mechanisms of retention in hplc - universitat de …...2 hplc’2013 (amsterdam) 1. retention in...

1

Mechanisms of retention in HPLCMechanisms of retention in HPLC

María Celia García-Álvarez-Coque

Department of Analytical Chemistry

University of Valencia

Valencia, Spain

https://sites.google.com/site/fuschrom/

HPLC’2013 (Amsterdam)

Part 2Part 2

2


1. Retention in reversed-phase, normal-phase and HILIC

2. Secondary equilibria in reversed-phase liquid chromatography: Part A

3. Secondary equilibria in reversed-phase liquid chromatography: Part B

4. Retention modelling (quantification or prediction): Part A

5. Retention modelling (quantification or prediction): Part B

6. Gradient elution

7. Peak profile and peak purity

8. Computer simulation

Index

Mechanisms of retention in HPLC

3


2.1. Introduction

2.2. Ion-interaction chromatography

2.2.1. Retention mechanism

2.2.2. Common reagents and operational modes

2.2.3. The silanol effect and its suppression

2.2.4. Addition of perfluorinated carboxylate anions, chaotropic ions and ionic liquids

2.2.5. Separation of inorganic anions with surfactant-coated stationary phases

2.3. Micellar liquid chromatography

2.3.1. An additional secondary equilibrium in the mobile phase

2.3.2. Hybrid micellar and high submicellar liquid chromatography

2.4. Recommended literature


4


2.1. Introduction


In RPLC with hydro-organic mixtures as mobile phases, which is the prevalent

chromatographic mode nowadays, the retention is theoretically explained by solute

partitioning between the mobile phase and the bonded phase, which depends on the

polarity: the more hydrophobic the solute, the longer its retention. To this, shape and

steric constraints should be added.

Partitioning Shape constraints Steric constraints

5


Limitations of RPLC

● Polar compounds (polar neutral or ionised organic compounds, and inorganic anions

or metal ions) show little or no retention.


o/w10 loglog Pcck

Rete

nti

on

facto

r

BH+

B

pH

HA

A−Rete

nti

on

facto

r

pH

Ionisable

compounds

This has been a challenge in environmental, clinical and food chemistry throughout

the development of RPLC.

6


● There is no ideal support for preparing RPLC stationary phases.

● The vast majority is still prepared with silica, due to its attractive

properties:

easy derivatisation

control of particle size and porosity

mechanical stability

incompressibility

● However, owing to steric problems in the derivatisation of silica,

silanol groups remain on the support in a non-negligible amount

and, when ionised, they interact with cationic solutes by ion-

exchange processes that increase significantly the retention.

● Protonated silanols can also interact with some solutes through

hydrogen bonding.

● The kinetics of the adsorption-desorption on silanols is slow,

which yields tailed and broad peaks.


Limitations of RPLC

BH

+

BH

+

BH

+

7


Types of interactions:

● Ion-exchange processes with ionic lipophilic reagents adsorbed on the stationary phase,

which attract solutes with an opposite charge or suppress the silanol activity.

● Formation of solute/additive ion pairs in the mobile phase.

● Acid-base reactions of ionisable compounds with changes of pH.

● Metal complexation

A : analysed solute (analyte) or silanol group on the support

X : a lipophilic ion, the hydrogen ion, a ligand, or other added species


A solution to avoid the poor interaction with polar compounds and the silanol activity is the

addition of reagents (additives) to the mobile phase, which give rise to diverse secondary

reactions on the support, or within the mobile phase.

C+ + X− C+ X−

A− + R+ R+ A−A− + H+ HA

M + L ML

R+

A −

R+

A−

A + X AX (2.1)

Addition of ionic reagents

8


A : molar fraction of A AX : molar fraction of AX

[X] : molar concentration of X in the mobile phase

K : formation constant (acid-base reaction: log K = pKa)

● Two or more secondary equilibria may exist inside the column

(even secondary equilibria of secondary equilibria).

● The aim is to enhance the chromatographic performance: change the absolute

and relative retention to convenient values and improve the peak profile.


The observed retention factor is a weighted average of the

retention of the solute species.

]X[1

]X[AXAAXAXAA

K

Kkkkkk

(2.2)

The addition of different types of reagents has given rise to

new chromatographic modes and an impressive increase in

the number of compounds that can be analysed by RPLC.

Cu2+

R−

L

H

H

9


2.1. Introduction












10


● The reagent typically contains a large organic ion that has a

lipophilic region that interacts with the bonded chains on the

stationary phase, and a charged region that interacts with an ionic

solute.

● The stationary phase is modified and interacts with ionic species,

but also with neutral species through polar interactions.

● The retention is regulated by the nature and concentration of the

adsorbed lipophilic ion and organic solvent in the mobile phase,

and by competing ions with the same charge as the analyte.




An RPLC mode with a broad scope of applications is achieved by

adding amphiphilic cations or anions to the hydro-organic

mixture (with both lipophilic and hydrophilic properties).

R3NH+ Cl−

R 3N

H+

R 3N

H+

R 3N

H+

A −

R−SO3− Na+

R−S

O3−

R−S

O3−

R−S

O3−

BH+

HA

C+

ACN

11


Adsorption of an ion pair

● In the origin of RPLC, bonded phases were

considered as equivalent to a mechanically

held liquid phase, and therefore, a liquid-liquid

extraction mechanism was postulated.

● The proposed mechanism assumed the

formation of an ion pair in the mobile phase by

combination of the solute and the lipophilic ion

of opposite charge, which partitions into the

non-polar liquid layer on the stationary phase.

● Hence, the name ion-pair chromatography

(IPC) taken from liquid-liquid extraction.


The retention mechanism that takes place by addition of amphiphilic ions is not currently

fully understood. Due to the complexity of the mobile phases, which contain the ionisable

or ionic solute(s), and at least, the additive and buffer ions (and their counterions), it is not

easy to explain their influence on the retention behaviour of ionic solutes.

R +

A −

Approaches

12


● Experimental facts further suggested that the lipophilic ion is distributed between the

mobile phase and stationary phase, where it is adsorbed (immobilised), behaving as an

ion exchanger for oppositely charged solutes.

● This model implies, essentially, an electrostatic interaction, and pioneered the

stoichiometric approach followed for decades.


R+

R+

R+

R+

A−

R+A−

Dynamic ion-exchange mechanism

13


Non-stoichiometric approaches

● Broader perspectives describe the ionic solute

as being under the summed influence of all ions

in the chromatographic system.

● Solute retention is influenced by its transfer

through the electrical double layer formed by

the lipophilic ion (primary charged ion region)

and counterion (diffuse outer region).

● This creates a surface potential, which depends

primarily on three parameters:

surface concentration of lipophilic ion

mobile phase dielectric constant

ionic strength

● The higher the surface concentration of

lipophilic ion, the larger will be the effective ion-

exchange capacity, and hence, the retention of

solutes with a charge opposite to the lipophilic

ion.


A-

A-

A-

14

HPLC’2013 (Amsterdam)2. Secondary equilibria in reversed-phase liquid chromatography: Part A

● The lipophilic ion is spaced over the stationary phase due to repulsion effects,

which leaves much of the original stationary phase surface unaltered and

available to interact with ionic and non-ionic solutes (dual mechanism).

● Small hydrophilic organic and inorganic anionic solutes probably interact

primarily by electrostatic forces.

● The actual mechanism is rather complex.

● Solutes need to be ionised to interact with the ionic reagent. Therefore, the

retention of ionisable compounds depends on the pH and pKa .

R−

R−

B+

B+

HA

pKa

HA

A−Rete

nti

on

facto

r

pH

Dual mechanism

15


Names

● Ion-pair chromatography is by far the most widely used term for this RPLC mode

but it is not descriptive of the retention mechanism

the term is usually associated with the addition of a small amount of the lipophilic

ion to avoid an excess in the mobile phase, but in some cases, large amounts of

reagent are added

● Paired-ion chromatography

● Ion-interaction chromatography (IIC)

● Ion-modified chromatography

● Hetaeric chromatography (hetaeron means counterion)

● Surfactant (or soap) chromatography (with ionic surfactants)

● Submicellar liquid chromatography (surfactants below the CMC)

perhaps, the most correct term !!!

2009

16




Reagents

● Any salt containing a lipophilic ion can be used as IIC reagent.

● Most applications imply salts of alkylammonium for anions, and

alkylsulphonates or alkyl sulphates for cations.

● The adsorbed ions may have different alkyl chain lengths:

the longer the chain, the more hydrophobic the reagent,

and the stronger its interaction with the bonded chains.

● The accompanying anion in alkylammonium salts can be:

inorganic (chloride, hydroxide or phosphate)

organic (salicylate or tartrate)

● The cation for alkylsulphonate and alkyl sulphate salts is usually:

sodium or potassium

● Newer reagents are chaetropic salts and ionic liquids.

+

AA−

alkyl sulphate

alkylsulphonate

R+

R +

17


Dynamic coating Permanent coating

The lipophilic ion is added to the

mobile phase.

The major advantage is the

possibility of controlling the

column ion-exchange capacity

by varying the mobile phase

composition.

The stationary phase is equilibrated

before the analysis with a highly

lipophilic ion.

The coating is strongly bound and

persists for long periods of

subsequent use.

To separate anions, the stationary phase must contain immobilised cations, and

to separate cations, it must contain immobilised anions. The same column can

be converted into an anion exchanger or a cation exchanger.

R3N

H+

R3N

H+

R3N

H+

A−

R−

SO

3−

R−

SO

3−

R−

SO

3−

BH+

Operational modes

18


At increasing concentration of IIC reagent, retention increases up to

saturation of the stationary phase surface. Meanwhile, at increasing organic

solvent concentration, retention decreases, due to desorption of the reagent

and competing equilibria in the mobile phase.


Benzyltrimethylammonium

cyanopropyl-silica

log

k

0 10 20 30 40 50

0.5

1.5

2.5

3.5

SDS, mM

0 10 20 30 40 500.5

1.5

2.5

3.5

1-Propanol, % (v/v)

Therefore, both IIC reagent and organic solvent should be kept constant in the mobile phase

at specified concentrations, in order to maintain a reproducible ion-exchange capacity.

R−

SO

3−

R−

SO

3−

R−

SO

3−

BH+

1-PrOHR−SO 3

−

19


● A longer equilibration time is required with respect to conventional RPLC, to get a

constant coating (to take especially into account in gradient elution).

● Relatively less-lipophilic IIC reagents are preferred, since they give rise to shorter

analysis times, and can be more easily removed from the stationary phase

surface. This can be done by washing the column with a polar organic solvent,

such as methanol.

● Some lipophilic ions tend to associate very strongly to the stationary phase,

changing the initial column properties permanently.

● It is not essential that the IIC counterion serves as the ion-exchange competing

ion. Other ions are often added to assist in the elution of strongly retained anions:

phosphate citrate

oxalate phthalate


R+

Something more to know …

A -

20


● For some IIC reagents, there is a need to saturate the mobile phase with silica to

avoid stationary phase solubilisation. This is carried out by inserting a pre-

column.

● System peaks corresponding to the added reagent will appear in the

chromatograms.

● Traditional lipophilic reagents are not usually compatible with evaporative light

scattering (ELS) and mass spectrometry (MS) detection.


R+

Something more to know …

A -

MSELS

Injection

21


The RPLC separation of nitrogen-containing basic compounds with silica-based

columns present several problems, including long retention, peak tailing, poor

efficiency, and strong dependence of retention on sample size.



In order to reduce the silanol effect (silanol problem), much effort has been invested in the

chemistry of bonded phases to eliminate metal impurities and residual silanols.

silanols

BH

+

BH

+

BH

+

The effects are due to ion exchange of the protonated

cationic solute on active (dissociated, anionic) silanols

on the support.

0 10 20 30 40 50

Acebutolol

Alprenolol

Atenolol

Celiprolol

Esmolol

Metoprolol

Oxprenolol

Pindolol

Propranolol

Timolol

SpherisorbC18

Time, min

22

HPLC’2013 (Amsterdam)1. Retention in reversed-phase, normal-phase and HILIC

Silanol activity: Underivatised silanols can interact with neutral solutes by

hydrogen bonding, and with positively charged basic compounds by electrostatic

attraction. This increases their retention and deteriorates the peak profile.

Depends on: amount of available silanol groups

relative acidic character

type (isolated, geminal or vicinal)

presence of metal impurities

Increasing acidity

23


Time, min

The extreme differences in the behaviour toward basic compounds of packing materials labelled

as being of the same type, such as bonded octadecyl-silica, is due to differences in the carrier

silica, type of bonded silane, and coating level, which give rise to different amounts and different

availability of surface silanols.

0 10 20 30 40 50

Acebutolol

Alprenolol

Atenolol

Celiprolol

Esmolol

Metoprolol

Oxprenolol

Pindolol

Propranolol

Timolol

SpherisorbC18

0

10 20 30 40 50 60 70

Alprenolol+ Propranolol

OxprenololCeliprolol

Esmolol

Metoprolol

Acebutolol

TimololPindolol

Atenolol

+

NucleosilC18

Time, min0 10 20 30 40 50

AcebutololAte

no

lol

CeliprololEsmolol

Metoprolol

Oxprenolol

Pin

do

lol

Propranolol

Timolol

Alprenolol

ZorbaxC18

Time, min

The brand-to-brand variation in selectivity of bonded-phase materials is, however, attractive.

RPLC would never have reached so broad applicability if only hydrocarbon-like stationary

phases were available: the separation chemistry becomes richer !!!

ACN / water

24


Solutions (and drawbacks)

● Reducing the pH below 3 to protonate residual silanols

An extreme pH can damage the silica packing

● Increasing the pH to obtain neutral solutes

Simultaneously more silanols are dissociated

● Masking the electrostatic interaction with IIC reagents

(silanol blockers, silanol suppressors or anti-tailing reagents)

An additional background for ELS and MS detection

Column properties may result permanently altered


With the newer generation of RPLC columns, based on ultra-pure silica and improved

bonding technologies, surface silanols have been significantly reduced, but the problem

has not been completely eliminated: some tailing still remains.

25


● Acidic mobile phases containing hydrophobic anions, such as alkylsulphonates or alkyl

sulphates, are used to cover the stationary phase and improve the peak profile.

Peak tailing suppression is not always successful, and the retention of basic compounds

can increase excessively.

● The use of amines is also widespread. Better silanol suppression is achieved with bulky

substituents. Quaternary amines or amines with long alkyl chains seem to be the best.

Concomitantly with the improvement in peak profile, the retention of basic compounds

may decrease excessively.

● A third option is the use of a combination of two ions of opposite charge, such as an

alkylsulphonate and an amine. While the alkylsulphonate acts as IIC reagent (increasing

the retention), the organic amine masks the residual silanols (which decreases the

retention). This yields an efficient separation within a reasonable analysis time.


Several alternatives with IIC reagents

BH +

BH

+

R3 N

H+

R3 N

H+

BH

+

26


Anionic reagents

● Alkylsulphonates

may strongly associate to the stationary phase making column regeneration difficult

● Perfluorinated carboxylates

are volatile, and thus, compatible with ELS and MS detection and also suitable for

preparative chromatography. Trifluoroacetic acid (TFA) is the most common reagent

due to its high purity, water solubility and transparency at 220 nm.


2.2.4. Addition of perfluorinated carboxylate anions,chaotropic salts and ionic liquids

Ionisation of carboxylic groups in amino acids and peptides can be suppressed at low pH.

However, this together with the suppression of the silanol charge may cause early elution

of these compounds and poor resolution, unless anionic reagents are added.

pKa1

0 147

pH

H2A+ A-HA+/- pKa2

(TFA)

27


● Other anions (most inorganic) used for separation of zwitterions and basic compounds

anions with a less localised charge, higher polarizability and smaller hydration degree

associate stronger to the bonded phase and yield longer retention and enhanced

peak profile, with the following trend:


Hofmeister series

PF6− > ClO4

− > BF4 − > CF3COO − > NO3

− > Cl − > CH3SO3 − > HCOO − > H2PO4

−

Chaotropicity

kosmotropicchaotropic Hydration degree

Chaotropicity or chaotropic effect is the ability to increase the disorder of water. This

explains the adsorption of chaotropic anions and the retention behaviour of cationic

solutes in their presence. The chaotropic effect also explains the retention behaviour in

the presence of different buffers.

28


Mechanism of retention with hydrophilic anions

● The adsorption capability of the most hydrophilic anions in the Hofmeister series is

small. Therefore, the retention mechanism with these anions has been explained by

considering that:

Cationic basic solutes are usually well solvated by the aqueous mobile phase,

with little affinity for the lipophilic phase.

However, cationic basic solutes can interact in the mobile phase with hydrophilic

anions to form an ion pair, which produces disruption of the solvation shell.

Since the ion pair is more lipophilic than the unpaired solute, it can be strongly

retained by the stationary phase.

BH +

A −

Ion-pair chromatography !!!

29


● Only the anion or only the cation is adsorbed on the stationary phase:

sodium hexanesulphonate and tetrabutylammonium hydroxide

● Both cation and anion are adsorbed (dual character):

hexylamine salicylate, butylammonium phosphate, and ionic liquids

the adsorption of cation or anion may be dominant !!!


A−

A−

A−

R+

R+

A−

A−

R+

A−

A−

R+ R

+

R+

A−

R+

R+

A−

A−R

+

R+ R

+

A−

R+

R+

A−

Mono and dual character of reagents

30


● Known mainly as green solvents, but in RPLC they behave just like dissociated salts.

● Although little research still has been done on the effect of ionic liquids on retention,

imidazolium tetrafluoroborates (BF4−) seem competitive against other common IIC

additives, with regard to retention and the silanol-masking effect of the cation.

● Retention is excessive with a strong chaotropic anion, such as pentafluorophosphate.


N N

butylCH3

+

1-butyl-3-methylimidazolium

tetrafluoroborate

BF4-

PF6− > ClO4

− > BF4 − > CF3COO − > …

R+

R+

BF

4-

BF

4-

R+

R+

R+

BF

4-

BF4

-R+BF

4-

charged bilayer

BH

+

Ionic liquids
















31

Ionic liquids

Ionic liquid

1-R-3-Methylimidazoliumcation

N N+

CH3R

Anion m.p.. (oC) d (g mL−1)Watersolubility

Physical stateat room

temperature

EMIM·PF6 1-Ethyl- PF6

−59 1.48 partially soluble solid

BMIM·BF4 1-Butyl- BF4

− - 71 1.21 soluble liquid

BMIM·PF6 1-Butyl- PF6

−11 1.38 non-soluble liquid

HMIM·BF4 1-Hexyl- BF4

− - 81 1.15 immiscible liquid


32


Peak profiles

0 5 10 15 20 25 30 35

Acebutolol

Celiprolol

Esmolol

Metoprolol

Oxprenolol

Pindolol

Propranolol

Timolol

Alprenolol

Ate

no

lol

Retention time, min

0 10 20 30 40 50 60 70

Alprenolol + Propranolol

OxprenololCeliprolol

Esmolol

Metoprolol

Acebutolol

Timolol

Pindolol

Atenolol

Retention time, min

Nucleosil

18.1% acetonitrile

Nucleosil

10.0% acetonitrile / 0.0244 M HMIM·BF4

33




Surfactant coating is an easy and inexpensive way of converting silica-

based RPLC packings into ion exchangers, offering different ion-

exchange capacities and selectivities. However, retention times may

drift, due to coating leakage, with a need of periodic column

regeneration. A reproducible behaviour needs a careful column

equilibration to its plateau capacity.A

ds

orb

ed

CT

AB

, m

mo

l·m

–2

0.00 0.05 0.10 0.15 0.200

1

2

3

4

5

6

CTAB, M

Silica

■ methyl

octadecyl

▲ cyanopropyl

○ octyl

● bare

34


Cationic surfactants with quaternary ammonium groups are frequently used for the

separation of inorganic anions.

● The stationary phase can be directly coated with the cationic surfactant.

● Coating first with a layer of nonionic surfactant, and then with the cationic surfactant,

can yield improved chromatographic performance.

anionic + cationic surfactant

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

HO

O

OO

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

H O

O

O

O

O

−

−

Brij-35

CTAB

cationic surfactant

−

−

CTAB

35


● A surfactant with a single functionality (anionic or cationic) requires a

competing ion to release ionic solutes from the Stern layer to the bulk

solution.

Need of competing ions


Sodium dodecylsulphate (SDS)

Cetyltrimethylammonium bromide (CTAB)

36


● With a zwitterionic surfactant (positive quaternary ammonium and negative sulphonate

groups close to each other), ionic solutes experience simultaneous attraction and

repulsion forces: there is no need of an ion-exchange competing ion.

● Coating with zwitterionic surfactant is termed electrostatic ion chromatography and is a

kind of green chromatography, since the mobile phase can be just pure water or an

electrolyte solution, such as NaHCO3 or Na2B4O7 .

● The addition of a cationic surfactant to the coating solution containing a zwitterionic

surfactant increases the retention of divalent anions with respect to monovalent anions,

and can change their elution order of both kinds of anions.


3-(N,N-dimethylmyristylammonio) propanesulphonate

Need of competing ions …

37


2.1. Introduction












38


Secondary interactions

● Displacement of the adsorbed solute by the IIC counterion.

● Formation of ion pairs between the solute and IIC counterion.

● With surfactants, interaction with dynamic aggregates called

micelles, formed above the critical micelle concentration (CMC).

● Above the CMC, the amount of adsorbed surfactant on the stationary

phase remains constant or is near saturation, which is an important

feature with regard to robustness.




Above a certain concentration of an IIC reagent in the mobile phase, the stationary

phase becomes saturated. Beyond this threshold, the retention, instead of further

increasing, may progressively decrease.

Micelles behave as a new phase (pseudophase) within the mobile phase, which leads into

the field of another RPLC mode, named micellar liquid chromatography (MLC).

MLC

39


k

0.00 0.02 0.04 0.06

0

20

40

60

80

BMIM·PF6 (M) SDS (M)

k

0.00 0.05 0.10 0.15

0

20

40

60

80

100

120

R+

R+ R

+

R+

A−

R+A−

BH+MLC

40


Cetyltrimethylammoniumbromide

Sodium dodecyl sulphate

41


● In the pseudophase LC modes (pseudo = false, imitation), the mobile phase contains

entities that interact with solutes, such as:

micelles cyclodextrins

vesicles nanometer-sized oil droplets in oil-in-water microemulsions

● MLC has achieved the greatest impact due to its simplicity and low cost.

● The unique selectivity of MLC is attributed to the ability of micelles to organise solutes at

the molecular level.

● However, the association of the surfactant monomers to the bonded phase creates a

surface similar to the exterior of an open micelle, with deep implications with regard to

retention and selectivity.


MLC is classified among the pseudophase liquid chromatographic modes.

42


● Surfactants with ionic, zwitterionic and non-ionic head groups are used to

separate ionic or neutral solutes that are able to interact with the surfactant.

● The anionic sodium dodecyl sulphate (SDS) is by far the most common

surfactant in MLC, followed by the cationic cetyltrimethylammonium bromide

(CTAB) and the non-ionic polyoxyethylene-(23)-dodecyl ether (Brij-35).

● Charged surfactants allow the separation of charged and neutral solutes, but

anionic solutes eluted with an anionic surfactant and cationic solutes with a

cationic surfactant will give peaks close to the dead time.

Types of surfactants


43


BH+

BH +

BH

+

BH

+

BH

+

BH

+

MLC Interactions

The surfactant chain is oriented to the mobile

phase, changing the stationary phase polarity

and type of interactions:

● hydrophobic

● electrostatic for charged surfactants

● specific interactions

Brij-35 interacts strongly with hydroxyls

in phenols and polyphenols

● shape contraints

● steric constraints

44


SDS CTAB

Solutes are separated on the basis of their differential partitioning between the bulk

aqueous phase and the micelles, or the surfactant-coated stationary phase. Both

equilibria can be altered for ionisable compounds by tuning the pH.


The mechanism of retention of solutes strongly associated to the surfactant

through hydrophobic interaction (highly apolar solutes), electrostatic attraction, or

specific interactions, should be explained by the direct transfer from the micelles

to the surfactant-modified stationary phase.

45

46




The idea of developing a chromatographic mode with aqueous micellar solutions as mobile

phases (without organic solvent) is highly attractive.

Green Chemistry: RPLC with water and soap

FIS

QUE

BAI

CRY

FLA

3OH

5OH

0 10 20 30

tR (min)

0.0

0.2

0.4

0.6

0.8

1.0Flavonoids

C18 / 0.04 M Brij-35

55oC

47


● With pure micellar mobile phases:

the effective stationary phase thickness in the packing increases significantly,

therefore, the strength of the interactions is larger and

solute mass transfer within the stationary phase is difficult (slower)

especially for highly hydrophobic solutes


In most cases, pure micellar mobile phases (without organic solvent) have two problems:

excessive retention and poor efficiency compared to conventional RPLC.

BH

+

BH +

BH

+

48


0 10 20

Time, min

0.1125 M SDS

15% (v/v) acetonitrile

0 50 100 150

Time, min

0.1125 M SDS

Basic drugs

49


● Organic solvents dissolve the surfactant coating and form hybrid micellar mobile

phases composed of surfactant and organic solvent.

● With a thinner surfactant layer on the stationary phase, the retention is decreased

and the efficiency improved to practical values.


The surfactant layer is decreased by addition of organic solvents to the mobile phase.

Ad

so

rbed

CT

AB

(m

mo

l·m

–2)

5.0

Organic solvent mole fraction

0.0 1.0 2.02.0

3.0

4.0Methanol

1-Propanol

1-Pentanol

Ad

so

rbed

SD

S (

mm

ol·

m–2)

0.0 1.0 2.03.0

3.8

4.6

Methanol1-Propanol

1-Pentanol

Organic solvent mole fraction


The separation mode is still predominantly micellar in nature, but the micelle is perturbed

by the organic solvent, giving rise to changes in the CMC and surfactant aggregation

number (a mixed micelle can be obtained).

tetrahydrofuran

% organic solvent (v/v)

SDS solutions

acetonitrile

Hybrid MLC

50

1-butanol

1-pentanol

methanol

1-propanol

ethanol

CM

C (

mM

)

% organic solvent (v/v)

0

2.5

5.0

7.5

10.0

12.5

15.0

51


● For historical reasons, alcohols (mainly

propanol) are the most common organic

solvents in hybrid MLC.

● Butanol and pentanol, which are stronger

solvents, are used to elute strongly

retained compounds.

● Acetonitrile (the most common solvent in

conventional RPLC) has been scarcely

used, but it gives rise to interesting

separations !!!


The presence of surfactants at high concentration increases the miscibility of organic

solvents. This allows a wider range of organic solvents at concentrations larger than

those in aqueous solution MLC expands the range of possible mixtures in RPLC.

Retention factor for benzene

5 6 7 8 9

-3

-2

-1

0

1

2

3

log

Po

/w

cyclohexanolhexanol

2-pentanol

1-butanol 2-butanol

2-methyl-1-propanol1-propanol

acetonitrileethanol

methanol2,3-butanediol

1,2-propanediol

formamide

2-methyl-1-butanol

3-methyl-1-butanol

1-pentanol


In the first reports on MLC, the probe solutes were hydrophobic, which

show poor performance in this chromatographic mode, especially in

the absence of organic solvent. This may be the reason of the

generalised idea that the peak profile in MLC is always poor, but in the

presence of organic solvent, the peak profile can be similar or even

improved with respect to conventional RPLC.

About the efficiency …


● Mobile phases containing SDS give rise to

highly symmetrical peaks for basic drugs.

● The suppression of the silanol effect is not due

to a direct electrostatic interaction with the free

silanols (case of amines), but to the protecting

covering of the stationary phase, which

prevents very efficiently that positively charged

solutes penetrate into the bonded alkyl-chains

to interact with the buried silanols.

● Meanwhile, the ion-exchange mechanism with

the sulphate group in the surfactant is a fast

process.


53

BH +

BH

+

BH

+

BH

+

A

12 13 14 15

Time (min)

BA = B

54

SDS: effective silanol suppressor

0 10 20 30 40 50 60

1

2

3

4 5

6

7 89

10

Time, min

Kromasil column


pH 3

0 10 20

1

23

4

5

67

8

910

Time, min

Kromasil column

0.1125 M SDS


pH 3


55


● The organic solvent concentration (v/v) that still preserves the integrity of

micelles is approximately:

15% for propanol and acetonitrile, 10% for butanol, and 6% for pentanol

● Higher concentrations of organic solvent can sweep out completely the

adsorbed surfactant molecules from the bonded phase surface, or at least,

avoid the formation of micelles (only surfactant monomers remain in the

mobile phase).

● Without micelles (no more MLC !!!), as long as the stationary phase is covered

by the surfactant layer, there will be a differentiated behaviour compared to

hydro-organic RPLC.


In principle, a high percentage of organic solvent is not desirable,

since it leads to micelle disruption.

56

Wrongly classified MLC procedures

0.03 M SDS / 55–70% (gradient) methanolAlkylbenzenesulphonates, ground and waste water

0.1 M SDS / 15% 1-butanol, pH 7Azithromycin, formulations

0.4 M SDS / 30-42% acetonitrile (gradient), pH 3

Biogenic amines, food substrates

0.01 M SDS / 30% 1-propanol, pH 2Antioxidants, olive oil

0.035 M SDS / 20–30% 1-propanol, pH 6.4Human growth hormone, fermentation broth

0.02 M SDS / 38% methanol / 2% 1-propanol, pH 6

Cortisol, urine

0.04 M SDS / 55% methanol, pH 30.04 M SDS / 70% acetonitrile, pH 3

Diverse drugs, biological fluids

Mobile phase compositionCompounds and sample


57


● A new chromatographic mode is achieved:

high submicellar liquid chromatography (HSLC).

● Performance is even improved with respect to

conventional RPLC or MLC: peak profiles and

resolution can be better, and analysis times,

shorter.

● This name indicates that the mobile phase

contains a surfactant at a concentration where

micelles are formed in water, but the high

concentration of organic solvent does not allow

their formation.


CH3 –C≡N

CH3 – C≡N

CH

3 –C

≡N

CH3 – C≡N

CH3 – C≡N

CH3 – C≡N

CH3 –C≡N

CH3 – C≡N

CH

3 –

C≡

N

CH

3 –C

≡N

CH3 – C≡N

CH 3 –C≡N

CH3 – C≡NCH

3 – C≡N

CH3 –C≡N

CH3 – C≡N

HSLC

SDS, MA

ceto

nit

rile

, %

(v/v

)

0 0.04 0.08 0.12 0.160

10

20

30

40

50

60

transition region

high submicellar

micellar

low submicellar

hydro-organic

a

bc

d

e

f

g

h

i

0 50 100 150 2000 10 20 30 40 50 60 0 1 2 3 4 5

0 2 4 6 8 10 0 20 40 60 80

0 20 40 60 0 20 40 0 10 20

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Time, min

12

3

4 5

67 8 9

10

1 2 3

4

5

678

9 10

12

3

4

5

678

9 10

1

23

4

5

678910

1 2

34

56

789 10

1

23

45

6 78910

1

23

4

5678

910

23

1

5 6 7 9 10

1

2 3

4

5 6 7

8910

8 4

0 40 80 120


β-Blockers

HSLCHSLC

MLC

IIC

MLC

59


● The variety of interactions between solutes, stationary phase, aqueous phase, and

micelles, which give rise to unique selectivity, often favourable to get good resolution.

● The possibility of separating both charged and neutral solutes in a single run.

● The separation of solutes in a wide polarity range with retention time windows

narrower than in classical RPLC. This makes gradient elution less necessary.

● The low organic solvent concentration in hybrid MLC, which means lower toxicity and

environmental impact of wastes with regard to conventional RPLC.

● The smaller evaporation of organic solvents, associated to the surfactant. This makes

micellar mobile phases stable for a longer time and recirculation of mobile phase

possible.

● Enhanced luminescence detection.


Advantages of MLC

60

Tarragona, Secyta’2012High submicellar liquid chromatography

The only real limitation of MLC is that direct on-line coupling to ELS and MS detection is

hindered by the presence of the high concentrations of surfactant in the mobile phase.

Injection of physiological fluids

Major advantage: The high solubilisation capability of micelles, which facilitates

dissolution of most matrices. This saves time in sample preparation, and allows the direct

on-column injection of physiological fluids or other liquid samples containing proteins.

0.1 M SDS

1−2% butanol

0.1 M SDS10−25% butanol

mechanisms of retention in hplc - universitat de …...2 hplc’2013 (amsterdam) 1. retention in...

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