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)