INTRODUCTION:

Few years Ago, the term “hyphenation” was coined by Hirschfeld in 1980, for the coupling of separation technique and on-line spectroscopic detection technique. The objective of coupling of chromatographic technique and spectroscopic technique was to obtain information rich source compared to information achieved from a distinct source.¹

One main effort of scientist involved in chromatography field over last few years has been mainly focused on increase in separation power of chromatographic technique. An important step was made by Golay in 1958, who supplant packed columns with capillary columns, acquiring almost a 10-fold increase in separation power.² Hyphenated technique combines two different analytical techniques and accomplished the benefits of both. Recently hyphenated technique achieved an on growing attention as the solution for the complex analytical problems. To obtain the structural idea about identification of compound present in crude sample, liquid chromatography (LC),³ usually High performance liquid chromatography (HPLC), Gas chromatography (GC), or Capillary electrophoresis (CE) are related to spectroscopic detection, e.g., Fourier-transform infrared (FTIR), photodiode array (PDA), UV-visible absorbance or Fluorescence emission, Mass spectroscopy (MS), and Nuclear magnetic resonance spectroscopy (NMR), which results in hyphenated techniques, e.g., CE-MS, GC-MS, LC-MS, etc.⁴The hyphenation does not always mean combination of two technique; the coupling of separation and detection technique can involve more than one separation or detection technique, e.g., LC-PDA-MS, LC-MS-MS, LC-NMR-MS, etc. where trace analysis is important, and the analyte enhancement is cardinal, on-line coupling with solid phase extraction (SPE), solid phase microextraction or large volume injection (LVI) can be fused to build in a more powerful integrated system, e.g., SPE-LC-MS.⁵

The Hyphenated techniques are as follows:

GC-MS

LC-MS

LC-NMR

MS-MS

LC-NMR-MS

1. GC-MS:

GC can differentiate the volatile and semi volatile substances, but GC cannot acknowledge them, whereas MS can categorize the substances by providing molecular level structural details, but MS cannot distinguish them. In 1957, Holmes and Morrell manifest the first coupling of gas chromatography with mass spectroscopy. Few years ago, GC-MS instruments were marketed with the improvement of computer-controlled quadrupole mass spectrometer for fast achievement to accommodate the separation in gas chromatography.⁶ Gas Chromatography-Mass spectroscopy (GC-MS) is the hyphenated analytical technique that connects the separation properties of gas-liquid chromatography with the detection factor of mass spectroscopy to diagnose different substances within a test sample. As the name suggests, a GC-MS instrument is made up of the following two major building blocks: a Gas Chromatography and a Mass spectroscopy.⁷ GC-MS differentiate chemical mixtures into individual components (using a gas chromatograph) and identifies/ quantifies the components at a molecular level (using a MS detector).⁸ It is one of the most appropriate tools for analyzing volatile organic samples.⁹

Instrumentation and working:

The Vapors get analyzed when they pass across the GC column with the support of heated carrier gas, the section occurs in the column, only the carrier gas (helium) can also be called as the mobile phase. The collaboration between analyte, mobile phase, and stationary phase leads to separation of the compounds.¹⁰ The sample navigate through the column, the contrast in the boiling point and other chemical properties lead to separation of the compound’s mixture. The components have variations in elution time and retention time due to their different adsorption or difference in the partition between the mobile phase and the stationary phase respectively. Then the separated components of the mixture get entered into the MS through an interphase or transfer line. This process is then further followed by ionization, mass analysis and detection of mass-to-charge ratios of ions yield from each analyze by the mass spectrometer.¹¹ Here the process of ionization ionizes the molecules and break the molecule. The process of ionization not only ionize the molecule but also break the molecule into the positive or negative modes.¹²

Interface:

The interfaces must provide between the two instruments. When the chemical ionization reagent gas is used as the carrier gas, the effluent gas be introduced directly into the mass spectrometer.

a. Jet\Orifice separator:

A precisely aligned, supersonic jet\orifice system is effective in removing the carrier as by effusion. Effluent from the gas chromatograph is pass through a fine orifice, where it is rapidly expands into a vacuum chamber. During this expansion, the faster diffusion rate of helium results in the higher sample concentration in the core of the gas stream, which is directed towards the second jet or orifice aligned with the first jet. Alignment and relative spacing of the expansion and collector orifice are very critical. The distance between the jet must be changed for a change in flow rate. Yields are about 25%. An all-glass jet separator is frequently used for packed columns operations. The short path through the interface or the ion source reduces dead volume, which gives better peak separations.

2. LC-MS:

LC-MS is the liquid chromatography- mass spectroscopy technique. This technique now has become a standard technique that provides simple and robust interface for the design of electrospray ionization (ESI). LC-MS relate the chemical separating power of LC with the ability of MS to selectively detect and confirm molecular identity.¹³

Instrumentation and working:

LC-MS is a technique that merge the physical separation of liquid chromatography (or HPLC) with the mass spectroscopy. Usually the automated LC-MS system consists of double three-way diverter within- line with an autosampler, LC system and the Mass spectrometer. The diverter generally works as an automatic switching valve to divert disagreeable portions of eluting from the LC system to waste before the sample enters the MS. The ionization techniques which are used in LC-MS are generally simple techniques that mainly shows the molecular ion species with only some fragmented ions. The knowledge obtained from a single LC-MS run is not enough to confirm the identity of the compound.¹⁴

Fig 2: - Schematic representation of Liquid chromatography- Mass spectroscopy

Interface:

a) Electrospray interface:

In this interface the eluant from LC is shifted to metallic capillary needle into a mild zone at atmospheric pressure. A high potential of up to 8kV relative to a counter electrode close to the capillary generates a strong electrical field that activates charges on the droplets as they emerge from the capillary. The charge droplets are then evaporated by a stream of heated (80-150ºC) nitrogen gas. As the liquid evaporate from the charged droplets, they shrink until the surface charge is accomplish on moving through the capillary is enough to break down the cohesion forces in the molecule and ionization occurs (still at atmospheric pressure). The ions are then introduced into a high vacuum mass spectrometer via a tiny aperture or skimmer.¹⁵

b) Atmospheric pressure chemical ionization (APCI) interface:

APCI uses a corona discharge around an electrode in heated zone apart from a capillary. The eluant from HPLC deliver through a heated capillary, where pneumatic nebulization occurs through a flow of hot gas to generate a fine mist of droplets. These droplets pass into a heated desolation / vaporization zone and heated up to 450ºC. However, these ions stay in desolvation zone for several seconds, they are able to collaborate with the sample molecules, creating a secondary ionization analogous to low pressure CI source. The source produces pseudo molecular ions (M+H) in the positive CI mode and (M-H) in the negative CI mode depending on whether it is operated in positive or negative CI mode. The source is connected to the mass spectrometer analyzer, which, like ESI, operates at high vacuum, through an aperture or skimmer, as the entire method works at atmospheric pressure. Unlike ESI, APCI is not well uniform to the analysis of polar components, as it also does not give multiply charge icons, so it can’t be used for high mass substance. Although, it does ionize non polar compounds much more effectively than ESI and can be use with normal- phase HPLC system as well as reverse phase (Aqueous base) system.¹⁶

3. LC-NMR:

Liquid chromatography, or HPLC, and nuclear magnetic resonance are combined to form LC-NMR. The most valuable structural information for determining the structures of natural compounds is provided by NMR, even though it is probably only slightly sensitive. Technology improvements had authorized the straight parallel linkage of HPLC systems to NMR, leading to the development of the new practical approach HPLC-NMR or LC-NMR¹⁷, which has been well-known for more than 15 years. The first HPLC-NMR research to make use of superconducting magnets was announced in the mid-1970s. The study of complex mixtures of various kinds, particularly the investigation of natural products and drug-related metabolites in biofluids, would undoubtedly benefit much from the use of LC-NMR.¹⁷

Instrumentation and working:

An autosampler, an LC pump, a column, and a non-NMR detector are typically included in LC unit of LC-NMR system. This detector sends the flow through the LC-NMR interface, which has additional loops for the short-term storage of selected LC peaks. The flow from the LC-NMR interface is then either directed to the flow-cell NMR probe-head or to the garbage. After passing through the probe-head, the flow is then routed to a fraction collector for recovery and further investigation of the various fractions. The main prerequisites for on-line LC-NMR, in contrast to the NMR and HPLC equipment, are the prolonged probes and a valve inserted before the probe for collecting alternatively continuous-flow or stopped-flow NMR spectra.¹⁸ Additionally, the primary detector for LC functioning is a UV-vis detector. For a typical HPLC-NMR coupling, magnetic field strengths greater than 9.4 T—or a 1H resonance frequency of 400 MHz—are advised. The analytical flow cell was actually planned for constant NMR detection. On the other hand, the demand of full structural identification for unknown compounds, especially innovative natural items have given rise to the stopped-flow mode's utilization. Following that, the flow from the LC-NMR interface is directed either to the flow-cell NMR probe head or to the trash. The flow is then directed to a fraction collector for recovery and additional research on the distinct fractions evaluated by NMR after passing through the probe head. The mobile phase solvent protons pose serious obstacles to getting a good NMR spectrum.¹⁹ The powerful solvent signals and the faint substance signals cannot be processed by the NMR spectrometer's receiver simultaneously. One of the three main approaches, presaturation, soft-pulse multiple irradiations, or water suppression enhancement through T1 effects (WET) presaturation using a z-gradient, can be used to produce solvent signal suppression.

Fig 3: schematic representation of LC-NMR

4. MS-MS:

Tandem mass spectrometry, sometimes referred to as MS/MS is an instrumental analysis technique that involves connecting two or more mass analyzers utilizing an additional reaction step to improve their ability to examine chemical samples. The examination of biomolecules like proteins and peptides is a frequent use of tandem mass spectrometry. The first spectrometer (named MS1) ionizes the molecules in the sample and separates the ions according on their mass-to-charge ratio (commonly expressed as m/z). Select MS1 ions with a specific m/z ratio are then made to fragment into smaller ions, for example, through collision-induced dissociation, ion-molecule interaction, or photodissociation. The second mass spectrometer (MS2) is then used to analyses the fragments by separating them according to their m/z ratio and detecting them. When using conventional mass spectrometers, ions with very similar m/z ratios might be difficult to distinguish and separate.

Instrumentation:

Although there is a physical link between the elements to maintain high vacuum, in tandem mass spectrometry in space, the separation elements are physically separated and distinct. These components could be time-of-flight, transmission quadrupole, or sectors. They can serve as collision chambers and mass analyzers when using several quadrupoles. Through the use of tandem mass spectrometry, numerous separation stages can be carried out throughout time while keeping the ions confined in one location. Such an analysis can be performed with a quadrupole ion trap or a Fourier transform ion cyclotron resonance (FTICR) device. Multiple phases of analysis can be performed using trapping equipment, which is commonly referred to as MSn (MS to the n). The number of steps, n, is frequently not stated, but on rare occasions the value is; for instance, MS3 denotes three stages of separation.²⁰

5. LC-NMR-MS:

Here are essentially two methods in series and in parallel for connecting HPLC to two detector systems. With the first setup, each detector receives effluent in turn from the column. This has the drawback that when each detector analyses the same component of a mixture at a different time, it is considerably more challenging to correlate the data from each system for that component. Additionally, pressure variations among the NMR and MS systems can result in leaks from the NMR flow probe when using a series connection. The flow from the HPLC is divided between the two detectors in a parallel arrangement. This makes it simple to modify the split flows to fit the needs of the experiment. An example is the simultaneous detection of an analyte in both the systems. Alternately, the NMR investigations could be focused on a specific eluting peak by using the MS data, which can be quickly gathered. If the mass spectrometer is needed for different investigations, it is quite simple to detach it from the NMR spectrometer in series and parallel systems. Disconnection can be necessary since a dedicated LC/NMR/MS apparatus may be viewed as a luxury in some labs. Installing a UV detector after the HPLC column has additional benefits. When an analyte is detected, the UV cell, if placed before the splitter, can be utilised to start NMR or MS detection, or synchronise peak delivery to the MS and NMR detectors.²¹ The eluent can be routed to the mass spectrometer and a peak observed before that peak reaches the UV cell if the UV detector is positioned after the splitter.²² The identical peak can be utilised to start a stopped-flow experiment on the peak in the NMR instrument when it is later discovered at the cell. Aqueous trifluoroacetic acid is an alternative modifier for the solvent, however it again causes issues in the mass spectrometer by inhibiting the ionisation of acidic analytes. Because it gives a single proton resonance in NMR away from the majority of analytes (about 9ppm) and does not prevent the ionisation of acids in the mass spectrometer, formic acid (pH-adjusted using ammonium format) is utilised in many experiments.²³

Fig 4: Schematic representation of LC-NMR-MS

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Received on 08.04.2023 Modified on 13.05.2023

Asian J. Pharm. Ana. 2023; 13(3):205-209.

DOI: 10.52711/2231-5675.2023.00033