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Liquid Chromatography

Instrumentation

Detection in LC Method development Environmental applications

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Classification of Different Chromatographies

According to Mobile and Stationary Phase Type

Gas

Liquid

Liquid

GLC

Solid

Liquid

LLC

Adsorbent GSC Mol sieve GSC

Adsorbent

LSC

Bonded

Phase

BPC

Size Exclusion

SEC

Ion exchange

IEC

Solid

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Choosing Liquid and Solid Phases

Liquid phase variables Aqueous (polar)

Salt solutions pH

Non-aqueous (non-polar) Mixed

Stationary phase characteristics: Thin layer and columns

Particle size: small to coarse Pressure: capillary action, gravity, low to

high Quantity: analytical (ng) to preparative (kg)

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Schematic of HPLC Unit

Gradient Controller

Pump

SampleIntroduction

Column Detector

Dataoutput

%MeOHin Water

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High Pressure Loop-Injector

Sampleloop

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Separation Modes

Adsorption chromatography Reversed-Phase chromatography

Ion-exchange chromatography Ion-pair chromatography Size-exclusion chromatography

Affinity chromatographyhttp://www5.gelifesciences.com/aptrix/upp00919.nsf/Content/LabSep_EduC

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Structure of water and water clusters

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www.chem1.com/acad/sci/aboutwater.html

Water/surface interactions

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Hydrophobicity

Liquid hexane in equilibrium with water

Hexane solubility in water: 1.8 x 10-4 mol/l at 303 K. Free energy gain for the transfer of one mole of hexane frompure hexane into aqueous solution:

+ 33 kJ/mol. Enthalpy and entropy change both negative. Rationalization in terms of the flickering water model: hexane

interacts with water less strongly than water with itself; hexaneforces the creation of clusters, which are represent a higherorder, i.e., less entropy.

Hydrophobicbonds are associations that are drivenby a gain in entropy of the water structure.

(Hexane)org = (hexane)liquid

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Molecular Mass > 2000

in organic solvents in water

< 30 nm 30 400 nm

Non-ionic orIon-paired Ionic

< 30 nm 30 - 400

BondedReversed

phase

Size ormolecularexclusion

Size ormolecularexclusion

BondedReversed

phase

Analyte properties and type of chromatography

Ionexchange

Molecular Size

Solubility

Ionization

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Roadmap to type of chromatography

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Columns and Stationary Phases for HPLC

Columns: Stainless steel I.D. 2-5 mm, length 5, 10, 15 or 25 cm

(for general analytical purposes)

Packings Porous particles (3, 5 or 10 Qm), preferably

spherical in shape Normal phases: polar compared to non-

polar solvents, silica, alumina, florisil Reverse phases: non-polar compared to

water: chemically modified silica, styrene-divinylbenzene resins, agarose

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Porous silica as a stationary phase SiO2

Most important chromatographic adsorbent Important properties: shape, pore width,

specific surface area

Macroporous spherical silica particle.[K.K.Unger, Porous silica, Elsevier, 1979]

Chemical structure of silica

Surface hydrolyzes in water:Water deactivates silica surfaceBecomes less sorbing

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Adsorption chromatography(Normal as opposed to reversed phase)

Polar stationary Non-polar mobile phase:

Hexane<CH2Cl2<MeOH

Normal phase was first described Reversed phase in now more common

Dipole-dipole interactions, hydrogen bonding,T-complex bonding

Asorbent Functional group

Alkane moiety

Adsorbent

polarity:Silica>Alumina>C-18 bonded silica

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Separation of a Test Mixture on Silica with Eluents of Different Strength

observe retention time and order!

A)

Strong solvent (THF)

C)

Weak solvent(hexane)

B)

Medium strong

solvent or mixture

min0 5 min

0 20

min0 2010

12

34

1 = p-xylene2 = nitrobenzene3 = acetophenone

4 = 2,6-dinitrotoluene

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Reversed-Phase Chromatography

Non-polar adsorbent surface and polareluent (introduced 1974)

Most widely used HPLC mode

Chemically bonded octadecylsilane (ODS)or C18 is most frequently used C8 and shorter alkyl chains are

alternatives

Silica with alkyl brush surface

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Making a silica surface hydrophobic

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Reverse Phase Chromatography:

Effect of Eluent Composition

min minmin min0 0 0 05 10 2010 2010 30

60% MeOH 30% MeOH 20% MeOH 10% MeOH

1,2,3

4

1

2

3

4

Separation of benzene (1), chlorobenzene (2), o-dichlorobenzene (3)and iodobenzene (4) on a C18 column with different methanol-watermixtures

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Current Protocols in Protein Science

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1: catalase

2: aldolase3: bovine serum albumins4: ovalbumine5: chymotrypsin6: ribonuclease A

Was used to evluate the sizeof BSA as 34.8Å

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Ion-Exchange Chromatography

Stationary phase has an ionically chargedsurface Ionic groups such as SO3, COO, NH3 or NR3 are

incorporated into resin or gel

Parameters that affect separation: Type of ion exchanger pH of mobile phase Ionic strength of mobile phase Type of counter ions in mobile phase

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Affinity Chromatography

Most specific method, interaction isbiochemical

antigen m antibody enzyme m inhibitor hormone m carrier

Suitable elution provides pure compound(s) Elution containing product with greater affinity Change in pH Change in ionic strength

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Detector requirements

High sensitivity, low detection limits, linearity,reproducibility

Should recognize when a substance zone is elutedfrom the column Changes in chemical or physical properties in solution

Bulk property: overall change in mobile phase Solute property: measures solute specific property

Almost all LC detectors are on-stream monitors (flow-through cells) under continuous flow conditions and the sample is always

dissolved in the eluent during detection.

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Detector types

Refractive index UV/Vis

Fixed wavelength Variable wavelength Diode array

FluorescenceLess common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering

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UV/Vis Detector

Most commonly used type of detector: Sensitive, suitable for gradient elution, relatively

unaffected by temperature fluctuations

Records compounds that adsorb UV (190-400

nm) or visible light (400-800 nm) Lambert-Beer Law: A(P) = log Io /It

= IC L Units of IP= cm-1 M-1

A is additive for compounds x + y: A(P) = IxCxLx + IyCyLy

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UV/Vis Detector

Fixed-wavelength detector low-pressure mercury vapor lamp emits very

intense light at 253.7 nm

Variable-wavelength detector Deuterium lamps emit continuous UV spectrum(up to ~ 340 nm); Tungsten lamps in the near-UVand visible ranges (340-850 nm)

offer best sensitivity by selecting an appropriate

wavelength Diode-array detector allows to simultaneously monitor a range of P or

to obtain a complete spectra

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Fluorescence Detector

Most sensitive (10 -1000 times higher than UVdetector)

~15% of all compounds have a natural

fluorescence Compounds are excited by shorter wavelength

energy and emit higher wavelength radiation Emission is measured at right angle to the

excitation Modern detectors allow fast switch of excitationand emission wavelength

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Refractive Index (RI) Detector

Only universal detector

~1000 times less sensitive than UV det. Presence of solute changes refractive

index of solvent

Cell must be thermostatically controlled Unsuited for gradient elution

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Detector types and limit of detection(LOD)

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Method Development

Selection of close example Selection of adsorbant type

Reverse phase columns are good staring point

Selection of mobile phase Variation of eluent composition provides the great

flexibility of HPLC separationsp adjusting the retention times

p

optimize resolution and total run time to maximizesample throughput

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Isocratic separation of six steroids

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Separation using gradients

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Polarity of Pure Solvents:

effect of P on k

Normal phase:k2 /k1 = 10

Reversed phase:k2 /k1 = 10

(P1 P2)/2

(P2 P1)/2

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Environmental Applications (HPLC)

ChlorophenolsChromatographic conditions:Column: 4µ Genesis C18, 150 x 4.6 mmMobile phase: 75% MeCN/25% 10mM KH2PO4 buffer

pH = 7.0 Flow rate: 1.5 ml/minDetection: UV at 280nm Temperature: RT

Sample:1. Phenol

2. 2-Chlorophenol

3. 2,4-Dichlorophenol4. 2,4,6-Trichlorophenol5. Pentachlorophenol

Source: VWR International

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Example: Analysis of Long-Chain Alkyl Aromaticsin Heavy Crude Oil, Dutta and Harayama, EST,2001, 102

Environmental Problem: Crude oil is frequentlyspilled into the marine environment, but itsdegradation is poorly understood, especially the non-

volatile fraction.Research Objective: Develop a method tocharacterize long-chain n-alkylbenzenes (C7 - C22)

Approach: Prepare a test sample by distilling at 230Cof the light fractions of 38L of light Arabian crude oil.

Use column and TLC to pre-separate and isolatefractions that are amenable for NMR and GC-MSanalysis.

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Lecture 5

Sample:non-distillable fraction of crude oil

Extract maltenes with n-hexane (residues asphaltenes)

Dissolve sample in chloroform and dry in vacuum

Column chromatography on activated silica:Fraction 1: n-hexane, Fraction 2: 1:1 n-hexane-benzene

TLC separation of n-hexane fraction: development for

40 min w/ n-hexane and 13 min n-hexane:toluene (20:80 v/v)

Collect fraction 1: Rf> 0.57 and 2a: Rf = 0.34 - 0.45,

extract with chloroform

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Maltenes constitute the fraction of asphalt which is soluble in n-alkane solvent such aspentane and heptane, consisting of:

�Smaller molecular weight versions of asphaltenes.�

Aromatic hydrocarbons with or without O, N and S.�Straight chained or cyclic unsaturated hydrocarbons�Cyclic saturated hydrocarbons.�Straight or branch chain saturated hydrocarbons

("saturates

).

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Asphaltenes are complex hydrocarbons

having the following components:�Condensed aromatic hydrocarbons with side-chainsup to C30�Hetero-aromatic compounds with sulfur present in

benzothiophene rings and nitrogen in pyrrole andpyridine rings�Bi- or polyfunctional molecules with nitrogen asamines, amides, and oxygen in groups such as:

ketones, armides, phenols, and carboxylic acids�Metals nickel and vanadium complexed with pyrrolenitrogen atoms in porphyrin ring structures

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Application

Fluoroquinolone

antibacterialagents inurban wastewater

Anal. Chem. 2001,73,3632-3638

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