INTRODUCTION:

The term Hyphenated techniques was first coined by Hirschfield¹. Hyphenation – linking together of "standard" analytical techniques - generally leads to enhanced analytical performance. Advanced chromatographic methods are based on the basic principle of conventional methods. But, due to advances being taking place into such methods, conventional methods become advanced currently. Basic separation methods like GC, LC, TLC are assembled with spectroscopic methods like UV, IR, NMR, MS. Presently all market leaders in analytical instruments are offering these combinations of analytical methods via sophisticated and advanced instrumentation. These results in array of advance instruments have made wide range of discoveries². The term Hyphenated techniques was first coined by Hirschfield.

Focus on Hyphenated techniques:

Marriage of two separate analytical techniques via appropriate interfaces leads to hyphenation in conventional separation analytical instruments. Combinations of these techniques produce "hybrid" or "hyphenated" techniques^3-7. Most often the other technique is some form of chromatography. Hyphenated techniques are widely used in chemistry and biochemistry. A slash is sometimes used instead of hyphen, especially if the name of one of the methods contains a hyphen itself. Sample preparation is often the most time consuming step in these analyses, exerting a huge influence on the speed, accuracy and precision of the analytical results.

Overview on different hyphenated analytical techniques

1. Liquid chromatography-Mass spectrometry (LC-MS):

Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity. Generally its application is oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture).

Problems in interfacing LC with MS:

The problem encountered when interfacing HPLC with MS is the mismatch between the mass involved in conventional HPLC (1ml/min), which are two or three orders of magnitude larger than can be accommodated by conventional MS vacuum systems. Another problem is the difficulty of vaporizing volatile and thermally labile molecules without degrading them excessively ⁸.

Instrumentation:

It consists of three major components: HPLC, Interface and MS as shown in fig. 1.

Various interfaces are used in LC-MS such as electrospray, thermospray, particle beam, FRIT-FAB ^9-14.TSP interface developed by M. Vestal has ability to handle the high flow-rates delivered by LC (up to 2 ml/min). particle beam allow flow-rates from 0.1-0.5 ml/min. Ion source used are Electrospray ionization, Atmospheric pressure chemical ionization, Atmospheric pressure photoionization. The main function of the mass analyzer is to separate, or resolve, the ions formed in the ionization source of the mass spectrometer according to their mass-to-charge (m/z) ratios. Popularly used mass analyzers are Quadrupole, MALDI-TOF, Ion trap, Fourier transform-ion cyclotron resonance (FT-ICR). The detector monitors the ion current, amplifies it and the signal is then transmitted to the data system where it is recorded in the form of mass spectra. The m/z values of the ions are plotted against their intensities to show the number of components in the sample, the molecular mass of each component, and the relative abundance of the various components in the sample^{15, 16}. Most widely used detectors for LC-MS are:

1) Dynode Particle Multiplier.

2) Electron Multiplier.

3) Photomultiplier.

4) Faraday Cup.

5) Micro Channel Plate.

6) Photo Diode Array.

7) Ion-to-photon Detector.

Fig.1 Diagrammatic representation of LC-MS

2. Liquid chromatography-Nuclear magnetic resonance spectroscopy (LC-NMR):

Albert et al reported coupling of LC with NMR as powerful and time saving technique^17-18.

Fig. 2 LC-NMR System

The combination of liquid chromatography (LC) and nuclear magnetic resonance (NMR) offers the potential of unparalleled chemical information from analysts separated from complex mixtures. A SPE trapping interface between the LC column and the NMR^19-21allows online analysis of low level minor components into a low volume flow cell for high quality NMR spectral data. In 1978, Watanabe reported the coupling of LC effluent to NMR using a stopped flow approach, and within 1 year, an on-line system had been reported. The major advantages of on-line as opposed to off-line NMR detection of LC are improved chromatographic resolution, consistent response, on-line data analysis, and rapid data acquisition.

Disadvantage: The principal drawback with these approaches has been the relatively poor mass sensitivity of the NMR detection system, especially when the observation time is limited for each analyte peak.

3. Liquid chromatography-Nuclear magnetic resonance spectroscopy-Mass spectroscopy (LC-NMR-MS):

The world’s first fully integrated commercial LC-NMR/MS setup was first launched by Bruker BioSpin GmbH in 1999 as shown in fig 3.

Fig 3. The Flow System in LC-NMR/MS.

An LC-NMR system including a Bruker Peak Sampling Unit (BPSU-36) was coupled with a Bruker Daltonics esquire series ion trap mass spectrometer via a Bruker NMR-MS interface (BNMI). Since October 2004 the Bruker Daltonics microTOF-LC time-of-flight mass spectrometer can also be integrated in an LC-NMR setup. Hailed as the ultimate bioanalysis tool for the pharmaceutical and biotechnology industries, this new technology took advantage of mass spectrometry’s rapid and ultra-sensitive screening capabilities, which can identify peaks of interest in complex mixtures for further structural analysis by NMR spectroscopy²².

Instrumentation:

The most common way of interfacing HPLC to two detectors is in parallel mode, the elute being split between both detectors. Splits flows can be adjusted easily. MS data may be used to direct the NMR experiments, concentrating on one particular eluting peak. In on-flow NMR mode, the elute is diverted into the NMR probe and “on-the-fly” data is collected to obtain a quick assessment of the sample. Since this happens in the chromatography time and at lower sensitivity, ¹H NMR is generally the tool of choice. However recent advances in sensitivity have enabled 2D NMR or ¹³C NMR to be used in this mode for certain applications. Stopped-flow mode is more sensitive and allows 2D NMR experiments of longer acquisition time for lower concentration components. Upon peak recognition, perhaps with a signal from the MS. Pumping stops so that the samples resides in the NMR cell while spectra are acquired. A third mode is loop storage, in which the HPLC fractions are collected in a loop for later analysis.

4. Tandem Mass Spectrometry (MS-MS):

Tandem mass spectrometry (MS-MS) is used to produce structural information about a compound by fragmenting specific sample ions inside the mass spectrometer and identifying the resulting fragment ions. This information can then be pieced together to generate structural information regarding the intact molecule. Tandem mass spectrometry also enables specific compounds to be detected in complex mixtures on account of their specific and characteristic fragmentation patterns.

Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD). An important application using tandem mass spectrometry is in protein identification²³.

The analysers can be of the same or of different types, the most common combinations being:

Quadrupole - Quadrupole
Magnetic sector - Quadrupole
Magnetic sector - Magnetic Sector
Quadrupole - Time-of-Flight.

Instrumentation:

One of the most commonly available tandem mass spectrometers is the triple quadrupole (QQQ) instrument. There are many other varieties and configurations of tandem instrument.

In a triple quadrupole mass spectrometer, there are several types of experiment that can be performed. The figure shows a schematic representation of three common types of MS/MS experiment.

(1) Product ion scan. In this case, the precursor ion is focussed in Q1 and transferred into Q2-the collision cell-where it interacts with a collision gas and fragments. The fragments are then measured by scanning Q3. This results in the typical MS/MS spectrum and is the method most commonly employed with ESI ionisation and/or LC-MS.
(2) Precursor ion scan. In this case Q3 is held to measure the occurrence of a particular fragment ion and Q1 is scanned. This results is a spectrum of precursor ions that result in that particular product ion - this is especially useful when used with EI or CI ionisation and/or GC-MS.
(3) Neutral loss scan. In this case Q1 is scanned as in (2) but this time Q3 is also scanned to produce a spectrum of precursor ions that undergo a particular neutral loss. Again this mode is especially useful for EI and CI ionisation. All the resulting daughter ions are scanned and recorded according to their m/z ratio^{24, 25}.

Fig.4 Triple Quadrupole Tandem Mass spectrometer

5. Inductively coupled plasma mass spectroscopy (ICP-MS)

Inductively coupled plasma mass spectroscopy (ICP-MS) was developed in the late 1980's to combine the easy sample introduction and quick analysis of ICP technology with the accurate and low detection limits of a mass spectrometer. Although crude by today’s standards, the system showed the enormous possibilities of the ICP as an excitation source and most definitely opened the door in the early 1980s to the even more exciting potential of using the ICP to generate ions³⁸. The resulting instrument is capable of trace multielement analysis, often at the part per trillion level. ICP-MS has been used widely over the years, finding applications in a number of different fields including drinking water, wastewater, natural water systems/hydrogeology, geology and soil science, mining/metallurgy, food sciences, and medicine^26,
35. In addition to ICPs, some of the other novel plasma sources developed were direct current plasmas (DCP) and microwave-induced plasmas (MIP) In fact, for those who want a DCP excitation source coupled with an optical emission instrument today, an Echelle-based grating using a solid-state detector is commercially available^{36, 37}.

Instrumentation:

ICP technology was built upon the same principles used in atomic emission spectrometry. Samples are decomposed to neutral elements in high temperature argon plasma and analyzed based on their mass to charge ratios. An ICP-MS can be thought of as four main processes, including sample introduction and aerosol generation, ionization by an argon plasma source, mass discrimination, and the detection system. The schematic below illustrates this sequence of processes^32-34.

Fig.5 Inductively coupled plasma mass spectroscopy (ICP-MS)

Solid samples are introduced by way of a laser abalation system and Aqueous samples are introduced by way of a nebulizer. The aerosol then passes into a spray chamber where larger droplets are removed via a drain (Jarvis et al., 1992). Typically, only 2% of the orginial mist passes through the spray chamber (Olesik, 1996). Because atomization/ionization occurs at atmospheric pressure, the interface between the ICP and MS components becomes crucial in creating a vacuum environment for the MS system. Detector found in an ICP-MS system is the channeltron electron multiplier, which has a high voltage applied to it opposite in charge to that of the ions being detected^27-34.

6. Liquid chromatography-Electrospray ionization-Mass spectroscopy (LC-ESI-MS):

The ESI process employed in this type of MS method is currently the softest ionization technique known (Cole, 1997) such that even non-covalent interactions in proteins can be measured (Last and Robinson, 1999). Thus ESI-MS is ideal for the analysis of biomacromolecules, as well as of small molecules because it allows fine structural information to be obtained directly from the biomolecules. Moreover, because of the analytes ability to be multiply charged after ESI the accurate molecular weight (10-100 kDa) of large proteins can be determined^{39- 40}.

Instrumentation:

In electrospray ionization, a liquid is pushed through a very small, charged and usually metal, capillary⁴¹. This liquid contains the analyte, dissolved in a large amount of solvent, which is usually much more volatile than the analyte.

Because like charges repel, the liquid pushes itself out of the capillary and forms an aerosol, a mist of small droplets about 10 μm across. Nitrogen is used to help nebulize the liquid and to evaporate the neutral solvent in the droplets. As the solvent evaporates, the analyte molecules are forced closer together, repel each other and break up the droplets. This process is called Coulombic fission because it is driven by repulsive Coulombic forces between charged molecules. The ions observed may are created by the addition of a proton (a hydrogen ion) and denoted $[M + H] +$ , or of another cation such as sodium ion, $[M + Na] +$ , or the removal of a proton, $[M - H] -$ . Multiply-charged ions such as $[M + 2 H] 2 +$ are often observed.

Fig. 6 Electrospray ionization process

7. Gas Chromatography-Mass Spectrometry (GC-MS):

This method combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. The use of a mass spectrometer as the detector in gas chromatography was developed during the 1950s by Roland Gohlke and Fred McLafferty^42-43. GC can separate volatile and semi volatile compounds with great resolution and MS can provide detailed structural information on most compounds such that they can be exactly identified after their separation⁹. These sensitive devices were bulky, fragile, and originally limited to laboratory settings. In 1996 the top-of-the-line high-speed GC-MS units completed analysis of fire accelerants in less than 90 seconds, whereas first-generation GC/MS would have required at least 16 minutes. This has led to their widespread adoption in a number of fields^44-46.

Problem in interfacing GC-MS:

Gas chromatography and mass spectrometry are, in many ways, highly compatible techniques. In both techniques, the sample is in the vapor phase, and both techniques deal with about the same amount of sample (typically less than 1 ng). Unfortunately, there is a major incompatibility between the two techniques: The compound exiting the gas chromatograph is a trace component in the GC’s carrier gas at a pressure of about 760 torr, but the mass spectrometer operates at a vacuum of about 10–6 to l0–5 torr.This is a difference in pressure of 8 to 9 orders of magnitude, a considerable problem⁹.

Instrumentation:

In practice, most GC-MS interfacing is now done by simply inserting the capillary column directly into the ion source. Fig. 7 is a diagram of one such system. The fused silica column runs through a 1/16-in.-diameter tube directly into the ion source.

The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules take different amounts of time (called the retention time) to come out of (elute from) the gas chromatograph, and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass to charge ratio. The GC columns most widely used for GC-MS are those in which the stationary phase has been chemically bonded to the fused silica; DB-5 is a common trade name ^{9, 48-49}.

The Data System:

In a typical GC-MS experiment, the mass spectrometer might be scanned every 2 sec during a 90-min GC run, whether GC peaks are entering the mass spectrometer or not. Assuming that each mass spectrum has an average of 100 mass/intensity measurements, one such GC-MS experiment will give 270,000 mass/intensity pairs. The data output from a typical GC-MS experiment is about 1 megabyte^{9, 48-51}.

Common Applications:

1] Quantitation of pollutants in drinking and wastewater using official U.S. Environmental Protection Agency (EPA) methods.

2] Quantitation of drugs and their metabolites in blood and urine for both pharmacological and forensic Applications.

3] Identification of unknown organic compounds in hazardous waste dumps

4] Identification of reaction products by synthetic organic chemists.

5] Analysis of industrial products for quality control.

8. Gas Chromatography-Infra red Spectroscopy (GC-IR):

The GC/IR Interface blends the separation capability of gas chromatography with the identification power of FT-IR to provide an efficient tool for analyzing complex mixtures. It is optimized for the analysis of small elution volumes found in today's high-resolution capillary columns, providing excellent sensitivity for compounds typically in the low nanogram range for volatile species. It is also effective for the analysis of multicomponent organic samples such as solvents, chemicals, pharmaceuticals, petrochemicals and environmental samples.

Instrumentation:

Effluent gas emerging from a GC at atmospheric pressure is let directly into a heated IR gas cell via a heated transfer line.

Fig.8 Diagrammatic overview of GC-IR

Griffiths et al ⁵² reported the use of an internally gold- coated glass tube, or light pipe as an interface for GC-FTIR. Capillary GC column is coupled with a FTIR. The modulated IR beam is focused into a heated light pipe cell through which the GC effluent is directed. The module's independent programmable temperature controllers for the light pipe and transfer lines allow the user to precisely control the experiment. The insulated transfer line and light pipe prevent cold spots and condensation. Detection limit of GC-IR is in between 10 and 100ng, columns of 0.3-0.5 i.d. are used.

9. Gas Chromatography-Infra red Spectroscopy-Mass spectroscopy (GC-IR-MS):

In 1980 first time GC-IR-MS was implemented. When GC-IR and GC-MS are used together in combination as GC-IR-MS, more GC peaks can be identified due to complementary nature of IR and MS data. Light pipes, a parallel interface between the GC and the two spectrometers, carrying most of the effluents to the IR spectrometer and the remaining to the mass spectrometer are the method of choice⁵³.

10. Gas Chromatography-Thin Layer Chromatography (GC-TLC):

It permits 2D chromatographic separation. This combination offers the possibility of independent and multiple qualitative identification as well as quantitative determination of individual compounds. The carrier gas leaving the GC is directed straight on the thin layer placed some millimeter away from the column exit, which allow 10^-5 to 10^-10 g/sec of the substance on the thin layer. Around 30-80% substance is retained, depending on temperature, gas flow, polarity, concentration and adsorption conditions. Characteristic hRf- values may be determined after development, so that each constituent of the original mixture can be characterized by two values, the retention index (GC) and hRf- values (TLC)⁵⁴.

11. Capillary Electrophoresis-Mass spectroscopy (CE-MS):

In 1976, the use of a small capillary tube as a compartment in which to carry out zone electrophoresis was first reported by Everaerts et al. Capillary electrophoresis can be described as the separation of a mixture in a capillary tube, by virtue of differing ionic mobilities induced by the application of a high potential along the capillary and mass spectroscopy provides structural information^55-60. The first report on CE-MS in combination as a powerful and important tool was reported in 1987^61-63.

Instrumentation:

The detection of the narrow CE peaks requires the use of a fast and sensitive mass spectrometer. Very small volumetric flow rates of 1µl/m from CE make it feasible to couple the effluent to sample ionisation interface. Generally Ion trap and TOF are used as detectors for CE-MS⁶⁴.

12. Capillary Electrophoresis-Nuclear magnetic resonance spectroscopy (CE-NMR):

Numerous obstacles for online coupling of CE with NMR were encountered. In 1994 Wu et al reported first CE-NMR but, not so used in routine operation^65-66. Two different microcoil NMR configurations have been used for online CE-NMR. One approach is based on a solenoid rf coil wound directly on the capillary and the other configuration is based on a saddle-type rf coil, which should have reduced diameter size^67-68.

13. Ion mobility spectrometry-mass spectrometry (IMS-MS)

Ion mobility spectrometry-mass spectrometry (IMS-MS) is a method that combines the features of ion mobility spectrometry and mass spectrometry to identify different substances within a test sample. On the one hand, ion mobility spectrometry was developed in the 70s and typically separates charged particles on a millisecond scale. On the other hand, time-of-flight mass spectrometry was developed in the 50s and typically separates charged particles on a microsecond scale. The combination of both instruments was pioneered in the end of the 90s at Indiana University by David E. Clemmer and co-workers^69-70.

Instrumentation:

The IMS-MS is composed of two major building blocks: the ion mobility spectrometer and the mass spectrometer. Whereas the ion mobility spectrometer is usually made of a drift region at atmospheric pressure or lower, the mass spectrometer is under a high vacuum.

Applications:

The IMS-MS technique can be used in proteomics, for analyzing complex mixtures of peptides.

14. Thin Layer Chromatography- Infra red Spectroscopy (TLC-IR):

TLC is another very fast and convenient method to separate samples. In the past unknown substances were scraped off from the TLC/HPTLC plate, eluted into a tube and transferred into either IR or MS. Combination of TLC with FTIR is useful for evaluation and identification of complex mixtures. It is also useful for quantitation of substances with no suitable UV responce¹.

15. Thin Layer Chromatography-Mass Spectrometry (TLC-MS):

The versatile instrument to extract compounds from a TLC/HPTLC plate and feed into a mass spectrometer for substance identification or structure elucidation. As an example as shown in fig. 9

Why a TLC-MS Interface?:

Surveys have shown that not all samples may be processed by HPLC-MS or HPLC-DAD systems due to no or low detectability of the compounds or impurities in the UV range, a heavy matrix load or a lack of MS compatible solvents. Now a very convenient and universal TLC-MS Interface is available which can semi-automatically extract zones of interest and direct them online into any brand of HPLC-MS system.

Instrumentation:

Fig. 9 CAMAG TLC-MS Interface

Questioned substances are directly extracted from a TLC/HPTLC plate and sensitive mass spectrometric signals are obtained within a minute per substance zone. The interface extracts the complete substance zone with its depth profile and thus allows detections comparable to HPLC down to the pg/zone range^71-83. As per Busch⁸⁴the interface has been proven to be one of the most reliable and versatile interfaces for TLC-MS coupling. Prosek explained computer controlled on-line TLC-MS interface ⁸⁵.

Technical functionality:

The instrument extracts circular zones of 4 mm diameter from a TLC/HPTLC plate, e.g. with methanol or any other appropriate solvent, using the standard flow speed of the HPLC-MS system (e.g. 0.1 mL/min)^75-82.

MALDI, TOF and desorption electrospray ionization are generally used in TLC-MS ⁸⁶.

Future of Hyphenated Analytical Techniques Instrumentation in Market Research:

Many leading participants, especially in the life sciences and pharmaceutical industries, have reported a rise in profits in recent years. Analytical instrumentation vendors now come up with new products and marketing strategies to generate higher revenue streams from these and other more lucrative end-user industries such as chemicals and petrochemicals and environmental. Vendors need to focus on providing competitively priced devices with multiple features that will not only improve end users; productivity levels but also increase the price-performance ratio of the products. In the hyphenated analytical instrumentation market, most technology innovations, product designs, and developments are centered on the requirements of life sciences and pharmaceutical industries.

As a result the popularity of advanced hyphenated techniques will continue to grow in future and accelerate the performance and productivity in advance analytical world.

CONCLUSION:

This review on “Advanced hyphenated techniques in Analytical chemistry” briefly describes hyphenated techniques used in the various areas of science and scientific research. It depicts various separation techniques assembled with latest spectroscopic methods. Introduction of commercial version of these equipments thus expected to find demand in industrial applications as well.

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