Liquid Chromatography 1

8/21/2019 Liquid Chromatography 1

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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 HPLCMethod Development, 2nd Ed., Wiley, 1997.


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Basic LC Terminology

Adsorption chromatography

The stationary phase is an adsorbent (like silica gel or any othersilica-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 ortetrahydrofuran).

Polar samples are retained on the polar surface of the columnpacking 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 waterand 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 f iltered according toits solvated molecular size.

Larger molecules are rapidly washed through the column;smaller molecules penetrate inside the pores of the packingparticles 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 taketo elute

The mobile phase is an aqueous buffer, where both pH andionic strength are used to control elution time

Basic LC Terminology


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Analytical Appl ications of LC

The branches of the LC family:Note this means analyte polarity

Basic Mechanisms used in LC Separations


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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 thenforced to flow through a packed small-particlechromatographic column, where the mixture is resolvedinto its components

HP = high pressure and high performance

Resolution depends upon the extent of interaction

between the solute components and the stationaryphase

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 newsubstances to be used as stationary phases

Relatively high inlet pressures and controlled flow of themobile phase

Precise sample introduction without the need for largesamples

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


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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 theirhydrophobicity 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)


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The Packed Column and the Stationary Phase

Packed LC columns, usually made of stainless steel andcarefully filled with material, are the heart of the LCexperiment

The stationary phase fills the column its properties arecritical 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-typeinteractions (e.g. hydrogen

bonds) to much weaker

Non-Polar Van der Waals

(dispersion)1-5 Weak, induced dipole


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Retention Mechanisms in LC

HPLC is a dynamic adsorption process. Analyte molecules, whilemoving through the porous packing bead, tend to interact with thesurface adsorption sites. Depending on the HPLC mode, the differenttypes of the adsorption forces may be included in the retention process

Hydrophobic interactions are the main ones in reversed-phaseseparations

Dipole-dipole (polar) interactions are dominant in normal phase mode.

Ionic interactions are responsible for the retention in ion-exchangechromatography.

Retention in LC is competitive:

Analyte molecules compete with the eluent molecules for theadsorption sites. So, the stronger analyte molecules interact withthe surface, and the weaker the eluent interaction, the longeranalyte will be retained on the surface.

Retention Mechanisms in LC

Remember the elution order! Normal-phase vs. reversed-phase LC


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Physical Properties of Stationary Phase Particles

HPLC separations are based on the surface interactions,and depends on the types of the adsorption sites (surfacechemistry). Modern HPLC adsorbents are the small rigidporous particles with high surface area.

Key parameters: Particle size: 3 to 10 m

Particle size distribution: as narrow as possible, usually within 10%of the mean

Pore size: 70 to 300 Surface area: 50 to 250 m2/g

Bonding phase density (number of adsorption sites per surfaceunit): 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, 1 979]

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

Si

O

DehydratedOxide Siloxane

O

H

O

H

Si

Si

O

Bound andReactiveSilanols


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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 theanalytical problem

Residualsilanols

SiO

SiO

SiO

SiO

SO

Si

Si OSi

OSiOH

S

OHOH

OH ii

O

O

Diagram from Crawford Scientific

Functionalizedgroups

Chemical Modifications to Silica

Groups are usually attached via reactionof an organosilane (which can be pre-polymerized in solution)

Besides attaching groups, it is alsopossible to polymerize the silica (or theattached group)

Purpose: stability at low pH, morecoverage

High-carbon load

Monomeric phases are morereproducible (easier reactions to control)

Monomeric phases are also knownas sterically-protected

Endcapping: fully react the silicasurface, remove silanols and theiracidity, more coverage

Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861


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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 beused 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 separatecarbohydrates

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 amore robust alternative to silica

(not ruined by trace water)

NitroNormal-phase, separates aromaticand alkene-containing molecules

Si NO2

Si O

OH

OH

Si CH2CH2CH2


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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


PRS

CBA

SAX

Electrostatic

Electrostatic

Electrostatic

H3+N

SO3-Si

Si

H3+N

O-

O

N+(CH3)3Si

-O3S



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Non-Polar Stationary Phase Interactions


C8

PH

C2

van der Waals

van der Waals

van der Waals

Si

Si

Si


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



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More Subtle Effects

Shape selectivity (correlates with stationary phase order), temperature,coverage (and the role of bonding chemistry):


More Subtle Effects

The effects oftemperature on theorder of the stationaryphase are oftensurprising:



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Chiral Stationary Phases

Interactions between chiral analytes (enantiomers and

molecules with more than 1 chiral center) and chiralstationary 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 stationaryphases 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 itcontains an aromatic. RP only.

Cyclodextrin Inclusion complexation, H-bondingPolar and aromatic groups, RP

and NP.

Polymer-basedcarbohydrates

Inclusion interactions, attractiveinteractions

H-bonding donors/acceptors, stericbulk at chiral center, RP and NP.

PirkleH-bonding, interactions, dipole-

dipole interactionsH-bonding donor/acceptors, mostly

NP.


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A Chiral LC Separation

Example: separation ofnaproxen enantiomers

Chiral AGP column AGP = 1-acid glycoprotein

(orosomucoid), 181 aminoacid residues and 14 sialicacid residues

Isocratic (no change in mobilephase composition duringseparation)

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.


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Typical IC Results

Example: an isocratic method formonovalent cations in ammonium

nitrate based explosives Detection limits 50-100 ppb, max

working range 40 ppm Method:

Sample Loop Volume: 50 L Columns: IonPac CS3 Analytical,

IonPac CG3 Guard Eluent: 25 mM HCl, 0.1 mM DAPHCl,

4% Acetonitrile Eluent Flow Rate: 1.0 mL/min Suppressor: Cation MicroMembrane Suppressor (CMMS) Regenerant: 100 mM

Tetrabutylammonium Hydroxide

Detector: Conductivity, 30 S fullscale Injection Volume: 50 L

From Dionex Application Note 121 R

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 somepolar organic solvent such as acetonitrile.

Size-exclusionLC 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 mobilephase (eluent) is one of the variablesinfluencing LC separations

Desirable properties: Purity Detector compatibility Solubility of the sample Low viscosity Chemical inertness Reasonable price

Figure from P henomenex technical literature


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Isocratic elution: the eluent composition remains constantas it is pumped through the column during the wholeanalysis.

Gradient elution: the eluent composition (and strength) issteadily changed during the run.

Control of Eluent Polarity

time

%m

obilephase

k

kNR

s

11

4

*

*

11

4 k

kNRs

where k* is the k at the midpoint of the column

LC Instrumentation

Pumps, Mixers

and InjectorsColumn Detector(s) Computer


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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

detectorsignal transductionamplification/scalingfiltering

A/Ddata acquisitiondigitization

tubing to detector flow cell

analog output

digital output

chromatogramdigital processingdata analysis


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The LC Pump(s)

Modern pumps have the following parameters:

Flow rate range: 0.01 to 10 ml/minPressure range from 1-5,000 psiPressure pulsations : less than1 %

Types of PumpsConstant pressure pumpsConstant flow pumps

Reciprocating Piston Pump (90% of HPLCs)small internal volumepulsed flow

Syringe type pumps (Displacement Pumps)limited solvent capacity

Pneumnatic Pumps (pressure)

Temperature Control in LC

Thermoelectric heating/cooling

the ability of a surface toproduce or absorb heatwhen current is appliedacross the junction of twodissimilar conductors orsemicondeucted

The effect can be reversed (i.e.heating turned to cooling) byreversing the DC currentthrough the junction

Also known as the Peltier effectafter its 1834 discoverer, aFrench watch maker


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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


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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 time

R = hw/sM

Concentration sensitive mV/mass/unit volume

R = hwF/sM

h = peak height mV

W = width at .607 of heightF = flow rate

M = mass of solute

s = chart speed


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Fixed / Variable Wavelength Detectors

mercury vapor lamp emit very intense light at 253.7nm. By filtering out all other emitted wavelengths,manufacturers have been able to utilize this 254 nmline to provide stable, highly sensitive detectorscapable of measuring subnanogram quantities ofany components which contains aromatic ring. The

254 nm was chosen since the most intense line ofmercury lamp is 254 nm, and most of UV absorbingcompounds have some absorbance at 254 nm.

Diode Array Detectors

Diode array detectors can acquire all UV-Visiblewavelengths at once.

Advantages:

Sensitivity(multiplex)

Speed

Disadvantages:

Resolution

Figure from Skoog, et al., Chapter 13


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Other Detectors

Fluorescence Detector

Electrochemical Detector

Evaporative Light Scattering

Putting i t 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:


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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 anappropriate method

If LC is best, thendetermine nature ofthe sample

Exploratory RPruns, i.e. fast simplegradients with C18phases, are usuallyhelpful in assessingretention and polarity


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Solid-phase Extraction (SPE)

What is SPE? The separation of an analyte or analytes from a mixture of

compounds by selective partitioning of the compoundsbetween 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)

SampleApplication

InterferenceElution

AnalyteElution

ColumnConditioning

ColumnEquilibration


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Solid-phase Extraction

Conditioning the cartridge:

Not conditioned Conditioned

SPE cartridges have a range of chemistries that are oftensimilar to those of LC stationary phases, but are optimizedfor adsorption/desorption

Solid-phase Extraction

Automated SPE systems for sample cleanup the SparkSymbiosisTM

Images from www.sparkholland.com

Can be hyphenated

with LC, MS, NMR,etc or used as astand-alone samplepretreatment


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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, PracticalHPLC 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 ofreversed-phase HPLC gradient elution)

Liquid Chromatography 1

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