Injection Volume: 50 µL
From Dionex Application Note 121R
Mobile Phases in LC
Mobile phases differ for each LC mode– Normal phase solvents are mainly nonpolar– Reversed-phase eluents are usually a mixture of water with some
polar organic solvent such as acetonitrile. Size-exclusion LC has special requirements for mobile phases
– Must dissolve polymers– Must also suppress all possible interactions of the sample molecule
with the surface of the packing material
The type and composition of the mobile phase (eluent) is one of the variables influencing LC separations
Desirable properties:– Purity– Detector compatibility – Solubility of the sample – Low viscosity– Chemical inertness – Reasonable price
Figure from Phenomenex technical literature
Isocratic elution: the eluent composition remains constant as it is pumped through the column during the whole analysis.
Gradient elution: the eluent composition (and strength) is steadily changed during the run.
Control of Eluent Polarity
time
% m
obile
pha
se
k
kNRs
11
4
*
*11
4 k
kNRs
where k* is the k at the midpoint of the column
LC Instrumentation
Pumps, Mixersand Injectors
Column Detector(s) Computer
LC Instrumentation The Agilent 1100, a typical modern LC system
Solvent reservoirs
Solvent degasser
Pump
Autosampler
Column oven
DAD
Review: The Purpose of Key LC Components
column •separation chemistry
detector•signal transduction•amplification/scaling•filtering
A/D•data acquisition•digitization
tubing to detector flow cell
analog output
digital output
chromatogram•digital processing•data analysis
The LC Pump(s)
Modern pumps have the following parameters: Flow rate range: 0.01 to 10 ml/minPressure range from 1-5,000 psi
Pressure pulsations : less than1 %
Types of PumpsConstant pressure pumpsConstant flow pumps
Reciprocating Piston Pump (90% of HPLC’s) small internal volume pulsed flow
Syringe type pumps (Displacement Pumps)limited solvent capacity
Pneumnatic Pumps (pressure)
Temperature Control in LC
Thermoelectric heating/cooling
– the ability of a surface to produce or absorb heat when current is applied across the junction of two dissimilar conductors or semicondeucted
The effect can be reversed (i.e. heating turned to cooling) by reversing the DC current through the junction
Also known as the Peltier effect after its 1834 discoverer, a French watch maker
Overview of LC Detectors
Common HPLC detectors– Refractive Index– UV/Vis
Fixed Wavelength Variable Wavelength Diode Array
– Fluorescence Detector
Less common:– Conductivity– Mass-spectrometric (LC/MS)– Evaporative light scattering (ELSD)
Desirable Features of an LC Detector
1. Low drift and noise level2. High sensitivity (ability to discriminate between
small differences in analyte concentration)3. Fast response4. Wide linear dynamic range5. Low dead volume6. Cell design that eliminates remixing of separated
bands7. Insensitivity to changes in types of solvent, flow
rate, temp8. Operational simplicity and reliability9. Non-destructive
Baseline Noise and Drift
Detector Response
The definition of detector response depends on whether it is mass sensitive or concentration sensitive
Mass sensitive mV/mass/unit timeR = hw/sM
Concentration sensitive mV/mass/unit volumeR = hwF/sM
h = peak height mV W = width at .607 of heightF = flow rateM = mass of solutes = chart speed
Cell Efficiency
Example:column 15,000 plates15 cm long2 min tR
2 ml at 1 ml /minpeak width of 80uLflow cell of 20 ul
only four measurements
Things to note:parallel light beamflow cell volume <1/10 of peak volumeoptimization of cell geometry
Ultraviolet/Visible Spectroscopic Detectors
infrared (IR) 2,500 - 50,000 nm
near infrared 800 - 2,500 nm
visible 400 - 800 nm
ultraviolet (UV) 190 - 400 nm
Any chemical compound could interact with the electromagnetic field. Beam of the electromagnetic radiation passed through the detector flow-cell will experience some change in its intensity due to this interaction. Measurement of this changes is the basis of the most optical HPLC detectors.
Name Chromophore Wavelength [nm] Molar extinction,
acetylide -C=C 175-180 6,000
Aldehyde -CHO 210 1,500
amine -NH2 195 2,800
azo -N=N- 285-400 3-25
bromide -Br 208 300
carboxyl -COOH 200-210 50 - 70
disulphide -S-S- 194 5,500
ester -COOR 205 50
ether -O- 185 1,000
ketone >C=O 195 1,000
nitrate -ONO2 270 12
nitrile -C=N 160 -
nitrite -ONO 220 - 230 1000-2000
nitro -NO2 210 strong
Fixed / Variable Wavelength Detectors
mercury vapor lamp emit very intense light at 253.7 nm. By filtering out all other emitted wavelengths, manufacturers have been able to utilize this 254 nm line to provide stable, highly sensitive detectors capable of measuring subnanogram quantities of any components which contains aromatic ring. The 254 nm was chosen since the most intense line of mercury lamp is 254 nm, and most of UV absorbing compounds have some absorbance at 254 nm.
Diode Array Detectors
Diode array detectors can acquire all UV-Visible wavelengths at once.
Advantages:
– Sensitivity (multiplex)
– Speed
Disadvantages:
– Resolution
Figure from Skoog, et al., Chapter 13
Other Detectors
Fluorescence Detector
Electrochemical Detector
Evaporative Light Scattering
Putting it All Together: LC Method Development
The importance – without a good method:– Co-elution can be missed– Unable to detect/assay key components
Basic consequences of method changes:
Choosing an LC Approach
Goals of a separation:
– Resolution (Rs) > 1.5
– Short separation time (5-30 minutes)
– Good quantitative precision/accuracy
– Acceptable backpressure
– Narrow peaks
– Minimal solvent use
Overall Strategy
First select an appropriate method
If LC is best, then determine nature of the sample
“Exploratory” RP runs, i.e. fast simple gradients with C18 phases, are usually helpful in assessing retention and polarity
Solid-phase Extraction (SPE)
What is SPE?– The separation of an analyte or analytes from a mixture of
compounds by selective partitioning of the compounds between a solid phase (sorbent) and a liquid phase (solvent)
Comparison with conventional liquid-liquid extraction (e.g. the organic sep funnel approach):
– SPE: selective towards functional groups (better)
– LLE: selective towards solubility
– SPE: more choices because no miscibility (better)
– LLE: must avoid miscible solvents
– SPE: concentrates analytes (better)
– LLE: can concentrate analyte after stripping
The Typical SPE Process
Conditioning: solvates the sorbent
Equilibration: removes excess conditioning solvent, matches with analytical conditions (prevents “shock”)
Sample Application
Interference Elution
Analyte Elution
Column Conditioning
Column Equilibration
Solid-phase Extraction
Conditioning the cartridge:
Not conditioned Conditioned
SPE cartridges have a range of chemistries that are often similar to those of LC stationary phases, but are optimized for adsorption/desorption
Solid-phase Extraction
Automated SPE systems for sample cleanup – the Spark SymbiosisTM
Images from www.sparkholland.com
Can be hyphenated with LC, MS, NMR, etc… or used as a stand-alone sample pretreatment
Homework and Further Reading
Homework problems (for study only):
– 28-2, 28-3, 28-11, 28-14
For a detailed discussion of method development in LC:
– L. R. Snyder, J. J. Kirkland, and J. L. Glajch, “Practical HPLC Method Development”, 2nd Ed., Wiley, 1997.
For recent advances in understanding gradient elution, see:
– P. Nikitas and A. Pappa-Louisi, Anal. Chem., 2005, 77, 5670-5677 (a new derivation of the equation of reversed-phase HPLC gradient elution)
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