Topic 13 Liquid Chromatography & Solid-Phase Extraction

7/29/2019 Topic 13 Liquid Chromatography & Solid-Phase Extraction

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SKA6014ADVANCED ANALYTICAL CHEMISTRY

TOPIC 16

Liquid Chromatography and Solid-phase

Extraction

Azlan Kamari, PhDDepartment of Chemistry

Faculty of Science and Mathematics

Universiti Pendidikan Sultan Idris


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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-desorptionsteps.

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 water

and methanol or acetonitrile.

The more nonpolar the material is, the longer it will be retained.


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Size exclusion chromatography (SEC) column filled with material having precisely controlled pore

sizes, and the sample is simply sieved or filtered 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 and

ionic strength are used to control elution time

Basic LC Terminology


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Analytical Applications of LC The branches of the LC family:

Note this means analyte polarity


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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-particle

chromatographic column, where the mixture is resolvedinto its components

HP = high pressure and high performance

Resolution depends upon the extent of interactionbetween the solute components and the stationaryphase


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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) particlesand the continual development of new substances to be usedas 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 smallflow 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


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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 and

carefully filled with material, are the heart of the LCexperiment

The stationary phase fills the column its properties arecritical to the separation


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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 retentionprocess

Hydrophobicinteractions are the main ones inreversed-phase

separations Dipole-dipole (polar) interactions are dominant innormal phasemode.

Ionic interactions are responsible for the retention inion-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
http://hplc.chem.shu.edu/HPLC/glossary/g_fgh.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_mno.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_ijl.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_ijl.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_ijl.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_ijl.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_mno.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_fgh.html


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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 surface

unit): 1 to 5 per 1 nm2


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Electron micr ophotograph of spherical and ir regular sili ca 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

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 the analytical problem

Residualsilanols

S iO

S iO

S iO

S iO

SO

S i

S i OS i

OS iO H

S

O HO H

OH i

i

OO

Diagram from Crawford Scientific

Functionalizedgroups


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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 theattached group)

Purpose:stability at low pH, more

coverage 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 PhasesName Structure Description

Silica Normal phase, for separating polar, non-ionicorganics

PropylReversed-phase, for hydrophobic interaction

chromatography (proteins, peptides)

C8 Reversed-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


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Common LC Stationary Phases

Name Structure Description

PhenylReversed-phase, retains

aromatic molecules. Alsoused for HIC (proteins)

Diol

Both reversed-phase andnormal-phase utility. Used for

RP SEC, also used for NPseparations as a more robustalternative to silica (not ruined

by trace water)

Nitro

Normal-phase, separates

aromatic and alkene-containing moleculesSi 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.


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Ionic Stationary Phase Interactions

Sorbents InteractionsPRS

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

Sorbents Interactions

C8

PH

C2

van der Waals

van der Waals

van der Waals

Si

Si

Si



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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):



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More Subtle Effects

The effects of

temperature on theorder of thestationary phaseare often surprising:



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Chiral Stationary Phases

Interactions between chiral analytes (enantiomers andmolecules 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).


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Chiral Stationary Phases

Interactions between chiral analytes and chiral stationaryphases are also possible.

Common chiral stationary phases:

Name Chiral Recognition MechanismAnalyte and Mobile Phase

Requirements

Protein

based

Hydrophobic and electrostatic

interactions

Analyte must ionize, helpful if it

contains an aromatic. RPonly.

CyclodextrinInclusion complexation, H-

bondingPolar and aromatic groups, RP

and NP.

Polymer-

basedcarbohydrat

es

Inclusion interactions, attractiveinteractions

H-bonding donors/acceptors,steric bulk at chiral center, RPand NP.

PirkleH-bonding, interactions,dipole-dipole interactions

H-bonding donor/acceptors,mostly NP.


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A Chiral LC Separation Example: separation of

naproxen enantiomers

Chiral AGP column AGP = 1-acid glycoprotein

(orosomucoid), 181 aminoacid residues and 14 sialicacid residues

Isocratic (no change in mobile

phase composition duringseparation)

O

HO

(S)

O

(S)-naproxen

O

HO

(R)

O

(R)-naproxen


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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 ammoniumnitrate based explosives

Detection limits 50-100 ppb, maxworking 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 mMTetrabutylammonium Hydroxide

Detector: Conductivity, 30 S fullscale

Injection Volume: 50 L

From Dionex Application Note 121R


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Mobile Phases in LC

Mobile phases differ for each LC mode Normal phasesolvents are mainly nonpolar Reversed-phaseeluents are usually a mixture of water with some

polar 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
http://hplc.chem.shu.edu/HPLC/glossary/g_mno.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_s.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_s.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_s.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_s.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_pr.htmlhttp://hplc.chem.shu.edu/HPLC/glossary/g_mno.html


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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) issteadily changed during the run.

Control of Eluent Polarity

time

%m

obilephase

kkNR

s11

4

*

*11

4 k

kNR

s

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


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LC Instrumentation

Pumps, Mixers

and InjectorsColumn Detector(s) Computer

LC Instrumentation


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LC Instrumentation

The Agilent 1100, a typical modern LC system

Solvent reservoirs

Solvent degasser

Pump

Autosampler

Column oven

DAD


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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/min

Pressure range from 1-5,000 psiPressure pulsations : less than1 %

Types of PumpsConstant pressure pumps

Constant flow pumps

Reciprocating Piston Pump (90% of HPLCs)

small internal volumepulsed flow

Syringe type pumps (Displacement Pumps)limited solvent capacity

Pneumnatic Pumps (pressure)


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Temperature Control in LC

Thermoelectric heating/cooling

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

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)


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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 range

5. 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

D t t R


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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 volumeR = 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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Cell Efficiency

Example:

column 15,000 plates15 cm long

2 min tR2 ml at 1 ml /min

peak width of 80uL

flow cell of 20 ul

only four measurements

Things to note:parallel light beam

flow cell volume


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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, eacetylide -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


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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. The254 nm was chosen since the most intense line ofmercury lamp is 254 nm, and most of UV absorbingcompounds have some absorbance at 254 nm.


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Diode Array Detectors

Diode array detectors can acquire all UV-Visible

wavelengths at once.

Advantages:

Sensitivity

(multiplex) Speed

Disadvantages:

Resolution


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Other Detectors

Fluorescence Detector

Electrochemical Detector

Evaporative Light Scattering


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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:


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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


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Overall Strategy

First select an

appropriate method If LC is best, then

determine nature ofthe sample

Exploratory RP runs,

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 ofcompounds 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


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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



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Automated SPE systems for sample cleanup the SparkSymbiosisTM

Can be hyphenatedwith LC, MS, NMR,

etc or used as astand-alone samplepretreatment

Topic 13 Liquid Chromatography & Solid-Phase Extraction

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