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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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