Development of methods to improve sensitivity and portability of capillary electrophoresis for the analysis of stabilizers and drugs 2

Chapter 1 Introduction 1.1 Basic Theory of Capillary Electrophoresis Analytical techniques, when based on the migration of electrically charged particles of ions in solution in an appli

Chapter 1 Introduction

1.1 Basic Theory of Capillary Electrophoresis

Analytical techniques, when based on the migration of electrically charged particles of ions in solution in an applied electric field, can be classified as electrophoresis Capillary electrophoresis (CE), also designated as high performance capillary electrophoresis (HPCE), normally carried out in fused silica capillaries, is based on the same basic theory as conventional electrophoresis

As CE is a liquid-phase separation technique, it has close resemblance to high performance liquid chromatography (HPLC), particularly ion chromatography and reversed-phase HPLC although there are significant differences The comparison is in terms of the following aspects:

1, The basic separation principle is different CE is mainly based on different migration speeds of charged particles under an electrical field to achieve the separation of components, which are the ionic differences between the analytes While reversed-phase HPLC, as well as gas chromatography (GC), is mainly based on polarity differences of the analytes

2, In contrast to HPLC where the mobile phase is constantly being pumped into the system, in CE, the ideal experimental conditions do not require a physical flow of the buffer across the column, since the driving force for the actual migration of species in the column in CE are their charges, the applied potential and EOF

3, Since the capillary applied in CE is normally empty (except Capillary Gel Electrophoresis (CGE) or Capillary Electrochromatography (CEC)), while the HPLC column is normally filled with particles so the longitudinal molecular diffusion (Eddy diffusion) and mass transfer restrictions encountered in liquid chromatography (LC) are not relevant in CE As a result, the separation efficiency of CE is significantly better than with HPLC

4, CE columns, whose internal diameters (ID) normally range from 25μm to 150μm, are much smaller than HPLC columns, whose IDs are normally 1mm to 4.5mm This difference mainly results in two advantages and one disadvantage The first advantage

is that the solvent/buffer consumption of CE is very much lower than the amount of organic mobile phase usually required for HPLC Consequently, it is not only more economical to use CE than HPLC for separations but also more environmentally friendly, as aqueous-based buffers instead of organic solvents in HPLC alleviates the problem of waste disposal Therefore, from a long term perspective, the use of CE seems both economically and environmentally desirable The second advantage is the smaller amount of sample loading in CE, which is especially useful in bioanalysis

because a lot of bio-samples are very expensive or precious However, the small column also results in much shorter detection pathlength in CE, which will strongly decrease the concentration sensitivity of CE technique So the CE sensitivity is normally 10 to 20 times less than that of HPLC for absorbance detection while all the other conditions are the same

The basic concept of CE is that an electrically charged species moves in a certain speed under the influence of an electric field Compounds are separated due to their different migration speeds

The velocity of an ion is given by the following equation:

ν = μE = Leff / tm [unit: m/s] (1.1)

where,

ν: the velocity of the component;

μ: electrophoretic mobility of the component;

E: the electric filed strength;

Leff: the effect length of capillary, the capillary length from inlet to the detector

tm: migration time of the component

After steady state is reached, the ions move with a constant velocity ν The velocity ν

is proportional to the applied electric field as well as the electrophoretic mobility μ, which is a characteristic property of a given ion in a given medium and at a given temperature So the electrophoretic mobility μ of the ions relates to ν and E The

velocity of an ion is determined by dividing the traversed capillary length Leff, by the migration time tm of the peak

The electrophoretic mobility μ can also be expressed as

L: distance from injection point to detection point;

σ: total spatial variance of the concentration profile of the zone

If the peaks acquired are symmetric and have Gaussian profile, the theoretical plate number can also be calculated from the electropherogram using the following equation:

N = 5.54 (t/w1/2)2 (1.4)

Where;

t: migration time;

w1/2: temporal peak width at half of the peak height

Compared with HPLC, the efficiency of CE is normally higher mainly due to the plug-shaped flow profile [1] However there is still zone broadening in CE technique The most important 6 causes of zone broadening in CE are the following:

1 Longitudinal diffusion: It corresponds to the theoretical limit, increases directly with analysis time and the diffusion coefficients, and inversely with the molecular weight

2 Thermal effects: The thermal effects lead to convection and to local changes in buffer viscosity

3 Injection length: If the injection length is too big, then broad peak and poor resolution will result, though the detection limit may be increased So it should be smaller than the zone generated by diffusion, normally less then 1% of the effective length of the capillary

4 Wall adsorption of samples: It causes peak tailing and poorly reproducible migration times, and can be decreased by using coated capillary and other more complicated method

5 Electrodispersion (Mobility difference): The electrodispersion causes “triangular” peak shapes

6 Difference in the liquid levels: This may cause hydrodynamic flow, that’s why one

of the basic requirements of a CE instrument is to make the inlet and outlet vial at the same level

1.2 Electroosmotic Flow (EOF)

Electroosmotic flow (EOF), which causes the buffer solution to flow in an electric field, is very important in CE It depends upon the distribution of charge in the proximity of the capillary surface Generally, nearly all surfaces carry a charge, and in the case of quartz capillaries wall, there are negative charges from the dissociation of the silanol groups In the solution these surface charges are counterbalanced by counterions, which means oppositely charged ions As Figure 1.1 shows, in the double layer, due to the negative charges of the fused silica surface, the positive ions predominate in solution are arranged in a rigid and a diffuse layer According to Stern’s theory, the potential built up on account of the charge distribution is divided into two regions: the rigid boundary layer with adsorbed ions and Stern boundary layer (diffuse boundary layer) A linear decrease in the potential in the region of the first layer and an exponential decrease in the rear layer were observed in experiments The exponential decrease is responsible for the electroosmosis and is designated as the ζ–potential

Figure 1.1: Charge distribution at the surface of fused silica and formation of the ζ–potential

Figure 1.2: Profile of the ζ–potential at the buffer/fused silica interface

a: rigid boundary layer with adsorbed ions; b: Stern boundary layer (diffuse boundary layer); c: Electrolyte

The migration velocity ν of the EOF can be described with following equation:

νeo = ε E ζ / 4πη [unit: m/s] (1.5)

Where;

ε: the dielectric constant of electrolyte

E: the applied field strength

ζ: the ζ–potential (zeta-potential)

η: the viscosity of the electrolyte

νeo is also described by the equation

100 μm the flow profile can be regarded as nearly ideally plug-shaped On the other

hand in hydrodynamic flow, the parabolic Hagen-Poiseuille flow profile appears This round head shaped (parabolic) flow profile is strongly dependent upon the capillary radius and the flow velocity The difference in flow profile results in different peak shapes as in Figure 1.3, which shows a comparison of both flow profiles in HPLC and

CE as well as the peak shapes in both techniques

(A) Flow profile in HPLC (B) Flow profile in CE Figure 1.3: Representation of a pressure-generated flow profile (A) and an ideal plug flow profile (B)

The EOF appears in nearly all electrophoretic separation methods because surface charges cannot be completely eliminated As the EOF is normally from anode to cathode, the detector in CE is usually placed on the cathode side All the cations, neutral components and anions move with the EOF as shown in Figure 1.4 But cations

will move faster than the EOF as their velocity direction is the same as that of the EOF,

so very rapid analysis times are therefore achieved with positively charged compounds The neutral compounds move at the same speed as EOF, so they cannot be separated in Capillary Zone Electrophoresis (CZE) However for anions, though they migrate opposite to the direction of the EOF, are also transported to the detector (on the cathode side) Since their migration velocities are typically lower than the velocity of the EOF, they can still be detected in CZE Therefore, under suitable conditions cations and anions can be separated from each other in a single analysis Only the anions that migrate faster than the flow velocity of the EOF migrate into the anode compartment and escape detection These ions can be detected by reversing the polarity, but then the cations and the slowly migrating anions escape detection

Figure 1.4: Separation in Capillary Zone Electrophoresis

It is possible to detect cations and anions in the same run by controlling the EOF to elute more components of the sample There are several frequently used methods to change the EOF, such as adjustment of pH value, varying electrolyte concentration of

the buffer and introducing buffer additives

The buffer concentration and the pH value represent the most important parameters for optimizing separation Variation in the buffer concentration presents one of the simplest and most effective means of influencing the EOF of the separation system Normally the EOF increases with decreasing buffer concentration and enables analysis

of highly negatively charged solutes that actually migrate counter to the EOF

pH value, as it will strongly influence the charges on the wall surface, have a big effect

on the EOF (The rough trend can be seen in Figure 1.5)

Figure 1.5 pH dependence of the electroosmotic flow

Secondly, addition of organic components also affects the EOF Polymers can, in part,

be adsorbed so strongly that they are not flushed out during a buffer change Such

cases, as well as the adsorption of surfactants, are termed “dynamic coatings” It will change the charge situation on the wall surface, as a result changing the EOF

Furthermore, it has become evident that organic solvents also exert a strong influence

on the velocity of the elctroosmotic flow They can change the EOF as well as the buffer viscosity, so that changes in separation efficiency and additional changes in selectivity may appear

In summary, the EOF normally diminishes with increasing electrolyte concentration, with the addition of organic components, as well as with the increases in the degree of dissociation of the surface silanol groups, i.e., with the pH value, for quartz capillaries

Through chemical modification of the capillary surface, the EOF can be not only controlled, but also reversed The EOF can be reversed by adding long-chained cationic detergents, such as cetyltrimethylammonium salts that are adsorbed on the surface silanol groups A double layer of the detergent is formed, with the positive charges directed toward the electrolyte (The formation of a double layer is shown schematically in Figure 1.6.) Another kind of additive which can help to attain reversal

of the EOF is polyamines, such as spermine With such coated capillaries, together with reversal of the field, separation of slowly and rapidly migrating inorganic anions can be achieved in a single analysis

Figure 1.6 Illustration of double layer and reversal of EOF by addition of a quaternary amine to the buffer

1.3 Different Modes of CE

1.3.1 Capillary Zone Electrophoresis (CZE)

CZE, which at present represents the most frequently used method of capillary electrophoresis, is carried out exclusively with electrolyte-filled capillaries The separation is based on the mobility differences of the solutes in free solution under the effect of an electric field (see Fig 1.4) The difference can be caused by different charges, masses or structures of chemical compounds CZE is the simplest mode of CE and it is also very powerful, but it has at least one shortcoming in that it cannot be applied to separate neutral compounds

1.3.2 Micellar Electrokinetic Chromatography (MEKC)

MEKC, which can be used to separate uncharged molecules, is another frequently used

CE mode Pseudo-phases, e.g surfactants are added to the buffer and the neutral molecules distribute themselves between the buffer and the micelles according to their hydrophobicities Besides this interaction, other specific (e.g hydrogen bonding) or universal interactions (e.g dipole-dipole, dispersive) can be also involved in the distribution of analytes between two pseudophases

Figure 1.7 Scheme of MEKC mode

The separation of neutral compounds is in principle impossible in free solution CZE, because of the lack of self-electrophretic mobilities of neutral analytes and thus the only driving force for neutral analyutes is the EOF, which is equal for all neutral

components of a sample On the other hand, in MEKC, the neutral components are differently distributed in the pseudophase which has a different mobility to the EOF They acquire different effective mobilities from the EOF and also from each other The range of separation is frequently called separation window in MEKC It lies between the compounds that do not reside within the micelles and hence migrate with the EOF, and those that are permanently enclosed within the micelles

1.3.3 Capillary Gel Electrophoresis (CGE)

Figure 1.8 Scheme of CGE mode

In CGE, the capillary is filled with a gel-forming medium, such as cross-linked polyacrylamide and agarose, (see Figure 1.8) CGE is the closest technique to traditional slab-gel electrophoresis, both of which separate the analytes according to their charge and size CGE is mostly used for the analysis of large molecules of biological interest such as proteins and nucleic acids The gel matrix hinders the electrophoretic migration of macromolecules The transport of the solutes through the

capillary is based on the charge of the macromolecules, but the separation is dependent

on the molecular size Besides the gel forming medium, other additives can also be added in CZE together with sieving materials to achieve the desirable selectivity

1.3.4 Capillary Isoelectric Focusing (CIEF)

Figure 1.9 Scheme of CIEF mode

In CIEF, separation occurs in a pH gradient and it also has the function of pre-concentration, (See Figure 1.9) The pH gradient is formed by the additions of ampholytes into the buffers, which contains both an acidic and a basic moiety and typically have pI values between pH 3 and 9 With a basic solution at the cathode and

an acidic solution at the anode, upon application of the electric field the charged ampholytes and sample components migrate through the medium until they reach a region where they are uncharged This process is known as “focusing” After that the current decreases and the capillary contents can be mobilized to the detector by applying pressure or by addition of an electrolyte to one of the reservoirs CIEF is most

commonly used in bioanalysis, such as the separation of immunoglobulins, hemoglobins and the measurement of the pI of proteins,

1.3.5 Capillary Electrochromatography (CEC)

Figure 1.10 Scheme of CEC mode

CEC is based on the partitioning of analytes between mobile and stationary phases So

it has the same separation principle as in chromatography The difference is that in contrast to the latter, the migration principle is electrophoretic in CEC, which means the migration and separation principle of CEC and MEKC is the same The potential advantages of CEC comparing with other separation technique have generated a lot of interest Firstly, as the ID of capillary is normally much smaller than that of the column

in HPLC CEC just need nano-liter amounts of samples, which is especially useful in

bioanalysis comparing with the much larger sample injection amount of HPLC Secondly, the separation result of CEC can be significantly different and mostly better than other separation methods by pressure-driven flows, such as GC, supercritical fluid chromatography (SFC) and HPLC It is because of the plug-like profile of electrokinetic flow in contrast to the parabolic profile of laminar flow Thirdly, the peak efficiency will also be higher in CEC than in HPLC, because mass transfer between the stationary and mobile phases can be substantially faster in some modes of CEC than in HPLC CEC also has a significant advantage compared with other mode

of CE since selectors insoluble in aqueous buffer can be used to improve elution and to enhance separation, which is impossible in CZE and MEKC

1.3.6 Capillary Isotachophoresis (CITP)

In CITP, a combination of two buffers (leading and terminating electrolytes) is used to create a state in which separated zones all move at the same velocity The velocities of all analytes are defined by the velocity of the leading anion The principle of separation

is the variation of the electric field in each zone, which causes the self-adjustment of the field to maintain a constant velocity, with the lowest field across the one with the highest mobility If an ion diffuses into a neighboring zone, its velocity changes and forces it to return to its own zone The concentration in each zone is also determined

by the concentration of the leading electrolyte and remains constant in CITP This principle can be used as a pre-concentration step in other modes However, there are

two shortcomings of CITP Firstly cations and anions cannot be analyzed in the same run Secondly the neutral compounds are unsuitable to be pre-concentrated with CITP

Table 1.1 Different modes of HPLC

Charged small and large

molecules

Modified/

Unmodified

Controlled/ uncontrolled

Charged and neutral analytes

to their charge and size in a carrier electrolyte

containing sieving gel-forming media

Useful for charged analytes

Modified and filled

Suppressed

Capillary Isoelectric

Focusing (CIEF)

Distribution of charged analytes according to their

pK or pI values and the pH gradient of the BGE

Mostly used for large-sized charged molecules

Modified/

Unmodified

Suppressed/ weakly present

Capillary

Electrochromatograp

hy (CEC)

Partition of analytes between stationary and mobile phases

Charged and neutral analytes

Useful for charged analytes

Unmodified Present

1.4 Sample Introduction and Concentration

Siphoning injection, i.e sample introduction by gravity, is more commonly used with non-commercial systems It relies on the siphoning of sample into the capillary by elevating the inlet end of the capillary relative to the outlet end

These two injection methods can be generalized as hydrodynamic injection The volume, v, of the sample injected into the capillary by using Poiseuille’s equation is given by

V = (Δpπr4t)/ (8ηL) [unit: Liter (L)] (1/7)

Where,

Δp: the pressure difference;

r: the inner diameter of the capillary;

t: the injection time;

η: the viscosity of the buffer;

L: the total length of the capillary

In the case of all hydrodynamic sample introductions, it is necessary that the material

in the capillary is free to flow So it is not suitable for viscous solutions, which is the shortcoming of this injection method Normally, 1% of the total capillary length of sample solution is injected

With electrokinetic loading, the inlet end of the capillary is immersed in the sample, the outlet in the separation buffer and a low voltage (1-10kv) is applied for durations of less then 1 min depending on the capillary length and ID The quantity, Q, of a component of the sample injected to the capillary can be represented by

Q = (μep + μeo) π r2 Ui ti c / L (1.8)

Where,

μep: electrophoretic mobility;

μeo: electroosmotic mobility respectively;

Ui: magnitude of the applied potential

ti: injection time

c: the concentration of the component in the sample solution

Electrokinetic introduction has two characteristics One is that the quantity of sample

introduced into the capillary is dependent on the electrophoretic mobilities of the sample components (μep), the EOF (μeo) and so on So the sample components with higher electrophoretic mobility will be preferentially introduced over those with lower mobilities This phenomenon is named as “electrokinetic bias” sometimes Another characteristic is that in the case of capillaries filled with a fixed gel or a rigid matrix, where the hydrodynamic injection is not available, electrokinetic injection is the only way to introduce sample

1.4.2 On-line Concentration Techniques in CE

One of the greatest shortcomings of CE is the general lack of sensitivity due to the short optical path length of detection and the low sample injection volume which just ranges from 2 to 20 nl To increase the sensitivity, two approaches, either to increase the amount of analyte added to the capillary or to improve the sensitivity of the detector, may be applied However, due to the characteristics of the CE technique, both

of these two approaches are limited For example, as the volumes of narrow-bore capillaries are very small, the introduction of a large sample plug without any special treatment may lead to unacceptably broad peaks and poor resolution The sensitivity of

a detector is also too hard or expensive to be improved A more practical and moderate way to address this concern would be to use on-line concentration techniques [7-9], which is done by manipulating the composition of the sample and background solutions together with sample injection procedures without modification of the

instrumentation

The existing on-line concentration method applied in CE can be categorized into two groups based on the physical phenomenon used to concentrate analytes One group involves manipulating the electrophoretic velocity of the analyte and includes techniques such as sample stacking [11], isotachophoresis [12], pH-mediated stacking [13] and matrix switching [14] The other group utilizes the partitioning of analytes into the pseudo stationary phase to affect analyte preconcentration, for example, sweeping [15]

1.4.2.1 Sample Stacking

Sample stacking, a well known phenomenon in electrophoresis, is based on the difference in electrophoretic velocities of solutes in a non-continuous buffer system, which has different electric field strengths [10]

Figure 1.11 General sample stacking model of anions

The basic principle of sample stacking of anions is summarized in Figure 1.11[33] The sample is prepared in a low conductivity solution while the background electrolyte (BGE) is a high conductivity solution Then normally the high electric field gradient (low-conductivity) sample zone and a low electric field gradient running electrolyte solution (high-conductivity zone) will be introduced into the capillary (Figure 1.11-A) When the high voltage is applied, the analyte ions will experience high electric field gradient strength and move faster in sample zone while they move slower in the low electric filed gradient buffer zone Thus the velocity of the ions will change suddenly once they cross the concentration boundary (Figure 1.11-1), resulting in the stacking of sample solution at the boundary (Figure 1.11-B)

Sample stacking can be performed in both hydrodynamic and electrokinetic injection modes In the latter mode, it is also called field amplified sample injection (FASI), which will be explained in 1.4.2.2

In hydrodynamic mode, there are also several variations which have been developed for the analysis of anions or cations Normal stacking mode (NSM) is the simplest among these It is performed by dissolving the sample in a low conductivity matrix and

by injecting it hydrodynamically Concentration factors of around 10 are usually obtained with NSM, improving the concentration LOD by one order of magnitude The shortcoming of NSM is the short optimum sample plug length that can be injected into the capillary without loss of separation efficiency or resolution This is due to the

broadening of stacked zones that result from the mixing of low and high conductivity zones in the concentration boundary and the laminar flow generated inside the capillary resulted from the mismatch of local and bulk EOF velocities [12, 13]

In order to overcome this shortcoming, another mode which is called large volume sample stacking (LVSS) was developed by Chien and Burgi[13] After injection of a larger volume of sample than found optimum in NSM, the sample matrix is pumped out from the capillary in order to preserve separation efficiency The EOF pump is used for this purpose The direction of pumping is always opposite that of the electrophoretic movement of the charged solutes The velocity of pumping should be lower than the electrophoretic velocity of the charged solutes

1.4.2.2 Field Amplified Sample Injection (FASI)

Figure 1.12 Field amplified sample stacking model of anions

Sample stacking, when applied with electrokinetic injection, [14-23] is usually termed field amplified sample injection (FASI) The basic theory of FASI is that when high voltage is applied to the two ends of the capillary, sample ions prepared in a more diluted buffer will experience higher electric field strength and move faster than the ions inside the BGE But when the sample ions reach the boundary between the sample and the BGE compartments, they will slow down and stack into a zone much narrower than the original sample plug Figure 1.12 illustrates the mechanism of FASI for anion stacking

FASI provides larger sensitivity enhancements compared with hydrodynamic injection [15] This is explained by the fact that in hydrodynamic injection, the volume of the sample solution that can be injected into the capillary limits the injected amounts of samples This is not a problem in electrokinetic injection since analyte molecules are introduced electrokinetically

In theory, a maximum signal enhancement is achieved when one switches the column directly from the high-conductivity support buffer to the low-conductivity sample solution However, during electrokinetic injection the buffer boundary may be disturbed, possibly by ions migrating from the buffer into the sample zone To overcome this, Chien and Burgi [23] showed that the injection of a small water plug at the injector site before electrokinetic injection establishes a high electric field at the injection point and, consequently, high stacking factors can be achieved

To inject sample ions into the column, the combination of the ion’s electrophoretic velocity and bulk electroosmotic velocity must be positive In FASI, the amount of ions is proportional only to their electrophoretic mobility As a result, there will be a bias in favor of high mobility analytes[14, 23] Furthermore, usually only one kind of ions can be concentrated effectively using a single electrokinetic injection For example, only positive ions can be injected and concentrated with the anode at the inlet end during electrokinetic injection

1.4.2.3 Isotachophoresis (ITP)

Figure 1.13 Isotechophoresis pre-concentration model

Isotachophoresis (ITP), another kind of stacking method in CE, is always performed

with the sample zone between the BGE of higher and lower electrophoretic mobilities

The former BGE is the leading electrolyte and the rear is the terminating electrolyte

Typically, either cations or anions can be separated in one run with some exceptions

[24, 25]

For cation analysis, the leading buffer is positioned at the cathodic end of the sample

zone while the terminating buffer is introduced after the sample zone When high

voltage is applied, a potential gradient develops and each of the analyte zones migrates

with the same velocity, where cations with lower mobility are present, electric field is

higher, making the velocity of the zone match the rest of the sample If a solute moves

too fast and enters the band before it, i.e a region of lower field strength, the analyte

will slow down until it enters its own zone again Eventually a steady state is reached where each analyte moves in a discrete band according to its mobility, Bands with the highest mobility will elute first And during this procedure, the sample is pre-concentrated and higher sensitivity of the detection can be gained

ITP is applied to many compounds ranging from small charged analytes to proteins [26-28]

1.4.2.4 pH Related Stacking

Figure 1.14 pH mediated stacking model

Sample stacking is normally performed with the sample either dissolved in water or diluted with a low conductivity buffer However biological and environmental samples

frequently have high concentration of salt such as NaCl etc so that they have high conductivities Therefore, desalting is often required for these samples, which is usually time-consuming as well as likely to decrease the precision One way for concentrating these samples is named pH-mediated stacking, as neutralization of the high-conductivity sample matrix with acid or base is applied The basic procedure of pH-mediated stacking is as following First, a sample in a high-strength biological medium is electrokinetically injected As the sample is injected, the anions of the strong acid such as Cl- are displaced by the anions of the weak acid in the BGE, such

as acetate Next, a plug of strong acid (HCl) is injected electrokinectically The protons from the acid injection migrate quickly through the sample zone, titrating with the acetate ions and creating a region of neutral charge and high resistivity This allows the sample cations to migrate quickly through the zone to the boundary with the BGE, where they stack into a narrow band [29-31] This method can also be used for anions

by incorporating an EOF modifier in the basic BGE and running under reverse polarity [32]

Another way to focus analytes is by mobility difference via a dynamic pH junction between sample and BGE zones [33-34], which is a specific focusing technique dependent upon the chemical properties of the analytes The differences in the electrophoretic mobility of the analyte are not caused by conductivity differences or higher electric field distributed within the sample zone but dependent upon changes in the ionization of the analyte at the junction between the sample and the BGE, for

example from neutral to fully ionized as a function of pH This method is useful for zwitterionic and weakly acidic analytes that display significant velocity differences in the sample and buffer matrix

1.4.2.5 Sweeping

Sweeping, a unique focusing phenomenon in MEKC, is the accumulation of analytes molecules by the pseudostationary phase that penetrates the sample zone Sweeping is normally under conditions of relatively constant electric fields throughout the capillary, negligible EOF, and neutral analytes prepared in a matrix void of the pseudostationary phase The enrichment efficiency is dependent on the anlyte’s ability to partition into the pseudostationary phase in MEKC [35-37], which causes a unique focusing effect The injected length of a neutral analyte zone was found to be theoretically narrowed by

a factor equal to 1/(1+k) (k, retention factor) and the concentration can be increased approximately by a factor, 1+k Detection sensitivity of some analytes can be improved 5000-fold without off-line treatment, by MEKC with UV detection

1.5 Different Detection Methods with CE

The typical CE set consists of the following 5 units: the anode and cathode reservoirs (Figure 1.15-A, B) with the corresponding electrodes, the separation capillary (Figure 1.15-C), the injection system (Figure 1.15-E), the high voltage (Figure 1.15-G) and the

work station (Figure 1.15-H)

Figure 1.15 Basic scheme of the CE instrument

A capillary tube (Figure 1.15-C) filled with the buffer solution is placed between two buffer reservoirs (Figure1.15-A, B) after introducing the sample from the sample reservoirs (Figure 1.15-E) High voltage, which is up to 30kv, is applied on the capillary and generates the electric field The results are measured by the detector (Figure 1.15-D) Then the signal is transferred to the work station (Figure 1.15-H), which acquires the data as well as controls the system

Detection is an important part in CE technology Sensitive, selective and universal detectors are highly demanded for CE The most frequently used CE detectors include ultraviolet-visible (UV-Vis) absorbance, fluorescence, electrochemistry and mass spectrometric detections A general challenge to all of the CE detection techniques is to

maximize sensitivity without loss of separation efficiency

to improve the sensitivity

Photodiode array detection, which can acquire chromatograms of multiple wavelengths

at the same time, has become more and more popular in UV detection nowadays

1.5.2 Fluorescence Detection

Fluorescence detection is also performed on-column But in contrast with UV

detection, fluorescence detection is more selective and potentially more sensitive, which is a result of the low background noise and the direct proportionality between excitation power and emission signal intensity For fluorescence detection, two orders

of magnitude lower detection limits are typically possible to be obtained

Jorgenson and Lukacs accomplished their pioneering work in CE with fluorescence detection in 1981[40] Besides the usual deuterium and xenon arc lamps, much interest

is put in using laser-induced fluorescence (LIF) for ultra trace detection of substances

in CE As low as 10-12 M (which corresponds to approximately 1000 analyte molecules introduced into the capillary) of concentration level can be achieved with LIF detection

At present, LIF is the best detection scheme in CE as far as sensitivity is concerned

But comparing with UV detection, fluorescence detection is more selective and less universal, because not many compounds contain fluorophores As a result, the derivatization step of combining the sample with other fluorescent compounds is necessary for compounds without fluorophores

10-5~10-9 M LOD, which is similar to UV-Vis detection ECD can be carried out in either on-column or end-column format in CE However ECD have a shortcoming that it’s difficult to decouple detector signal from interference of separation voltage, which

is the field a lot of scientists are investigating Normally ECD in CE is classified into amperometry, conductivity and potentiometry according to the operation principles

a constraint on limit of detection (LOD) Suppressed conductivity detection allows lower LOD to be achieved since a suppressed system enlarges the difference between the conductometric signal of the analytes and the background of buffer electrolytes [49-50]

Conductivity detection, when operated under a direct current (DC) mode, is sometimes termed as potential gradient detection (PGD) [52] It works on the basis of the

changes in the electric field strength between zone boundaries during electrophoresis The electric field strength is inversely proportional to the ionic mobility Therefore, the potential gradient originates from the differences in mobility between migrating zones, and is applicable to all charge-carrying compounds It is similar to conductivity detection, but it does not involve measurement of alternating or direct current Experimental demonstration [53] showed that the detection limit of an analyte would

be improved by using buffer containing low-mobility counterions [51]

Conductivity detection has a lot of advantages However the contact conductivity cells have mostly metal electrodes that are immersed directly in the electrolyte solution, which will cause problems in CE because of the small ID of the capillary The problem was solved when contactless conductivity detector (CCD) was introduced in 1998 [41,42,54,55] Since then, this detection method has become more and more popular because of its three main advantages: Firstly, the electronic circuitry is decoupled from the high voltage applied for separation (no direct DC coupling between the electronics and the liquid in the channel) Secondly, the formation of gas bubbles at the metal electrodes is prevented, and thirdly, electrochemical modification or degradation of the electrode surface is prevented [44] In CCD detection, an ac-excitation voltage is

applied through the capillary wall as the tubular electrode fitted on the outside forms a capacitor with the solution inside Then on the second electrode downstream, the

resulting cell current is picked up [42] When the frequency of the measuring voltage is considerably higher than is usual in contact conductivity cells, better coupling can be

achieved [43]

2 Potentiometric detection

Potentiometric detection is based on membrane potential response of an ion selective electrode to the activities of ionic solutes In 1991 Haber et al made the first potentiometric detector with end-column design in CE [56] Later, on-column detection was introduced to reduce baseline drift and noise [57] However potentiometric detection has not yet gained its popularity, as some disadvantages hampers its application such as tedious sensor preparation procedures and limited lifetime

3 Amperometric detection

Under the influence of a constant potential, when electron transfers to or from electroactive solutes at the surface of a solid electrode, current is directly related to concentrations of electroactive solutes The measurement of amperometric detection is based on this principle End-column amperometric detection using a small ID capillary does not require the decoupler because essentially all of the applied separation voltage

is dropped across the capillary itself [58, 59] Amperometric detection has advantages

of high sensitivity and good selectivity So it is probably the most widely used electrochemical detection scheme in CE nowadays Amperometric detection is particularly useful in detection of solutes easily oxidized or reduced Chemically modified electrodes [60] or pulsed amperometric detection (PAD) have been demonstrated to be useful for expanding the applications of amperometric detection

PAD is also useful for solving the problem of electrode fouling which is often the main reason for the loss of reproducibility and sensitivity in amperometric detection

1.5.4 Other Detection

Other detection methods, such as Mass Spectrometry detection (CE-MS), Raman, on-line radioactivity measurements, circular dichroism, refractive index, thermooptical absorbance and capillary vibration, have also been introduced in some journals.[4, 61] Except for CE-MS which will be introduced in the next section, the potential of these other techniques to be used to gather with CE in routine applications cannot be assessed at this time, but a significant improvement in the detection sensitivity was not expected [62]

first time applied zone electrophoresis in glass tubes of 200-500μm bores in 1974 [63] Several years later, Mikkers, Everaerts and Verheggen [46] performed zone electrophoresis in narrow-bore Teflon tubes with an internal diameter of 200 μm But until James Jorgenson and Lukacs helped to achieve the rapid development of this only method in the early 1980s [64-66], CE was shown to be a viable analytical technique

In their work, CE demonstrated the potential for producing high resolution separations

of bio-polymers, as well as smaller pharmaceutical agents, and the advantage of using small amounts of both sample and reagents In the 1990s, the horizon of CE was expanded greatly Hjerten etc pursued improvement in capillary coatings to prevent analyte adsorption to the capillary surface [67] Terabe used micellar solution to separate neutral and charged species, which was the pioneering work on MEKC [68-70] Additionally, Zhu, Bruin et al., and Ganzler et al developed non-gel sirving matrices for size-based separation of biopolymers [71, 72] Furthermore, Progress in instrumentation has led to the development of new detectors and mass spectrometer interfaces In the first half of 2000s, diversified developments of CE were also achieved For example, a lot of attentions was been paid on electrochemical detection methods, especially the capacitively coupled contactless conductivity detection (C4D); miniaturizations of CE technique also gained more and more attention because of its outstanding advantages in further miniaturization and the earning integration of multiple functions, as will also be mentioned in following sections

1.6.2 General Introduction of Several Trends in CE Field

There are several major trends in CE developments according to the analysis of recent journals The first trend is the combination of new detection method with CE, so that not only the quantitative information but also the qualitative information of samples can be provided Mass spectrum (MS) is the most interesting detection system to be used with CE technology Besides that some interests were also shown in CE-nuclear magnetic resonance (CE-NMR) combination The second trend is the miniaturization

of CE, so that the analysis can be performed faster, and the parallel dimensions analysis can also be carried out easily This lab-on-chip idea is very exciting and more and more researchers are interested in this new trend The third trend is the improvement of sensitivity by stacking and similar on-column strategies Besides these three major trends, there are also other trends such as chiral separations, nonaqueous

CE, immunoassays etc

1.6.3 CE-MS

The use of a mass spectrometer as detector for CE has increasingly gained acceptance, complementing or replacing conventional detection methods such as UV absorbance, electrochemical detection, or laser-induced fluorescence Of all CE detection methods reported to date, MS has the greatest potential [73] CE is a separation technique of high separation efficiency, high speed and small sample injection MS is a detection