Mechanisms of retention in HPLC

Mechanisms of retention in HPLC María Celia García-Álvarez-Coque Department of Analytical Chemistry University of Valencia Valencia, Spain https://sites.google.com/site/fuschrom/ 1 Part 2

Mechanisms of retention in HPLC Index 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 2

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 3

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 4

Limitations of RPLC Polar compounds (polar neutral or ionised organic compounds, and inorganic anions or metal ions) show little or no retention. log k c 0 c1 log P o/w HA Retention factor A Ionisable compounds Retention factor BH + B ph ph 5 This has been a challenge in environmental, clinical and food chemistry throughout the development of RPLC.

Limitations of RPLC 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 BH + BH + BH + 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 ionexchange 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. 6

Addition of ionic reagents 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. A + H + HA Types of interactions: A A + A 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 C + + X C + X A + X AX (2.1) A : analysed solute (analyte) or silanol group on the support A M + L ML 7 X : a lipophilic ion, the hydrogen ion, a ligand, or other added species

The observed retention factor is a weighted average of the retention of the solute species. k k A ka kax K [X] A kax AX (2.2) 1 K [X] A : molar fraction of A AX : molar fraction of AX L H R Cu 2+ H [X] : molar concentration of X in the mobile phase K : formation constant (acid-base reaction: log K = pk a ) 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. 8 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.

2.2. Ion-interaction chromatography 2.2.1. Retention mechanism R 3 NH + Cl R 3 NH + R 3 NH + R 3 NH + A 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). 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. C + BH + R S3 R S3 R S 3 Na + ACN 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. HA R S3 10

Approaches 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. 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. A 11 Hence, the name ion-pair chromatography (IPC) taken from liquid-liquid extraction.

Dynamic ion-exchange mechanism 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, stoichiometric approach followed for decades. and pioneered the A A 12

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). A - A - A - 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 ionexchange capacity, and hence, the retention of solutes with a charge opposite to the lipophilic ion. 13

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 pk a. B + Dual mechanism pk a R B + R HA HA Retention factor A 14 ph

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) perhaps, the most correct term!!! Ion-modified chromatography Hetaeric chromatography (hetaeron means counterion) Surfactant (or soap) chromatography (with ionic surfactants) Submicellar liquid chromatography (surfactants below the CMC) 15 2009

2.2.2. Common reagents and operational modes Reagents A A 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. alkylsulphonate 16 alkyl sulphate

perational modes 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. A 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. BH + R 3 NH + R 3 NH + R 3 NH + R S 3 R S 3 R S 3 17 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.

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. 3.5 3.5 Benzyltrimethylammonium cyanopropyl-silica log k 2.5 2.5 1-PrH R S 3 BH + 1.5 1.5 R S 3 R S 3 R S 3 0.5 0 10 20 30 40 50 SDS, mm 0.5 0 10 20 30 40 50 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. 18

Something more to know 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. A - It is not essential that the IIC counterion serves as the ion-exchange competing ion. ther ions are often added to assist in the elution of strongly retained anions: phosphate oxalate citrate phthalate 19

Something more to know 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 precolumn. System peaks chromatograms. corresponding to the added reagent will appear in the Traditional lipophilic reagents are not usually compatible with evaporative light scattering (ELS) and mass spectrometry (MS) detection. A - Injection 20 ELS MS

2.2.3. The silanol effect and its suppression 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. silanols BH + BH + BH + Atenolol Pindolol Acebutolol Timolol Metoprolol Celiprolol Esmolol Spherisorb C18 xprenolol Propranolol Alprenolol 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 Time, min 21 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.

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. Increasing acidity 22 Depends on: amount of available silanol groups relative acidic character type (isolated, geminal or vicinal) presence of metal impurities

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. Atenolol Pindolol Timolol Metoprolol Acebutolol Esmolol Celiprolol xprenolol Zorbax C18 Alprenolol Propranolol Atenolol Pindolol Acebutolol Timolol Metoprolol ACN / water Celiprolol Esmolol xprenolol Spherisorb C18 Propranolol 0 Atenolol Pindolol Timolol Acebutolol Metoprolol Esmolol Celiprolol xprenolol Nucleosil C18 Alprenolol+ + Propranolol 0 10 20 30 40 50 Time, min 0 10 20 30 40 50 Time, min Alprenolol 10 20 30 40 50 60 70 Time, min 23 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!!!

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. 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 24

Several alternatives with IIC reagents 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. BH + BH + BH + R3 NH+ R3NH + 25

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. Anionic reagents Alkylsulphonates H pk a1 2 A + HA +/- pk a2 A - 0 14 7 ph may strongly associate to the stationary phase making column regeneration difficult (TFA) 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. 26

Hofmeister series ther 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: Chaotropicity PF 6 > Cl 4 > BF 4 > CF 3 C > N 3 > Cl > CH 3 S 3 > HC > H 2 P 4 chaotropic Hydration degree kosmotropic 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. 27

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. A BH + Ion-pair chromatography!!! 28

Mono and dual character of reagents nly 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 A A A A A A A A A 29

Ionic liquids 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 (BF 4 ) 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. PF 6 > Cl 4 > BF 4 > CF 3 C > BF 4 - BF 4 - N CH 3 + N BF - 4 butyl charged bilayer BF 4 - BH + BF 4 - BF 4-1-butyl-3-methylimidazolium tetrafluoroborate 30

Ionic liquids Ionic liquid 1-R-3-Methylimidazolium cation N + N Anion m.p.. ( o C) d (g ml 1 ) Water solubility Physical state at room temperature R CH 3 EMIM PF 6 1-Ethyl- PF 6 BMIM BF 4 1-Butyl- BF 4 BMIM PF 6 1-Butyl- PF 6 HMIM BF 4 1-Hexyl- BF 4 59 1.48 partially soluble solid - 71 1.21 soluble liquid 11 1.38 non-soluble liquid - 81 1.15 immiscible liquid 31

Peak profiles Atenolol Pindolol Timolol Acebutolol Metoprolol Esmolol Atenolol Pindolol Timolol Metoprolol Acebutolol Esmolol xprenolol Celiprolol Celiprolol xprenolol Alprenolol Propranolol Alprenolol + Propranolol 0 10 20 30 40 50 60 70 Retention time, min 0 5 10 15 20 25 30 35 Retention time, min Nucleosil 18.1% acetonitrile Nucleosil 10.0% acetonitrile / 0.0244 M HMIM BF 4 32

2.2.5. Separation of inorganic anions with surfactant-coated stationary phases Surfactant coating is an easy and inexpensive way of converting silicabased RPLC packings into ion exchangers, offering different ionexchange 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. 33 Adsorbed CTAB, mmol m 2 6 5 4 3 2 1 0 0.00 0.05 0.10 0.15 0.20 CTAB, M Silica methyl octadecyl cyanopropyl octyl bare

H 2. Secondary equilibria in reversed-phase liquid chromatography: Part A 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. H H H H H H H Brij-35 CTAB CTAB 34 cationic surfactant anionic + cationic surfactant

Need of competing ions 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. Sodium dodecylsulphate (SDS) Cetyltrimethylammonium bromide (CTAB) 35

Need of competing ions 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 NaHC 3 or Na 2 B 4 7. 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 36

2.3. Micellar liquid chromatography 2.3.1. An additional secondary equilibrium in the mobile phase 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. 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). MLC 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. 38 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).

A A MLC BH+ 80 120 k 60 k 100 80 40 60 20 40 20 39 0 0.00 0.02 0.04 0.06 BMIM PF 6 (M) 0 0.00 0.05 0.10 0.15 SDS (M)

Sodium dodecyl sulphate Cetyltrimethylammonium bromide 40

MLC is classified among the pseudophase liquid chromatographic modes. 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. 41

Types of surfactants 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. 42

MLC Interactions BH + The surfactant chain is oriented to the mobile phase, changing the stationary phase polarity and type of interactions: BH + hydrophobic electrostatic for charged surfactants BH + specific interactions Brij-35 interacts strongly with hydroxyls BH + BH + BH + in phenols and polyphenols shape contraints steric constraints 43

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. SDS CTAB 44

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

2.3.2. Hybrid micellar and high submicellar liquid chromatography 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 1.0 0.8 QUE FIS BAI Flavonoids C18 / 0.04 M Brij-35 55 o C FLA 0.6 CRY 3H 5H 0.4 0.2 0.0 0 10 20 30 t R (min) 46

In most cases, pure micellar mobile phases (without organic solvent) have two problems: excessive retention and poor efficiency compared to conventional RPLC. 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 BH + BH + BH + 47

Basic drugs 0 50 100 150 Time, min 0.1125 M SDS 0 10 20 Time, min 0.1125 M SDS 15% (v/v) acetonitrile 48

The surfactant layer is decreased by addition of organic solvents to the mobile phase. rganic 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. Adsorbed SDS (mmol m 2 ) 4.6 3.8 1-Propanol 1-Pentanol Methanol 3.0 0.0 1.0 2.0 rganic solvent mole fraction Adsorbed CTAB (mmol m 2 ) 5.0 Methanol 4.0 1-Propanol 3.0 1-Pentanol 2.0 0.0 1.0 2.0 rganic solvent mole fraction 49

Hybrid MLC 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). 15.0 SDS solutions 12.5 methanol 10.0 CMC (mm) 7.5 5.0 ethanol 1-propanol acetonitrile 2.5 1-butanol 1-pentanol tetrahydrofuran 0 50 % organic solvent (v/v) % organic solvent (v/v)

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. log P o/w 3 2 1 0-1 -2-3 51 2-methyl-1-butanol cyclohexanol hexanol 3-methyl-1-butanol 2-pentanol 1-pentanol 1-butanol 2-butanol 2-methyl-1-propanol 1-propanol acetonitrile ethanol methanol 2,3-butanediol 1,2-propanediol formamide 5 6 7 8 9 Retention factor for benzene 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!!!

About the efficiency 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.

Mobile phases containing SDS give rise to ABH+ B A = B 12 13 14 15 Time (min) 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. BH + BH + BH + Meanwhile, the ion-exchange mechanism with the sulphate group in the surfactant is a fast process. 53

SDS: effective silanol suppressor 1 2 3 1 2 3 5 4 5 6 8 7 4 6 9 10 7 8 9 10 0 10 20 30 40 50 60 Time, min Kromasil column 15% (v/v) acetonitrile ph 3 0 10 20 Time, min Kromasil column 0.1125 M SDS 15% (v/v) acetonitrile ph 3 54

In principle, a high percentage of organic solvent is not desirable, since it leads to micelle disruption. 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. 55

Wrongly classified MLC procedures Compounds and sample Mobile phase composition Diverse drugs, biological fluids 0.04 M SDS / 55% methanol, ph 3 0.04 M SDS / 70% acetonitrile, ph 3 Cortisol, urine 0.02 M SDS / 38% methanol / 2% 1-propanol, ph 6 Human growth hormone, fermentation broth Antioxidants, olive oil 0.035 M SDS / 20 30% 1-propanol, ph 6.4 0.01 M SDS / 30% 1-propanol, ph 2 Biogenic amines, food substrates 0.4 M SDS / 30-42% acetonitrile (gradient), ph 3 Azithromycin, formulations 0.1 M SDS / 15% 1-butanol, ph 7 Alkylbenzenesulphonates, ground and waste water 0.03 M SDS / 55 70% (gradient) methanol 56

A new chromatographic mode is achieved: high submicellar liquid chromatography (HSLC). HSLC Performance is even improved with respect to conventional RPLC or MLC: peak profiles and resolution can be better, and analysis times, shorter. CH 3 C N CH 3 C N CH 3 C N CH 3 C N CH 3 C N CH 3 C N This name indicates that the mobile phase contains a surfactant at a concentration where CH 3 C N CH 3 C N CH 3 C N CH 3 C N micelles are formed in water, but the high concentration of organic solvent does not allow their formation. CH 3 C N CH 3 C N CH 3 C N CH 3 C N CH 3 C N CH 3 C N 57

60 Acetonitrile, % (v/v) 50 40 30 20 10 d low submicellar b c hydro-organic a high submicellar transition region micellar i h g f e β-blockers 0 0 0.04 0.08 0.12 0.16 SDS, M 1 23 4 5 6 7 8 9 10 2 3 5 4 1 2 (a) 1 (b) (c) 6 8 7 10 9 3 5 4 6 8 7 IIC 9 10 0 10 20 30 40 50 60 0 1 2 3 4 5 0 50 100 150 200 1 (d) 1 (e) 1 (f) 2 8 4 MLC 2 53 2 6 7 3 3 9 5 10 5 6 7 8 7 6 8 4 4 10 9 10 9 0 2 4 6 8 10 0 40 80 120 0 20 40 60 80 1 1 (g) 1 (h) 2 MLC 2 3 (i) 2 3 HSLC HSLC 5 3 5 8 5 7 8 6 4 8 7 6 7 6 4 9 4 10 10 9 9 10 0 20 40 60 0 20 40 0 10 20 Time, min

Advantages of MLC 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. 59

High submicellar liquid chromatography Tarragona, Secyta 2012 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 SDS 10 25% butanol 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. 60