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Isala klinieken Gas chromatography

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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 kliniekenVoordelen

Vluchtige componenten in organischeoplosmiddelen

Zeer hoge resolutie

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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 kliniekenTypical Modern HPLC System

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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 kliniekenIncreasing k, N, and

Inital Chromatogram

Increasing k

Increasing N

Increasing

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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 kliniekenResolution f(L) - Constant dp & F

L = 5 cm

L = 10 cm

L = 15 cm

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Isala kliniekenRs f(L) -Constant dp & Pressure (~3000 psi)

L = 5 cm

L = 10 cm

L = 15 cm

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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 kliniekenRP Solvent Properties

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Isala kliniekenRP Solvents - Viscosity

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Isala kliniekenRP Binary Mixture Solvent Strengths

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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 kliniekenRP Retention Chain Length Effects

de Galan, J. Chromatogr., 196, 21 (1980)

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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)

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Isala kliniekenSerotonine en 2 metabolieten… wat nu?

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Isala kliniekenOntwikkel een methode

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Isala kliniekenStel RPLC C18. Voorspel de positie