Capillary Electrophoresis

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Introduction

Today the most powerful analytical tools used as separation techniques is capillary electrophoresis and ultra high performance liquid chromatography. Capillary Electrophoresis (CE) is the first technique used to carry out liquid sample detection using a capillary; it is also called high- performance capillary electrophoresis (HPCE). (1) This technique is carried out in very thin capillary tubes and is used to separate and analyze the purity of a wide spectrum of biological molecules including amino acids, peptides, proteins, nucleic acids as well as small organic molecules such as drugs or metal ions. CE can identify point mutations in DNA and can also be used to quantify DNA in human diseases. (1)

Chromatography is the term used for a variety of related techniques used for the separation of substances in a mixture (1). One such technique is ultra high pressure liquid chromatography (UHPLC). This is a modern technique that improves upon standard HPLC by utilizing higher pressure and packing material consisting of smaller particles. This results in an improved resolution and faster separation time (2). Specialized instrumentation, that can withstand the increased heat generation and pressure of this technique, is used. UHPLC has various applications in a variety of fields such as the analysis of dose formulations and the analysis drug and pesticide metabolites (2).

1. Principles on which each technique is based

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In CE the molecules in the injected sample migrate at different rates along the length of the capillary, either by hydrodynamic flow or electromigration. (3) If an uncoated open- tube fused silica capillary is used as the separation chamber, 2 electrokinetic actions occur under the influence of the electric field. First, electrophoresis of the ions take place, and secondly electroosmosis, due to the immovable charge of the capillary walls being effective from basic to weak acid pH range of the ions, will occur. (3) Positive, negative and neutral sample molecules migrate at different rates and they are all, regardless of charge sign, drawn towards the cathode by electroosmosis. The electroosmosis flow (EOF) is much greater than the electrophoretic velocity of the analytes. The combination of electrophoretic migration and electroosmotic flow cause the positive molecules to reach the cathode first. (3)

The polarity of the capillary wall charge determines the direction of the EOF. (3) Charges accumulate to the inner surface of the capillary. The positive charged ions of the buffer solution will form two inner layers of cations (called the electrical double layer on the capillary wall).

The first layer is fixed and is known as the static layer (stern layer), while the outer layer is referred to as the mobile layer (outer Helmholtz plane). (3) The mobile cation layer is driven to the direction of the negatively charged cathode when an electrical field is applied. The bulk buffer solution migrates with the mobile layer, resulting in EOF. The EOF is the result of the adsorption of the electrically charged ions of the buffer to the capillary walls. (3)

The velocity of the EOF is directly proportional to the applied electrical field strength [V/cm] as given in the Smoluchowski equation:

VEOF = μEOF x E (3)
VEOF is the electrophoretic migration velocity towards the electrolyte of opposite charge. μEOF is the electroosmotic mobility (EOM), which is directly proportional to the ionic charge of the sample and indirectly proportional to the friction forces present in the buffer solution. (3)
The EOM is dependant on the zeta potential (ξ), the viscosity of the solution and the permittivity of the medium and is given in the following equation:
μEOF= ξε/η (3)
ε = the permittivity (dielectric constant) of the medium [F/m] and η= the viscosity [poise]

In CE the liquid flow caused by endoosmosis shows a plug profile because of the uniformly distributed driving force along the capillary. As a consequent, a uniform flow velocity vector occurs across the tube. Only in the double layer region the flow velocity approaches zero.

The efficiency is dependent on the number of theoretical plates (N) achieved by the capillary: (3)
N=16 (t/w)2
t is the migration time of the component(s)
w is the temporal peak width at the base line(s)

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The zone broadening by diffusion process is described by Einstein’s equation:
σ2= 2Dt [cm2] (3)
σ2 is the spatial variance [cm2]
D is the diffusion coefficient [cm2/s]

The height of the theoretical plate (H) for CE can be calculated by the following equation
H= σ2/x = 2D/v; where it corresponds to a linear peak spreading (height of theoretical plate (cm) vs. flow rate (sec/cm) ) (4)
x is the distance a charged specie moves (cm) at a migration time (sec)
σ is the standard deviation of the curve (the half width of the peak at half height)

(add fig 2.48 CE)…………………………………………………………p 61 (3; page61 )

Resolution for CE is given in R= 0.177 (μ1-μ2) √v/ D (μmean+ μeof) (3)
μ1, 2 is the mobility of the component 1 and 2 separately
The equation shows that resolution of two peaks decrease with an increase magnitude of EOF it they both share the same direction.

In chromatography, the selectivity is given by the separation factor α, which is defined by the capacity factors of the two analytes:
α = k2 / k1 = t2- t0/ t1- t0, where t2≥t1 (3)
k1’ and k2’ is the capacity factors of component 1 and 2 separately
t0 is the dead time of the column
t1,2 is the retention time of component 1 and 2 separately

In comparison with chromatography, no dead volume exists in CE and α can be defined as following:
α = t2/ t1 (3)
t1 and t2 is the migration time of component (s) 1 and 2 separately
α is always ≥1, because t2≥t1 (3)

The most critical factor for optimization in CE is selectivity. (3) In CE the method of optimization for selectivity is limited to modifications of the electrolyte system, whereas for chromatography, a variety of stationary and liquid phases are available which cover almost all purposes. (3)

Chromatography relies on the distribution of a substance between two non-mixing phases (1). Distribution between the stationary- and mobile phase varies from compound to compound, depending on their physical and chemical properties. This differential distribution therefore allows for the separation of various compounds in a mixture (1).

As UHPLC is derived from standard HPLC, they share many common principles. Eluent is passed through a column at high pressure. The column is packed with a stationary phase consisting of tiny, approximately spherical, rigid particles (2). The compounds to be separated interact with the eluent, as it passes thought the column, and the matrix particles(1). Compounds with more interaction with the matrix are eluted later than those with more interaction with the eluent(1).

The resolution of the separation of substances in a mixture is influenced by a variety of factors, one being the number of theoretical plates(1). Theoretical plates are neighbouring zones in the column(1). These zones allow for the equilibration between the mobile and the stationary phase. The higher the number of plates, the better the resolution for an analysis(1). The number of plates is given by the following equation:

N=L/H

Where L signifies the column length and H the plate height. Clearly the number of plates, and therefore the resolution, is directly proportional to the column length while being inversely proportional to plate height(2).

The van Deemter equation describes the relationship between plate height and eddy diffusion, longitudinal diffusion and resistance to mass transfer (A, B and C respectively in equation below).

H = A + B/Ux + Cux

The A term is proportional to the particle diameter (dp) whereas C is proportional to dp2. Therefore, by decreasing the particle diameter, the plate height decreases subsequently leading to an increase in plate number and resolution of an analysis. In addition to reducing the overall plate height, the reduction in particle size also decreases the effect that a high eluent flow rate has on plate height. Therefore increasing the flow rate will increase plate height by a lesser magnitude when smaller particles are used.

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The use of small particles increases the resistance to the flow of eluent. This problem can be overcome by making use of high pressures to drive eluent through the column, thus allowing for sufficient flow rates. This is in contrast to CE where electroosmotic flow is used to to move solvent through the capillary.

2. Instrumentation needed for each technique

The instrumentation needed to perform CE include polymide coated capillary, a high voltage power supply ( maximum 60kV), two buffers that can assist both the capillary and the electrodes connected to the power supply and a detector. (x) The capillary is filled with an appropriate separation buffer at a desired pH and the sample is introduced at the inlet via a syringe- to capillary adaptor or a pressure- driven rinse. A high voltage power supply is then switched on causing the ionic species in the sample plug to migrate with an electrophoretic mobility to the cathode and pass a detector, which use UV/Vis absorption, where information is collected and stored by a data analysis system. (x)

The UHPLC system consists of some basic components. These components include a solvent reservoir, a high pressure pump, a sample injection system, the chromatography column, a detector, a data recorder and a sample collector.

Capillary tubes are used as columns in UHPLC. These tubes, as in CE, are commonly made out of fused-silica and have inner diameters of 30 to 50 um. These diameters fall into approximately the same range as those used in CE . These tubes are packed with the matrix material and attached to the sample injection valve. Columns range in length depending on various factors, such as time restraints or the resolution requirements but are also generally similar to the lengths used in CE.

In HPLC silica particles with a diameter of 3um to 5um are generally used as a packing material. This size is reduced to between 1 and 2 in UHPLC. These non-porous silica particles can withstand high pressures without breakdown.

As previously mentioned, particle size has a detrimental effect on eluent flow rate. The high pressures needed to overcome flow rate retardation are upwards of 400 bars and are commonly in the region of 1200 - 1500 bars. Pressures of 7000 bars have been reached. These high pressures provide some practical challenges and specialized pump systems are required. Pneumatic amplifier pumps are commonly used. These pumps utilize a series of pistons of decreasing area to amplify an initial pressure.

Other components of the chromatography system are also specialized to cater for the high pressure requirements. These components need to be able to resist the enormous pressures. Fittings, sample injection equipment and valves used have to be able to seal very tightly and are designed to fulfil this requirement. These components are constructed from materials such as stainless steel and brass. Valves may even be diamond coated to aid in pressure resistance.

Various detection methods are available such as TOF-MS and UV detection. Each of these methods have their own advantages and drawbacks.

4. One important application for each technique

Metabolomics is the quantitative and qualitative study of all the metabolites present in an organism. Discoveries in this field have many applications in fields such as disease diagnostics, toxicology and clinical research. Metabolites in the cells and tissues are present in very small quantities, therefore the use of high resolution analysis techniques are imperative if accurate results are to be obtained. Both CE and UHPLC fall into this class of analytical techniques and each of them have different strong points that can be exploited for use in the study of metabolomics.

The increase in resolution, effeciency and speed in UHPLC over HPLC has made it an attractive and powerful tool in studies of the metabolism. Evidently the use of UHPLC in metabolism studies has increased in recent years. One example of the increased quality in results obtained by UHPLC over HPLC is in a study by Wilson et al. where the number of metabolites detected in a mouse bile sample increased from 1800-2000 using HPLC coupled with MS to 10 000- 13 0000 when UHPLC-MS was used. A study by Plumb et al. on drug metabolites in mouse bile showed that peak width reduction, as a result of the use of UHPLC-MS, increased analytical sensitivity by 3-5 fold. UHPLC is powerful for studies in both animal and plant metabolites. From the above it can be seen that UHPLC is very effective in the separation of various metabolites when it is coupled with mass spectometry.

In CE, analytes are separated based on their mass over ratio. In metabolite fingerprinting, CE is used as a complimentary alternative to UHPLC, because it is suited to ionic and polar compounds, which might not be retained by reverse- phase columns. With CE, fast separations with high efficiencies are achieved with minimal sample volumes, which are advantageous when the sample is limited, e.g. human plasma.

In addition to good resolution, sensitivity and short analysis time; CE is a complementary to HPLC for metabolic studies. In the above mentioned comparison, one can clearly indicate that both these techniques are important for the progress of metabolic studies, e.g. gene function determination and pharmaceutical research and development.

5. Advantages and disadvantages of each technique

One advantage in using capillaries in CE is that the small diameter of the tubing- there is a large surface-to-volume ratio, which results in enhanced heat dissipation which ultimately leads to the reduction of heating effect- problems. (1) The result of this advantage is the fact that it helps to eliminate both convection currents and zone broadening owning to increased diffusion caused by heating. In addition to the practical procedure- no stabilizing medium in the tube is needed and free-flow electrophoresis will be a positive practical outcome. (1)

The disadvantage which occur in CE; is linked to the generation of ionized groups on the capillary wall which could be a extreme risk factor for an unwanted experimental outcome; for example, when using proteins for separation, protein cationic groups will adsorb to the ionized silanols of the silicon. (1) This will ultimately lead to the smearing of the protein as it moves through the capillary or complete loss of protein, due to total adsorption to the walls.(1)

The main advantage of UHPLC is that it has a very fast elution time and high resolution. This saves time in the lab, meaning less work has to be repeated as a result of weak resolution, while more samples can be analysed in a given time than with other LCs. I has a very wide field of applications.

UHPLC can be a dangerous method. High pressures mean that capillary tubes can rupture and their shards can form projectiles. Liquid jets can also occur. The high temperatures also need to be dissapated to prevent overheating of the equipment.

6. Summary

The comparative basic theory between CE and UHPLC starts off in CE sample introduction when sample solvent is introduced by means of hydrostatic sample introduction or electromigrative sample introduction of the carrier electrolyte. (4) In comparison with CE, which makes use of carrier electrolyte to identify the distance moved, UHPLC makes use of eluent and mobile phase to do the latter. The sample is injected into UHPLC, most different from CE. (4) In CE molecules and ions move by means of EOF and an electropherograms is needed to measure this. UHPLC is dependant on flow rate for movement of particles and a chromatogram is needed for measurement. Without a high voltage supply system CE will not occur, whereas UHPLC is dependant of a pump for successful analytical separations. The choice of operating parameters for sample introduction in CE influences the final result to a higher degree than in UHPLC. (4)

Reference

1. Wilson, K., Walker, J., (2007) Principles and Techniques of Biochemistry and Molecular Biology, 6th Ed., School of life science, University of Hertfordshire, UK

2. Jerkovich A.D., Mellors J.S., Jorgonson J.W. (2003) LCGC North America 270, 600-611

3. Kuhn, R., Hoffstetter- Kuhn, S., (1993) Capillary Electrophoresis: Principles and Practice, Fachlochschule für Technik und Wirtschaft, Reutlingen, Germany

4. Jandik, J., Bonn, G., (1993) Capillary Electrophoresis of Small Molecules and Ions, Johannes- Kepler- University, Linz, Austria

x. Landers, J.P., (1997) Handbook of Capillary Electrophoresis, 2nd Ed.,