21 Capillary electrophoresis laser-induced fluorescence detection (CE-LIF)

Dr. Anchal Srivastava

Contents of this Unit

1.  Introduction

1.1 Capillary Electrophoresis (CE)

1.2 Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF)

2.  Modes of CE

3.  Principle Operation of CELIF

4.  Different Mode of Detection

5.  Summary

Learning Outcomes

• After studying this module, you shall be able to learn
• The basic concept of Capillary Electrophoresis (CE) and its role in CE-LIF Different types of CE and its application
• The characteristics of CE detection

1.  INTRODUCTION

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a versatile and sensitive analytical tool with many potential applications. It is successfully employed for the separation of small ions, neutral molecules and large biomolecules. It is being utilized in widely different fields, such as analytical chemistry, forensic chemistry, clinical chemistry, organic chemistry, natural products, pharmaceutical industry, chiral separations, molecular biology, and others.

1.1 Capillary Electrophoresis (CE)

Capillary Electrophoresis (CE) is a modern high performance separation method. The separation is based on different migration of analytes in a capillary over which a high voltage (typically 10-30 kV) is applied. Typical inner diameters of commonly used capillaries are in the range of 25-75 μm and the detection is usually on-column.

Capillary zone electrophoresis (CZE) is one of the CE modes that is characterized by the use of a single background electrolyte (BGE) (isocratic), which enables the generation of a constant electrical field. The detector response in the end of the capillary (the electrophoregram), has a characteristic peak profile that is a function of many factors (type of sample, injection, detection, sorption, mobility differences, etc.). The qualitative characteristics are related to the migration time of the peaks and the quantitative characteristics are represented by the peak height or the peak area.

Electrically charged particles are moving in the applied electrical field in a direction determined by its charge and the field orientation. Positively charged particles are moving towards the negative pole and negatively charged particles are moving towards the positive pole.

A charged particle is characterized by an electrophoretic mobility µ defined as:

where E is the intensity of the electrical field, U is the applied voltage, L is the total distance between the electrodes (the total length of the separation capillary).

The relationship between the mobility and the charge of the particle follows from the balance between the electric driving force and the retarding friction force. The electric force is given by the product of the particle charge and electric field intensity; the friction force is given by the Stokes relationship F = 3πμdv, where μ is the liquid viscosity, d is the hydrodynamic particle diameter and v is the particle velocity. The electrophoretic mobility is conventionally defined positive for cations and negative for anions.

The electrophoretic mobility is calculated from the migration time tm, which is the time during which the analyte migrate from the injection end of the capillary to the detector:

where lef is the effective length of the capillary (distance between the injection end of the capillary and the detector).

In the case of commonly used fused silica capillaries, basic electromigration is influenced by the electroosmotic flow (EOF; Fig. 1). EOF is independent on the charge of the analytes and the resulting mobility µ can be defined as:

where t₀ is the migration time of neutral (non-charged ) particles.

Figure 1. Electroosmotic flow : The inner surface of the fused silica capillary carries fixed negative charges (dissociated silanol groups). In consequence of the law of electroneutrality conservation, there is an excess of positively charged ions in the BGE near the inner capillary wall. Movement of these cations towards the cathode generates a flow of bulk liquid inside the capillary in this direction

Many analytes that can be separated by CE has an acid-base character. Because the ionization equilibrium is established in a much shorter time compared to the electromigration time, all forms of a certain ion are moving with the same speed in a single zone. The effective mobility, is the sum of the electrophoretic mobilities µi of each ion form multiplied by the respective molar fraction xi:

Analytical chemists usually solve the task how to separate all analytes (or the major analytes at least) efficiently. The chemists try to optimize the separation, which usually means finding the conditions at which the differences of the ion mobilities of the analytes are maximal. The parameters to optimize are: pH, ionic strength and polarity of BGE, voltage and current during analysis, injection process, etc. Complexation with ligands e.g. crown-ethers or cyclodextrins or enhanced modes of CE, such as micellar electrokinetic chromatography (MEKC) can be used to improve separation efficiency. This is especially useful for separation of neutral compounds (by MEKC, where separation is based on differential distribution into micelles) or enantiomers (by use of cyclodextrin. Enantiomers interact differentially with cyclodextrins), which are not separated by conventional CE. Modifiers of capillary walls, such as organic solvent, gel or linear polymer can be used to suppress sorption of analytes to the capillary wall and suppress or even reverse the direction of the EOF. For instance, addition of a cationic surfactant cetyltrimethylamoniumbromid (CTAB) forms a positively charged layer on the silica surface resulting in a reversed EOF.

1.2 Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF)

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a versatile and sensitive analytical tool with many potential applications. Its advantages over other chromatographic methods include minimal solvent and sample requirements, low waste volumes, simple instrumentation, and diversity of analytes studied. A wide variety of analytes can be studied because the separation is based on the size and charge of the analyte. The separation conditions can be easily altered using different buffers and buffer additives. However, since few analytes are naturally fluorescent, LIF detection requires analytes to be tagged with fluorescent probes, which can increase sensitivity and yield additional separation from other sample components that do not bind the probes. LIF is advantageous because not all dyes interact with all analytes so this allows for increased selectivity.

2.  DIFFERENT MODES OF CE

Different modes of capillary electrophoretic separations can be performed using a standard CE instrument. The origins of the different modes of separation may be attributed to the fact that capillary electrophoresis has developed from a combination of many electrophoresis and chromatographic techniques. In general terms, it can be considered as the electrophoretic separation of a number of substances inside of a narrow tube. Even though most applications have been performed using liquids as the separation media, capillary electrophoretic techniques encompass separations in which the capillary contains electrophoretic gels, chromatographic packings or coatings.

The distinct capillary electroseparation methods include:

(A)  Capillary zone electrophoresis (CZE)

(B)  Capillary gel electrophoresis (CGE)

(C)  Micellar electrokinetic capillary chromatography (MEKC or MECC)

(D)  Capillary electrochromatography (CEC)

(E)  Capillary isoelectric focusing (CIEF)

(F)  Capillary isotachophoresis (CITP)

In electrophoresis a mixture of different substances in solution is introduced, usually as a relatively narrow zone, into the separating system, and induced to move under the influence of an applied potential. Due to differences in the effective mobilities (and hence migration velocities) of different substances under the electric field, the mixture then separates into spatially discrete zones of individual substances after a certain time.

3. PRINCIPLE OPPREATION OF CE-LIF

Figure 2. Scheme of CELIF instrumentation.

The instrument is equipped with a DPSS Nd:YAG laser (diode pumped solid state neodymium-doped yttrium aluminum garnet, 1064 nm) with a frequency double emitting light with the wavelength 532 nm. The laser beam is focused with a quartz lens (with the focal length of 10 mm) into the center of the capillary held in an adjustable clamp positioned on micrometric xy stage. Fluorescent radiation is collected with a microscope objective (60×) and directed through an optical slit (spatial light filtration) towards a photomultiplier tube (PMT). Two filters are inserted in front of the PMT to assure optical filtration.

The entire optical setup is covered with a light-protective cover (shield). The carousel with injection and background electrolyte BGE vials and the high voltage electrodes is placed in a protective plastic cover equipped with a safety switch, which turns off the HV if the protective cover is removed to protect operators from electrical shocks.

4. MODE OF DETECTION

The most common mode of detection in CE is on-capillary detection. In CE, migration velocity of each solute through the capillary is a function of its electrophoretic mobility in conjunction with the electroosmotic flow EOF. Since detection occurs on capillary, these forces are operative as the solute is traversing the detection window. As a result, slower moving components spent more time migrating past the detector window than their more rapidly moving counterparts. Currently, there are many detection techniques which are available for the CE detection, such as absorbance detection, fluorescence detection, electrochemical detection, conductivity and mass spectrometry etc. In this module, UV-Vis absorbance detection and fluorescence detection were employed.

4.1 UV-Vis detection

UV-Vis absorption detection is by far the most popular technique used today because it is simple to use and most analytes containing chromophore can be observed with it. Several types of absorption detectors are available on commercial instruments. The principle of UV-Vis detection is based on the Lambert-Beer law which is an empirical relationship that relates the adsorption light to the properties of the materials through which the light is traveling. In essence, the law states that there is a logarithmic dependence between the transmission of light through a substance and the concentration of the substance. The law tends to break down at very high concentrations. UV-visible absorption is useful for a large number of compounds that contain a chromophore. If analytes do not contain a chromophore, indirect detection can be employed for detection. This involves using a buffer in the capillary which actually absorbs the radiation from the lamp along with analytes which do not absorb UV radiation. As analytes move past the detector the amount of light passing through the capillary increases as UV absorbing buffer is excluded. However, indirect UV detection has lower sensitivity, around 10-100 times less than direct UV detection. UV detection is performed on column for miniaturized detection volume and convenience in operation, but the optical path length is determined by the inner diameter of the capillary. Thus, it limits the sensitivity of absorbance detection techniques since the signal of this type of detector is proportional to the optical path length.

4.2 Fluorescence detection

The second most widely used CE detector is based on fluorescence, using either an arc lamp or laser as an excitation source. This highly sensitive and selective detector is especially important in biological applications. Fluorescence is expected in molecules moles. The fluorescence detector with lamp-based incoherent light sources are of low cost and good commercial availability, but further improvement on detection sensitivity is limited by low excitation power intensity and considerable light scattering from the capillary wall. In 1985, Gassmann et al. reported the first application of laser-induced fluorescence detection in CE. LIF produces remarkable improvements on detection sensitivity because of the monochromaticity and coherent nature of the laser light. At present, LIF is the best detection scheme in CE as far as sensitivity is concerned. Although on-column fluorescence detection can provide excellent detection limits, the technique is less versatile than UV detection and must be derivatives with some type of fluorophores. An alternative to derivatization of nonfluorescent compounds is to perform indirect fluorescence detection. The procedure is performed on-column, by incorporating a fluorescent dye into the background electrolyte. When ionic analytes interact with the fluorophore, the result is either displacement of the fluorophore or ion pairing with i that are aromatic or contain multiple conjugated double bonds with high degree of resonance stability. The main advantage of fluorescence detection compared to alternative detection techniques based on absorption measurement such as UV-Vis, lies in the greater sensitivity. This greater sensitivity results from the fact that the background emission due to the buffered solution is very low by comparison with the emission from the fluorochromes. In another word, signal to noise ratio is not a function of detection cell path length. For those fluorescent molecules, fluorescence detection provides selective excitation of the analytes to avoid interferences. Fluorescence is an important CE detection scheme mainly because it aids in analyte specificity and can provide extremely low detection limits. Detection limits range from approximately 10-15 to 10-20 Indirect fluorescence detection is typically less sensitive than direct fluorescence detection. However, it is usually more sensitive than direct UV detection. Another main disadvantage of indirect detection is that its universal nature could become a hindrance with extremely complex samples which may contain interfering species.

5. SUMMARY

In this module you study

CE displays an enormous efficiency and possesses inherent advantages over conventional separation techniques. The technique has fundamentally better capability for high-resolution separation as a result of its characteristic flat flow profile. Although currently CE is at an early stage of development and there are still needs to improve column technology, to enhance selectivity in separations, and to refine the instrumentation for CE work a t this stage, it can be certain that there will be an immense potential for further developments in the area of CE.

Lasers can be used to increase the amount of light focused onto the small detection volume in on-column detection systems. There are potential benefits in their use in both UV absorbance and fluorescence detection systems, despite the fact that only few wavelengths are available from current lasers sources. In particular, laser-induced fluorescence detection has generated tremendous excitement in recent year. The main reason for the growing interest is the impressive sensitivity achievable by this method. The detection of several hundred molecules in CE employing laser induced fluorescence has already been demonstrated, and the ultimate goal of detecting a single molecule will probably be attempted in the near future.

Suggested Reading

• Landers JP (1994) (ed) Handbook of capillary electrophoresis, CRC Press, Boca Raton, FL
• Guzman NA (1993) (ed) Capillary electrophoresis technology, Marcel Dekker, New York