Partition Liquid Chromatography

Principles of Separation

Liquid-liquid extraction:Partition of a solute between two non-miscible liquid phases

Partition coefficient

inf

sup

C

CK

To totally extract the desired soluteK must be either very large or very small

Liquid-Liquid Extraction

Static representation (dynamic in reality)

Zone 1 Zone 2 Zone n

Mobile phase

Stationary phase

Zone 3

Solute molecules

Each solute partitions in the two phases according to its own K= according to its relative affinity for the two phases

mobile

stationary

C

CK

Principles of Separation

Partition Chromatography

Principles of Separation

Interactions

Solute

SP MP

Principles of Separation

Partition Chromatography

K must not be too large otherwise the retention is too long(too much affinity for the stationary phase)

K must be large enough for the solutes to be a little retained in the stationary phase otherwise no separation is possible(too much affinity for the mobile phase)

mobile

stationary

C

CK

The equilibrium is respected in the whole columnEach zone is called a « theoretical plate »

For a separation to occur: K ≠ K

If K > K, remains longer in the column than

Retention of the solute depends on:

- The nature of the solute - The nature of the stationary and mobile phases- The relative affinity of the solute for SP and MP- The specific surface area of the stationary phase (more SP = more interactions)- The temperature (can influence the thermodynamic equilibrium K)

Retention does not depend on:

- The geometrical parameters of the column (length, internal diameter…)- The particle diameter of the SP- The flow rate- The amount of solute injected

Principles of Separation

Separation depends on:

- The nature of the solutes- The nature of the stationary and mobile phases- The relative affinity of the solutes for SP and MP- The specific surface area of the stationary phase (more SP = more interactions)- The temperature (can influence the thermodynamic equilibria K1 and K2)

Separation does not depend on:

- The geometrical parameters of the column (length, internal diameter…)- The particle diameter of the SP- The flow rate- The amount of solute injected

Principles of Separation

Principles of Separation

SP is a liquid

Separation is based on relative solubilities in MP and SP

Normal Phase-partition was first described but Reversed Phase-partition is now more common

NP RP

Polar Stationary PhaseNon Polar Mobile Phase

Non Polar Stationary PhasePolar Mobile Phase

Si O Si Si OO Si O Si O Si O Si O Si O

O

H

O

H

O

H H

O O

H

O

Si

N≡C

O

Si

O

HO

HO

O

N

Si

H

H

CYANO DIOL AMINO

Basic

Acidic

Dipole

NPLC-Partitioning

Polar interactions

Si O Si Si OO Si O Si O Si O Si O Si O

O

H

O

H

O

H H

O O

H

C8 C18 PHENYL

O

Si

O

Si

O

Si

Alkyl chains from C1 to

C30

Polymers: PS-DVB

London forces

London forces+

π-π interactions

RPLC-Partitioning

Hydrophobic interactions

RPLC-Partitioning

OctaDecyl-Siloxane phases (ODS)

A great variety of stationary phases

None of them is identical to the other!!

Example ODS phases

Trathnigg et al., J. Chromatogr. A, 1128 (2006) 39-44

PEG 300

PEG 300, 24.61% (w/w) methanol on different C18 columns

First step is to determine which mode to use, NP or RP?

If sample is water insoluble or non polar, use NP

If sample is water soluble or not soluble but polar, use RP

MP is never a single solvent, always a blend of two or more

hexane CCl4 THF acetonitrile methanol water

Optimum polarity is obtained by mixing solvents

Partitioning

Not all solvents are usable

MeOH, ACN, THF, H2O are the most widely used in RPLCHXN, CH2Cl2, iPrOH are the most widely used in NPLC

All are low viscosityavailable in high puritynot too expensiveUV transparentmiscible in each other

Mobile phase

Determining the optimum RP solvent blend

Start with a single solvent and waterAdjust the % of water from 0% on up until the best separation is obtained(optimum k for peaks of interest)

Create blends using each of the other solvents and water that have the same polarity

Evaluate each solvent for improvements in peak shape or movement of selective peaks

A mix of any of the blended solvents is then evaluated for optimum resolution

Possible elution gradient (similar to T gradient in GC)

Mobile phase

elution gradient

-Total analysis time is reduced- overall resolution is improved- better peak shapes are possible- improved sensitivity

Requires a compatible detector

Total flow rate is held constantOnly the proportion of the solvents are changed

Mobile phase

Example: carbohydrate analysis, NPLC

fructose

glucose

Maltose = di-glucose

Maltotriose

oligosaccharides

OH

CH2OH

OH

CH2OH

OH H

H OH

O

OH

OH

H

OH

H

OHH

OH

CH2OH

H

OH

OH

H

OHH

OH

CH2OH

H

O

H

OHH

OH

CH2OH

HH

O

OH

H

OH

OH

H

OHH

OH

CH2OH

H

O

H

OHH

OH

CH2OH

HH

O

O

H

OHH

OH

CH2OH

H

O

OH

H

H

Example: carbohydrate analysis, NPLC

Noguetra et al., J. Chromatogr. A, 1065 (2005) 207-210

Typical chromatogram for separation of five carbohydrates

(1) fructose (2) glucose (3) maltose (4) maltotriose (5) maltotetraose

Spherisorb NH2 (250 x 4.6 mm, 5 μm)ACN-H2O gradient elution, 1 mL/minDetection: ELSD

Carbohydrates in beer

Beer sample

Example: homologous series, RPLC

15 homologous n-alkylbenzenes, linear gradient of MeOH in H2O on ODS

P. Jandera, J. Chromatogr. A, 845 (1999) 133-144

Example: homologous series, RPLC

30 homologous oligostyrenes, linear gradient of dioxane in n-heptane on silica gel

P. Jandera, J. Chromatogr. A, 845 (1999) 133-144

1 2 3

45 6

RPLC chromatograms of standard mixture, Pingtangan capsules, Zhiwuyidaosu capsules, Gliclazide tablets

Yao et al., J. Chromatogr. B, 2007 (in press)

Example: anti-diabetic sulfamide drugs

Pharmaceutical conterfeiting

Alltima C18 (150 x 4.6 mm, 5 μm)MeOH- pH 3 phosphate buffer (70:30), 1 mL/minUV-detection

LC-MS coupling

Studied from 1974

Most important difficulties:

Important quantity of solvent to eliminate

Fragile molecules

Wide range of polarity and molecular weight of analytes

LC-MS coupling

Capillary3-5 kV

Drop containing

the ions

During the evaporation of the solvent, th electric field inside the drop increases and ejects the ions (electrostatic repulsion)

++

++

++

+-- - -

--

-

-

+++

+

+

-- - --

-++

++

++

+-

---

--

-

-

-

-+

Electrospray Ionisation (ESI)

LC-MS coupling

Atmospheric Pressure Chemical Ionisation (APCI)

Heated Nebuliser

Corona needle

N2

N2

SolutesSolvent Molecules

Liquid

Formation of an aerosol

Vaporisation of the solvent and sample Solvent molecules are ionised

Charge transfer and collision

Solute Ions [M+H]+

are formed

+

++

++

++

LC-MS coupling

Atmospheric Pressure Chemical Ionisation (APCI)M

olec

ular

Wei

ght

Non-Polar Polar

EI /

CI

EI /

CI

Electrospray ( ESI )Electrospray ( ESI )

APCIAPCI

100.000

1.000

Example LC-UV / MS: penicillin in urine

1.00 2.00 3.00 4.00 5.00 6.00Time0

100

%

13

100

%

UV 268nm

MS - SIMm/z 333 + m/z 349

Example LC-ESI-MS: vitamins analysis

LC/ESI-SIR chromatogram of a 0.2 mg/L standard mixture.

(1) Taurine(2) nicotinic acid(3) Nicotinamide(4) pantothenic acid(5) pyridoxal;(6) Pyridoxine(7) hippuric acid(8) Thiamine(9) Biotin(10)Riboflavin(11)ascorbic acid(12)folic acid

Chen et al., Analytica Chimica Acta, 569 (2006) 169-175

Example LC-ESI-MS: vitamins analysis

(1) Taurine(2) nicotinic acid(3) Nicotinamide(4) pantothenic acid(5) pyridoxal;(6) Pyridoxine(7) hippuric acid(8) Thiamine(9) Biotin(10)Riboflavin(11)ascorbic acid(12)folic acid

Example LC-ESI-MS: vitamins analysis

LC/ESI-SIR chromatogram of a multivitamin tablet sample.

(1) Taurine(2) nicotinic acid(3) Nicotinamide(4) pantothenic acid(5) pyridoxal;(6) Pyridoxine(7) hippuric acid(8) Thiamine(9) Biotin(10)Riboflavin(11)ascorbic acid(12)folic acid

Chen et al., Analytica Chimica Acta, 569 (2006) 169-175

Example LC-APCI-MS: Phenolic acids in industrial wastewater

Example LC-APCI-MS: Phenolic acids in industrial wastewater

Column: Porous Graphitic Carbon (10 x 2.1 mm, 5 μm)

Mobile Phase: Gradient elution with A- MeOH-ACN-Formic acid 0.2M (40:40:20) and B- THF, flow rate 1 mL/min

Detection : APCI, negative mode

Organic molecules are "embraced" by the carbon chains of the stationary phase

Unlike the typical organic target molecule peptides and proteins adsorb to the stationary phase often by multi-point attachment

In contrast to bulk water, hydrophobic surfaces are covered by a shell of highly ordered water molecules.

The chromatograms show the effect of varying the organic solvent concentration in isocratic experiments. Note that no concentration is capable of eluting all four components in the same run

Classical" hydrophobic organic molecules are sensitive to the carbon chain length, while more or less identical results are obtained for proteins and larger peptides, regardless of the carbon chain length

This picture shows hydrophobic and hydrophilic parts on the surface of lysozyme. The most hydrophobic parts are dark red, the less hydrophobic lighter red. The most hydrophilic parts are shown in dark blue, while the less hydrophilic parts are lighter blue

Purification by partitioning the sample between two liquid phases. The distribution is controlled by the difference in polar properties of the respective phases

Reversed Phase Chromatography utilises solubility differences between the sample components by a continuous re-partitioning mechanism

The adsorption of hydrophobic moleculesis a reversible reaction whose equilibriumis controlled by the salt concentration

The desorption curve is shifted to the right with increasing net hydrophobicity

Between NP and RP, the elution order will be somewhat reversed but not exactly, other factors must be considered

Stationary phaseBonded silica

Polar ligandsNormal-Phase LC

« Classical Partition Chromatography »

Non polar ligandsReversed-Phase LC

Partitioning

Mixed stationary phases

May have both bonded ligands

In RP, polarity of the solvent determines how long the solutes are retained (k)

Snyder classed solvents according to acidic, basic, dipole characters

Proton acceptor

Proton donor dipole

MeOH

THF

H2O

Partitioning

Partitioning

T has a minimal effect on the separation

It allows obtaining thiner peaks = higher resolution

High temperatures require resistant columns

Temperature

Particular vs. monolithic