On-Line Concentration Techniques in Capillary Electrophoresis and the Experimental Investigation of Electroextraction

ON-LINE CONCENTRATION TECHNIQUES IN

CAPILLARY ELECTROPHORESIS AND THE

EXPERIMENTAL INVESTIGATION OF

ELECTROEXTRACTION

Riikka-Mari Haara Master's thesis 4.8.2016 University of Helsinki Laboratory of Analytical Chemistry

Tiedekunta/Osasto Fakultet/Sektion – Faculty Laitos/Institution– Department Faculty of science Chemistry

Tekijä/Författare – Author Riikka-Mari Haara

Työn nimi / Arbetets titel – Title On-line concentration techniques in capillary electrophoresis and the experimental investigation of electroextraction

Oppiaine /Läroämne – Subject Analytical chemistry

Työn laji/Arbetets art – Level Aika/Datum – Month and year Sivumäärä/ Sidoantal – Number of pages Master's thesis 08/2016 94

Tiivistelmä/Referat – Abstract

Capillary electrophoresis is a great option for analyzing metabolomics compounds since the analytes are often charged. The technique is simple and cost-efficient but it is not the most popular equipment because it lacks high concentration sensitivity. Therefore, on-line concentration techniques have been developed for capillary electrophoresis. The aim of this thesis is to give an introduction to the most common on-line concentration methods in capillary electrophoresis, and to demonstrate a novel on-line concentration technique termed electroextraction.

Until now, the research of on-line concentration techniques in capillary electrophoresis is mainly focused on methods based on field amplification, transient isotachophoresis, titration incorporated methods or sweeping, which are presented in the literature section.

In a two-phase electroextraction, the electrodes are placed in an aqueous acceptor phase and in an organic donor phase, in which the analytes are dissolved. When the voltage is applied, the conductivity difference in the two phases cause high local field strength on organic phase leading to fast migration of the cationic analytes towards the cathode. As soon as the analytes cross the solvent interface, their migration speed decrease and they are concentrated at the phase boundary. In these experiments, a normal capillary electrophoresis analyzer was used with a hanging aqueous phase droplet at the tip of the capillary inlet.

The experimental part was carried out at Leiden University, Division of Analytical BioSciences in the Netherlands. An electroextraction-capillary electrophoresis system was built for the analysis of biological acylcarnitine compounds. After the method parameters were assessed with ultraviolet detection, the method was coupled with mass spectrometric detection, and the selectivity and repeatability were briefly tested.

Sensitivity was enhanced with the electroextraction procedure but the extraction factors were not satisfactory yet. Selectivity of electroextraction was discovered when the extraction of acylcarnitines was performed using different solvents. All parameters affecting the electroextraction procedure were not tested, and therefore the instability of the method was not completely understood. Thus, the method should be further investigated and optimized. In fact, all on-line concentration methods ought to be optimized for the target analytes in their existing matrix. Avainsanat – Nyckelord – Keywords On-line concentration, capillary electrophoresis, electroextraction

Säilytyspaikka – Förvaringställe – Where deposited Laboratory of Analytical Chemistry and Helsinki University Library

Muita tietoja – Övriga uppgifter – Additional information

Table of contents Page

1. ABBREVIATIONS 3

2. INTRODUCTION 6

I THEORY 7

3. CAPILLARY ELECTROPHORESIS 7

3.1. Electro-osmotic flow 8

3.2. Separation of compounds 9

3.3. Injection 10

3.4. Detection 11

4. ON-LINE CONCENTRATION TECHNIQUES 13

4.1. Field amplified sample stacking 14

4.2. Field amplified sample injection 18

4.3. Pressure assisted electrokinetic injection 20

4.4. Large volume sample stacking 21

4.5. Transient isotachophoresis 26

4.6. Electrokinetic supercharging 34

4.7. Titration incorporated methods 36

4.7.1. Dynamic pH junction 37

4.7.2. pH-mediated stacking 40

4.8. Sweeping 46

4.8.1. Sweeping in electrokinetic chromatography 46

4.8.1.1 Pseudostationary phases 46

4.8.1.1.1 Charged pseudostationary phases in a homogeneous electric field 49

4.8.1.1.2 Charged pseudostationary phases in a heterogeneous electric field 50

4.8.1.2 Parameters for optimal sweeping 53

4.8.2. Sweeping in capillary zone electrophoresis 54 1

4.9. Electroextraction 58

4.10. Combination of on-line concentration techniques 59

II EXPERIMENTAL 64

5. BACKGROUND 64

5.1. Electroextraction procedure 64

5.2. Chemicals 66

5.2.1. Crystal violet 66

5.2.2. Acylcarnitines 67

5.2.3. Solvents 69

6. EXPERIMENTS 70

6.1. Preparation of solutions 70

6.1.1. Background electrolyte solution 70

6.1.2. Crystal violet samples 70

6.1.3. Acylcarnitine samples 71

6.2. Analytical conditions 71

7. RESULTS AND DISCUSSION 73

7.1. Samples 73

7.2. Electroextraction 73

7.3. Visual monitoring of the stability of a droplet 75

7.4. Electroextraction-capillary electrophoresis-ultraviolet detection 77

7.5. Capillary zone electrophoresis-mass spectrometry 79

7.5.1. Electroextraction parameters 79

7.5.2. Different solvents 81

7.5.3. Repeatability 83

8. CONCLUSIONS 84

9. ATTACHMENTS 86

10. REFERENCES 86

1. ABBREVIATIONS

5-FAM 5-carboxyfluorescein Ab antibody AD amperometric detection APFO ammonium perfluorooctanoate ARG phenylthiohydratoin arginine ASEI anion selective exhaustive injection BGE background electrolyte, running buffer BPDE benzo(a)pyrene diol epoxide Brij 35 polyoxyethylene (23) lauryl ether Brij 58 polyoxyethylene (20) cetyl ether C4D capacitively coupled contactless conductivity detector CAPS 3-(Cyclohexylamino)1-propanesulphonic acid CE capillary electrophoresis CEC capillary electrochromatography CGE capillary gel electrophoresis CIEF capillary isoelectric focusing CITP capillary isotachophoresis CMC critical micelle concentration CSEI cation selective exhaustive injection CTAB cetyltrimethylammonium bromide CTAC cetyltrimethylammonium chloride CV crystal violet CZE capillary zone electrophoresis DETA diethylenetriamine DG deoxyguanosine DLLME dispersive liquid-liquid microextraction DTAB dodecyltrimethylammonium bromide EDTA ethylenediaminetetraacetic acid EF extraction factor EKC electrokinetic chromatography EKI electrokinetic injection

EKS electrokinetic supercharging EKSI electrokinetic stacking injection EOF electroosmotic flow ESI electrospray ionization EtOAc ethyl acetate FA formic acid FASS field amplified sample stacking FASI field amplified sample injection FESI field enhanced sample injection FL fluorescein disodium salt GSH glutathione GSSG glutathione disulfide HDI hydrodynamic injection HCB high conductivity buffer HIS phenylthiohydratoin histidine HPLC high performance liquid chromatography HV high voltage ICP inductively coupled plasma ID internal diameter IDP indapamide IEF isoelectric focusing ITP isotachophoresis L length LE leading electrolyte LIF laser-induced fluorescence LLE liquid-liquid extraction LOD limit of detection LVSS large volume sample stacking MEEKC microemulsion electrokinetic chromatography MEKC micellar electrokinetic chromatography MeOAc methyl acetate MeOH methanol MES 2-(N-morpholino)ethanesulfonic acid miRNA microRNA MS mass spectrometry

MSPE magnetic solid-phase extraction MW molecular weight n-BuOH n-butanol NACE non-aqueous capillary electrophoresis NaDC sodium deoxychlolate NPS neutral pseudostationary phase NRB neutralization reaction boundary PAEKI pressure assisted electrokinetic injection PEI-Mal maltose-modified hyperbranched poly(ethylene imine) PDA photodiode array detector Poly-SUS poly(sodium 10-undecenyl sulfate) proFACE protein-facilitated affinity capillary electrophoresis PS pseudostationary phase pSAm-f poly(sodium 2-acrylamido 2-methyl 1-propane sulfonate-co-stearyl acrylamide) SB-12 N-dodecyl-N,N-dimethylammonium-3-propane-1-sulfonic acid SDME single drop microextraction SDS sodium dodecyl sulfate SOT sotalol SPE solid phase extraction SVZ sample vacancy zone TE terminating electrolyte TFA trifluoroacetic acid TG tris-glycine tITP transient isotachophoresis Tris tris(hydroxymethyl)aminomethane TTAB tetradecyltrimethylammonium bromide UV ultraviolet

2. INTRODUCTION

Capillary electrophoresis (CE) has a long history as a separation technique and it is applied to pharmaceutical, environmental, biological, food, forensic and toxicological samples. Power of the technique is in high separation efficiency, simplicity, low cost and the small need for sample and solvent volume. However, CE has never gained similar popularity as high performance liquid chromatography (HPLC) because CE suffers from low concentration sensitivity due to small sample injection volume (nL). Addition of an on-line preconcentration technique to CE analysis has given rise to better sensitivity. New preconcentration methods are being developed and recently the combination of more than one technique has become popular.

Many metabolomics compounds are charged making CE a potential instrument for analyses. In metabolomics studies, often analytes in low concentrations are the most significant. To understand metabolic processes, the number of samples to be analyzed is large. Fast and sensitive analytical techniques are needed to keep up with the high sample throughput. None of the existing analytical methods is capable of measuring all the desired analytes in one run. Sample preparation will always result in loss of some compounds. Analytical devices have also limitations with sample matrices and analyte characteristics. Combination of analytical techniques ought to be tested to increase the information from a sample in a single run.

The purpose of this thesis is to give an introduction to the most widely used on-line concentration methods in CE and additionally present a novel preconcentration method named electroextraction (EE). Focus is on on-line concentration techniques combined to capillary zone electrophoresis (CZE), and to some extent to micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). Non-aqueous capillary electrophoresis (NACE) is not discussed here. Experimental was done at Leiden University in the Netherlands. There the Analytical BioSciences group is focused on metabolomics research. The aim of the experimental work was to build an automated EE-CE-MS (mass spectrometry) system and thereafter investigate the selectivity of EE with biological analytes.

I THEORY

3. CAPILLARY ELECTROPHORESIS

Electrophoresis is the movement of charged compounds in an electric field. Electrophoresis was introduced as separation technique in 1937 by Tiselius.1 One of the early electrophoretic formats was the slab gel model but a more efficient and automated technique was needed. Electrophoresis was transferred into narrow-bore open tubular capillaries to prevent convection and improve dissipation of Joule heating; that is how initial capillary electrophoresis was developed in 1967 by Hjertén.

The main components of capillary electrophoresis equipment are shown in figure 1.1 The capillary is filled with background electrolyte (BGE) and its both ends are placed in BGE reservoirs. Due to the BGE solution the system is conductive. Electrodes are used to make electric contact with the high voltage (HV) power supply. During injection the inlet BGE vial is replaced with a sample vial allowing sample to enter the capillary either by the force of external pressure (i.e. hydrodynamic injection, HDI) or the mobility caused by an electric field (i.e. electrokinetic injection, EKI). CE separation is operated by applying an electric field after the sample vial is replaced with the BGE reservoir. Normally high voltages (10-30 kV) and electric fields (100-500 V/cm) are applied. A detector is located at the outlet side of the capillary. Modern instrumentation is controlled via a computer and data collected from a run is called electropherogram.

Figure 1. Schematic picture of a CE system.

3.1. Electro-osmotic flow

Capillaries used in CE separations consist mostly of uncoated fused silica. Internal diameter (ID) used is usually 25-100 μm and length 25-100 cm leaving the volume inside the capillary very small. Large surface area compared to volume is beneficial because the heat that is generated when the electric current flows through capillary (Joule heating) is efficiently dissipated.2 There are free silanol groups (SiOH) on the surface of the capillary. When the pH is high enough, these silanol groups start to dissociate.

SiOH → SiO- + H+

When positive ions from the BGE solution attach to the wall of a negatively charged capillary, a Stern´s layer is formed. Some of the cations close to the wall of the capillary are free to move towards the cathode due to the electric field. This freely moving layer is the diffusion layer and its movement is called electro-osmotic flow (EOF). Opposite charged ions near the wall of the capillary cause potential difference (the zeta potential) in the solution. EOF and analysis speed increase when a bigger potential difference is applied. A buffer solution is used as an electrolyte solution to make runs repeatable and to

prevent changes in pH. Decreasing the ionic strength of the electrolyte solution increases the potential since then Stern´s layer is tight and the diffusion layer is loose.

3.2. Separation of compounds

In CE, compounds are separated due to their unequal mobility in an electric field.1,2 Ions are moving either towards the cathode or anode depending on their electrophoretic attraction. Electric field (E) induces the speed of an ion, which is also influenced by the electrophoretic mobility of the ion (1).

v = μep E = → μep = = (1)

2 -1 -1 -1 where μep = electrophoretic mobility [m V s ], E = electric field [V m ], U = voltage -1 [V], v = speed [m s ], s = distance [m], t = time [s], Ltot = total length of the capillary [m] and Ldet = length of the capillary at detector [cm].

The total mobility of a compound is the sum of electrophoretic mobility and electroosmotic mobility (2). Mobilities can be calculated from the electropherogram. In a positive field electrophoretic mobility is negative for anions but also they will travel towards cathode because EOF is faster. Neutral ions are migrated with EOF because they do not have a charge.

μtot = μep + μEOF (2)

Small ions with high number of charges have the highest mobilities, which can be seen from the equation 3 of electrophoretic mobility presented in physical parameters:

μep = (3)

where z=ion charge, η=solution viscosity and r= ion radius

Magnitude of an electric field is dependent on applied voltage and the length of the capillary (4).

E = (4)

3.3. Injection

Usually a sample is introduced into the capillary using pressure or voltage.2 In the first case, the sample container is pressurized or negative pressure is applied to the collector container when sample solution begins to flow into the capillary. Another mean to inject a sample with pressure is siphoning. The sample container is set on a higher level than the collector reservoir, which makes the sample flow into the capillary because of difference in pressure. EKI is accomplished by applying a voltage over the capillary with the inlet in the sample vial. Compounds are transferred into the capillary by means of EOF and electrophoretic mobility. HDI is not applicable to very viscous samples but it is more repeatable and simple way to inject than EKI. The strength of EKI is the ability to inject samples in viscous media. Also, either positive or negative compounds can be selectively injected by choosing the polarity in EKI.

The injection zone should be shorter than dispersion caused by diffusion to have maximal resolution and efficiency.1 That is why injection plug length should be 1-2 % of the total capillary length. This small injection volume and also small dimensions of the capillary

cause challenges to detection. Volume injected in HDI is derived from Hagen-Poiseuille equation (5), which is used to calculate pressure difference in flowing fluid in a cylindrical pipe:

V = (5)

where V=injected volume [nL], ΔP=pressure difference in capillary [mbar], d=inner diameter of capillary [μm], t=injection time [s], η=viscosity of BGE [Pa s] and Ltot=total length of capillary [cm].

EKI is not as reproducible because it is dependent on electrophoretic mobility of an ion, its concentration, and also the EOF. Variations in sample conductivity cause a change in the sample resistance, and thus to the injection amount Q (6).

Q = (6)

where Q=quantity injected [g or mol], μEOF=EOF mobility, U=voltage [V], r=capillary radius, c=analyte concentration [mol/L], t=time [s] and Ltot=total length of capillary [cm].

3.4. Detection

The most common detector used in CE is ultra violet (UV) visible absorption.2,1 It is robust and efficient due to on-capillary detection without using interfaces that cause band broadening. The optical window is made by burning away a piece of the coating of the capillary. Sensitivity of UV detector is limited by short path length caused by the small diameter of the capillary. Better sensitivity is obtained when mass spectrometry is used as detection technique. Electro-spray ionization (ESI) is best suited to CE-MS systems because ESI works well with small flow rates and with polar or charged compounds, which are used in CE.

Liquid coming from the CE separation has to be altered into gaseous ions before MS detection.1,3 The capillary is inside a steel tube (spray needle) and the sample is exiting capillary through a conical tip of the needle. High voltage is applied on the inlet of the capillary, however, also ESI needs an electrical field. Usually outlet of the capillary is at ground and voltage is applied at the MS inlet. When the potential of the capillary is positive and the potential of the MS is negative, positive ions enter the MS (positive ion mode). Negative ions react with the needle (oxidation). Fine spray is emitted when capillary pushes away ions with the same charge. Charges are located on the surface of the droplet and charges end up closer to each other as the solvent evaporates. When the Coulombic repulsion of charges exceed the surface tension, a droplet explodes into smaller droplets (figure 2). There are two theories how a single analyte is ionized during electrospray. Charge residue mechanism assumes that these explosions occur until solvent is evaporated and a gas-phase ion forms.3,4 This is happening most likely to small molecules. Large molecules might be sprayed according to the ion evaporation theory, which states that analytes can escape from the droplet into gas phase because of an electric field.3,5

Figure 2. ESI positive ion mode.

The interface used in the experiments in this thesis was sheath-liquid assisted to maintain electrical contact independent of EOF.1,6 Schematic of an Agilent Technologies sheath- liquid interface is shown in figure 3.7 CE capillary is placed inside a larger tube and sheath-liquid is flowing between them. The sheath-liquid normally consists of methanol and dilute formic acid. Methanol has a low surface tension, which promotes evaporation.

CE effluent and sheath-liquid merges at the end of the capillary outlet. The outermost tube is set for hot nitrogen gas flow to assist spray formation and solvent evaporation.

Figure 3. (A) CE-ESI-MS coaxial sheath-flow interface. (B) Design of the sprayer tip. Reprinted with permission of the publisher from reference 7.

4. ON-LINE CONCENTRATION TECHNIQUES

CZE is the most commonly used capillary electroseparation method, yet other separation techniques may be used such as capillary isotachophoresis (CITP), capillary isoelectric focusing (CIEF), capillary electrochromatography (CEC)8, capillary gel electrophoresis (CGE), MEKC and MEEKC.9,10 Novel methods may contain more than one separation mode. CZE has become an attractive separation technique due to its high resolution power. Usually separated analytes are ions but some modifications allow separation of neutral analytes. Small columns enable heat dissipation with high field strengths causing effective separation millions of theoretical plates and short analysis time. Small columns also minimize the solvent consumption and sample volume but alternatively they cause limitations. In the case of on-column UV detection, short optical path length restricts

detection according to the Beer-Lambert law. This brings on the concentration sensitivity less than with HPLC. Therefore, alternative capillary geometries and optical designs are developed. Furthermore, the use of highly sensitive detectors, such as MS or laser-induced fluorescence (LIF), has decreased limits of detection (LOD). However, the study of metabolomics, proteomics and glygomics is focused on trace level analysis which cannot be achieved without the combination of sample preconcentration and sensitive detection.

Along with detection, on-line concentration methods are tempting since they might reduce the need of sample pretreatment. Samples with high saline, protein or carbohydrate content may affect CE separation, and thus sample pretreatment is usually mandatory. On-line preconcentration methods suitable for samples with high salt content have had lots of interest.11-15

Chromatographic methods suffer from band broadening producing bandwidth at detection greater than the initial injection bandwidth.16 In addition to diffusion, the mobility of an analyte is affected by the properties of the BGE in CE. Analytes can be stacked into sharp zones when migrating through altered BGE composition segments. Changes are made, for instance, with differences in the salt content, buffer pH or additive concentration of the sample and BGE solvents. Focusing resists band broadening processes.

4.1. Field amplified sample stacking

In one of the most simple and widely used stacking technique a sample is dissolved in a low-conductivity buffer solution and hydrodynamically injected into a capillary where the running buffer has higher conductivity.9,10 Ions experience higher electric field in the sample zone than in the buffer zone because the sample zone has higher resistivity. Thus, sample ions quickly approach the buffer zone where their migration speed decline and they stack at the phase boundary. This field amplified sample stacking (FASS, or sometimes referred as FESS, which indicates field enhanced sample stacking) can be implemented in the easiest way by no more than varying the pH or concentration of the solutions. Positive ions stack up in front of the sample buffer plug and negative ions stack up in the end of the plug. After the stacking process these thin zones are separated with conventional free zone capillary electrophoresis. FASS was first introduced by Mikkers et al. in 1979.

The flux of the ions out of the concentration boundary is the same as the flux into it if there is no electroosmotic flow.10,17 In other words, a pseudo-stationary boundary is formed and there will be no diffusion. This stationary concentration boundary is the most apparent difference between field-amplified CZE and ITP since in the latter the boundary is moving at a constant velocity. However, usually in field amplified CZE the pH is in the region where electroosmotic flow exists, and the concentration boundary is moving at an average flow of the electroosmotic velocities of the different solutions in the capillary.

Field amplification depends on resistivity differences in the buffer solutions.10,17 Unfortunately, field amplification cannot be done endless by raising the concentration difference of the buffer solutions. Like analyte velocity, EOF is faster in the sample zone than in the buffer zone. Due to this mismatch, the field amplification is restricted by resistance coming from a laminar flow at the concentration boundary. Laminar flow will enhance sample diffusion and the stacked sample zone will be broadened.

Chien and Helmer17 investigated electroosmotic and laminar flow in field amplified CZE. They used 2-(N-morpholino)ethanesulfonic acid (MES) and histidine buffers at pH 6.2. Samples of 0.731 mM phenylthiohydratoin arginine (ARG) and 0.734 mM phenylthiohydratoin histidine (HIS) in distilled water were injected into a capillary half filled with 100 mM MES-histidine and half with various concentrations of the same buffer (figure 4).

The results show that the lower the sample concentration was the faster the analytes migrated. Impact of the laminar flow can also be seen in figure 4 since peaks were much wider with lower concentrations. It was calculated that the field amplification was almost three. Chien and Helmer also did the same experiment with a shorter low-concentration buffer plug and saw that it made peaks even more broader but faster.10,17 They decided that the optimum is somewhere between stacking and broadening. Burgi18 continued optimization with Chien and they concluded that the sample concentration should be 10 times less than that of the electrophoretic separation buffer. In addition, optimal sample plug length would be 10 times the width of the diffusion-limited peak.

Figure 4. Field amplified sample stacking. Electropherograms of phenylthiohydratoin arginine (ARG) and phenylthiohydratoin histidine (HIS) samples from a system where half of the capillary is filled with 100 mM MES-HIS buffer and another half with the same buffer of concentration: a) 100 mM b) 50 mM c) 25 mM and d) 12.5 mM. Sample was injected at the lower concentration end of the capillary with gravity injection, capillary length was 75 cm and separation voltage 25 kV. Figure from reference 17. Reprinted with permission from the publisher.

FASS is applicable to dilute samples with small ionic strengths but unfortunately physiological samples usually contain approximately 1 % of salts.11,19 As a consequence, bio-samples ought to be desalted prior to field amplification. Shihabi11 demonstrated that physiological amounts of salt can be tolerated if 66 % of acetonitrile is present in the sample solution. At the same time, acetonitrile deprotonizes samples, hence preventing absorption of the proteins onto the capillary wall. In spite of acetonitrile, larger amount of salts decreased peak heights. In this case, standards should be added to the samples with varying salt content like urine samples. Friedberg et al.20 also reported better stacking with acetonitrile (figure 5). In addition, they concluded that pH and ionic strength had effect on

stacking and that different factors had varying influence on magnitude of each component. Organic solvents can enhance stacking when they are added to sample solution but in the BGE they may suppress the EOF, raise solubility (as in MEKC), or reduce the conductivity of the solution.21

Figure 5. Increasing stacking efficiency with acetonitrile. 6 mg/L nitrite (Ni) and nitrate (Na) in: (A) water, (B) 0.9 % NaCI, (C) 66 % acetonitrile in water and (D) 66 % acetonitrile in 0.9 % saline. Sodium bromide was used as internal standard. Other conditions: capillary L 50 cm and ID 75 µm, voltage 6 kV (reversed polarity) and detection at 214 nm. Reprinted with permission from the publisher.20

FASS method has been employed to biological11,20,22, pharmaceutical23 and environmental24 applications among other things. Khan et al. obtained a 1000-fold enrichment from blood serum.22 They analyzed cancer biomarkers (miRNAs) in blood samples by double stranded binding protein (p19) coated magnetic beads followed by FASS in the protein-facilitated affinity capillary electrophoresis (proFACE). FASS is mostly used with other separation or concentration techniques, which is why its enrichment quantity itself is not always estimated. Examples of FASS concentration methods are listed in table 1.

4.2. Field amplified sample injection

When FASS mechanism is used with EKI, the method is called field amplified sample injection (FASI, or FESI as field enhanced sample injection).10 Positive and negative ions can be injected into the capillary if polarity is switched at proper times. In a similar way, as in EKI compared to HDI, also FASI is not as repeatable as FASS. This is because conductivity varies according to sample concentration.

Table 1 contains a few examples where FASI has been utilized. FASI does not tolerate as much salts as FASS but in many cases it may lead to a better stacking as shown in the table. Sensitivity enhancement of FASI is around 50-1000. Shihabi26 was able to receive up to 70-fold concentration factors when he injected a cationic drug sample electrokinetically from acetonitrile. To further increase the sensitivity enhancement, modifications to a FASI-CZE-UV method are usually required. This can be done, for example, by changing the detector and/or using an additional off-line extraction method. Miquel and colleagues27 increased sensitivity enrichment from 25 to 6500 with an off-line solid phase extraction step when they were analyzing benzophenone UV-filters from river water samples.

Table 1. Analytes that have been on-line concentrated via FASS, FASI and PAEKI.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement Peptides FASS-CZE-UV Acetonitrile 20 11 and NaCl

Nitrite and nitrate Serum FASS-CZE-UV Acetonitrile 20 Cerebrospinal fluid Urine Plant tissue

MicroRNAs Blood serum offline p19 bead precipitation- 0.5 fM 1000 22 FASS-CZE-LIF

Sertraline Urine (DLLME-)FASS-CZE-UV 2.5 nmol/L (74 and) 11 23

SE (IV), Se (VI) FASS-CZE-UV CTAB 57 and 71 ng/L 24

Imatinib mesylate FASS-CZE-UV 2-hydroxypropyl- 25 β-CD

Basic drugs: propranolol and quinine FASI-CZE-UV Acetonitrile 40-70 26

Benzophenone UV-filters River water FASI-CZE-UV 21-59 µg/L 9-25 27 off-line SPE-FASI-CZE-UV 0.06-0.6 µg/L 2400-6500

Benzimidazole drugs Swine muscle MSPE-FASI-CZE-UV Acetonitrile 1.05-10.42 ng/g 28 Swine liver 1.06-12.61 ng/g

Inorganic contaminants Liquefied petroleum gas FASI-CZE-UV CTAB 2-25 ng/mL 100-1000 29

As (III), As (V), Se (IV), Se (VI) Drinking water PAEKI-CZE-MS Acetonitrile 1-3 ng/mL 21 and bromate ions

DNA oligonucleotides PAEKI-CZE-MS 0.01-0.08 µM 300-800 30 and their adducts

L-Arginine, L-lysine, imidazole PAEKI-CZE-MS 18–28 pg/mL 3000 31

Verteporfin drug Artificial urine PAEKI-MEKC-UV Chiral selector 10.3 µg/L, 116 (FASS) 32 39 (FASI)

4.3. Pressure assisted electrokinetic injection

In addition to the stacking in FASI, pressure assisted electrokinetic injection (PAEKI) utilizes countercurrent for better sensitivity.21 EOF is balanced by applying an opposite external hydrodynamic pressure during injection. A stationary boundary is formed at the inlet of the capillary and analytes accumulate in zones according to stacking. Zhang et al.21 created a counter-ion layer theory to explain the mechanism of PAEKI. The stationary boundary prevents analytes from migrating out of the capillary. When anionic analytes are concentrated at the boundary via stacking, a cation layer is formed at the BGE side due to electrostatic attraction. Voltage is switched alternately from positive to negative to migrate the plug intact. At halfway of the capillary, ion layers begin to physically separate because of differences in electrophoretic mobilities, and finally the counter-ion layer is fully damaged. At this point, diffusion due to electrophoretic mobilities of ions becomes stronger than the electrostatic attraction holding the counter-ion layer together. The advantage of the counter-ion layer is the hindering effect of band broadening resulting in a narrow sample zone. PAEKI allows potentially unlimited injection time, requires no leading electrolyte, and stacking is enabled simply by diluting the sample.

PAEKI has been implemented in some applications ranging from enrichment of inorganic ions21 to DNA oligonucleotides30. Originally PAEKI was developed for anionic analytes but lately it has been expanded for cationic analytes as well31. Table 1 lists experiments where PAEKI has been used for sample concentration.

As other concentration methods, also PAEKI is not completely straightforward. Xu et al.32 applied PAEKI for verteporfin drug and noticed that balance conditions varied with matrix, and that high concentration of salts in the sample degraded sensitivity. Therefore, pressure and applied voltage were adjusted to balance bulk flow of the BGE. Injection time (2.0 min) was sustained and analytes were stacked at almost motionless boundary using 0.8 psi at -10.3 kV for a 0.10 mg/L sample. They also compared PAEKI with FASS and FASI for verteporfin enantiomer analysis (figure 6). A huge sample vacancy zone (SVZ) was detected using FASS (figure 6 A) since the sample plug was long but analytes were not separated and detected. In figure 6 B analyte peaks did not appear because of the opposite EOF. In this instance, verteporfin drugs were detected only using PAEKI (figure 6 C).

Limit of detection (LOD) was determined for PAEKI, FASS and FASI methods. Sensitivity enhancement of PAEKI was estimated to be 116 and 39 in comparison with FASS and FASI, respectively.

Figure 6. Comparison of three different concentration techniques for 0.10 mg/L verteporfin drug enantiomers (named Ia-1, Ia-2, Ib-1 and Ib-2). 2 min injection of A) FASS at 0.8 psi, B) FASI at -10.3 kV, C) PAEKI 0.8 psi at -10.3 kV. Figure modified from reference 32. Adapted with permission from the publisher.

4.4. Large volume sample stacking

In conventional sample stacking sample volume is restricted due to disturbances originating from the low-concentration sample buffer. Large volume cause more laminar flow inside the capillary and decrease the field strength in the support buffer. Consequently, electrophoretic velocity of the analytes will be minimal after stacking at the boundary and separation will occur only with EOF.

To increase sample volume, a large volume sample stacking (LVSS) technique, introduced by Chien and Burgi,33 is available. In LVSS, a large sample volume up to the entire capillary volume is hydrodynamically injected into the capillary. Disturbances are eliminated after FASS with removal of the remaining sample matrix from the capillary using external pressure or EOF.

In LVSS, with polarity switching, reverse polarity is applied, which makes the sample matrix exit the capillary with EOF.33 Polarity is chosen based on the ion of interest and it is switched between stacking and detection. For example, this method makes anions migrate toward the positive electrode at the outlet and stack at the interface of buffer solutions, whereas cations and neutrals exit into the waste buffer reservoir. The velocity of the boundary is toward the negative electrode, and therefore the polarity is returned to normal before the stacked zone moves out of the inlet. This means that the method needs current monitoring. Polarity is switched when the current reaches 99 % of the original support buffer value. Separation and detection takes place with normal polarity.

LVSS can be applied to cations when EOF is suppressed by coating the capillary or it is reversed by adding an organic modifier to the buffer.33 Cetyltrimethylammonium bromide (CTAB) at low concentrations in the buffer forms hemimicelles on the oxide surface of the uncoated capillary resulting a change in zeta potential and reversing EOF (figure 7).34

Normal LVSS is limited to low mobility analytes but variations of the method can be applied to faster ions. LVSS without polarity switching is carried out by suppressing EOF with low pH or reversing it with an EOF modifier (e.g. cationic surfactant).35 Dynamic coating is performed by adding the EOF modifier to the running buffer and injecting hydrodynamically a large sample plug without the modifier into the capillary.36 When negative voltage is applied to the injection end, EOF begins to push the sample plug out of the capillary. At the same time, running buffer containing the modifier is pulled into the capillary from the outlet and anions in the sample plug are migrating towards the outlet. After the sample matrix is pushed out, the modifier has coated the whole capillary. Finally, the system is equilibrated for a while, EOF is reversed, and separation begins without polarity switching.

Figure 7. Reversing EOF with a modifier. A) Normal CE separation mode and B) CTAB present in the BGE forms hemimicelles on the capillary surface leading to reversed EOF. Figure adapted with permission from the publisher.34

Studies of LVSS with EKI have also been made. In this case, the method is named FASI- LVSS (or FESI-LVSS). Quirino and Terabe35 and also Polikarpov et al.37 gained around 100 fold improvement in detection sensitivity when they compared LVSS with HDI. Further investigation led to around 1000 fold enhancement in peak size using FASI-LVSS. Polikarpov's group injected a water plug hydrodynamically prior to EKI to improve focusing effect. Figure 8 illustrates how peak intensity was grown in their experiments. Proteins were analyzed with a maltose-modified hyperbranched poly(ethylene imine) (PEI- Mal) coated capillary first without concentration and then with FASI-LVSS.

Figure 8. Concentration effect of on-line FASI-LVSS on protein intensity signals. A) HDI for 2 s; sample concentration 1 mg/mL B) FASI-LVSS injection at 10 kV for 90 s, sample concentration 10 µg/mL. Peak identification: 1) albumin, 2) lysozyme, 3) myoglobin, and 4) insulin. Conditions: PEI-Mal coated capillary, L 45.5 cm, ID 50 µm; running buffer 100 mM phosphate at pH 2.2 and detection UV at 214 nm. Figure reprinted with permission from the publisher.37

Quirino and Terabe35 named their technique simply as FESI, although they used long injection time for their bromide, nitrate and bromate model compounds. They also removed a part of the sample matrix and water plug using electroosmosis. Similar sample concentration techniques have various names and abbreviations according to authors, which sometimes makes it tricky to compare them.

Advantage of LVSS and its variations in sample concentration have been used in many applications, especially for biomolecules. Different systems adopting LVSS as a concentration technique are listed in table 2. As can be seen from the table, the sensitivity enhancement usually varies between 50 and 1000, although for sure someone has already reached better concentration factors. Disadvantage of the method is that positive and negative ions cannot be stacked simultaneously.

Table 2. Analytes that have been on-line concentrated via LVSS.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement Phenylthiohydantoin(PTH) aspartic acid, LVSS-CZE-UV TTAB several 33 PTH-glutamic acid, PTH-arginine and hundredfold PTH-histidine

Drugs LVSS-CZE-UV CTAB 0.092-0.82 µg/mL 34

Bromide, nitrate and bromate LVSS-CZE-UV 2.22, 1.83 and 100 35 6.51 ppb Arsenic acid, monomethylarsonic acid LVSS-CZE-UV DETA 15, 22 and 20 36 and monophenylarsonic acid TTAB 30, 35 and 40

Albumin, lysozyme, myogobin and insulin LVSS-CZE-UV PEI-Mal coated 2.0, 1.0 , 2.0 79, 50, 56 37 column and 2.5 µg/mL and 68 FASI-LVSS-CZE-UV PEI-Mal coated 0.2, 0.1, 0.2 1320, 970, 1040 column and 0.5 µg/mL and 340

Glyphosate, glufosinate, and Drinking water LVSS-CZE-C4D CTAB 0.01, 0.05 53, 48 and 51 38 aminophosphonic acid and 0.1 µM Drinking water FASI-CZE-C4D CTAB 0.0005, 0.01 1002, 245 and and 0.02 µM 257

Secobarbital, amobarbital, barbital Rat plasma LVSS-CZE-UV MeOH 0.048, 0.057, 0.039 172, 170, 202 39 and phenobarbital and 0.015 µg⁄mL and 169

γ-aminobutyric acid, glycine and glutamate LVSS-CZE-C4D INST capillary 9, 10 and 15 nM 100 40 coating solution Microdialysate 29, 29 and 37 nM

4.5. Transient isotachophoresis

CZE and isotachophoresis (ITP) are both separation techniques based on the influence of an electric field but their processes differ from each other.41 ITP employs a leading electrolyte (LE) and a terminating electrolyte (TE) surrounding the sample plug. LE contains a fast co-ion whereas TE contains a slow co-ion. A potential gradient is formed under separation voltage and the zones with sharp boundaries between them migrate with the same velocity. Sample stacking occurs to analytes whose mobility is between the leader and terminator. Isotachophoretic stacking is illustrated in figure 9.

Figure 9. Isotachophoretic sample stacking. A) Sample adjusted between the leading and terminating electrolytes and B) sample separates into zones with sharp boundaries after the voltage is applied. Figure adapted from reference 41. Adapted with permission from the publisher.

Field strength is inversely proportional to the mobility of the ions. If an ion migrates too slowly and enters the zone behind it, its migration will be accelerated due to higher field

strength in the lower mobility zone and the ion will return back to its own zone. In addition to the migration velocity dependence on mobility, capillary length and applied voltage, ITP is accompanied by the concentration of the ions in the leading and preceding zones.42 Therefore, the analyte concentration is adjusted to the same level according to the LE ion. Another ruling factor is the BGE, which can have significant influence on resolution and separation efficiency. Technique is termed transient isotachophoresis (tITP) when ITP is used for preconcentration following another separation mode, CZE for instance. ITP-CZE concentrates and separates sample zones from each other making detection better than using just one of the techniques. If a compound in the sample matrix is used as the LE/TE, the uncertainty of the method increases due to concentration variations in different samples.

Stacking with tITP is mainly classified into the following three modes:42 a) tITP induced by the separation medium: a sample plug is injected into a capillary filled with BGE, which contains the LE. TE is injected behind the sample plug. Alteration from ITP to CZE can be made by placing the inlet in LE. When the LE reaches the sample plug, the field gradient disappears and CE separation begins. b) tITP induced by the sample composition: a sample containing the LE is injected into a capillary filled with BGE, which contains the TE. Roles of the LE and TE can be vice versa as well. ITP stacking and CZE separation are simultaneous. Process is shown in figure 10. c) ITP-CZE combination: two capillaries are coupled together (CC, column coupling) as ITP is performed in the first capillary and CZE is run in the second one.

Figure 10. Shematic picture of tITP induced by the sample composition. Sample plug contains the leading electrolyte A for analytes X1 and X2. BGE acts as terminating electrolyte. Figure from reference 42. Reprinted with permission from the publisher.

During the ITP process, sample component zones migrate in the order of their mobilities and the whole stack of sample zones migrate with a constant velocity.42 Detection times are ruled mainly with the selection of LE and its migration time. Analyte concentrations change during ITP, and are finally adjusted according to LE. Hence, stacking can be several orders of magnitude.

Figure 11 represents the separation power of tITP against plain ITP using thiamin and ferroin as model compounds.43 Electropherogram of ITP is different from tITP since in ITP the analyte concentration is calculated from the isotachophoretic step length and in ITP- CZE from the peak area. This stair-like electropherogram of ITP is seen in figure 11 a. When the sample is further diluted, stacked sample zones become narrower than the detector cell, therefore making separation impossible as in 11 b. However, analysis of the

same diluted sample with combination of ITP-CZE enables separation of the two analytes and Gaussian shaped peaks are formed (11 c).

Figure 11. Separation power of tITP against ITP. A) ITP of 7 nmol thiamin and 2 nmol ferroin B) ITP of 35 pmol thiamin and 10 pmol ferroin and C) tITP of 35 pmol thiamin and 10 pmol ferroin. Conditions: LE 0.02 M sodium acetate and acetic acid, TE 0.01 M acetic acid, UV detection. Figure from reference 43. Reprinted with permission from the publisher.

It is common for biological samples to contain one or more coionic component in bulk levels that may affect the stacking progress in ITP.44 Gebauer et al. simulated this kind of procedure by computer for tITP induced by the sample composition. Simulation was based on the initial isotachophoretic migration model and it was verified by experimental work. They presented that in the most straightforward case the sample contains both the analyte and the bulk component. Denoting C, X and A to the BGE coion, analyte and the bulk component, respectively, it applies that the analyte's mobility must be between the other two. Equation 7 is for leading type of stacking where u is the mobility of a component. Opposite equation applies for terminating type of stacking.

uC < uX < uA (7) 29

Adding another bulk component, B, to the sample may change the system. When the mobilities of both bulk components are highest, stacking is the same for the analyte X as it would be without the other bulk component. Here the lower mobility bulk component acts as the stacker. But in a situation where uB < uX, stacking can be prevented. Computer calculations led to a controlling parameter which is the concentration ratio of the bulk components in the sample. Ratio cA/cB has to exceed a critical value to ensure stacking. The critical value is a function of the mobilities of the compounds X, A, B and of the BGE counterion.

Intriguing bulk component in the BGE acting as a LE was found by Liu and co-workers.45 They examined the tITP of ﬂuorescently labeled DNA-protein complexes (benzo(a)pyrene diol epoxide deoxyguanosine, BPDE-dG, adducts) in genomic DNA. Two antibodies (Ab) were used to recognize adducts in the immunoassay, and LIF was used for detection. When they made tests with fresh and stored BGE, they observed that the signal of the immunocomplexes of BPDE-dG adducts increased when they used stored BGE (Tris- glycine buffer, TG). The group predicted that aerial carbon dioxide was penetrated through the capped bottle and spontaneously dissolved into the alkaline BGE because no chemical reaction should have been taking place in the stored BGE itself. Since dissolved aerial − 2− carbon dioxide dissociates to bicarbonate (HCO3 ) and carbonate (CO3 ) in the BGE, tITP was investigated by adding varying concentrations of ammonium bicarbonate (NH4HCO3) or sodium carbonate (Na2CO3) into the BGE. Finally, their hypothesis was proven true, when signals of the immunocomplexes of BPDE-dG adducts were increased using both additional compounds in the BGE. This was expected and explained by the fact that these compounds share the same major anion, HCO3 in the BGE, which will act as the leading anion. Their results of the increasing focusing effect using NH4HCO3 in the BGE are shown in figure 12.

Utility of tITP is the possibility to inject a large amount of sample into a capillary, and during the process the large analyte zones are sharpened leading to high sensitivity.46 Table 3 summarizes experiments where tITP has been used. The concentration power of tITP is similar to the other on-line concentration methods discussed earlier. Usually enhancement is around 50-500-fold but a positive exception was obtained by Zheng et al.46 Another benefit of tITP is that it can be applied to complex sample matrices especially of biological

Figure 12. Increasing tITP focusing of immunocomplexes of BPDE-dG adducts using

NH4HCO3 in the BGE. Peak identification: 1) BPDE-dG adduct-1°Ab2°Ab immunocomplexes and 2) mixture of unbound 2°Ab and 1°Ab2°Ab complexes. BGE was TG in pH 8.5. Sample in tris-acetate buffer, pH 7.8. Figure from reference 45. Reprinted with permission from the publisher. origin including urine and blood. Zheng et al.46 chose CE over GC and LC techniques when they needed a low-cost and simple test method for analyzing various doping analytes from urine. GC-MS method is the official method for analyzing doping, yet it requires derivatization. LC-MS would call for purification and concentration prior to analysis because of the low concentration of analytes in a complex matrix. Adding tITP step to the analysis of doping with CE-AD, Zheng et al. gained better sensitivity, resolution and peak height enhancement factors up to 5500-fold. Figure 13 illustrates their findings. Healthy volunteers took oral doses of sotalol (SOT, 80 mg) and indapamide (IDP, 2.5 mg), after which their centrifuged and filtered urine samples were analyzed. Figure 13 A shows the electropherograms of the blank urine sample (a) and the urine sample after taking SOT and IDP (b) without concentration using tITP. It indicates that rather high concentrations of

SOT can be detected in the normal injection mode but the shape of the peak is bad. In addition, IDP cannot be detected at all. When tITP was added to the method (figure 13 B), both analytes could be successfully detected.

Figure 13. Electropherograms of doping substances without concentration (A) and with tITP (B). (a) Blank urine sample and (b) urine sample containing SOT and IDP doping substances. Analytical conditions: LE 10.0 mmol/L HCl-Tris at 15 kV for 20 s, TE 15.0 mmol/L glycine at 15 kV for 20 s, sample injection with EKI at 15 kV for 45 s, capillary L 70 cm ID 25 µm and separation voltage 15 kV. Figure from reference 46. Reprinted with permission from the publisher.

Table 3. Analytes that have been on-line concentrated via tITP and electrokinetic supercharging (EKS).

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement

Trace metal ions: Fe2+ , Ni2+ , Zn2+ Urine tITP-CZE-UV Fluorinated carbon 0.7, 0.4 and 475-510 12 polymer neutral 1.2 ppb

Doping substances: methylephedrine, Urine tITP-CZE-AD 0.04-1 pmol/L 5100-5500 46 celiprolol, sotalol and indapamide (IDP)

Heavy metal cations: Cd(II), Pb(II), Snow tITP-CZE-UV 40-120 µg/L 50 47 Cu(II), Ni(II) and Zn(II)

Bromate Drinking water ITP-CZE-UV Methylhydroxy- 0.6 µg/L 48 (column coupling) ethylcellulose

Paralytic shellfish toxins Mussel tITP-CZE-C4D 74-1020 ng/mL 8-97 49 Mussel tITP-CZE-UV 141-461 ng/mL 19-84

Thrombin protein and its 29mer and 15mer DNA tITP-CZE-LIF 50

Trastuzumab and cetuximab Digested peptide tITP-CZE-MS/MS 51

Rare-earth chlorides EKS-CZE-UV 0.3 µg/L 3000 52

Rare-earth metal ions EKS-CZE-UV 0.4-1.3 ng/L ≥100 000 53

Non-steroidal anti-inflammatory drugs EKS-CZE-UV HDMB 10.7-47.0 ng/L 9100-11 800 54

Hypolipidaemic drugs EKS-CZE-MS HDMB 180 ng/L 1000 55

Fe(II), Co(II) and Ni(II) EKS-CZE-UV 30 ppt 56

Peptides Protein EKS-CZE-UV HPC capillary coating nM range 1000-10 000 57

Flavonoids: naringerin, hesperetin, Aqueous extract of EKS-CZE-UV HDMB capillary 2.0-6.8 ng/mL 824-1515 58 naringin and hesperidin Chinese drug coating Clematis hexapetala

Biogenic amines: dopamine, epinephrine Mice brain EKS-MEKC-UV Tween-20 0.42-0.57 ng/mL 2193-2976 59 and norepinephrine

4.6. Electrokinetic supercharging

Electrokinetic supercharging (EKS), introduced by Hirokawa and co-workers,52 incorporates FASI and tITP. They examined the EKS process and saw that as other CZE preconcentration methods, EKS demands optimization too. They concluded that disadvantage of the method is that the moving boundary process was not ruled. The method was employed to rare-earth elements leading up to 3000-fold better sensitivity. Zhongqi et al.53 made a similar experiment with some improvements to the preconcentration procedure by changing the electrode's shape to a ring and the volume of the sample vial to a bigger one with stirring. Detection limits were greatly improved and they concluded that in comparison to normal CE injection EKS could increase detection sensitivity at least 100 000-fold.

Dawod and co-workers54 were quite successful with their preconcentration when they developed an EKS-CZE-UV method for analyzing non-steroidal anti-inflammatory drugs from drinking and wastewaters. They dynamically coated the capillary with hexadimethrine bromide (HDMB) to reverse and stabilize EOF. Compared to FASS, sensitivities of the analytes were enhanced up to 11 800-fold in pure water. Unfortunately, one of the analytes co-migrated with the ITP-boundary, and therefore could not be determined. When the method was applied to real water samples, the samples needed dilution. This is explained through the conductivity of the sample in EKI system since unwanted matrix ions will decrease the amount of analytes. Proper dilution was found for drinking water but the high conductivity of wastewater demanded higher dilution, which made LODs high. Hence, wastewaters obligated liquid-liquid extraction (LLE) prior to analysis. Later their group combined EKS-CZE with MS to analyze hypolipidaemic drugs from drinking and wastewaters but the result were similar (figures 14 and 15).55 Above discussed EKS results and additional outcomes are found in table 3.

Figure 14. EKS-CZE-MS electropherograms of drinking water spiked with (A) 50 µg/L of hypolipidaemic drugs and (B) 50 µg/L of the drugs and diluted 1:4. Conditions: L 88 cm and ID 50 µm fused silica capillary, BGE 60 mM ammonium bicarbonate containing 60 % methanol, pH 9.00, EKI of sample at 10 kV for 170 s, hydrodynamic injection of 1 mM TE 3-(Cyclohexylamino)1-propanesulphonic acid (CAPS) at 50 mbar for 10 s and separation voltage 25 kV. Figure from reference 55. Reprinted with permission from the publisher.

Examples of EKS applications are given in table 3, including the above mentioned results. EKS seems to be quite efficient concentration method since the sensitivity enhancement factors obtained in researches are great, from thousand onwards.

Figure 15. EKS-CZE-MS electropherograms of wastewater spiked with (A) 50 µg/L of hypolipidaemic drugs and (B) 50 µg/L of the drugs and LLE. Conditions as in figure 14. Figure from reference 55. Reprinted with permission from the publisher.

4.7. Titration incorporated methods

Manipulating pH of the sample and BGE can end up in stacking. Utilization of pH in stacking comprises different techniques, which are referred to as pH-mediated stacking, dynamic pH junction and neutralization reaction boundary (NRB) among others.

4.7.1. Dynamic pH junction

Focusing via dynamic pH junction originates when an analyte migrates differently in two separate buffers with deviant pH values. This is created by preparing the sample in a different pH than the BGE. pH junction is dissipated after stacking where the term dynamic refers to.

Episodes of dynamic pH junction are elucidated in figure 16 where peptides are concentrated under alkaline conditions.60 An acidic sample plug is injected into a capillary filled with basic BGE (A). When voltage is applied, the H+ ions from the sample and OH- ions from the BGE begin to interact rising the pH at the boundary. As cationic analytes reach this point, they are converted to anions, which changes their direction against EOF and their migration velocity is reduced. The pH boundary sweeps through the whole sample plug focusing analytes into narrow zones (B), that are separated and detected via normal CZE.

McGribbin16 and co-workers examined focusing induced by velocity-difference with pH junction of nucleotides. They diluted a deoxyguanosine (dG) sample in the same borate buffer that was used as the BGE, and fixed its pH value to 8.0. pH of the BGE was varied between runs (figure 17). Sodium chloride was also added to the sample to show that the method was applicable to biological samples.

Deoxyguanosine is almost neutral at pH 8.0 and therefore migrates close to EOF as in a).16 In higher pH, dG becomes more ionized and its migration velocity is decreased. In figure 17 a) and f) the sample and the BGE have the same pH value. In those cases poor separation is resulted due to the large injection volume. A pH difference is able to stack dG into a sharp zone and the optimum was found at pH 9.7. This experiment was successful with or without addition of sodium chloride, which implicates that different electrophoretic mobilities are not caused by conductivity differences or a potential gradient. Stacking is based on the ionization change of the analyte at the junction between the sample and the BGE, thus making it a selective method and dependent on analytes characteristics. After the stacking, inlet was replaced with the BGE, and pH junction was dissipated by hydroxide and borate ions.

Figure 16. Dynamic pH junction of acidic peptides and proteins. A) A long low pH sample plug is introduced into an uncoated capillary filled with a high pH BGE. B) A pH boundary is formed at the front end of the sample zone sweeping throughout it during electrophoresis. Cationic analytes are converted to anions, and their migration velocity is decelerated. C) Focused analytes migrate to the detector. Figure from reference 60. Reprinted with permission from the publisher.

Figure 17. Series of electropherograms illustrating the stacking effect of pH junction. Sample solution was 20 µM dG in 160 mM borate buffer with 150 mM sodium chloride added. pH of the sample was 8.0 except at f) where it was 9.5. The pH of the borate buffer was a) 8.0, b) 8.5, c) 9.0, d) 9.5, e) 9.7 and f) 9.5. Injection volume was 180 nL, voltage 15 kV and capillary length 57 cm. Figure from reference 16. Reprinted with permission from the publisher.

Monton60 and Ye61 with their groups also stated that dynamic pH junction is independent from above mentioned conductivity-based stacking techniques where sample and BGE have high conductivity difference. The statement was based on results that improved with increasing conductivity of the sample matrix up to a certain point. At higher concentrations, peaks began to broaden. Both groups continued to investigate pH junction further. They tested the effect of sample matrix pH on focusing analytes and noticed that the pH of the sample would cause a great change on analyte's ionization state, and as a result, in its mobility. Interfering effects arising from sample properties may be induced from incomplete titration. If the sample concentration is too high for the BGE, the changes in ionization and mobility might be less striking.

Dynamic pH junction has been piloted for various analytes (table 4). Analytes of biological origin, like peptides, are usually in charged states, and altering the pH can change their ionization significantly. Because of this, dynamic pH junction is a desirable choice for

focusing them.60 If properly optimized, dynamic pH junction is a potential stacking method for other analytes too. Zhang62 and colleagues tested dynamic pH junction for analyzing preservatives from food products and concluded that the proposed method was faster, simpler and consumed less reagents in comparison with reports of LLE and solid phase extraction (SPE)-CZE. Tang et al.63 came to the same conclusions when they analyzed biogenic amines from urine samples. In order to use dynamic pH junction in a routine analysis, pH and concentration conditions at the pH boundary ought to be optimized for each sample type to avoid incorrect identification and quantification due to the changes in migration time. In spite of studies made in optimum conditions, the signal enhancement is usually around 50-100. This implies that dynamic pH junction is not the most powerful on- line concentration method.

4.7.2. pH-mediated stacking

Another titration based concentration method is termed pH-mediated stacking according to Zhao and Lunte.14 In this technique, the sample zone is titrated to neutral, which generates a low-conductivity region and therefore launches FASS. Limitation of bare FASS is the demand for sample matrix to be less conductive than the BGE. Since BGE with more than 100 mM of salt is not normally used, physiological samples with high salt content would need pretreatment to trigger FASS. Fortunately, pH-mediated stacking allows high-ionic strength matrices. Both anionic and cationic analytes can be stacked with it, referred to as base stacking and acid stacking, respectively.

The mechanism of base stacking is demonstrated in figure 18. EOF and polarity are reversed to attract migration of the sample anions toward the detector.14 The BGE consists + of a salt of a weak base, which is in this instance ammonium ion (NH4 ). During EKI, cations from the BGE start to displace sample cations (figure 18 A). Another EKI follows introducing a basic (NaOH) solution (figure 18 B). Rapid hydroxide ions (OH-) from the base begin to titrate the BGE cations to ammonia, whereupon a zone of low conductivity is formed. Field amplification takes place in the titrated zone and analyte anions are stacked

Table 4. Analytes that have been on-line concentrated via dynamic pH junction and pH-mediated stacking.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement

Nucleotides Dynamic pH junction- 40 nM 50 16 CZE-UV

Four peptides Dynamic pH junction- 65-124 60 CZE-UV

Four peptides Urine Dynamic pH junction- 0.2-2.0 nmol/L 61 CZE-MS

Benzoic acid and sorbic acid Beverage, vinegar, Dynamic pH junction- 0.03 and 62 fruit jam CZE-UV 0.02 mg/L

Biogenic amines: Urine Dynamic pH junction- 1 nM 100 63 Tyr, Try, 5-HT, DA, E and NE CZE-AD

Phenylalanine and tyrosine Dynamic pH junction- PB-PVS 0.036 and 64 CZE-UV capillary coating 0.049 μM Urine 0.054 and 0.019 μM

Plant peptide hormones: systemin Plants Dynamic pH junction- 0.5 nM 90-123 65 CZE-MS

Table 4, continues.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement Phenolic acids: p-hydroxybenzoic acid, Ringer's solution pH-mediated stacking- TTAB 0.3 µM 66 14 vanillic acid, p-coumaric acid CZE-UV and syringic acid

Melamine Milk pH-mediated stacking- 0.01 µmol/L 67 CZE-UV

Glutathione and Ringer's solution pH-mediated stacking- TTAB 0.75 µM and 26 68 glutathiode disulfide CZE-UV 0.25 µM

Anionic nucleosides Ringer's solution pH-mediated stacking- TTAB 69 CZE-UV

Tricyclic antidepressant and pH-mediated stacking- ≤14 and ≤30 ng/mL 10 70 beta-blocker drugs CZE-UV

Derivatized amino acids Ringer's solution pH-mediated stacking- SDS 1.5-3 μM 20-32 71 MEKC-UV

in front of the zone. After the base injection, focused anions are separated electro- phoretically in the remaining BGE zone. Several injection combinations can be used in base stacking but EKI+EKI is the best because it is consuming least of the capillary length for the stacking process.66 Nevertheless, pH-mediated stacking requires a long capillary length for the stacking process.14 Acid stacking is implemented as basic stacking but with normal polarity and EOF.14 Apart from the BGE in base stacking, it must be made from the salt of a weak acid. Zhao and Lunte14 showed evidence for the pH-mediated mechanism by monitoring the current behavior during the analysis.

+ Figure 18. The mechanism of base stacking. A) During EKI cations from the BGE (NH4 ) start to displace sample cations. B) Second EKI of NaOH begins the titration of the BGE cations to ammonia. Field amplification occurs in the titrated, low conductivity zone and analyte anions are stacked in front of the zone. Figure adapted from reference 66. Adapted with permission from the publisher.

To gain the best stacking effect and separation efficiency, the amounts of sample and hydroxide (base stacking) or proton (acid stacking) have to be tuned.14,66-67 If not enough hydroxide or proton is injected, a part of the sample analytes will not be stacked and that part will appear as a broad zone in the electropherogram in front of the sharply stacked

peaks. This cannot be overcome by adding excess of hydroxide or proton since it would use too large a portion of the capillary for stacking and leave insufficient length for the separation. It would also leave a weak electric field for the separation. The amount of the sample has to be proper for the same reasons. Furthermore, both injection times are dependent on the BGE features. As earlier mentioned, pH-mediated stacking is suitable for samples with high ionic strength and actually it increases the stacking efficiency.14,66 However, the cost of growing ionic strength is the decrease in sensitivity due to the weaker electric field during EKI.

A drawback of pH-mediated stacking is the need for a long capillary length, leaving little portion for the separation.14 Zhao and Lunte14 solved this problem with a double capillary system; one capillary was used for stacking and the other for separation. They compared pH-mediated stacking of phenolic acids in Ringer's solution with normal EKI, and resulted in 66-fold increase in sensitivity. With the double capillary stacking system they were able to increase sensitivity to 300-fold. pH-mediated stacking in CZE has been applied to many sample matrices and analyses (table 4). Hoque and colleagues68 compared the analysis of glutathione (GSH) and glutathione disulfide (GSSG) in Ringer's solution with and without stacking, and found 26- fold increase in sensitivity using pH-mediated stacking. Without stacking the GSH and GSSG peaks were hardly separated from the baseline (figure 19). They also successfully used the technique to analyze GSH and GSSG in liver microdialysates of rats.

Figure 19. Sensitivity enhancement of pH-mediated stacking of glutathione (GSH) and glutathione disulfide (GSSG) in Ringer's solution. (A) GSH without stacking: 3 s EKI of 10 µM GSH in Ringer’s solution (B) GSH with stacking: 30 s EKI of 10 µM GSH in Ringer’s solution/60 s EKI of NaOH, (C) GSSG without stacking: 3 s EKI of 10 µM GSSG in Ringer’s solution and (D) GSSG with stacking: 30 s EKI of 10 µM GSSG in Ringer’s solution/60 s EKI of NaOH. Capillary diameter 50 µm and length 60 cm, BGE of

100mM NH4C1 with 0.5 mM TTAB at pH 8.4, EKI at -10 kV, separation at -10 kV and detection at 214 nm. Figure from reference 68. Reprinted with permission from the publisher.

4.8. Sweeping

Sweeping is a technique designed by Quirino and Therabe in 1998 that is based on the interaction between an additive and analyte.72 Additive is a pseudostationary phase (PS) in electrokinetic chromatography (EKC) or a complexing agent in CZE, and the accumulation of the additive is caused by electrophoresis. Sweeping allows both neutral and charged analytes to be concentrated.

4.8.1. Sweeping in electrokinetic chromatography

EKC is a capillary electromigration separation mode including a PS which interacts with the analytes without affecting their migration velocity.73 Because PS is added into the separation buffer, analytes begin partitioning between PS and the surrounding phase as the buffer passes the sample solution zone.74 For neutral analytes, separation is based on partitioning alone but charged analytes are separated due to partitioning and electrophoresis.

4.8.1.1 Pseudostationary phases

The fundamental requirements for sweeping are to prepare a sample matrix free of PS and to select a PS towards which analytes possess highest possible affinity.74 A high affinity towards PS will increase partitioning and concentrating effect. Affinity can be altered by adding an organic solvent to the sample matrix or to the separation buffer13.

First carrier used in sweeping was charged PS micelle named sodium dodecyl sulfate (SDS) (figure 20).72 In addition to the most commonly used sweeping carrier SDS, other anionic micelles are bile salts,13 and ammonium perfluorooctanoate (APFO)75. Among cationic micelles dodecyltrimethylammonium bromide (DTAB),76-78 cetyltrimethylammonium bromide (CTAB), 79 cetyltrimethylammonium chloride (CTAC),75,80 and tetradecyltrimethylammonium bromide (TTAB)81 have been successfully applied to sweeping. In general, ionic micelles attract sweeping of analytes with opposite charge.76

Figure 20. Examples of pseudostationary phases, SDS (upper compound, MW 288) and Brij 35 (lower compound, MW 1225), used in sweeping.

In order to form micelles and use MEKC separation, micelles have to be added above the critical micelle concentration (CMC). Gong et al.78 found out that sweeping is possible to achieve below CMC if aggregate formation can be induced. They analyzed oligonucleotides below and above CMC of DTAB as shown in figure 21. Below CMC analytes were separated according to CZE (figure 21 a), whereas above CMC analytes were separated by MEKC (figure 21 b). The analyte aptamers are not strongly affected by the DTAB because they have low hydrophobicity, and therefore are weakly partitioned into micelles. CZE mode resulted in higher peak of aptamers but MEKC was indicating better separation power since two peaks were visible. The best signal enhancements were obtained above CMC for 5-carboxyfluorescein (5-FAM) and fluorescein disodium salt (FL), when also the peaks had longer retention times due to the strong interactions with micelles. This led to the recommendation to use DTAB below CMC for oligonucleotides and above CMC for hydrophobic small molecules.

Apart from ionic surfactants, also nonionic,82 polymeric,83 mixed micelles,84 and microemulsions85 have a capacity for sweeping. Polyoxyethylene (23) lauryl ether (Brij 35, figure 20) and polyoxyethylene (20) cetyl ether (Brij 58) are neutral PSs but the use of these neutral PSs is limited to charged analytes. Polymeric PSs, such as poly(sodium 10- undecenyl sulfate) and poly(sodium 2-acrylamido 2-methyl 1-propane sulfonate-co-stearyl acrylamide) abbreviated as poly-SUS and pSAm-f, respectively, have given promising

Figure 21. Electropherograms below and above the CMC of DTAB. BGE was 20.0 mM

Na2HPO4 with 11 % acetonitrile (v/v) and either (a) 10 mM DTAB or (b) 20 mM DTAB.

Sample solutions in Na2HPO4 buffer with conductivities adjusted to be similar to BGE. Injection: at 2.0 psi for 60 s. Peak identification: 1. 5-FAM, 5.0 nM, 2. FL, 5.0 nM, and 3. aptamers. Conccentration was 12.5 nM. Figure from reference 78. Reprinted with permission from the publisher. results for hydrophobic solutes in sweeping.83 Stability of polymeric PSs allow the use of organic modifiers, and thus the sweeping and separation of hydrophobic solutes using MS detection. Polymeric PSs also lack the CMC. Mixed micelles have been used for MEKC to alter selectivity and in a similar way they may be advantageous for sweeping.84 For one, SDS combined with a zwitterionic N-dodecyl-N,N-dimethylammonium-3-propane-1- sulfonic acid (SB-12) has shown to work. Microemulsions are mixtures of surfactants and

alcohols with modified selectivities and they allow separation of highly hydrophobic compounds.86 Besides the above mentioned PSs, there are more than 200 PS systems reported in the literature for EKC.

Fuguet et al.86 used a chemometric tool for 55 selected PSs in order to study the selectivity and properties of the phases. From the data they concluded that hydrophobicity and hydrogen bond acidity or basicity were the crucial factors for the differences in selectivity of surfactants.

4.8.1.1.1 Charged pseudostationary phases in a homogeneous electric field

Liquids with similar conductivities will sustain homogeneous electric field. The sweeping in a homogenous electric field and separation of anionic analytes using a cationic micelle CTAC is demonstrated in figure 22.80 A capillary is filled with micellar BGE and a large volume of analytes free from micelles is injected into the capillary (figure 22 A). Both ends of the capillary are immersed into the micellar BGE and negative voltage is applied. Cationic surfactants are adsorbed onto the wall of the capillary creating a positively charged capillary wall and reversed EOF directing towards the anode. CTAC has electrophoretic mobility towards the cathode, it enters the sample zone from the anodic side and sweeps the analytes (figure 22 B). Micelles interact with analytes in the sample zone, pick up and concentrate analytes into a narrow zone. A zone without micelles is formed at the interface of sample and BGE zones. After all analytes are completely swept (figure 22 C), MEKC separation occurs. Although CTAC has the mobility towards the cathode, EOF will bring analytes to the detector since its electrophoretic migration is faster than the opposite migration of the micelle.

Basics of sweeping with an anionic surfactant are the same as with cationic but the electrode polarity is reversed.80 If analytes are neutral, they gain electrophoretic mobility via complexation with a charged micelle.13 Highly hydrophobic analytes have a high affinity for micelles, consequently they spend a lot of time complexed with the PS. Therefore, these analytes have lower velocity than EOF. Velocity of neutral analytes with less affinity toward micelles is more dependent on EOF.

Figure 22. Evolution of analyte zones in sweeping MEKC of anionic metal-CDTA complexes using CTAC micelles. A) A large sample plug devoid of micelle is injected into the capillary filled with micellar BGE. B) CTAC begin to migrate toward the cathode and enters the sample zone sweeping analytes. C) Completely swept analytes.

4.8.1.1.2 Charged pseudostationary phases in a heterogeneous electric field

Sweeping occurs regardless of the conductivity differences with sample matrix and BGE.74 Palmer et al.13 were the first ones to test sweeping in a heterogeneous field. They created reduced electric field in the sample region by adding salt to the sample to create 2-3 fold higher conductivity than the BGE. Figure 23 depicts sweeping of neutral analytes in a reduced electric field in the sample solution.15 First (figure 23 A), sample solution with a high conductivity is injected into the capillary filled with BGE. Analytes that are found at the cathodic and anodic ends of sample-BGE interface, are aa and ac, respectively.

Corresponding micelles are mca and mcc. Difference to the homogeneous electric field model is that the velocities of the micelles are not the same. Migration of mcc is lower than that of mca. Arrows in the figure illustrates magnitudes of electrokinetic velocities.

Application of voltage (figure 23 B) will stack micelles mcc at the interface of sample and

BGE zones (Ic) and sweep the analytes. Stacking will occur only if the mobility of the micelle is less than the mobility of the co-ion of the salt according to the self-sharpening effect in ITP. In this case they are both anions. The area between mcc and Ic has now higher micelle concentration than the BGE and the area between ac and Ic contains no analytes.

The high conductivity zone is situated between Ia and Ic. When the analyte ac interacts with micelle, its migration velocity is either equal or faster than that of the micelle. The migration velocity of aa is the same with EOF until the time aa reaches mcc. In figure 23 C, the injected analyte zone is completely swept having the length of difference of distances traveled by ac and mcc. In figure 23 D micelles begin to destack and swept analyte zones are broaden at the interface Ia because micelles and swept analyte zones separate from the high-conductivity region. Destacking occurs when micelles migrate through the high- conductivity sample region, enter the BGE area and adjust again. This decreases micelle concentration and the area is broadened. In figures 23 D and E, the broadened analyte molecules are between the area of aa and ac. However, it is necessary for the swept analyte zones to leave the sample zone to gain separation. Injected sample plug length and amount of the salt added to the sample determines the length required for the destacking. Note, that too much salt and/or too long sample plug may prevent destacking and subsequent separation.

Cao and colleagues87 demonstrated how electropherograms of flavonoids seem under excess salt (figure 24). The best preconcentration was achieved by adding 50 mM salt in the sample. Beyond that, zone broadening began to dominate and higher concentrations increased current, which was harmful to sweeping.

Figure 23. Chain of events of micelles and neutral analytes during sweeping with high EOF and high salt concentration matrix. A) In the beginning a sample plug with greater conductivity than the BGE is injected. B) Applying voltage at positive polarity results in stacking of micelles from the cathodic side at the interface between the sample and BGE zones (Ic). C) Analyte zone is completely swept. D) Stacked micelles exit the sample zone and destack at Ia making analyte zones broader. E) Swept analyte zones leave the entire sample zone. Figure from reference 15. Reprinted with permission from the publisher.

Figure 24. Electropherograms of flavonoids in different salt matrixes using sweeping- MEEKC. Salt concentrations: (a) 25 mM, (b) 50 mM, (c) 100 mM, and (d) 150 mM. Peak identification: 1.quercetin, 2. calycosin, 3. ononin, 4. rutin and 5. calycosin-7-O-β-D- glucoside. Injection pressure was 50 mbar and time 100 s, sample concentration was 1.25 µg/mL and BGE was 0.5% (w/v) ethyl acetate, 2.0% (w/v) SDS, 9 mM DTAC, 4.0% (w/v) 1-butanol and 25 mM phosphoric acid (pH 2.0). Figure from reference 87. Reprinted with permission from the publisher.

4.8.1.2 Parameters for optimal sweeping

All analyses, especially when using additional on-line sample concentration techniques, ought to be performed under optimal conditions in order to achieve successful and reliable results. Cao and co-workers87 determined some optimum parameters of sweep-MEEKC of flavonoids. Concentration of surfactants, sample matrix, effect of salt or organic modifier, sample injection volume, and the polarity of the voltage were examined. Additionally, BGE concentration and pH were varied, which influenced the mobility of analytes, the dissociation of the solutes and the interaction between analytes and PS. Cao's experiments87 with different injection volumes are shown in figure 25. As one might 53

expect, increasing injection time will raise peak areas as well. The increase of peak areas is usually not that straightforward since what Cao noticed was that peaks broadened and baseline was disrupted with longer injection times than 350 s. Hence, compromises have to be made between parameters when selecting optimal conditions. Gong et al.78 examined sweeping conditions for 5-carboxyfluorescein and sodium fluorescein model analytes. In addition to Cao's parameters, capillary coating was used and injection voltage was optimized. Analyses using sweeping as a preconcentration method under optimized conditions are listed in table 5.

Figure 25. The effect of injection time on peak areas of flavonoids. Conditions as in figure 24. Figure from reference 87. Reprinted with permission from the publisher.

4.8.2. Sweeping in capillary zone electrophoresis

Sweeping in CZE offers less possibilities for different analytes than sweeping in EKC with its many PSs. Sweeping via complexation in CZE is mostly applied with borate or ethylenediaminetetraacetic acid (EDTA).

Table 5. Analytes that have been on-line concentrated via sweeping.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement

Alkyl phenyl ketones: hexanophenone, Sweep-MECK-UV SDS 15 butyrophenone, propiophenone, valerophenone, and acetophenon

Several test analytes; steroids, sweep-MECK-UV SDS 1.7-9.6 ng/mL 1.5-2.6 72 valerophenone and quinine 88-5044

5-carboxyfluorescein and EKSI-sweep-MECK DTAB 75 fM 4500 and 76 sodium fluorescein 4000

Fluorescein and SDME-sweep-MECK-LIF DTAB 0.1 pM 28 000 and 77 6-carboxyﬂuorescein 32 000

Oligonucleotides Sweep-MECK-LIF DTAB 112 78

Flunitrazepam, 7-aminoflunitrazepam, Sweep-MECK-PDA CTAB 13.4, 5.6 and 110, 140 and 79 N-desmethylflunitrazepam 12.0 ng/mL 200

Metal-CDTA complexes: Sweep-MECK-UV CTAC 0.2, 1.2, 0.6, 15, 17, 26, 80 Pb(II), Cu(II), Co(II), Mn(II) and Zn(II) 1.3 and 1.8 µM 42 and 40

Naphthalenesulfonic acids: 1-NSA, Sweep-MECK-UV TTAB 0.47-0.96 ppb 670-760 81 2,6-NDSA, 1,5-NDSA, 2,7-NDSA, 2,7-NDSA

4-chlorophenol, 4-ethylphenol and Sweep-NPS-EKC-UV Brij 35 218, 164 and 54, 100 and 82 3-methylphenol 176 nM 87

Table 5, continues.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement

Quinine Sweep-RF-EKC-UV pSAm-24 3 ppb 10 000 83 pSAm-28 42 ppb 580

p-tert. butylphenol and Sweep-MECK-UV SDS-SB-12 158 and 122 and 360 84 2,4,6-trichlorophenol 97 nM

Flavonoids MEECK-sweep-UV Microemulsion 2.1-14 ng/mL 35-79 85

Flavonoids MEECK-sweep-UV Microemulsion 0.004-0.022 µg/mL 185-508 87

Pyridine and adenine nucleotide metabolites Bacillus subtilis Sweep-CZE-UV Borate 20 nM 88 cell extract

Ca(II), Mn(II) and Zn(II) Sweep-CZE-MS EDTA <0.15 mg/L ≥10 89

Pd(II), Cu(II), Zn(II), Pb(II), Ni(II), Co(II), Sweep-CZE-UV EDTA 18-234 nM 60-180 90 Cd(II) and Mn(II)

Glucosides Dual sweep-MEKC-UV Borate and brij 35 1.6-4.8 µg/mL 50-130 91

Cationic drugs: promethazine, dibucaine, Purified water offline EC-sweep-MECK-UV SDS 40-90 pg/mL 10 400-45 000 92 doxepin, verapamil, and alprenolol Wastewater 1.20-6.97 ng/mL 1300-6500 Purified water Sweep-MECK-UV SDS 74-322 Wastewater 74-322

Neutral analytes containing vicinal diol groups can be selectively separated owing to the complexation agent borate.88 With diol sites borate anions form anionic complexes making their electrophoretic mobility higher. Following these events, the complexing agent acts as a carrier in sweeping and separation. Sweeping is operated by preparing a sample matrix devoid of borate. Applying potential sets off borate to enter the sample zone and to form complexes, which accumulate to a concentrated zone. Markuszewski and colleagues88 determined the optimum separation conditions for pyridine and adenine nucleotide metabolites in Bacillus subtilis cell extract at nM levels using borate complexation. They examined the best conditions for BGE concentration and pH as well as sample ionic strength, pH and injection length. Under optimum conditions, approximately a 4-fold increase in peak narrowing was achieved in comparison to LVSS. Also, the method demonstrated excellent linearity and reproducibility. Therefore, the researchers concluded that the method was useful for concentrating diol analytes.

Similar to complexation with borate, metal ions can be separated and swept via complexation with EDTA.89-90 EDTA forms 1:1 UV-absorbing chelate with most metals. Figure 26 demonstrates an electropherogram of cobalt(II) swept using EDTA. Because EDTA is UV-absorbing, there is a difference in absorbance between EDTA-BGE and EDTA vacancy regions.

In addition to UV detection, sweeping of metal-EDTA complexes can be run using electrospray ionisation (ESI) and MS detection because ESI is soft enough to keep the complexes intact.89 A high pH can cause solubility problems due to metal hydrolysis, and thus is unsuitable. Additionally, the compatibility of the sample matrix has to be considered when using ESI-MS for detection. Inductively coupled plasma (ICP) with MS or atomic absorption spectrometry are usually utilized when there is a need for high sensitivity analysis of metals. However, CE serves an attractive option for metal analysis due to its small sample size and reagent requirements, high speed, high separation efficiency, simplicity and environmental friendliness. Unfortunately, all this is gained with less sensitivity. Quirino and co-workers89 introduced a sweeping-CZE-ESI-MS method for metal analytes; calcium(II), manganese(II) and zinc(II). Peak heights were improved at least tenfold and LODs were at µg/L levels. Their LODs were an order of magnitude

Figure 26. Sweeping-CZE-UV of cobalt via complexation with EDTA. Conditions: EDTA-BGS was 1 mM EDTA in 30 mM sodium acetate (pH 5.5), sample solution at 100 ppb Co(II) in acetate buffer (pH 5.5) adjusted to the conductivity of the EDTA-BGE, injection length 20.4 cm, voltage -27 kV and capillary length 65 cm. Figure from reference 90. Reprinted with permission from the publisher. higher than what Isoo and colleagues90 determined in a similar experiment. They estimated that it was because higher EOF in sweeping is more powerful under suppressed EOF. They also suggested that the sensitivity of the method could be improved with the combination of preconcentration techniques. The above mentioned methods of sweeping in CZE are summarized in table 5.

4.9. Electroextraction

Apart from the most employed on-line preconcentration methods for CE, new techniques are piloted. One of them is electroextraction (EE), which was used in the experimental part of this study. As in other preconcentration methods, the aim of EE is to concentrate analytes in a fast manner into a small volume.93-96 When an electric field is applied, the extraction rate is increased. In this research a two-phase liquid-liquid system was used (figure 27).

Figure 27. The principle of a two-phase electroextraction (EE).

The sample is mixed with organic phase and electrodes are placed in both phases. When the voltage is applied, cationic analytes start to migrate fast towards the cathode, which is placed in the aqueous phase. The organic donor phase has low conductivity and high electric field strength, whereas the opposite dominates in the aqueous acceptor phase. The conductivity difference in two phases causes higher local electric field strength on organic phase, thus acting as the driving force of migration. As soon as analytes cross the solvent interface, their migration speed decrease and they are concentrated in the aqueous phase (equations 1-4). With a positive electric field, only cations are extracted into the acceptor phase.

4.10. Combination of on-line concentration techniques

A traditional method can be sometimes more powerful than newer, more complex methods. Maijó and co-workers compared preconcentration magnitude of LVSS, FASI, sweeping and in-line SPE for paraben analysis. By far the biggest concentration enhancement was achieved with in-line SPE (table 6). Choosing the best preconcentration method is not always enough. Due to the low concentration of some analytes of interest,

sensitivity of CE analysis might be too low. Detection sensitivity can be further improved utilizing dual or multiple preconcentration methods. Yet, resourcefulness is needed when designing combinations. Each individual technique has its own requirements and limitations making some connections challenging or impossible. Everything needs to match from the sample and BGE composition until detection.

Many combined techniques employ sweeping with another or multiple other techniques. Cation or anion selective exhaustive injection (CSEI or ASEI)-sweeping method is a fusion of FASI and sweeping, which has provided almost a million-fold increase in detection sensitivity.74 In CSEI, the capillary is filled with a low conductivity BGE. A plug of high conductivity buffer (HCB) devoid of organic solvent is hydrodynamically injected following a plug of water.74,97 The cationic sample, which is prepared in a low- conductivity solution, is electrokinetically injected by a positive voltage for a long time. Analytes are stacked into zones because they experience a high electric field strength due to the difference of concentrations in the sample solution and HCB. After the injection, both ends of the capillary are placed into a low-pH micellar BGE. Analyte cations in HCB are swept by negatively-charged micelles and the analytes are separated via MEKC at negative polarity.

Quirino and his colleagues74 reported magnificent, close to a million-fold enhancement factors using CSEI-sweep-UV for laudanoside and naphthylamine cations (figure 28). Xu et al.97 also got a great concentration factor with their similar experiment, when detection sensitivity increased about 5000-fold for cotinine in mouse serum sample. Lin's98 and Wang's99 groups decreased experimental steps and used chemometric tools in planning optimal conditions. Some of the conditions were tested experimentally and verified with computer simulations. Lin et al.98 reached 6000-fold signal enhancement for nicotine in tobacco with CSEI-sweep. Wang and colleagues99 were able to increase sensitivity 900- fold when they analyzed ractopamine in porcine meat.

Figure 28. Nearly a million-fold concentration of cations using CSEI-sweep. Sample contained 1. laudanosine and 2. 1-naphthylamine in water. Sample concentration was 240 ppm (A) and 240 ppt (B). injection: 0.6 mm of the sample solution (A), 30 cm of HCB and then 3 mm of water followed by 23 kV electrokinetic injection of the sample solution for 1000 s (B). Sweeping and MEKC voltage was -23 kV with the micellar BGE at both ends of the capillary. Nonmicellar BGE: 1 mM triethanolamine/15% acetonitrile/100 mM phosphoric acid. Micellar BGE: 100 mM SDS/1 mM triethanolamine/15% acetonitrile/50 mM phosphoric acid. HCB: 100 mM phosphoric acid. Figure from reference 74. Reprinted with permission from the publisher.

Additionally to ASEI/CSEI, at least FASS, LVSS, dynamic pH-junction and pH-mediated stacking have been combined with sweeping technique. Examples of combined concentration techniques are listed in table 6. In comparison to all single on-line concentration methods, the biggest sensitivity enhancement factors are attained with combined techniques.

Table 6. Analytes that have been concentrated via combinated on-line techniques.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement Pb(II), Co(II), Mn(II) and Fe(III) CSEI-sweep-CZE-UV EDTA 24-252 pM 50 000-140 000 90

Parabens: methylparaben, ethylparaben, LVSS-CZE-UV 3-4 ng/mL 53-77 100 propylparaben, isopropylparaben, FASI-CZE-UV 2 ng/mL 105-120 butylparabe,and benzylparaben Sweep-MEKC-UV SDS 18-27 ng/mL 17-29 in-line SPE-CZE 0.01-0.02 ng/mL 12 600-18 400

Laudanoside and naphthylamine CSEI-sweep-MEKC-UV SDS 12 and 56 pM 9 000 000 and 74 550 000

Cotinine CSEI-sweep-MEKC-UV SDS 0.2 ng/mL 5 000 97

Nicotine CSEI-sweep-MEKC-UV SDS 0.5 nM 6 000 98

Ractopamine CSEI-sweep-MEKC-UV SDS 5 ng/g 900 99

Phenoxy acidic herbicides ASEI-sweep-MEKC-UV SDS 0.1-0.5 ng/mL 100 000 101

Drugs: codeine, morphine, metamphetamine, Urine LVSS-sweep-MEKC-UV SDS 7.5-30 ng/mL 200 102 ketamine, alprazolam, clonazepam, diazepam, flunitrazepam, nitrazepam and oxazepam

Enrofloxacin and ciprofloxacin Food FASS-sweep-MEKC-UV NaDC 1.87 and 376 and 406 103

Table 6, continues.

Target analytes Sample matrix Preconcentration, separation Additive used LOD Sensitivity Ref. modes and detection enhancement 2.21 ng/mL

Benzoic and sorbic acids Dynamic pH junction- SDS 8.2 and 6.1 nM 930 and 920 104 sweep-MEKC-UV Wine 47.9 and 38.0 nM

Dipeptides Dynamic pH junction- SDS and Brij35 1.0-5.0 pM 1000 105 sweep-MEKC-LIF

Glutamic and aspartic acids Human serum Dynamic pH junction- β-CD 0.061 and 30 and 55 106 sweep-CZE-UV (complex formation) 0.032 µg/mL

Nucleosides: cytidine, adeosine, Dynamic pH-junction-sweep- Borate 0.017-0.255 µg/mL 7-84 107 5-methyluridine, uridine, guanosine, LVSS-CZE-UV inosine and xanthosine

5-hydroxy-tryptamines: ondansetron and FASI-dynamic pH-junction- 2 and 5 nM 357 and 345 108 tropisetron CZE-AD

Amino acids pH-mediated stacking- 0.1-2 µM 17-300 109 tITP-CZE-MS

Phenolic acids EKS-sweep-MEKC-UV SDS 0.01 -2.5 ng/mL 400-25 000 110

II EXPERIMENTAL

5. BACKGROUND

Many compounds of the metabolome are or can be charged, thus they are applicable for CE. Due to the low injection volume in CE, detection is challenging. Therefore EE is promising as concentrative technique prior CE.111 For a variety of compound groups, the suitability of EE is not yet known. The purpose of this work was to build and test an automated EE-CE-MS system in order to investigate the selectivity of EE. To study this, the influence of organic phase composition and log P value of the analyte were taken into account.

5.1. Electroextraction procedure

Initially EE was developed as a separation technique for chemical engineering.93-96 After that, different type of systems have been developed. For example, effect of convection has also been avoided in a similar technique called electrodialysis. This technique involves a membrane between the two phases. As an option to obtain an immiscible liquid-liquid system, two phases can be generated mixing two different polymers with water.112 Even an analytical EE device on a chip has been developed113.

In this study, EE was executed in a normal commercial CE apparatus. EE consists of four steps prior to separation. First, the inlet of the capillary is placed into a sample vial and a negative pressure is applied to form a hanging BGE droplet (figure 29, steps 1-2). Negative pressure describes pressure to opposite direction from a normal HDI. The pressure is stopped and EE begins by switching the voltage on (step 3). Finally, the droplet containing the extracted analytes is injected into the capillary using HDI (4). Normal CZE separation is performed after EE.

Figure 29. Four stages of EE. 1) Inlet of the capillary is in the sample vial, 2) a hanging BGE droplet is formed using pressure, 3) EE in action. Analytes are extracted in the BGE droplet. 4) The droplet is injected into the capillary.

Distribution coefficients are changed under an electric field.112 Effective partition coefficients do not change when the volume ratio is changed and extraction has taken place long enough to reach a balance in partitioning. Therefore, recovery is increased when acceptor phase volume is increased. Although the recovery is better, maximum concentration in acceptor phase is not increased when the volume is increased. That is why same concentrations should be gained with different droplet sizes if the system is in balance.

It has been investigated that recovery increases with an increase in the field strength until optimum is reached.114 The mass transfer is due to migration, diffusion and convection. When two phases are used and there is a stable interface between them, convection may be negligible. Mass transfer slows down gradually. Increased analyte concentration near the interface will lead to an inverse electric field, and therefore slowing down migration speed. Furthermore, concentration differences are evened due to diffusion, and concentration diffusion back towards the donor phase begins.112,115 In spite of the slower speed, migration towards the acceptor phase continues, and when the electric field strength is high enough, extraction is depleted.

5.2. Chemicals

List of the chemicals used in the experiments are in table 7. All reagents were analytical grade. Water was purified with Millipore system MQGradient A10.

Table 7. Chemicals used in the measurements.

Reagent Manufacturer Formic acid Acros Organics Trifluoroacetic acid Biosolve Acetic acid VWR Ethyl acetate Sigma-Aldrich Methyl acetate Merck n-butanol J.T. Baker β-alanine Aldrich crystal violet Sigma-Aldrich acyl-carnitines Sigma-Aldrich

5.2.1. Crystal violet

The cationic dye crystal violet (CV) was chosen as a model compound because of its violet colour, and hence the possibility to visually follow the colour change in experiments. The structural formula of CV is shown in figure 30. In highly acidic solutions two or all three nitrogen atoms of CV carry positive charge and the colour is green or yellow.116 In this work, the pH was less acidic leaving only one nitrogen atom charged with violet colour.

Figure 30. Structural formula of crystal violet; C25H30N3, molecular weight 372.5 g/mol.

5.2.2. Acylcarnitines

In addition to CV, some biological compounds, i.e. carnitine and its four acyl ester compounds (acylcarnitines), were selected. By analysing free carnitine and individual acylcarnitine concentrations unusual metabolic conditions can be indicated. Carnitine is a cofactor for several enzymes, and thus its functions are related to energy production.117 Acylcarnitines are found mostly in muscles but also in all organs and tissues, and they are excreted in the urine and in the bile. Urine would be the easiest sample type to collect but concentrations at that type of sample are the lowest.

Different carnitines were selected to examine the impact of EE to similar compounds with different polarities. Carnitine (4-N-trimethylammonio-3-hydroxybutanoate) is the backbone of all these compounds and they differ from each other according to the length of the residual fatty acid chain. Carnitine and acylcarnitines are zwitterionic; they contain a positive ammonium group and a negative carboxyl group.118 In this form their net charge is zero and they are not applicable to EE or CE analysis. Thus, they ought to be converted as positive charged compounds by acidic pH adjustment. As an example, the structural formulas of free carnitine and hexanoylcarnitine are given in figure 31. The fatty acid residue, which free carnitine is lacking, is circled in hexanoylcarnitine. The length of the fatty acid residue chain distinguishes acylcarnitines from each other.

Figure 31. Structural formulas of free carnitine (C7H15NO3 MW=161.2 g/mol, left) and hexanoylcarnitine (C13H25NO4, MW=259.3 g/mol, right). Fatty acid residue part is circled.

As the size of the acylcarnitine molecule and number of carbon atoms increase, its polarity decreases in the following order: carnitine (161.2 g/mol, highly polar) > acetylcarnitine (203.2 g/mol) > hexanoylcarnitine (259.3 g/mol) > octanoylcarnitine (287.4 g/mol) > lauroylcarnitine (343.5 g/mol). The ratio of concentrations of a compound between two immiscible phases is called partition coefficient. The logarithm of partition coefficient between n-octanol and water (log P) is used to express lipophilicity of a compound. Log P values are growing in the order of lipophilicity (table 8).

Table 8. Predicted log P values of model compounds.119

Compound predicted Log P

crystal violet 0.9

carnitine -2.9

acetylcarnitine -2.4

hexanoylcarnitine -1.7

octanoylcarnitine -1.2

lauroylcarnitine 0.03

5.2.3. Solvents

Methyl acetate (MeOAc), ethyl acetate (EtOAc) and n-butanol (n-BuOH) were used to dissolve model compounds. Properties of these three solvents are in table 9.

Dielectric constant (ε) is the amount ratio of energy stored in a solvent relative to vacuum when a voltage is applied. Molecular structure affects the value because dipoles orient along external electric field and charges separate in apolar molecules. Hence, molecules are polarized and electric field strength is decreased. The polarity of a solvent is usually estimated from the dielectric constant. The value of ε is high with polar solvents, for example water has the value of ε=80.1 (at 20 °C)120. In EE, dielectric constant and hence polarity of a solvent are important because they might affect extraction recoveries of different compounds.

MeOAc and EtOAc have similar properties but their log P values differ, whereupon their lipophilicities are also different. BuOH has much higher viscosity and dielectric constant than the esters.

Table 9. Properties of solvents used in the experiments.120

Molar weight Viscosity Dielectric Formula Name Log P Boiling point [° C] [g/mol] [mPas] constant

C3H6O2 Methyl acetate 74.08 0.18 0.36 57 7.1

C4H8O2 Ethyl acetate 88.11 0.73 0.42 77 6.1

C4H10O 1-Butanol 74.12 0.84 2.54 118 17.8

6. EXPERIMENTS

6.1. Preparation of solutions

6.1.1. Background electrolyte solution

1 M formic acid (FA) was used as the BGE solution to make sure that the pH was low enough to prevent EOF. EOF would create BGE movement towards detector, and therefore making droplet used in EE hard to control. It was calculated that the pH of 1 M FA was 1.87. EOF becomes significant above pH value 4 so there was hardly any EOF interfering1. The BGE solution was prepared dissolving 0.95 ml of formic acid into 25 ml of Milli-Q water.

6.1.2. Crystal violet samples

In CE-UV experiments CV was used as a model compound. It was chosen because some tests had been made with CV in the past and the colour of the compound is visible to eye. CV was dissolved into EtOAc because it is nontoxic, inexpensive and it was also used in the previous tests. EtOAc itself is nonconductive but some aqueous electrolyte solution can be added into it to make it conductive and improve solubility of ionic species. Van der Vlis et al.94 explored that β-alanine can be used as a carrier electrolyte to conduct current in EtOAc.

The sample consisted of CV in EtOAc that was saturated with β-alanine. Theoretically, 3.5 % of water can be added but EtOAc already contains a small amount of water. It was tested that 2.8 % of water can be added without letting two phases to form. First, a β-alanine stock solution (30 mM) was made by weighting 26.8 mg of β-alanine into 10 ml of water and adjusting pH to 5 with pH meter and acetic acid. The final concentration of the β- alanine solution was 10 mM and it was prepared diluting 5 ml of stock solution into 15 ml of water. Different concentrations of CV were made (5, 10, 100 and 250 μM) by diluting stock solution (50 mM in 1:1 MeOH/H2O). CV samples (100 μM and 250 μM) in the BGE solution (1 M FA) were also prepared and used as reference.

6.1.3. Acylcarnitine samples

All acylcarnitine samples were prepared from stock solutions (50 μM in 50:50

MeOH/H2O) that were stored in a freezer. 0.25 μM acylcarnitine samples were made by diluting stock solution into EtOAc, MeOAc and n-BuOH. Solutions were not saturated with 10 mM β-alanine because with MeOAc and BuOH it started to precipitate on the walls of the container after adding solvent. Only 5 μl of β-alanine was added into all samples and saturation of solvent was completed with water. Samples contained 2.8 % (EtOAc), 7.5 % (MeOAc) and 13.5 % (BuOH) of water.

6.2. Analytical conditions

Figure 32 shows the CE-MS setup used in these experiments. CE and MS both had their own computers for controlling. Analytical conditions used in the experiments are listed in table 10.

Figure 32. CE-MS equipment used in the project.

Table 10. Experimental conditions.

Capillary untreated silica capillary, Polymicro Technologies Capillary length 100 cm Capillary ID 50 μm Conditioning 5 min with BGE CE-UV CE apparatus Hewlett Packard 3D CE Wavelength 590 nm CE-MS CE apparatus Agilent Technologies 1600 series MS apparatus Bruker Daltonics MicrOTOF Spray CE-ESI spray source, sheath-liquid assisted sprayer delivered with Harvard apparatus syringe infusion pump 22 at rate 4 μl/min Sheath-liquid 1:1 methanol / 0.1 % or 1 M formic acid Drying gas Nitrogen Spray settings: End plate offset -500 V Capillary voltage -4500 V Nebulizer gas pressure 0.4 bar Dry gas flow 4 L/min Dry temperature 180 °C

Suction at the capillary outlet disturbs EE at the inlet. Therefore, when EE was used with CE-MS, drying gas and nebulizer gas were switched off. After the EE step they were switched on again. This was built in the method and done automatically. After every run the capillary was flushed for 5 minutes with BGE. When a new capillary was introduced, it was first flushed 5 minutes with 1 M sodium hydroxide, water and BGE. Cooling of the sample tray was applied with water circulation during runs.

7. RESULTS AND DISCUSSION

First, different EE times and voltages were tried with CE-UV but methods were working randomly and were not repeatable. There seemed to be no consistency in the results and optimal analytical conditions were not found. That is why visual tests were carried out and results were used to build a working method for CE-UV and CE-MS.

7.1. Samples

Although the method was not completely working, it was seen from the peak size that EE was working better with EtOAc saturated with β-alanine than without. Samples containing acid were also analyzed. 1 % formic acid, 1 M formic acid and 1 % trifluoroacetic acid (TFA) were tried. At this point, the method was not reproducible but it seemed like the acid had no positive influence on the results. That is why acid was not being used in later experiments.

7.2. Electroextraction

At first, EE was tried using voltages 5 and 10 kV and rather long extraction times with small droplets. For example, droplet was formed using pressure -35 mbar for 35 seconds and it was extracted for a minute. On most times EE failed, which was easily seen from the current profile (figure 33). When non-conductive solvent enters the capillary, current rapidly falls down. It seemed like the droplet was not stable.

Successful EE-CE current profile is shown in figure 34. During the droplet formation current is zero. Voltage is switched on to carry out EE. After EE, voltage is again zero when the droplet is sucked back into the capillary. Finally, high voltage is applied during CE separation until the end of the run.

Figure 33. Current profile when solvent enters capillary.

Figure 34. Normal current profile.

7.3. Visual monitoring of the stability of a droplet

The stability of a droplet was tested with different droplet sizes and voltages. On the first setup, the inlet of the CE apparatus worked as outlet and end of the capillary was put into a sample tube (25 ml) containing EtOAc saturated with water. A grounded electrode was placed into the solvent and the tube was used as inlet. Because of this opposite arrangement, polarity was changed vice versa as well. The size of a droplet was varied using pressure of -50 mbar and different times. The pressure was always the same because it is the largest pressure that can be used with Agilent Technologies CE apparatus and the droplet had to be large enough to be seen with eye. Times 2, 4, 6 and 10 minutes were used. After the droplet was formed, voltage was applied. Voltage of 10 kV was used many times when EE was tried blind with CE-UV but with such a high voltage the droplet disappeared immediately. Therefore, voltages of 0.25, 0.5, 1, 2 and 5 kV were applied. Time was measured until the voltage dropped close to zero and the droplet disappeared. Three replicates were made from every measurement.

First results claimed that droplets were really unstable, which did not explain why some of the random EE tests were successful. Another droplet test setup that illustrates better the real situation was also made. This time the end of the capillary was placed in a glass vial (2 mL) through the grounded electrode that was taken from the CE device. The inlet was adjusted close to the outlet height level. Visual detection was improved by adding CV into the solvent. EtOAc was saturated with β-alanine (10 mM). During EE the droplet changed colour from transparent to blue. The setup and the blue colour of a droplet is shown in figure 35.

Sample consisted of CV in EtOAc. BGE droplet was blue because CV was concentrated into it. The most important difference between the two setups was the placement of the inlet and outlet height levels. It is important for the droplet stability to avoid siphoning. On the first test the inlet was higher than the outlet, which is why the droplet was sucked into the capillary and current reduced. Also the electric field was different. On the first test electric field was smaller in the sample according to equation 4 because electrode was placed further away from the end of the capillary. The second setup illustrates better conditions that would dominate in a real situation. Measured droplet stability times are in

Figure 35. EE of crystal violet. Capillary is placed in a sample vial through an electrode. attachment 1. On the second test not as many replicate measurements were made as on the first test, because droplets were more stable and it took a lot of time to make the measurements. Most of the replicates differed on the second test setup, which is the reason a third measurement was made to those droplets. Results from the second test are gathered in figure 36. The graph was drawn using averages of the closest replicates. Droplet volume was calculated from analytical conditions using CE expert lite; a converter on the internet121.

There are variations on the results and curves are not linear as they almost were on the first setup (attachment 1). In spite of that, it seems like the bigger the droplet the more stable it is. All sized droplets vanished quickly with 5 kV voltage and with 1 kV they lasted the longest. For further development of the method, it would be crucial to understand why the droplet vanishes after a period of time. There might be still some EOF sucking the droplet back into the capillary. Other option is that the droplet has so many positive charges that they start to repel each other and the droplet explodes like in the ESI-MS-spray. Also, the droplet might dissolve slightly into the solvent if the energy of the current makes it more favourable.

Figure 36. Results from the second visual test. Droplet size as a function of droplet stability.

7.4. Electroextraction-capillary electrophoresis-ultraviolet detection

With the results of the droplet test cautious analysis conditions were chosen to test EE with UV detector (table 11). The sample selected was 250 μM CV in EtOAc (saturated with 10 mM β-alanine). Droplet was formed and sucked back into the capillary without voltage on to keep the droplet stable. After these three EE steps, pressure (50 mbar) was used during the CZE separation because no EOF was occurring. Voltage was 20 kV.

Table 11. Electroextraction procedure for CE-UV.

Forming a Unit Extraction Injection droplet Pressure [mbar] 50 0 50 Time [min] 2 1.5 0.5 Voltage [kV] 0 1 0

Using these analytical conditions EE was working and overloaded peaks were gained (figure 37). Areas of the peaks ranged from 18000 to 25000, average was 22000, standard deviation 3000 and relative standard deviation 14 %. As a comparison, sample with the same concentration was prepared in the BGE and analysed with HDI. The same amount of

sample was injected into the capillary as with EE (50 mbar, 0.5 min). This time no peak was seen. Concentration of the sample was high and also visible to eye so UV detector should have been able to detect that. Injection without CE voltage was also attempted with injection times 100, 50 and 30 s. At that time small peaks were seen and the size was changing according to the injection volume. The results obtained with our CE system are in agreement with Sun and Qi122, who noticed that under high acidity (pH ≤ 3.75) CV had low response and resolution with UV detector. Our results demonstrate the concentrating power of EE, since CV could only be detected with EE-CE-UV and not with CE-UV.

Figure 37. Four replicates of 250 μM crystal violet runs with EE-CE-UV. Length of the capillary at detector was 92 cm and ID 50 μm, EE conditions are given in table 12. Separation voltage was 20 kV and 1 M FA was used as BGE.

7.5. Capillary zone electrophoresis-mass spectrometry

Since the interest was on metabolomics, CV was not really a relevant compound and also it would contaminate MS. Therefore it was not used in the further experiments. Instead of CV, acylcarnitines were chosen to be used as a sample for CE-MS. Five acylcarnitines with different polar properties were selected.

7.5.1. Electroextraction parameters

The EE-method that gave good results with CE-UV was transferred to CE-MS. Initially, the method was not working with MS-equipment and EtOAc entered the capillary. Detectors differ from each other, hence requiring different analytical conditions. The method was tried without injecting the droplet back into the capillary. The droplet fell on the bottom of the sample vial after EE and ions that were already inside the capillary were separated. These ions migrated their way into the capillary similar to EKI. Since peaks were detected, it meant that already some ions started to migrate into the capillary during EE and not all ions were concentrated on the surface of the droplet. This method would not give good extraction coefficients because most of the extracted ions were in the droplet. New parameters for EE had to be tested. Voltage, extraction time and injection volume were changed and two droplet sizes were tried (table 12). During the CE separation voltage was 20 kV.

Table 12. EE parameters.

Unit Forming a droplet Extraction Injection Pressure [mbar] 50 0 50 Time [min] 1-2 0.5 0.25 Voltage [kV] 0 0.2 0

EE was working with both droplet sizes, only current profile was not looking normal because current declines in the beginning of the CE separation (figure 38.). There was no outlet vial in the MS-system. When a droplet was formed at the inlet of the capillary, it was pushing BGE away from the capillary and forming an empty space in the other end of the

capillary. Therefore, sheath-liquid was flowing (4 μL/min) into the MS-system close to the end of the capillary filling the empty space. Sheath-liquid, which was first used, was 50:50 v/v methanol/0.1 % formic acid. Methanol was added to the sheath-liquid because of its low surface tension to promote the formation of gas-phase ions in the ionization. This sheath-liquid composition was not good because when it entered capillary, it did not conduct as much as BGE solution in the capillary. That is why current dropped down in the beginning of the run and returned back to normal after applying positive pressure for some time. More conductive sheath-liquid, and thus more similar to BGE, was made by replacing 0.1 % FA with 1 M FA. The second sheath-liquid led to a much better current profile (figure 39). Using a 50 nL droplet size, the current was almost normal. With a bigger droplet size the current profile was more disturbed. When the bigger droplet was formed, more sheath-liquid accessed to the capillary.

Figure 38. Current profiles of runs with droplets of 50 nL (red line) and 100 nL (blue line). Sheath-liquid was 50:50 v/v MeOH/0.1 % FA and flow rate 4 μL/min.

Figure 39. Current profiles of runs with droplets of 50 nL (red line) and 100 nL (blue line). Sheath-liquid was 50:50 v/v MeOH /1 M FA and flow rate 4 μL/min.

7.5.2. Different solvents

0.25 μM acylcarnitine samples prepared in three different solvents were run with EE parameters that can be seen in table 12 using a 50 nL droplet. Additionally, a similar sample in the BGE was run with normal injection with the same injection volume (50 mbar for 0.25 min). Electropherograms of these runs are in figure 40. The most polar compounds migrate first because their sizes are the smallest.

The peak profile is different on every run and peak areas of different compounds vary also with HDI. The areas of EEs were divided with the areas of HDIs, and resulted extraction factors (EFs) are listed in table 13 . All compounds were extracted best using MeOAc. Polar carnitine and acetylcarnitine are hardly extracted at all with EtOAc. The opposite happened with BuOH. It dissolves those compounds best but the extraction in general succeeded worst because EFs are the smallest. EtOAc is saturated with smallest amount of water and its dielectric constant is the smallest, hence making it the most non-polar solvent and least conductive. That is assumably why it did not extract the most polar compounds almost at all. Poor EFs with BuOH might be explained by high viscosity of butanol. It takes longer time to extract compounds because compounds are travelling with lower 81

speed in the solvent. In this experiment analyte concentration was kept the same but EFs could be bigger if more sample volume was added into solvents. Influence of initial analyte concentration on EE ought to be tested.

Figure 40. Comparison of normal injection with EE. Length of capillary, 1 m and ID 50 μm; EE parameters are given in table 5 (50 nL droplet), CE separation voltage was 20 kV. Each sample contained 0.25 μM of acylcarnitines. 1. carnitine, 2. acetylcarnitine, 3. hexanoylcarnitine, 4. octanoylcarnitine, 5. lauroylcarnitine. A) Hydrodynamic injection 50 mbar 0.25 min, sample in 1 M FA. B) EE, sample in EtOAc. C) EE, sample in MeOAc. D) EE, sample in n-BuOH.

Table 13. Extraction factors of analytes using different solvents.

Injection, solvent Carnitine Acetyl- Hexanoyl- Octanoyl- Lauroyl- carnitine carnitine carnitine carnitine Normal injection, FA 1 1 1 1 1 EE, EtOAc 0 0 6 7 8 EE, MeOAc 8 9 9 6 3 EE, BuOH 3 3 1 0 0

Extraction factors obtained with MeOAc as the solvent are bigger than using HDI. With optimization of the method, these EFs could be even higher. Most crucial would be to solve why the droplet is not stable during EE. Largest amount of analyte cations are presumably on the surface of the droplet. Therefore, injecting the whole droplet back to the capillary would enable highest possible EFs.

Choosing a suitable solvent EE could possibly be used for selective extraction. If polar compounds were target analytes and sample matrix would contain disturbing lipophilic compounds, butanol could be used as an EE solvent. In that case disturbing compounds would hardly be extracted and CE run would result in clearer electropherogram. Besides this example, there are many other possibilities to be explored. Also the use of solvent mixtures could be one option.

7.5.3. Repeatability

Since MeOAc as a solvent gave the best extraction factors, the same experiment was later repeated three times (table ). HDI was not run on this day and therefore extraction factors were not determined. Areas varied between replicates although the sample was the same. This can probably be explained by the varying room temperature that influences the performance of the CE injection and separation, as well as EE. Using these conditions the repeatability of EE was reasonable, but not satisfactory yet. The repeatability can be improved by working under better temperature-controlled conditions and by adding an internal standard prior to analysis.

Table 14. Areas of acetylcarnitine replicates in MeOAc.

Area 1 Area 2 Area 3 average Compound sd [x106] rsd [x106] [x106] [x106] [x106] Carnitine 2,6 1,7 1,8 2,0 0,5 23,5 Acetylcarnitine 7,2 5,2 5,5 6,0 1,1 17,9 Hexanoylcarnitine 21,0 22,0 25,4 22,8 2,3 10,2 Octanoylcarnitine 10,0 12,4 15,3 12,6 2,7 21,4 Lauroylcarnitine 0,9 1,3 1,9 1,4 0,5 36,4

Transfer to the acceptor phase is due to migration and diffusion. Concentrations of the phases influence diffusion and at some point diffusion back to the organic phase begins.93 Impact of sample concentration should be tested if EE would be considered as a quantitative method. Also, migration might have influence on repeatability. It is known that EKI is not as reproducible as HDI. Migration of analytes on EE is similar to migration in EKI. Droplet shape and size are something to consider as well. Round shape of the droplet can cause different electric field alignment compared to a test made in a vessel with two immiscible liquids. This might have an influence on EE.

When an EE system is built, temperature ought to be stable because distribution factors vary according to temperature. A raise in temperature also increases the conductivity of the organic phase123. Temperature of the EE-CE-MS system in this experiment was partly stabilized. Samples were cooled using water circulation and the CE cassette was automated to a stable temperature by CE apparatus. On an EE-CE-UV system, this arrangement would be enough, but the case of the MS system was more complex. The end of the capillary before the MS interface was at room temperature and the room temperature varied almost every day. Temperature did not make much of a difference in the repeatability tests because three runs were operated on a sequence one after another.

8. CONCLUSIONS

On-line concentration techniques can facilitate and make CE analyses more sensitive. Time-consuming pretreatment methods may be completely replaced and lower concentration levels can be detected. On-line concentration methods have been successfully implemented for different sample matrices from environmental to human origin.

One on-line concentration technique was experimentally piloted. A novel EE-CE system was developed, coupled to MS and tested with acylcarnitines to concentrate and determine cationic analytes. First, the method was built for UV detector. When nice concentrative results were obtained, the method was transferred to MS detector. Due to different requirements of the two detectors, method could not be directly utilized. Some optimization was done with the MS detector and the selectivity of EE was studied with

three solvents and five acylcarnitines. Peak size distribution fluctuated with different solvents, which indicated selectivity. Repeatability of EE was briefly tested with three runs but the results were not satisfactory yet since there were some variations on peak areas. Based on the results in this research, it looks like EE could be a powerful technique to concentrate analytes. For the future, optimization and repeatability of the method is needed for better understanding its potential as a concentrative technique.

Unfortunately, all on-line concentration methods have their own restrictions, and there is no method that would be applicable to all kind of compound groups. Like EE, other methods need optimization too. Some methods are suitable only for either cations or anions. Moreover, inside the ion group, method might be suitable only to a certain compound group. Common to all of the on-line concentration methods is that the sample matrix and composition of the BGE have a huge impact on the functionality of the method. Seldom are the sample concentrations similar or known, and therefore the suitability of a method may also not be known in advance. For example, FASS and related techniques, such as CSEI-sweep, cannot be used to samples with high salt and low analyte concentrations. In those cases, diluting the sample makes it impossible to detect even lower analyte concentrations. Instead, tITP is better for samples containing high salt concentrations but the method is not free of difficulties either. Large concentration levels of other compounds may cancel the whole concentration process. Based on the tables in this thesis, it can be concluded that on-line concentration methods using EKI are the most efficient but they are not necessarily repeatable enough. Sometimes chemometric tools can be utilized to help evaluating interactions inside the capillary and reduce the number of trial runs. Combining preconcentration methods usually yields to better sensitivity but combinations might be cumbersome to setup and optimize.

Although on-line concentration methods need optimization, they do improve CE methods greatly. Once the piecework of optimization is done, the sensitivity of the method is better and in some cases pretreatment can be avoided. On-line concentration methods together with the high chromatographic qualities of CE can publicize and make CE more common technique in the future. The idea of miniaturizing CE with its on-line concentration channels on a microchip is palatable too.

9. ATTACHMENTS

1. Visual test results 2. Graph from the first visual test results

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Attachment 1. Visual test results.

250 V 500 V 1 kV 2 kV 5 kV Droplet size Replicate Time to lose the Time to lose the Time to lose the Time to lose the Time to lose the Current [μA] Current [μA] Current [μA] Current [μA] Current [μA] droplet [min] droplet [min] droplet [min] droplet [min] droplet [min]

2 min -50 mbar 1 0,55 0,30 0,40 0,50 0,40 1,00 0,30 1,10 0,05 2,20 100 nL 2 0,80 0,30 0,60 0,50 0,40 1,00 0,11 1,90 0,20 3,20 3 0,60 0,30 0,60 0,50 0,40 1,00 0,23 1,90 0,20 3,20 average x 0,65 0,30 0,53 0,50 0,40 1,00 0,21 1,63 0,15 2,87 4 min -50 mbar 1 1,20 0,30 1,70 0,50 1,50 1,00 0,70 1,80 0,05 4,80 200 nL 2 1,70 0,30 1,60 0,50 1,45 1,00 1,10 1,80 0,04 3,80 3 1,50 0,30 1,65 0,50 1,60 1,00 0,95 1,80 0,05 4,20 average x 1,47 0,30 1,65 0,50 1,52 1,00 0,92 1,80 0,05 4,27 6 min -50 mbar 1 2,84 0,30 2,80 0,50 2,00 0,90 1,80 1,80 0,70 4,80 300 nL 2 2,72 0,30 2,40 0,50 2,30 0,90 1,65 1,80 0,55 4,80 3 2,98 0,30 2,65 0,50 2,35 0,90 1,70 1,80 0,30 4,60 average x 2,85 0,30 2,62 0,50 2,22 0,90 1,72 1,80 0,52 4,73 8 min -50 mbar 1 3,60 0,30 3,60 0,50 2,90 0,85 2,40 1,70 1,05 4,70 400 nL 2 3,50 0,30 3,30 0,50 3,50 0,85 2,40 1,70 0,95 4,70 3 4,00 0,30 3,50 0,50 3,50 0,85 2,60 1,70 1,00 4,70 average x 3,70 0,30 3,47 0,50 3,30 0,85 2,47 1,70 1,00 4,70 10 min -50 mbar 1 3,80 0,20 4,40 0,50 4,93 0,80 3,70 1,70 1,40 4,50 500 nL 2 4,60 0,30 4,80 0,50 4,40 0,80 3,70 1,60 1,50 4,50 3 4,50 0,25 4,80 0,50 4,80 0,80 3,30 1,60 1,50 4,50 average x 4,30 0,25 4,67 0,50 4,71 0,80 3,57 1,63 1,47 4,50

Attachment 2. Graph from the first visual test results.

5 0.25 5 kV 4 0.5 kV 4 3 3 1 kV 2 2 2 kV 1 1 5 kV 0

Droplet stability stability time [min] Droplet 100 150 200 250 300 350 400 450 500 Droplet size [nL]