1. INTRODUCTION:

1.1. HYPHENATED ANALYTICAL TECHIQUES:

It is a separation technique it referred as the combination of two or more techniques to separate the chemicals from solutions and detect them. It was first. Introduce by HJERTON in the year 1967. The term “hyphenation” was first adapted by Hirsch FELD in 1980 to describe a possible combination of two or more analytical methods in a single run. (1).

ADVANTAGES:

i. Fast and accurate analysis.

ii. Better reproducibility.

iii. A higher degree of automation.

Types of hyphenated techniques:

I Double hyphenated technique.

E.g. LC-MS, LC-NMR, L C-IR, CE-MS.

II Triple hyphenated technique.

E.g. LC-API-MS, ESI-MS-MSI.

1.2. CE-MS:

CE-MS stands for capillary electrophoresis- mass spectroscopy.it is an analytical technique use for detecting and analysing substances. in this method, capillary electrophoresis (CE) is combine with mass spectroscopy (MS) as a detection method. CE is known for its high efficiency in separating different substances, while MS offers high selectivity in detecting and identifying this substances. This combination makes CE- MS a very useful and attractive technique in the field of analysis.it allows scientist to separate and detect substances with great precision and accuracy.

2. DEFINATION:

2.1 CAPILLARY ELECTROPHORESIS:

An analytical method called capillary electrophoresis divides ions according to their electrophoretic mobility with the use of an applied voltage 1000volts/cm. A capillary is present by anode and cathode together. It plays an ideal role in the analysis of highly polar charged analyte CE uses tubes 20-100µm diameter and 20-100 cm in length. High efficiency and narrow peaks are the outcome of higher electric fields.

2.2 MASS SPECTROMETRY:

Mass spectrometry is one of the widely used analytical technique that is used for determining molecular mass and clarification of structure of the organic and inorganic and may complex biomolecules. In this technique the sample under investigation is included process of ionization, separation, and detection results a mass spectrum. (2,3).

2.3 C E MS:

CE MS is also known as the capillary electrophoresis in mass spectrometry, which is a technique used in chemistry to analyze substances. Basically, mass spectroscopy is a method that’s becoming popular for detecting and identifying compounds during capillary electrophoresis.

ADVANTAGES:

i. High efficiency of separation.

ii. Ability to separate both charged and non-charged molecules.

iii. Offers new selectivity, an alternative to HPLC.

iv. Easy and predictable selectivity. V. Small size of sample (1-10 µl). V I. Faster separation (1-45 min).

v. It can be automated.

vi. Easily coupled to MS.

DISADVANTAGES:

i. Sticky compounds.

ii. Species that are difficult to dissolve.

iii. Reproducibility problems.

iv. Cannot do preparative scale separations.

APPLICATIONS:

i. It is use in the pharmaceutical analysis for purity and quality control.

ii. It is use in the bioanalysis for analysis of proteins, peptides, nucleic acid, carbohydrate and metabolites.

iii. It is also used in environmental analysis for determining the presence and concentration of various pollutants. (Heavy metals pesticides etc.)

iv. It is used in forensic analysis for identification and quantification of drugs, toxins, metabolites. (14).

v. It is widely used in food analysis for separation of food components, additives, contaminants, and allergens.

vi. Characterization of monoclonal antibodies.

vii. Use for chiral separation.

viii. Determination of drugs in human plasma.

ix. Use for amino acid analysis.

x. For synthetic in vitro glycolysis studies.

LIMITATION:

i. This technique is still not widely accepted for routine use.

ii. The limited sample volumes that can be analysed without compromising separation efficiency.

iii. Detection limits for most application are too high.

iv. The migration times tend to fluctuate with a change of temperature in the environment.

v. The reproducibility ruggedness of CE MS are not currently as good as those of L C MS. (4, 5).

3. PRINCIPLE:

3.1 CAPILLARY ELECTROPHORESIS:

The principle of capillary electrophoresis deals with the separation of a complex mixture of molecules based on the differential analyte ions under the influence of an applied electric field.

Capillary electrophoresis is a widely used analytical technique in various fields, such as chemistry, biochemistry, and pharmaceuticals. It is based on the principle of separating charged molecules in a capillary filled with an electrolyte solution under the influence of an electric field. One of the fundamental principle of capillary electrophoresis is electrophoretic mobility.

3.1.1 Electrophoretic mobility:

Electrophoretic mobility refers to the movement of charged particles in an electric field. The principle of electrophoretic mobility allows for the separation and analysis of various analyses within a mixture by controlling the experimental conditions, such as the PH of the electrolyte solution and the strength of the electric field. It is possible to optimize the separation of compounds with different electrophoretic motilities. It depend on the charge of molecules, viscosity and atoms radius.

It expressed as:

µEP = q/f =q/6πors

f = spherical particle given by stokes law

q= charge

r= radius of ion

π= viscosity of the solvent.

E = field strength

The electrical field can be determined by the following equation;

V = µE P E

3.1.2 Electro osmotic flow –

It is caused by applying high- voltage to an electrolyte – filled capillary. This flow occurs when the buffer running through the silica capillary tube has PH greater 3 and SIOH group loose a proton to become SIO. The applied electric field causes free cations to move towards cathode creating a powerful bulk flow. The rate of electroosmotic flow is governed by ;( 6, 7).

µEOF = €/4πn*E£

€= dielectric constant

n = viscosity

£= zeta potential

E = field strength

3.2 MASS SPECTROMETRY:

This technique works by exposing a vaporized sample to a high voltage electric current. the sample loses electrons and forms positively charged ions that are pushed along a circular path using magnetic and electrical field. The size of the path depends on the mass to charge ratio of the ions. (m/e). When the sample is exposed to an electron beam, it causes an electron to be knocked out from the sample molecule, resulting in the creation of a parent ion (M+).

M + e- M+ + 2e

This parent ion may further break down into smaller ions and free radicals if there is enough energy supply.

4. INSTRUMENTATION:

C E MS:

CE MS is the hyphenated technique where CE is connected to the MS with the help of the long capillaries which will increase the analysis time also there is a lack of suitable volatile buffer which has to be compatible with the mass spectrometer.

Fig.1: Schematic Representation of Instrumentation of Capillary Electrophoresis.

4.1 CAPILLARY ELECTROPHOESIS:

The CE apparatus consist of various major components they are;

I. INJECTOR

Ii. CAPILLARY

Iii. BUFFER RESERVIOR

IV. DETECTOR

V. BGE VESSEL

vi. HIGH VOLTAGE SUPPLY

vii. ELECTRODE

Viii. RECORDER

4.1.1 INJECTOR:

An injection of sample is made into the capillary tube. The most popular methods for introducing samples are hydrostatic and electrokinetic injections. When using pressure injection, the capillary's sample introduction end is also briefly inserted into a tiny cup that holds the sample. The sample solution is then injected into the capillary using a pressure differential. Either pressurising the sample or raising the sample end will terminate the pressure differential. Hydrostatic injections are not selective because of ion mobility, but they are not suitable for use in capillaries that are filled with gel.

4.1.2 CAPILLARY:

A 40–100cm long, buffer-filled fused silica capillary with an internal diameter of 10–100µm connects two buffer reservoirs that also contain platinum electrodes. One end of the sample is introduced, and the other end is used for detection. The right side, or next to the cathode electrode, is where the detector should be positioned. The capillary tube has a 0.5mm detector window that allows the injected sample to pass through. In the event that a provision is missing, the capillary tube is heated to 130 °C using 96–98% conc. H2SO4 or conc. KOH.

4.1.3 BUFFER RESERVIOR:

This method requires two buffer reservoirs, an anode and a cathode reservoir, connected to the capillary. The buffer solutions in these reservoirs facilitate the movement of analytes by providing necessary conductivity and PH conditions. The anode reservoir contains a positively charged electrode while the cathode reservoir contains a negatively charged electrode. Common buffer systems include phosphate, borate, and tris-based buffers.

4.1.4 BGE VESSEL:

If selecting the right buffer for electro-osmotic and electrophoretic mobility based on analyte behaviour and ph constants, buffer plays a crucial role. Selecting high-quality buffers and preparing them under optimisation focus are important. The most often used buffers in CE are phosphate, ethanoate, and borate buffers. Additionally utilised are buffer additives such as urea, surfactants, and organic and inorganic salts.

4.1.5 HIGH VOLTAGE SUPPLY:

Through modern technology, high potentials—up to 30 kV—can be used in CE to achieve incredibly quick and effective separations. The entire sample is drawn towards the cathode by electro endosmosis when the components are migrating at different rates along the length, despite being separated by the electrophoretic migration. The purpose of maintaining cooling systems is to lower the system's temperature or dissipate heat.

4.1.6 ELECTRODES:

An electrochemical cell's electrode is referred to as the cathode or the anode. It is now understood that the anode is the electrode where electrons exit the cell and undergo oxidation, while the cathode is the electrode where electrons enter the cell and undergo reduction. Depending on which way the current flows through the cell, each electrode has the potential to become the anode or the cathode. An electrode that serves as both the cathode and the anode of two cells is known as a bipolar electrode.

4.1.7 DETECTORS:

Many detection tools can identify capillary electrophoresis separation. There are various detector configurations that can be used. Given that each analyte's quantity is passing because the detector is so tiny, illuminating a small portion of the capillary with the source lamp increases the path length and lowers the detection limit if absorbance is being used. Detectors for UV absorption, fluorescence, conductivity, potential gradient, amperometric, diode array, inductive coupled plasma, refractive index, x-ray optical activity, Inductive coupled plasma, Raman spectroscopy, and Refractive index are the most often used types. Atomic absorption and the thermo optical absorbance detector.

4.1.8 RECORDER:

The CE electrophorogram is a plot of the detector signal on the y-axis against the time from injection on the x- axis, similar to a chromatogram. The example that follows uses indirect detection. Observe the y-axis. (8).

4.2 MASS SPECTROMETRY:

A typical mass spectrometer contain following components

I. Innlet System (Sample Handling System)

II. ION Source (Ionization Chamber)

III. Electrostatic Accelerating System

IV. Magnetic Field

V. ION Seperator

VI. ION Collector (Detector and Read Out Device)

VII. Vaccum System

Fig. 2: Mass Spectrometer

4.2.1 Sample Inlet System:

An interface between an ion source and a sample is called an inlet system. The inlet system's goal is to introduce the sample into the ion source with as little vacuum loss as possible. A port through which the sample is injected or placed into a chamber at high vacuum and heated to achieve vaporisation can serve as the basic inlet system. A GC inlet system will evaporate the sample and separate the mixture into its constituent parts if it is a mixture of compounds. As the components enter the mass spectrometer, their respective mass spectra are recorded one after the other. Therefore, it is possible to obtain the mass spectra of individual components of a complex mixture without first separating them.

4.2.2 ION SOURCE:

As the mass analyzer only uses gaseous ions, ionisation becomes necessary. The molecules are typically ionised by adding a proton or removing an electron when they enter the mass spectrometer in the gas phase.

By subjecting the molecules to a high energy electron beam—typically consisting of 70 eV electrons—an electron can be removed from them. The energy of this electron beam is far greater than the energy at which the molecules are ionised during the bombardment. Because it has one unpaired electron, the resultant ion is a radical ion.

4.2.3 Electrostatic Acelerating System:

Due to the potential difference between the first accelerator plate and the second repeller plate, a strong electric field exists between them, which accelerates the production of positively charged ions in the ionisation chamber. After going through the second accelerator plate, these ions reach their ultimate speeds. The first and second accelerating plates maintain a potential difference of roughly 400–4000V, which accelerates the ions of masses m1, m2, m3, etc., to their final velocities. The ions that emerge from the slit are a collimated beam of high-kinetic energy ions travelling at high speeds.

4.2.4 Magnetic Field:

The ions travel in a curved path as they enter the magnetic field after being accelerated by the electric field. The ion mass (m), accelerating voltage (V), electron charge (e), and magnetic field strength (H) all affect the radius of curvature (r). The mass-to-charge ratio (m/e) and curvature radius (r) are the foundations of mass spectrometry, which in turn have a mutual reliance. Any modification to the magnetic field's (H) strength or accelerating voltage in alters the values of m/e and r.

4.2.5 ION Seperator:

Another name for an ion separator is a mass analyzer. The core of a mass spectrometer is where ionised masses are taken and divided according to charge to mass ratios. There are numerous commercial types of mass analyzers available depending on the sorting technique.

4.2.5.1 Single focusing magnetic sector analyser:

It has an evacuated glass tube in the shape of a horseshoe with an electron bombardment source, accelerating plates on one end, and a collector slit on the other. There is a mechanism to apply the electric/magnetic field at the tube's curvature. The sample in the generated vapour is exposed to an electron beam at 70eV through an inlet. Every molecule lost one electron as a result, turning them into positively charged ions. These molecules travel in a straight line after becoming positively charged and are accelerated by accelerating plates. They move in a curved path when electric or magnetic fields are applied, and the molecular ions are collected and separated based on their mass. Various fragments land on the detector before the mass.

4.2.5.2 Double focusing magnetic mass analyser:

It is employed to distinguish the fragment's minute mass variations. It has two carefully chosen electrostatic and magnetic components that focus an ion beam. They offer a very high resolution. Energy must be decreased before ions are permitted to enter the magnetic field in order to improve focusing. By connecting two mass analyzers in series, resolving power can be increased. A mass analyzer beam is first passed through the radial electrostatic field in double focussing.

4.2.5.3 Quadra pole ion trap analyser:

It is the most typical form. All of the charged molecules will accelerate and move away from the centre line as a result of the DC bias; the rate of acceleration will depend on the charge to mass ratio of each molecule. They will be absorbed if their trajectory deviates too far and they strike the container's sides or metal rods. Therefore, the DC bias serves as the mass spec's magnetic field B and can be adjusted to certain charge to mass ratios that hit the discoverer. A will be produced by the two sinusoidal electric fields at a 90-degree phase shift and orientation. Electric field that gradually changes into a circle. Consequently, the charged particles will travel in a spiral as they descend towards the detector.

4.2.5.4 Ion cyclotron resonance:

ICRs are magnetic field-based ion traps that use ions to be drawn into an orbit within them. All of the ions in a given range are trapped inside this analyzer; an applied external electric field aids in the generation of a signal rather than separation occurring. As mentioned earlier, when a moving charge enters a magnetic field, it experiences a centripetal force making the ion orbit. Once more, the centripetal force acting on the ion is equal to the force exerted by the magnetic field.

4.2.6 ION Collector:

Ions travel from the analyzer to the detector, where they produce a signal. The m/z value is obtained by further amplifying the signal. Typically, a direct writing recording oscilloscope with three to five galvanometers is used as the readout system.

4.2.7 Vacuum System:

Vacuum is necessary to permit the ions to reach the detector without colliding with any extraneous materials including atmospheric gases which themselves undergo ionization and fragmentation giving their spectra. (9, 10, 11)

5. COUPLING OF CE MS:

Capillary Electrophoresis (CE) coupled with Mass Spectrometry (MS) combining CE’s high efficiency and high speed with the high sensitivity and high selectivity offered by MS detection is very attractive. There are several factors that must be considered when coupling the CE instrument to an MS detector.

5.1 Coupling Ce with Maldi- MS:

When using an off-line coupling method to couple CE to MALDI, the CE effluent is applied dropwise to the MALDI target plate, dried, and subjected to MS analysis. For online coupling, a moving target with continuous contact with CE capillary end is required. The moving target takes analyte into MS where it is desorbed and ionized. Musyimi et al. developed a new technique where a rotating ball was used to transfer CE to MS. The sample from CE is mixed with matrix coming through another capillary. As the ball rotates the sample is dried before it reaches the ionization region. This technique has high sensitivity since no makeup fluid is used.

5.2 INTERFACING WITH CE –MS:

Capillary electrophoresis is a separation technique that uses a high electric field to produce electro-osmotic flow for separation of ions. Analyte migrates from one end of capillary to others based on their charge, viscosity and size. The mobility increases with the strength of the electric field. The main issue with connecting CE to MS is a lack of knowledge about the basic processes involved in the interface of two techniques. The separation and detection of the analyte can be improved with a better interface. The most used ionization technique is ESI. The three setups commonly used in CE-ESI-MS Sheathless or nanospray, liquid junction, and coaxial sheath liquid are the three types of coupling. In each of these configurations, the high voltage circuit at the separation capillary's outlet is closed.

Fig. 3: Schematic Diagram of Coupling of CE-MS

5.2.1 Electrospray ionization interface:

The cathode end of the CE capillary was terminated inside a stainless steel capillary in the initial CE-MS interface. Electrical contact was made at that point completing the circuit and initiating the electrospray. There were a few issues with this interface system, such as the two systems' different flow rates. Since then, the interface system has been improved to have a continuous flow rate and good Electrical contact. At present, three types of interface systems exist for CE/ESI-MS which are discussed briefly.

5.2.2 Sheath less interface:

CE capillary is coupled directly to an electrospray ionization source with a sheathless interface system. The electric contact for ESI is realized by using capillary coated with a conductive metal. The system has minimal background, low flow rates, and high sensitivity since no sheath liquid is used. Nevertheless, there are several issues with these interface designs, such as poor repeatability and low mechanical robustness. The latest sheathless interface design features porous ESI emitter through chemical etching.

Fig. 4: sheath less interface diagram

This design successfully overcomes the issues with reproducibility related to earlier designs and offers reliable interface with mass spectrometry.

5.2.3 Sheath flow interface:

Fig. 5: Sheathflow Interface Diagram

When the CE separation liquid and sheath liquid are combined and flow coaxially through a metal capillary tubing, the sheath flow interface establishes the electrical connection. Commonly used sheath liquid is 1:1 mixture of water-methanol with 0.1% acetic acid or formic acid. The system is more reliable and has a wide selection range of separation electrolytes. There might be some decrease in sensitivity due to sheath liquid.

5.2.4 Liquid junction interface:

In this setup, an electrode is placed at the injector extremity close to the MS inlet at the junction of two capillaries, and a voltage is applied to the system. An advantage of this setup is that the electrode's electrical contact is stable. In this method, makeup liquid is combined with separation electrolytes from CE capillaries using a stainless steel tee. A small space is kept between the tee's opposite sides as the ESI needle and CE capillary are inserted. The electrical contact is established by makeup liquid surrounding the junction between two capillaries. This system is easy to operate. But the sensitivity is lower, and separation may be harmed by mixing two liquids. (12, 13).

6. FUTURE SCOPE:

i. Proteomics

ii. Metabolomics

iii. Pharmaceutical analysis

iv. Environmental analysis:

v. Forensic analysis

vi. Food and beverage analysis

vii. Clinical diagnostics

viii. Biochemical research

7. CONCLUSION:

i. Hyphenation includes both separation and identification which makes the analysis of samples easy. Nowadays the hyphenated techniques are more used than normal spectroscopic and chromatographic techniques.

ii. This review article concludes by highlighting the important role that hyphenated analytical techniques play in the field of mass spectrometry coupled with capillary electrophoresis (CE MS).

iii. The review also highlights how crucial it is to keep developing new instruments, techniques, and data analysis tools for hyphenated CE MS. These developments have made it possible to successfully analyse a variety of sample types, such as biomolecules, metabolites, pharmaceuticals, and environmental contaminants.

iv. The review article also discusses possible future directions for this field of study. It asks for extending the range of analyte coverage, investigating novel applications for hyphenated CE MS techniques, enhancing analytical performance and dependability, and lowering the cost and increasing the accessibility of these methods.

8. ACKNOWLEDGEMENT:

I am most grateful to my professor Ms. Prajakta Thete, who gave me the opportunity to carry out this work at the Department of Pharmaceutical Analysis. I am deeply grateful as well to Dr. Rishikesh, S. Bachhav Principal of K C T s R. G. Sapkal College of Pharmacy Anjaneri Nashik for being an inspiring supervisor, for constant support.

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Received on 22.01.2024 Modified on 02.03.2024

Asian J. Pharm. Tech. 2024; 14(2):128-134.

DOI: 10.52711/2231-5713.2024.00023