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Isala kliniekenKolom chromatografie
Gas chromatography
GC geschikt voor stoffen met een Mr < ca. 800 g/mol
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Isala kliniekenChromatografische principe
Scheiding is gebaseerd op verdeling van de analieten over twee verschillende fase, de stationaire en mobiele fase.
In GC is er geen mobiele fase, alleen een carrier gas
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Isala kliniekenDetectoren in GC
MS (massa spectrometrie) ECD (electron capture detectie) FID (vlam ionisatie detectie
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Isala kliniekenNVK-FAZ
Farmaceutische Chemie
Casus 1: Bepaling van aspartaam in cola-light
Theorie: LC: Principes, theorie en instrumentatie(Nadruk op praktische aspecten)
Deel I (45 min)
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Isala kliniekenAspects of HPLC
Amenable to diverse samples including labile organics, biomolecules,
and ions - Can handle > 60% of all existing compounds vs. 15% for GC
High-resolution - Resolves hundreds of components in complex samples
High sensitivity detection - pg - ng detection limits
Rapid and precise analysis - 1 - 60 min analysis, Precision < 1% RSD
Automated analysis - Using autosampler and data system for unattended analysis and
report generation
Quantitative sample recovery - Preparative technique from g to kg quantities
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Isala kliniekenBasic Concepts & Terminology
Chromatography is a dilution technique Migration distance versus peak dispersion [N = (L/)2]
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Isala kliniekenDifferential Migration
Analiet distributie tussen:– mobile phase (liquid)
– stationary phase (solid support)
Componenten wordengescheiden op basis van verblijftijd en voorkeur voorde stationaire fase
On-line detectors detecterende eluerende componenten
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Isala kliniekenVoorspel positie in RPLC
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8Time (min)
Abs
orba
nce
260
nm
pyridine
tert-butylbenzene
uracil
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Isala kliniekenTerminology / Figures of Merit
Column dead time (volume) (to) Retention time (tR) Peak width (Wb = 4 ) Retention factor (k) Selectivity () Column efficiency
– Plate number (N)– Height Equivalent of a Theoretical Plate (H or HETP)
Resolution (Rs)
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Isala kliniekenRetention Time (tR)
• to - unretained solute
• tR - retention time
• Wb - base width (4)
• W0.5 - 1/2 height width
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Isala kliniekenRetention (Capacity) Factor
peak retention in terms of column volumes k = tR - to / to
k=0 k=1 k=2k=3
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Isala kliniekenSeparation (Selectivity) Factor ()
= k2 / k1 = 2.9/1.6 = 1.81 depends on the column packing and the mobile phase must be > 1 for peak separation
k = 1.6 k = 2.9
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Isala kliniekenColumn Efficiency (N)
Migration distance versus dispersion N = (L/)2 = (tR/)2 = 16(tR/Wb)2 = 5.54(tR/W0.5)2 = 16(130/10)2 = 2704 Determined by particle size (dp), column length (L) and flow rate (F)
N=2704
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Isala kliniekenResolution (Rs)
Rs = (tR2 - tR1) / 0.5(w1 +w2) Rs = 2(tR2 - tR1) /(w1 +w2) Rs = 2(tR) /(w1 +w2)
alt., since wb = 1.7 w0.5h
Rs = 1.18(tR) / (w0.5,1 +w0.5,2)
Rs = 1.5 (baseline resolution)
Rs = 1.91
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Isala kliniekenThe Resolution Equation
Rs = 1/4 N)0.5 ( (k / 1+k) efficiency selectivity retention
The goal of most LC separations is to achieve baselineresolution for all key analytes (Rs > 1.5)
k should be kept between 2 to 10
is maximized with by selecting the column and mobile phase that resolve all critical analytes
N is increased using high-efficiency columns of a reasonable length
Review & Discussion of Rs eq's, J.P. Foley, Analyst, 116, 1275-1279 (1991)
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Isala kliniekenBeinvloeden van de retentie
Verhogen N
Verhogen
Verhogen k
Het verbeteren van scheiding mbvBasic Resolution Equation
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Isala kliniekenResolution - Effect of Increasing N
Langere kolom of kleinere deeltjes
Uiteindelijk gelimiteerddoor druk
Elke verdubbeling van het schotelgetal, verbetert de resolutie
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Isala kliniekenResolution - Effect of Increasing
Most effective approach
Most difficult to rationally identify
Must be > 1
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Isala kliniekenResolution - Effect of Increasing k
k = 0, geen retentie
k ~ 2-10, optimalesnelheid/resolutie
Makkelijkstbeinvloeden, mbvmobiele fase
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Isala kliniekenDispersion Processes
van Deemter Term
Eddy Diffusion(A-term)
Molecular Diffusion(B-term)
Mass Transfer(C-term)
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Isala kliniekenVan Deemter Plots
Plots show relationship of HETP with flow rate and dp
Small dp yields lower HETP
The optimum flow rate is higher with smaller dp
Small-particle columns have less efficiency loss at high flow rates
H = A + B/u + Cu
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Isala kliniekenStrong Solvent Injections
Mobile phase: Buffered to pH 3.5:acetonitrile (92:8)Sample dissolved in buffer with ( a) 0%, ( b) 30%, (c) 50%, (d) 70% ACN
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Isala kliniekenLC Column Geometry / Design
Resolutie vs tijd– Kolomlengte– deeltjesgrootte– Toegestane druk– Optimale deeltjesgrootte vs kolomlengte limiteert de druk
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Isala kliniekenDe toekomst????
UPLC?– Hoge flow-rate en kleine deeltjes grootte.
HTLC?– Hoge temperatuur
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Isala kliniekenLC Modes
Reversed-phase (RPC)– Scheiding op basis van hydrofobiciteit
Normal-phase or Liquid-solid (NP, LSC)– Scheiding op basis van adsorptie/desoprtie op een polaire
stationaire fase (bv silica)
Ion-exchange (IEC)– Separation based on ion-exchanging with the counter-ions and ionic
interaction with the bonded ionic group
Size-exclusion (SEC or GPC)– Scheiding op basis van grootte en vorm
Note: Ion chromatography is IEC with specialised equipment while ion-pairing chromatography is a special technique in RPC for ionizable compounds.
Analyte
Stationary Phase
Mobile Phase
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Isala kliniekenReversed-phase LC (RPLC)
Separation based on partitioning of the analyte into a hydrophobic stationary phase bonded on silica support (e.g., C18, C8)
– Uses polar mobile phase such as mixture of methanol/acetonitrile and water; Polar analytes elute first while non-polar analytes are retained more
Most common HPLC mode used in > 60% all LC separations
– For polar (water-soluble), medium polarity, and some non-polar analytes
– Ionic analytes can be separated using ion-pairing reagents
FLOW
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Isala kliniekenMobile phase
HPLC mobile phases are usually a mixture one or more solvents with these characteristics
Desirable Physical Properties– High purity, low cost, UV transparency, non-corrosive, low viscosity, low toxicity,
non-flammable, sample solubility Strength
– Strength is related to polarity of solvent; Water is a strong solvent in normal phase but a weak one in reversed-phase
– Solvent strengths under normal phase are characterised by Hildebrand's scale (Eo) Selectivity
– Depends on dipole moment, induced dipole, H-bonding, and dispersive characteristics of the solvents
Solvents should be filtered and degassed before used
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Isala kliniekenCommon Mobile Phase Modifiers
Buffers – Stabiliseert pH
Acidifiers – Vermindert ionisatie van zuren
Amine modifiers– Vermindert tailing bij RPLC (amines gaan interactie aan met silanol
groepen)
Ionic strength– Stabiliseert elutie van ionogene analieten bij IEC
Ion-pair reagents – Verbetering scheiding van ionogene stoffen bij RPLC (hexane sulfonate)
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Isala kliniekenNormal Phase Chromatography (NPC)
Uses silica, alumina, amino-, cyano-, or phenyl bonded phases
– Uses nonpolar mobile phase such as hexane, CH2Cl2 modified with isopropanol
– Polar analytes has longer retention times
Excellent for non-polar analytes, isomers, group separation, and for sample clean-up
FLOW
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Isala kliniekenIon-exchange Chromatography (IEC)
Used to separate ionic or ionizable analytes
Stationary phase is bonded cationic (sulfonate) or anionic (quaternary ammonium) groups on polymeric materials or silica
– Uses mobile phase consisting of buffered solutions of different pH and ionic strength
Common applications are analysis of ions, amino acids, proteins/peptides, polynucleotides, etc.
+-
SO Na3+
SO Na3
p+
Polymericor silica support
-
Cationic exchanger
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Isala kliniekenSize-exclusion (SEC or GPC)
Separation based on sieving action of cross-linked polystyrene with controlled pore sizes
– Common mobile phase is toluene, THF, CH2Cl2
– Large molecule elutes first while small molecules penetrate into the pores and elute later
Useful for molecular weight determination of polymers and biomolecules
FLOW
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Isala kliniekenMonolithic Silica
Large pore diameter is about 2 µm
Mesopore diameter is about 12 nm
Porosity value > 0.8 Allows high linear velocity with minimal pressure Short diffusional distances
allow large N with high linear velocity
Currently only available in 4.6 mm id.
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Isala kliniekenRestricted Access Media
~ 60 Å porous silica hydrophilic phase on
particle surface hydrophobic phase on
particle interior typically > 95%
surface area is interior allows direct injecton
of particulate free biological fluids
highly useful in 2D column switching methods
compatible with any detection mode
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Isala kliniekenColumn Type & Silanol Effects
Left: Lichrospher 100 RP-18, 5 µm
– provide symmetrical peaks for neutral substances
Right: Lichrospher 60 RP-Select B, 5 µm
Mobile phase: methanol/water (75/25), flow rate = 1.5 ml/min
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Isala kliniekenMinimizing Silanol Effects
Analytes: Mixture of tricyclic antidepressants
– Nordoxepin– doxepin– desipramine– protriptyline– imipramine– nortriptyline– amitriptyline
Left: C8 phase, mp of acetonitrile/water, pH 2.5
Right: C8 phase, mp of acetonitrile/water, pH 2.5 plus 5 mM nonylamine
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Isala kliniekenHPLC Operation
Flow rate ranges– 0.1 to 3.0 mL/ min for analytical columns– > 5 mL/min for preparative columns
Operating pressure (500 - 2000 psi) Sample size (1 to 100 L) Isocratic and gradient elution
– Isocratic - mobile phase composition remain unchanged – Gradient - mobile phase composition is changed from weak to strong solvents to
accommodate complex samples with analytes of wide polarities
Column temperature (ambient to 40oC)– Column temperature is controlled for more precise retention times, increased selectivity,
and improved sample solubility
Detector operating conditions– Detector type and operating conditions (e.g. monitoring wavelength)