ELECTRONIC TONGUE / ELECTRONIC NOSE

This post has been taken from http://csrg.ch.pw.edu.pl/tutorials/electronicT_N/

ELECTRONIC TONGUE / ELECTRONIC NOSE (ETongue, ENose) are systems for automatic analysis and recognition (classification) of liquids or gases, including arrays of non-specific sensors, data collectors and data analysis tools. Electronic tongues are used for liquid samples analysis, whereas electronic noses - for gases. The result of Etongue/Enose can be the identification of the sample, an estimation of its concentration or its characteristic properties.

This new technology has many advantages. Problems associated with human senses, like individual variability, impossibility of on-line monitoring, subjectivity, adaptation, infections, harmful exposure to hazardous compounds, mental state, are no concern of it.

Synonyms of an electronic tongue: artificial tongue, taste sensor

Synonyms of an electronic nose: artificial nose, olfactory system

APPLICATIONS OF E-TONGUES/E-NOSES:

Foodstuffs Industry

• food quality control during processing and storage (water, wine, coffee, milk, juice...)
• optimalization of bioreactors
• control of ageing process of cheese, whiskey
• automatic control of taste

Medicine

• non-invasive diagnostics (patient's breath, analysis of urine, sweat, skin odour)
• clinical monitoring in vivo
• identification of unpleasant odour of pharmaceuticals

Safety

• searching for chemical/biological weapon
• searching for drugs, explosives
• friend-or-foe identification

Environmental pollution monitoring

• monitoring of agricultural and industrial pollution of air and water
• identification of toxic substances
• leak detection

Quality control of air in buildings, closed accommodation (i.e. space station, control of ventilation systems)

Chemical Industry

• products purity
• in the future - detection of functional groups, chiral distinction

Legal protection of inventions - digital "fingerprints" of taste and odours

SENSING METHODS APPLIED

ETongue	ENose
Potentiometric sensors Measurements of conductivity Voltamperommetry Optical sensors Biosensors	Conductivity sensors: MOSFET (Metal-oxide-silicon field-effect-transistor) CP (Conducting Polymer) Piezoelectric sensors QMB (Quartz Crystal Microbalance) SAW (Surface Acoustic Wave) Optical sensors

PATTERN RECOGNITION

The electronic tongue or nose system performance is dependent on the quality of functioning of its pattern recognition block. Various techniques and methods can be used separately or together to perform the recognition of the samples. After measurement procedure the signals are transformed by a preprocessing block. The results obtained are inputs for Principal Components Analysis, Cluster Analysis or Artificial Neural Network.

Measurement

Sensors arrays' outputs are arranged in data matrix (Fig. 1).

Fig. 1. Data matrix

Each sample is characterized by unique and typical set of data, forming "fingerprint" of an analyte in m-dimensional pattern space.

Preprocessing

Preprocessing is the phase in which linear transformation on the data matrix is performed (without changing the dimensionality of the problem) in order to enhance qualitative information. Typical techniques include manipulation of sensor baseline, normalization, standarization and scaling of response for all the sensors in an array.

Principal Component and Cluster Analysis

A multi-sensor system produces data of high dimensionality - hard to handle and visualize. Principal Component Analysis (PCA) and Cluster Analysis (CA) are multivariate pattern analysis techniques reducing dimensionality of the problem and reducing high degree of redundancy.

PCA is a linear feature-extraction technique finding most influential, new directions in the pattern space, explaining as much of the variance in the data set as possible. This new directions - called principal components - are the base for a new data matrix. Usually 2 or 3 of them are sufficient to transfer more than 90% of the variation of the samples.

The base principle of Cluster Analysis is the assumption of close position of similar samples in multidimensional pattern space. Similarity between each 2 samples is calculated as a function of the distance between them - usually in Euclidean sense - and displayed on a dendrogram (Fig. 2).

Fig. 2. Cluster Analysis: a), b) different types of dendrograms

Artificial Neural Networks (ANN)

Neural Networks are information processing structures imitating behavior of human brain. Their main advantages, such as: adaptive structure, complex interaction between input and output data, ability to generalize, parallel data processing and handling incomplete or high noise level data make them useful pattern recognition tools. There are many possible architectures and algorithms available in the literature, but the most common in measurement applications is feed-forward network (multilayer perceptron MLP) and back-propagation learning algorithm.

The base units of artificial neural networks are neurons and synapses. Neurons are organized in layers and connected by synapses. Their task is to sum up their inputs and non-linear transfer of the result, which is then transmitted via synapsis with modification by means of the synapsis weights - this signal, in turn, is the input for the next layer of the network (Fig. 3).

Fig. 3. Neural Networks: a) single neuron, b) feed-forward network

The use of ANN involves 3 phases:

• The learning phase - after establishing number of neurons, layers, type of architecture, transfer function and algorithm, network is forced to provide desired outputs corresponding to a determined input. It is made by adjusting the synapses' weights in order to minimize the difference between desired and current output.
• The validation phase - verification of the generalization capability of network by means of data different (but with similar characteristics) from data used in the learning phase.
• The production phase - in which the network is capable of providing outputs corresponding to any input.

REFERENCES:

• Craven M. A., Gardner J. W., Electronic noses - development and future prospects, Trends in Analytical Chemistry, vol. 15 (1996), 486
• D'Amico A., Di Natale C., Paolesse R., Portraits of gasses and liquids by arrays of nonspecific chemical sensors: trends and perspectives, Sensors and Actuators B, 68 (2000), 324
• Nagle H. T., Schiffman S. S., Gutierrez-Osuna R., The how and why of electronic noses, IEEE Spectrum, September 1998, 22
• Di Natale C., Davide F., D'Amico A., Pattern recognition in gas sensing: well-stated techniques and advances, Sensors and Actuators B, 23 (1995), 111
• Gardner J. W., Detection of vapours and odours from a multisensor array using pattern recognition Part I. Principal Component and Cluster Analysis, Sensors and Actuators B, 4 (1991), 109
• Gardner J. W., Hines E. L., Tang H. C., Detection of vapours and odours from a multisensor array using pattern recognition Part II. Artificial Neural Networks, Sensors and Actuators B, 9 (1992), 9
• Toko K., Taste sensors with global selectivity, Materials Science and Engineering, C4 (1996), 69
• Vlasov Y., Legin A., Non-selective chemical sensors in analytical chemistry: from "electronic nose" to "electronic tongue", Journal of Analytical Chemistry, 361 (1998), 255
• Krantz-Ruckler C., Stenberg M., Winquist F., Lundstrom I., Electronic tongues for environmental monitoring based on sensor arrays and pattern recognition: a review, Analytica Chimica Acta, 426 (2001), 217
• Winquist F., Holmin S., Krantz-Ruckler C., Wide P., Lundstrom I., A hybrid electronic tongue, Analytica Chimica Acta, 406 (2000), 147

LINKS:

Commercially available e-noses/e-tongues:

• http://www.alpha-mos.com/
• http://www.detect-measure.com/neo.htm
• http://www.osmetech.plc.uk/
• http://www.appliedsensor.com/
• http://www.airsense.com
• http://cyranosciences.com/
• http://estcal.com/
• http://www.hkr-sensor.de/
• http://www.lennartz-electronic.de/

Chemometrics:

• http://ull.chemistry.uakron.edu/chemometrics/
• http://www.spectroscopynow.com/Spy/basehtml/SpyH/
  1,1176,2-0-0-0-0-home-0-0,00.html/

Fiber optic chemical sensors

This post is taken from http://csrg.ch.pw.edu.pl/tutorials/fiber/

INTRODUCTION

Fiber optic chemical sensors (FOCS) can offer several advantages over traditional sensors. The light weight and small size of fiber optic sensors are strongly complemented by their strong immunity to electromagnetic interference. Since the fiber sensors are made of glass they are environmentally rugged and can tolerate high temperatures, vibration, shock and they can operate in extremely harsh conditions. There are many excellent reviews on FOCS published in recent years [1,2,3].

There are many types of fiber optic chemical sensors which can measure concentration of neutral or charged species. The principle of operation of FOCS will be explained on the example of pH sensor because it is one of the most important sensor in analytical chemistry.

Fig.1 Schematic experimental set-up of FOCS.

The sensor consists of three main parts: light source, optrode and detector. The main part of the sensor, so-called optrode, contains an appropriate indicator which changes its optical properties in dependence on the analyte. In most cases, it is necessary to use an indicator because the analyte does not give or exhibit changes of optical properties. The indicator can change, for example, absorbance or fluorescence intensity. The light source is matched to the so-called analytical wavelength of the indicator then the best sensitivity of the sensor can be obtained. Detector, usually photodiode or PMT, converts optical signal into electric one which is next electronically processed.

OPERATING PRINCIPLE

Fig.2 shows operating principle of a pH sensor based on absorbance indicator.

The pulse of light from a light emitting diode (LED) is coupled into optical fiber and transmitted to a pH sensitive membrane. The membrane changes its absorbance (colour) in dependence on pH of the sample. If the absorbance is quite low the light is only slightly absorbed which is depicted by almost the same pulse of the light returning to the photodiode. When the pH of the sample is changed the absorbance of the membrane increases so the returning pulse of the light is smaller (see Fig.3).

Fig.3 Sensor based on absorbance indicator at solution of different pH.

Light is usually modulated to a square wave because the measurements are not influenced by an ambient light and in order to increase the signal to noise ratio.

Sensors can be also based on the use of fluorescence indicator immobilized in the membrane.

Fig.4 Sensor based on fluorescence indicator.

In this case, the light excites molecules of the indicator which emits light at different wavelength. The analyte can, for example, influence on the intensity of the fluorescence radiation which is depicted by different amplitude of the returning pulses (see Fig.4).

Fig.5 Sensor based on fluorescence indicator at solution of different pH.

SIGNAL CONVERSION IN THE SENSOR

Typical conversion process of the signal in the FOCS is presented in Fig.6.

Fig.6 Conversion of the signal in fiber optic chemical sensor.

Chemical signal caused by the analyte is converted into optical one in chemooptical interface. The chemooptical interface consists of membrane with an appropriate indicator. The indicator changes its optical properties (e.g. absorbance, fluorescence) in dependence on analyte. Such an optical signal with information about sample under test is converted into electric signal in an optoelectronic interface. The main part of this interface is the photodetector connected to an electronic circuit. The electric signal can be acquired and processed by PC-lab card. In the case of a pH sensor signal is processed according to procedure shown in Fig.7.

Fig.7 Signal processing in a fiber optic pH sensor.

The signal from the sensor is acquired by the card, then it is processed according to the calibration procedure and displayed on the monitor. The acquired data can be saved on the disk.

EXAMPLE OF FIBER OPTIC pH SENSOR

The fiber optic system can be controlled by a software developed in Lab Windows environment. The main idea of the software is to design user friendly and interactive application which can be applied in many sensor configurations. The user can set up its own application based on pre-design modules. There are many modules possible to use in each phase of signal processing. Some typical ones designed for signal acquisition are presented in fig.8.

Fig.8 Examples of modules for signal aquisition.

The user has to chose a module which is the most suitable for its application and place it on the screen. Similar modules are designed for each part of signal processing. It is possible to use as many modules as the user needs. Every module has its own name and in this way it can be recognized by any module. The modules shown in fig.8 allow to set the gain of the acquisition card, sampling rate, channel of the card etc. The output signal from the card is transmitted to further processing.

Fig.9 shows application software designed for a pH sensor with the use of different modules.

Fig.9 Fiber optic pH sensor controlled by LabWindows application.

The signal from the sensor is amplified by proper settings of control module of Lab-PC+ card. Next module is used for signal processing where calibration formula is applied. The calibration data can be saved and retrieved for each sensor. Last module gives graph with dependence of the signal versus time. When appropriate settings of the card and calibration procedure were done it is possible to switch off any display which is not important during the measurements without any influence on work of the system. The modules left will be rescaled automatically. For example, only pH value can be displayed on the monitor.

Main features of the application:

• software is user friendly and is based on typical Windows commands
• designed process can be saved
• display is rescaled automatically
• user can chose modules for configuration
• user can design his own project

Example of sensor configuration

The most basic version of the experimental set-up is shown in Fig.10.

Fig.10 Configuration of fiber optic pH sensors (GEN-square wave generator, LED-light emitting diode, PD-photodiode, AMP-transimpedance amplifier, AF-active filter).

Modulated light from a light emitting diode (LED) is transmitted to the optrode by one arm of the fiber optic bundle. The light matched to the maximum of the molar absorbance of neutral red (560 nm) is reflected in dependence on pH variations and then it is transmitted to a photodiode by the second arm of the bundle. The photodiode is connected with a transimpedance amplifier and active filter. The electrical signal obtained is acquired and processed by a pc-lab card with 12-bit A/D converter. The optrode was built on the common end of the bundle by the use of removable Teflon tube, which holds an optomembrane.

REFERENCES

1. W.R.Seitz Chemical sensors with fiber optics, Crit. Rev. in Anal. Chem., 19, 1988, 15
2. D.L.Wise Biosensors with fiber optics, Humana Press, New York, 1991
3. O.S.Wolfbeis Fiber optic chemical sensors and biosensors, Boca Raton, 1991
4. K. Seiler and W. Simon, Principles and mechanisms of ion-selective optodes, Sensors and Actuators B, 6 (1992) 295-298.
5. A. Dybko, W. Wróblewski, J. Maciejewski, Z. Brzózka, R. Romaniuk, Novel matrix for fibre optic chemical sensors made of particle track polymer, Proc. SPIE, 2508 (1995) 351-357.
6. A. Dybko, W. Wróblewski, J. Maciejewski, Z. Brzózka, R. Romaniuk, J. Kie^(3)kiewicz, Polymer track membranes as a trap support for reagent in fibre optic sensors, J. of Apll. Polym. Sci., 59 (1996) 719-723.
7. A.Dybko, W.Wróblewski, J.Maciejewski, R.Romaniuk, Z.Brzózka Efficient reagent immobilization procedure for ion-sensitive optomembranes, Sensors and Actuators B - in press
8. E. Bishop, Indicators, Pergamon, Oxford, 1972.

OPTICAL FIBER TECHNOLOGY

All optical fibers consist of a core having the refractive index higher than of surrounding cladding. They can be made of just glass or polymer, or combination of both. They also have protective polymer layers called buffer or jacket.

Fig.1 Cross-section of optical fiber

There are two methods to manufacture optical glass fiber: either directly drawing the fiber from molten glasses, which are placed in two concentric crucibles (Double Crucible method) or from a glass rod called preform. Nowadays most optical fibers are made from the preform. There are three steps in this method:

1. Fabrication of the preform
2. Drawing the fiber from the preform
3. Coating and jacketing process

The preforms are fabricated using chemical vapor deposition methods [1]:

1. Modified Chemical Vapor Deposition (MCVD)
2. Plasma Modified Chemical Vapor Deposition (PMCVD)
3. Plasma Chemical Vapor Deposition (PCVD)
4. Outside Vapor Deposition (OVD)
5. Vapor-phase Axial Deposition (AVD)

All these methods are based on thermal chemical vapor reaction that forms oxides, which are deposited as layers of glass particles called soot, outer rotating rod or inside glass tube. The same chemical reactions occur in both methods. Starting materials are solutions of SiCl4, GeCl4, POCl3, and gaseous BCl3. These liquids are evaporated within oxygen stream and form silica and other oxides. Chemical reactions proceed as follows:

SiCl₄ + O₂ → SiO₂ + 2 Cl₂
GeCl₄ + O₂ → GeO₂ + 2 Cl₂
4 POCl₃ + 3 O₂ → 2 P₂O₅ + 6 Cl₂
4 BCl₃ + 3 O₂ → 2 B₂O₃ + 6 Cl₂

Germanium dioxide and phosphorus pentoxide increase the refractive index of glass, whilst boron oxide - decreases. These oxides are known as dopands. Changing composition of the mixture during the process influences refractive index profile of the preform.

Modified Chemical Vapor Deposition (MCVD)

This method was developed by Bell Laboratories [2]. The gaseous mixture of reactants described above is fed at the end of a rotating silica tube. This tube is heated by a traversing oxygen-hydrogen burner (Fig.2). As a result of chemical reactions glass particles, called soot, are formed. These particles are deposited on internal wall of the tube. The soot is then vitrified by the traversing burner to provide a thin glass layer. The process is repeated many times as the cladding layers and core layers are formed. When the deposition is finished, the temperature of the burner is increased to collapse the tube into a solid preform. The entire process is highly automated and all process parameters are precisely controlled.

Fig. 2 Deposition by MCVD process

Plasma Modified Chemical Vapor Deposition (PMCVD)

A modification of MCVD method is a process known as PMCVD. In addition to the normal MCVD technique the radio-frequency coil around the tube generates an internal high temperature plasma.

Fig.3 Deposition by PMCVD process

Plasma Chemical Vapor Deposition (PCVD)

The PCVD method is similar to PMCVD. The radio-frequency coil is replaced by a microwave cavity resonator (Fig. 4). In this method reactions lead directly to of glass layer without forming the soot.

Fig. 4 Deposition by PCVD process

Outside Vapor Deposition (OVD)

This process is also called the "soot process". It was exclusively used by Corning since the 1970s, and the patent of such a technology has expired since July 2000.
Halogens and O2 react in a hot flame to form hot glass soot, which is deposited layer by layer on an aluminium oxide or graphite mandrel. The central mandrel is removed after deposition. In the last step, called sintering, a hollow porous preform is dehydrated and collapsed in controlled atmosphere, (e. g. helium) to form desired preform.

Fig. 5 Preform fabrication by OVD process

Vapor-phase Axial Deposition VAD

In VAD method (in contrary to above methods) the perform can be fabricated continuously. Starting chemicals are carried from the bottom into oxygen-hydrogen burner flame to produce glass soot which is deposited on the end of a rotating silica rot. A porous preform is then grown in the axial direction. The starting rod is pulled upward and rotated in the same way as that used to grow single crystals. Finally the preform is dehydrated and vitrified in ring heaters (Fig. 6). This process is preferred for the mass production.

Fig. 6 Preform fabrication by VAD process

Fiber Drawing

Optical fibers are obtained by drawing from the preform at high temperature. The drawing process must be integrated with the coating process to avoid contamination of fiber surface. These processes are shown schematically in Fig.7.

Fig. 7 Schematic of fiber drawing and draw tower solution

The tip of the perform is heated in a furnace to a molten state. Formed molten gob falls down under the force of gravity while shrinking in diameter into a proper diameter strand. It is controlled continuously during the drawing process. Diameter drift cannot exceed 0.1%. The strand is threaded through a series coating applicators immediately after drawing. Liquid prepolymer coatings are cured by thermal or ultraviolet apparatus. Dual coating, soft inner and hard outer, is needed to avoid microbending and protect against impact and crushing forces in either manufacturing process or installation. silicone coating and acrylate, Tefzel (ETFE), Teflon (PFA), nylon buffers are applied during the fiber drawing, while additional materials such as Hyrtel and PVC can be extruded after the draw process. The fiber with coatings is pulled down and wound on a winding drum. The drawing process must take place in air conditioned room, because air pollution influences fiber attenuation.

References

1. Fibre optics: theory and applications, Serge Ungar, Wiley, New York, 1990.
2. Handbook of fiber optics: theory and applications, Chai Yeh, Academic Press, San Diego, 1990.

Field effect transistors (FETs) as transducers in electrochemical sensors

This post has been taken from http://csrg.ch.pw.edu.pl/tutorials/isfet/

INTRODUCTION

Chemical sensors are microdevices that connect the chemical and electrical domains (i.e. transduction of the chemical information into electric signal). The response of the sensors should be fast and selective for the analyte. Moreover, these devices should have a lifetime in the order of months. The construction of chemical sensors requires the integration of a sensing receptor and a transducing element into a defined chemical system. Field effect transistors (FETs) are very interesting because they can be made very small with current planar IC technology and have the advantage of a fast response time.

FROM MOSFET TO ISFET

The FETs are able to measure the conductance of a semiconductor as a function of an electrical field perpendicular to the gate oxide surface. In the most simple version, (i.e. a metal oxide semiconductor field effect transistor, n-channel MOSFET), a p-type silicon substrate (bulk) contains two n-type diffusion regions (source and drain). The structure is covered with a silicon dioxide insulating layer on top of which a metal gate electrode is deposited (figure 1a).

Figure 1. Schematic representation of a MOSFET a) and an ISFET structure b).

When a positive voltage (with respect to the silicon) is applied to the gate electrode, electrons (which are the minority carriers in the substrate) are attracted to the surface of the semiconductor. Consequently, a conducting channel is created between the source and the drain, near the silicon dioxide interface. The conductivity of this channel can be modulated by adjusting the strength of electrical field between the gate electrode and the silicon, perpendicular to the substrate surface. At the same time a voltage can be applied between the drain and the source (Vds), which results in a drain current (Id) between the n-regions.

In the case of the ISFET, the gate metal electrode of the MOSFET is replaced by an electrolyte solution which is contacted by reference electrode (then the SiO₂ gate oxide is placed directly in an aqueous electrolyte solution, figure 1b) [1]. The metal part of reference electrode can be considered as the gate of the MOSFET.

In ISFET, electric current (Id) flows from the source to the drain via the channel. Like in MOSFET the channel resistance depends on the electric field perpendicular to the direction of the current. Also it depends on the potential difference over the gate oxide. Therefore, the source-drain current, Id, is influenced by the interface potential at the oxide/aqueous solution. Although the electric resistance of the channel provides a measure for the gate oxide potential, the direct measurement of this resistance gives no indication of the absolute value of this potential. However at a fixed source-drain potential (Vds), changes in the gate potential can be compensated by modulation of the Vgs. This adjustment should be carried out in such a way that the changes in Vgs applied to the reference electrode are exactly opposite to the changes in the gate oxide potential. This is automatically performed by ISFET amplifier with feedback which allow to obtain constant source-drain current. In this particular case, the gate-source potential, is determined by the surface potential at the insulator/electrolyte interface.

When SiO₂ is used as the insulator, the chemical nature of the interface oxide is reflected in the measured source-drain current. The surface of the gate oxide contains OH-functionalities, which are in electrochemical equilibrium with ions in the sample solutions (H⁺ and OH^-). The hydroxyl groups at the gate oxide surface can be protonated and deprotonated and thus, when the gate oxide contacts an aqueous solution, a change of pH will change the SiO₂ surface potential. A site-dissociation model describes the signal transduction as a function of the state of ionization of the amphoteric surface SiOH groups [2,3]. Typical pH sensitivities measured with SiO₂ ISFETs are 37-40 mV/ pH unit [3].

The selectivity and chemical sensitivity of the ISFET are completely controlled by the properties of the electrolyte/insulator interface. Other inorganic gate materials for pH sensors like Al₂O₃, Si₃N₄ and Ta₂O₅ have better than SiO₂ properties in relation with pH response, hysteresis and drift. In practice, these layers are deposited on the top of the first layer of SiO₂ by means of chemical vapour deposition (CVD).

ISFETs have been chosen as a transducing element because the SiO₂ surface contains reactive SiOH groups which can be used for covalent attachment of organic molecules and polymers.

MEMFET AND SURFET

The ISFET can be modified with a sensing membrane, that contains an ionophore, which determines the response of the sensor [4]. If the gate oxide is covered with an ion-sensitive membrane, the device is known as a MEMFET [5]. In this case, the ion-sensing layer is penetrable for ions (unblocked); the membrane potential is generated throughout the membrane, which is detected by the FET structure. The first ISFET modified with a sensing membrane containing an ionophore, which enables the detection of the activity of an ion by its complexation, was reported by Moss [6]. A K⁺ - sensitive FET was obtained by solvent casting of a conventional plasticized PVC membrane, containing valinomycin on the gate oxide surface. Other approaches proposed Ca²⁺ sensitive MEMFET (with ion exchanger in polymeric membrane) [7] or deposition of AgBr membranes (Ag⁺ or Br^- sensors) [8].

SURFET represents an ISFET with an ion-blocking layer, which covers the pH-sensitive sites of the gate insulator. At the surface of this layer a surface potential is established by selective association of ions. An example of a SURFET is the perylene gate ISFET with attached benzo-18-crown-6 ionophore molecules, that selectively complex potassium ions [9]. In contrast to the MEMFET, where the association coefficient of the ionophore with recognised ion in the membrane phase determines the selectivity, in SURFET the same process in the aqueous phase controls the selectivity.

It can be concluded from the literature reviews [10,11], that MEMFETs are readily fabricated by means of solvent casting of PVC membranes, with incorporated plasicizer and ionophore on the top of the ISFET gate oxide. Due to poor adhesion of the membrane to the gate oxide it can peel out easily and its electroactive components may leach out. The leaching out effect can be diminished by using extremely hydrophobic receptor or the ionophore can be covalently linked to the organic matrix at the ISFET gate oxide [12]. However, there is no thermodynamically well-defined membrane-ISFET interface and finally the pH sensitivity is not completely eliminated.

CHEMFET

ISFETs modified with plasticized PVC membranes lack a thermodynamically well-defined interface between the sensing membrane and the solid contact. Nevertheless, the PVC-modified ISFETs do not seem to suffer from the ill-defined inner contact and acceptable stabilities and drift values have been reported [7, 13, 14]. Up to now, no experimental efforts were made to improve this system because the properties of the devices are quite satisfactory. However, following studies showed, that changes of carbon dioxide concentrations in the sample solution influence strongly the measurements [15]. This was attributed to the diffusion of carbon dioxide through the membrane and the successive formation of carbonic acid at the membrane-gate oxide interface with traces of water present at the interface. Consequently, the concentration of protons, which determines potential at the membrane insulator interface, undergo large variations. This phenomenon explains why ISFETs modified with PVC membranes generally perform satisfactory (the PVC membranes usually contain reasonable amount of water and therefore H⁺ ions are present and control the membrane-insulator potential). Besides the CO₂ interference, the need for high amount of water inside the membrane matrix was the key reason that urged to develop a thermodynamically well-defined interface.

Several approaches have been described in the literature for FET based sensors as possible solutions for these problems. In most cases, an intermediate Ag/AgCl layer is applied on the gate-insulator surface [16], which at least eliminates the CO₂ interference. Various methods of deposition of a Ag/AgCl layer on a silicon substrate were reported with a conclusion, that different IC-compatible methods give satisfactory layers [17]. However, the Ag/AgCl-membrane interface becomes critical. The equilibrium state of this interface relies on the exchange of scarcely present Cl_- ions in the membrane. Therefore, a better approach seems to be a deposition of an additional layer (e.g. sodium glass) between the polymer and the gate insulator or poly(vinyl alcohol) between the polymer and the Ag/AgCl layer on the top of the gate insulator [18]. In this way common ions can be provided by the intermediate layer.

Another approach, a novel architecture - chemically modified FET (CHEMFET), is designed to solve the problems (figure 2).

Figure 2. Schematic representation of a chemically-modified FET-CHEMFET.
Top: cross-section through the various layers with potential determining species.

The attachment of the membrane can be improved by mechanical [19] or chemical [20,21] anchoring to the surface of the gate oxide. For chemical attachment of polymer films the gate oxide surface is silylated with 3-(trimethoxysilyl)propyl methacrylate. The methacrylate modified surface can subsequently react with vinyl or methacryl monomers or prepolymers. The use of a UV-photopolymerizable monomers, hydroxyethyl methacrylate (HEMA), is advantageous from the point of view of the ultimately desired mass production of the CHEMFETs, which is essentially based on photolithography. The introduction of such a hydrogel layer [22,23], in which an aqueous buffered solution of salts can be absorbed, between the gate oxide and the sensing membrane eliminates the interference of CO2 on the CHEMFET response. Moreover, this stabilizes the potential developed in the sensing membrane. Plasticized PVC membranes, that contain an ionophore, are widely used as sensing membranes. Leakage of plasticizer to the contacting aqueous solution and weak adhesion of the membrane to the ISFET prompted the search for other polymer membranes like polyurethane, silicone rubber, polystyrene, polyamide and several polyacrylates [20,23,24].

The problem of the thermodynamically ill-defined membrane-gate interface was solved by an application of chemically attached poly(2-hydroxyethyl methacryalate) (polyHEMA) hydrogel between a hydrophobic membrane and the gate oxide layer. This novel architecture of FETs allows to design new chemical sensors based on polymeric membranes containing molecular receptors. CHEMFETs selective to K⁺ [20,25-27], Na⁺ [28-30], Ag⁺ [31], some transition metals cations (Pb²⁺, Cd²⁺) [32-34] and some anions (NO^3-) [35-37] have been developed. However, the sensors exhibit limited lifetime which resultes from the leaching out of electroactive components, i.e. the ligand and the ionic sites. Electroactive components with an enhanced lipophilicity could be applied to increase durability of the sensor, but a more efficient method is based on covalent anchoring of these components to the membrane matrix. Application of membranes containing covalently bond ionophore and covalently bond ionic sites significantly improves the durability of CHEMFETs [26-29].

REFET

The use of a conventional reference electrode limites seriously the application of ISFETs with respect to the small size. Therefore the development of a miniature reference electrode made with the IC-compatible technology (reference field effect transistor - REFET) is of great interest for the wide-spread use of these sensors.

One of the approaches to solve this problem is the on-chip fabrication of an Ag/AgCl electrode with IC-compatible techniques, including a gel filled cavity and a porous silicon plug [38-42]. However, all the constructions have the disadvantage of a liquid-filled internal cavity with associated limited lifetime because of leakage of reference solution. A better approach to the problem of the reference electrode could be the application of two chemically unequally sensitive ISFETs operating in a differential mode with a common quasi-reference electrode (QRE) (e.g., a metal wire Pt), which can be easily integrated on the silicon chip [43-46]. This device have the additional advantage that external disturbances influencing both ISFETs (e.g. light and temperature) can be reduced. The accuracy of differential measurements depends on the difference in the ion sensitivity of both ISFETs, although total insensitivity of one ISFET (REFET) would be preferred. Such a reference FET should ideally case show insensitivity to all species present in the sample solution.

Originally, the oxide gate surface shows pH sensitivity, owing to the presence of hydroxyl groups, which can dissociate and can be protonated. It was reported that the total elimination of the pH-sensitive groups by chemical monolayer modification cannot be achieved [47]. However, the pH sensitivity can be suppressed by attaching to the gate surface an ion-blocking hydrophobic polymeric layer. In this modification the polymer is chemically bounded to the gate surface, which results in a long lifetime of the device. For ion-blocking layers, a stable attachment has been realized by plasma deposition [48-52]. However, potential variations with electrolyte compositions for such modified REFETs were observed [53]. Moreover, the deposition is limited to very thin polymeric layers because of diminishing electrical sensitivity (transconductance) with increasing insulator thickness [5].

In contrast to ion-blocking polymers, modification of REFETs with ion-unblocking (conductive) polymers would have the advantage of an equal transconductance of the REFET and ISFET [54,55]. Unfortunately, such ion-unblocking hydrophobic membranes result in a short lifetime of the sensor, if they are not chemically anchored to the surface. ISFETs have been modified in order to prepare REFETs with polymeric membranes which are covalently linked to the gate oxide surface [56,57].

Two types of REFET structures can be distinguished with respect to the penetration of ions into the polymeric layer, resulting in two different mechanisms of the REFET operation. In a non-ion-blocking REFET structure there is ion exchange between the solution and the polymer; consequently a thermodynamical equilibrium between ions in the solution and in the polymer is achieved and the membrane electrical potential is a membrane potential. In an ion-blocking REFET structure this ion exchange is negligible and in this case the electrical potential measured is a surface potential resulting from reversible ion-complexation reactions at the surface of the polymer.

CONCLUSION AND REFERENCES

Conclusion

The application of field effect transistors (FETs) as transducers in electrochemical sensors was firstly described in 1970 by Bergveld. These devices can transduce an amount of charge present on the surface of the gate insulator into a corresponding drain current. The fast expantion of these transducing elements was possible due to the introduction of IC-technology in their construction, which allowed mass fabrication.

This modern technology provide a possibility to design multi-ion sensors integrated with the reference cell (REFET). These sensors are very small, longliving and use only very small amounts of ion-sensing compounds.

References

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Coated wire and solid state electrodes

This post has been taken from http://csrg.ch.pw.edu.pl/tutorials/solid/

In the conventional ion-selective electrodes (ISE), the ion-selective membrane is in electrical contact with the inner reference electrode through the inner reference solution. The convertion from ionic conductivity (in the membrane and the inner reference solution) to electron conductivity (in the inner reference electrode and external instrumentation) is provided by the reversible reaction of the inner reference electrode resulting in an ISE exhibiting a stable and reproducible standard potential.

The desire to miniaturise and mass fabricate sensors has led to the development of potentiometric solid-state sensors with new sensing systems, namely solid contact electrodes (SCEs) [1] such as solid crystal membranes and coated wire electrodes (CWEs) [2,3].

Coated-wire electrodes refer to a type of ISE in which an electroactive species is incorporated in a thin polymeric support film coated directly on a metallic conductor. This move to the total elimination of the internal filling solution provides new advantages. The substrate in the wire type electrodes is usually platinum wire, but silver, copper and graphite rods have also been used. CWEs are manufactured by dipping a metal wire into a solution of the membrane mixture [3]. The scheme of CWE is presented in the Fig. 1.

Fig. 1. The scheme of coated wire electrode

Sensors for Ca²⁺, NO^3-, K⁺, Cl^-, Li⁺ and ClO^4- have been developed and sometimes these electrodes exhibited better selectivity than conventional type electrodes with an internal solution. Simplicity of design, lower costs, mechanical flexibility of miniaturisation and microfabrication widened the application for wire type electrodes, especially in the fields of medicine and biotechnology.

However, the configuration of CWE involves some crucial drawbacks. Commonly observed phenomena in such solid-state ion-sensors are a poor mechanical adhesion of the PVC-based sensitive layer to the transducer surface and insufficient electrochemical stability i.e. shift and drift of the EMF [4]. The standard potential of CWEs is often unstable varying for the electrode during its lifetime. These systems cannot provide very reproducible potentials due to the poorly defined charge transfer process at the interface between the ionically conducting membrane and the electrically conducting substrate. In the case of CWEs, drift characteristics were reported to be dependent on the kind of solids used [5,6] and on dissolved oxygen [6]. It has been found that usually an oxygen half-cell is set up on the metal surface so that the electrodes are susceptible to the oxygen content of the solution [7,8].

ISEs with direct contact of the membrane to a metal substrate constructed in a different manner have also been described. Some improvement has been made for this type of electrode with regard to the adhesion of the membrane by using a metal loaded epoxy as substrate [9], but these electrodes also did not possess a defined interface and were found to suffer from oxygen interface.

A more stable electrode potential can be obtained by contacting the ion-selective membrane to the solid substrate via an intermediate layer. Polymeric materials exhibiting mixed ionic and electronic conductivity, containing extended p-conjugated back-bones, such as poly(pyrrole) [10], poly(aniline) [11-13], poly(thiophene) [12] can be applied for this purpose. These materials may be prepared electrochemically by oxidation of their monomer.

Owing to the introduction of such electrically conducting polymers (CPs) t he ionic response of an ion-selective membrane (ISM) is converted to an electric signal.

To facilitate the charge transfer across the interface in solid contact ISE (SC-ISE), improve the potential stability and prevent oxygen interference the introduction SAM of a lipophilic redox-active compound was also proposed [14]. The structures of compounds used for this aim are presented in the Fig. 2.

Fig. 2. The structures used compounds for SAM preparation

In such case a lipophilic and redox-active compound is attached by self-assembly to the inner gold electrode. The redox properties of the SAM guarantee a stable potential, while their lipophilicities prevent the formation of an aqueous layer between membrane and the metal electrode.

MINIATURISED SOLID-STATE SENSORS BASED ON SILICON TECHNOLOGY

The first miniaturized potentiometric solid-state sensors based on silicon technology were developed at University of Michigan several years ago [15,16]. Silicon structures employed for this purpose possessed both: the sensing and electrical contact sites on the front side of the chip (FSC). The scheme of such a sensor is presented in the Fig. 3.

Fig. 3. The scheme of the front-side solid-state miniaturized sensor

Such sensors' design requires an additional encapsulation layer that covers the electrical contact pads. Insufficient quality of this encapsulation layer might cause a solid problem when sensors are used in an electrolyte solution. In order to eliminate this problem, potentiometric sensors with a back-side electrical contact (BSC) and front-side sensing site, fabricated with silicon technology, have been designed and applied for the fabrication of miniaturized BSC ion-sensors [17]. The structure of BSC sensor is presented in the Fig. 4.

Fig. 4.The structure of BSC sensor

The back-side contact silicon-based chips were fabricated using IC technology, by photolithography processes. The structures possess the miniaturised Ag/AgCl sensing site situated on the front of the chip. Such Ag/AgCl electrode shows the changes in EMF as a function of chloride concentration according to the Nernst equation:

E = E⁰ - 59.16 lg a_Cl^-

The potentiometric response (the calibration curves) of 10 randomly chosen sensors toward changes in chloride concentration is presented in the Fig. 5.

Fig. 5. The response of 10 randomly chosen BSC sensors toward changes in chloride concentration

Based on the potentiometric characteristics of BSC chip it can be concluded that Ag/AgCl sensing site can play role of the internal reference electrode. Such sensors are useful for miniaturised ion-selective sensor preparation.

However it was found that both kinds of sensors with front and back-side electrical contact suffer from instabilities of potential values. Several approaches (e.g. the incorporation of lipophilic silver-ligand complexes within polymeric films, the intermediate pHEMA layer introduction) have been suggested to improve the stability of such sensors by establishing a reversible electron transfer pair at the membrane/solid contact interface. The scheme of miniaturised solid-state chemically modified BSC sensor with intermediate pHEMA layer and polymeric membrane is presented in the Fig. 6.

Fig. 6.The structure of BSC chemically modified sensor

Such configuration involves the charge transfer processes at both sides of the IS membrane well thermodynamically.
Ion-selective miniaturised BSC sensors with polymeric membranes are typically investigated in a galvanic cell:

Ag/AgCl/KCl_(sat)/1M CH₃COOLi//sample solution//liquid membrane/
/internal filing solution (pHEMA)/Ag/AgCl

It is common to divide the membrane potential (E_M) into several separate potential contributions, namely the phase boundary potentials at both interfaces and the diffusion potential within the ion-selective membrane. The potential at the membrane/inner filling solution (pHEMA layer) interface can usually be assumed to be independent of the sample. The boundary potential (membrane/sample solution) depends on the ion-exchange processes between the solution and membrane phase. The diffusion potential within the membrane may become significant if considerable concentration gradients of ions with different mobilities arise in the membrane.

The selectivity of classical ISE and miniaturised sensors in the presence of the primary and interfering ions can be described by the selectivity coefficient according to the Nicolskii-Eisenman equation:

Where: E - the membrane potential; E⁰ - constant; a_I, a_J - the activity of primary and interfering ions, respectively; Z_I, Z_J - charge of primary and interfering ions, respectively; I - primary ions; J - interfering ion;

The miniaturised chemically modified back-side contact chips can be designed as anions as well as cations selective sensors. The calibration curves for NO^2- - selective sensors with membranes based on linear polyurethane (Tecoflex) and containing the tetraphenyl porphyrin nitrite (CoTPPNO₂) as a nitrite-selective ionophore are presented in the Fig. 7.

Fig. 7. The calibration curves for the BSC sensors based on Co(III)[TPP]NO₂/TDMACl//PU/o-NPOE

The sodium selective chips based on back-side silicon sensors have also been described [18]. The potentiometric response of the BSC structures with polymeric membrane containing isodecyl acrylate/acrylinitryle and calix[4]arene as a sodium selective ionophore is presented in the Fig. 8.

Fig. 8. The calibration curves of Na⁺ - selective miniaturised BSC sensors with intermediate pHEMA layer and membranes based on copolymer isodecyl acrylate/acrylonitryle and calix[4]arene as a sodium selective ionophore

Plasticised PVC is commonly used as the membrane material for classical ISEs and CWEs. However it can not be applied as a polymeric matrix for miniaturised silicon-based sensors due to the lack of its adhesion to Si3N 4 surface. Insufficient adhesion may cause detachment of ion-selective membrane based on linear polymer and shortening of sensor's lifetime. The application of the photocurable polymeric matrix is recommended in order to fulfil the requirements related to membrane adhesion to sensor's support and its mechanical properties.

REFERENCES

1. B.P. Nikolskii, E.A. Materova, Ion Sel. Electrode Rev. 7, (1985), 3.
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15. H.D. Goldgberg, The Batch Fabrication of Integrated Chemical Sensors Arrays, Ph. D. Dissertation, University of Michigan, 1993.
16. G.S. Cha, M.E. Meyerhoff, H.C. Cantor, A.R. Midgley, H.G. Goldberg, R.B. Brown, Anal. Chem. 63, (1991), 1666.
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18. K. Wygladacz, E. Malinowska, J. Jazwinski, Z. Brzozka, SPIE, Vol 4616, (2001), 32.

Raman Spectroscopy

Raman Scattering: the Basics

Raman scattering is a powerful light scattering technique used to diagnose the internal structure of molecules and crystals. In a light scattering experiment, light of a known frequency and polarization is scattered from a sample. The scattered light is then analyzed for frequency and polarization. Raman scattered light is frequency-shifted with respect to the excitation frequency, but the magnitude of the shift is independent of the excitation frequency. This "Raman shift" is therefore an intrinsic property of the sample.

Because Raman scattered light changes in frequency, the rule of conservation of energy dictates that some energy is deposited in the sample. A definite Raman shift corresponds to an excitation energy of the sample (such as the energy of a free vibration of a molecule). In general, only some excitations of a given sample are "Raman active," that is, only some may take part in the Raman scattering process. Hence the frequency spectrum of the Raman scattered light maps out part of the excitation spectrum. Other spectroscopic techniques, such as IR absorption, are used to map out the non-Raman active excitations.

Additional information, related to the spatial form of the excitation, derives from the polarization dependence of the Raman scattered light. The shape of an excitation in a material, for example a vibration pattern of the atoms in a molecule, and the polarization dependence of the scattering, are determined by the equilibrium structure of the material through the rules of group theory. By this route one gleans valuable and unambiguous structural information from the Raman polarization dependence.

As an example of some actual Raman data taken here, consider the intensity/frequency spectra presented in the figure below.

Here, the frequency is plotted relative to the laser frequency, so the frequency scale represents the Raman shift. The peaks in the intensity occur at the frequencies of the Raman active modes. The spectra differ because of the different polarization conditions enforced on the incident and scattered light. Different polarization conditions select different sets of Raman active excitations.

Experimental Considerations: the NIST Raman Apparatus
Raman scattering is, as a rule, much weaker than Rayleigh scattering (in which there is no frequency shift) because the interactions which produce Raman scattering are higher order. Therefore most experiments require an intense source which is as monochromatic as possible--a laser with a narrow linewidth is usually used--and the collected light must be carefully filtered to avoid the potentially overwhelming Rayleigh signal. Other potentially large sources of non-Raman signal include fluorescence (the decay of long-lived electronic excitations) and of course light from ambient sources. Fluorescence can be particularly pernicious to a Raman measurement because the fluorescence signal is also shifted from the laser frequency, and so can be much more difficult to avoid. (Note that although the fluorescence spectrum is shifted from the laser frequency, the fluorescence shift depends on the laser frequency whereas the Raman shift does not).

So the relative weakness of the Raman signal dictates the organization of the data-taking apparatus. The original Raman setup here at NIST is fairly standard. The photo below shows the basic Raman configuration.

The laser beam from the Argon-ion laser is filtered for monochromaticity and directed by a system of mirrors to a focussing/collecting lens. The beam is focussed onto the sample; the scattered light which passes back through the same lens is then passed through a second lens into the first stage of the spectrometer. One point to notice is that a sample should be oriented such that the specular reflection from the sample passes outside of the collection lens--otherwise, the laser signal might damage the detector which is sensitive enough to see the weak Raman signal.

The Spectrometer and Detector
The spectrometer itself is a commercial "triple-grating" system. Physically, it is separated into two stages which are shown schematically here.

The first stage is called a monochromator, but is really used as a filter. Its structure is basically two diffraction gratings, separated by a slit, with input and output focussing mirrors. The incoming signal from the collecting lenses is focussed on the first grating, which separates the different wavelengths. This spread-out light is then passed through a slit. Because light of different wavelengths is now travelling in different directions, the slit width can be tuned to reject wavelengths outside of a user-defined range. This rejection is often used to eliminate the light at the laser frequency. The light which makes it through the slit is then refocussed on the second grating, whose purpose is only to compensate for any wavelength-dependence in the dispersion of the first grating. This grating is oriented such that its dispersion pattern is the mirror image of that from the first grating. Finally the light is refocussed and sent out to the second stage.

The second stage focusses the filtered light on the final grating. The dispersed light is now analyzed as a function of position, which corresponds to wavelength. The signal as a function of position is read by the system detector. In the present case the detector is a multichannel charge-coupled device array (CCD) in which the different positions (wavelengths) are read simultaneously. The wavelength/intensity information is then read to a computer and converted in software to frequency/intensity. This is the Raman spectrum which appears as the raw data.

Frequency Resolution; Intensity Limits
The Raman data comes out as an intensity/frequency plot. To resolve a Raman peak of a certain width, the resolution of the spectrometer should be smaller than the peak width. In the system described above, the resolution is determined by a final slit between the third grating and the CCD array. The final dispersed image of the sample spot is focussed in the plane of the CCD array; the slit width determines the extent to which the image may shift along the face of the CCD array, and hence the frequency resolution. When the apparatus is properly aligned, the intensity is a function of four factors: the applied laser power, the sample properties (how absorptive/reflective the sample is, and the intrinsic strength of the Raman modes), the width of the spectrometer's admission slit, and the width of the resolution slit. There is a tradeoff between resolving power and intensity. As for signal noise, statistically speaking, Raman is like a random decay process, so the noise in the Raman spectrum follows Poisson statistics. Finally, the CCD array has a certain dark current which is a function of the detector temperature. Typically, reducing the CCD array temperature to about 150 K with liquid nitrogen as a cryogen reduces the variation in dark current to about 20 counts per CCD pixel. Hence 20 counts is the practical limit of a measured signal.

Spectrometer Response; Raman Intensity Standards
Ideally, first stage of the spectrometer filters out the laser frequency, while leaving the rest of the frequencies unaffected, and the second stage spreads the filtered light onto the CCD array, which then reponds uniformly to each frequency. Of course, nearly the opposite is true: every spectrometer in its parts and as a whole has a wavelength (or frequency) dependent transmittance. The actual spectrum displayed by the software is the product of the spectrometer frequency response with the actual spectrum of the scattered light. To know not only the energies of the Raman-active excitations, but also the relative magnitudes of the scattering at different frequencies, one needs a calibration of the spectrometer response to a source with a known spectrum. Typically, one uses a NIST-traceable standard lamp; a recent NIST project concentrated on using the spectrum from a well-characterized piece of luminescent glass, and this is one of the calibrations we use with our instrument.

The material presented in this post have been taken from the webite mentioned below.For more in-depth coverage,please visit : http://physics.nist.gov/Divisions/Div844/facilities/raman/Ramanhome.html

Supercritical fluid chromatography

The following is an article obtained from the website http://www.pharmainfo.netreviews/super-critical-fluid-chromatography-fundamentals-and-applications.html and is a fairly simple and easy-to understand explanation of supercritical fluid chromatography. The references have not been included here and the interested students can go to the above web page to view the references ( labeled as 1,2 etc. throughout the article)

When supercritical fluid chromatography was first developed in 1960, it was considered as a science fiction chromatography and a revolutionary separation technique. But its reputation has slowly ebbed over the years and it is now moving forward by leaps and bounds as a stable analytical method with many advantages over the existing chromatographic methods.

Supercritical fluid chromatography is more versatile than high performance liquid chromatography, more cost-efficient, user friendly, with higher throughput, better resolution and faster analysis times than general liquid chromatographic methods. The instrumentation that is required for supercritical fluid chromatography is versatile because of its multi-detector compatibility. Due to this, supercritical fluid chromatography has formed a niche in the pharmaceutical industry. The present article reviews the fundamentals, instrumentation and varied applications of supercritical fluid chromatography in the analytical arena.

Introduction

The phenomenon and behavior of supercritical fluid (SCF) has been the subject of research right from 1800’s. Hanny and Hogarth in 1879 first demonstrated solubility in (SCF) but first suggestion of supercritical fluid chromatography (SFC) was put forward in 1958 by Lovelock. In 1962 Klesper Corvin and Turner used SFC for separation of porphyrins. Giddings in 1966 and sie rijender in 1967 were responsible for further developments of SFC. jentoft and gouw in 1972 successfully carried out analysis of petroleum derived mixture by SFC. Novotny and Lee et al. demonstrated the first experiments on capillary SFC in 1982. The first commercial packed column of SFC was made available in 1981 and the first commercial capillary column SFC instrument was introduced in 1985 ¹ .

Super Critical fluids: Fundamentals and Properties

Supercritical fluid may be defined from a phase diagram for a pure substance (Fig.1), in which the regions corresponding to solid, liquid and gaseous state are clear. A substance such as CO ₂ can exist in solid, liquid and gaseous phases under various combinations of temperature and pressure. For every substance there is a temperature above which it can no longer exist as a liquid, no matter how much pressure is applied. Likewise, there is a pressure above which the substance can no longer exist as a gas no matter how high the temperature is raised. These points are called critical temperature and critical pressure respectively ² and are the defining boundaries on a phase diagram for a pure substance. At this point, the liquid and vapour have the same density and the fluid cannot be liquefied by increasing the pressure. Above this point, where no phase change occurs, the substance acts as a supercritical fluid. So SCF can be described as a fluid obtained by heating above the critical temperature and compressing above the critical pressure ³ . There is a continuous transition from liquid to SCF by increasing temperature at constant pressure or from gas to SCF by increasing pressure at constant temperature. The term, compressed liquid is used frequently to describe a supercritical fluid, a near critical fluid, an expanded liquid or a highly compressed gas ⁴ .

Fig. 1: Phase Diagram for Pure Substance ⁴

Important Properties of Super CRITICAL fluids ⁵

SCFs have high densities (0.2-0.5gm/cm ³ ) due to which they have a remarkable ability to dissolve large, non-volatile molecules, for example, SC - CO ₂ readily dissolves n-alkanes containing 5 to 30 carbon atoms, di-n-alkyl phthalates with dialkyl group containing 4-16 carbon atoms and several polycyclic and aromatic compounds with many rings. Solvation strength of SCF is directly related to the fluid density. Thus solubility of solid can be manipulated by making slight changes in temperatures and pressures. Certain important processes are based upon the high solubility of organic species in SC -CO ₂ , for example; it has been employed for extracting caffeine from coffee beans to get decaffeinated coffee and for extracting nicotine from cigarette tobacco.

A second important property of SCFs is that dissolved analytes can be easily recovered by simply allowing the solutions to equilibrate with the atmosphere at low temperatures, for example an analyte dissolved in the SC- CO ₂ can be recovered by simply reducing the pressure and allowing to evaporate under ambient laboratory conditions. This property is particularly useful with thermally unstable analytes.

Another advantage of many SCFs is that they are inexpensive, innocuous, ecofriendly and non-toxic. With SCFs at hand, there is no need of any organic solvents. Finally SCFs have the advantage of higher diffusion constants and lower viscosities relative to liquid solvents. The low viscosity means that pressure drop across the column for a given flow rate is greatly reduced. The greater diffusibility means longer column length can be used. Higher diffusion coefficient means higher analysis speed that increases in the order HPLC, SFC and GC. These advantages are important in both, chromatography and extractions with SCFs.

SCFs are finding applications in fractionation of low vapour pressure oils, in several reactions in different areas of biochemistry, polymer chemistry, environmental sciences as well as food, polymer and material industries ⁵ .

Table I: SCFs have densities, viscosities and other properties that are intermediate between those of a substance in gaseous and liquid state ⁶

Property	Gas (STP)	SCF	Liquid
Density (g/cm 3 )	(0.6-2) x 10 -3	0.2-0.5	0.6-2
Diffusion coefficient (cm 2 /s)	(1-4) x 10 -1	10 -3 x 10 - 4	(0.2-2) x 10 -5
Viscosity (G Cm -1 s -1 )	(1-4) x 10 - 4	(1-3) x 10 - 4	(0.2-3) x 10 -2

Table II: Critical properties of some commonly used SCFs ^{7, 8}

Fluid	Critical Temperature (K)	Critical Pressure (bar)
Carbon dioxide	304.1	73.8
Ethane	305.4	48.8
Ethylene	282.4	50.4
Propane	369.8	42.5
Propylene	364.9	46.0
Trimethoflurane	299.3	48.6
Chlorotrifluoromethane	302.0	38.7
Trichloromethane	471.2	44.1
Ammonia	405.5	113.5
Water	647.3	221.2
Cyclohexane	553.5	40.7
n-Pentane	469.7	33.7
Toluene	591.8	41.0

The two supercritical fluids of particular interest are , carbon dioxide and water.

Carbon dioxide ⁹

It is a non-flammable, nontoxic and ecofriendly solvent with low critical temperature of 304K and moderate critical pressure of 73bar.It is miscible with variety of organic solvents and is readily recovered after processing. As it’s a small and linear molecule, it diffuses faster than conventional liquid solvents. It is often used to replace freons and certain organic solvents.

Water ⁹

It has a critical temperature of 647K and critical pressure of 220bar due to its high polarity. The character of water at supercritical conditions changes from one that supports only ionic species at ambient conditions to one that dissolves paraffins, aromatics, gases and salts. Due to this unique property, research has been carried out on supercritical water for reaction and separation processes to treat toxic wastewater. Control of reactions that depend on the dielectric constant of a medium is also possible in supercritical water as its dielectric constant changes from about 78 at room temperature and atmospheric pressure to roughly 6 at critical conditions.

The final choice of SCF depends on the specific application as well as other factors like safety, flammability, phase behavior, solubility at operating conditions and cost of fluid.

Supercritical Fluid Chromatography

Chromatography is an analytical technique used for the separation of complex chemical mixtures into individual components. In SFC, the sample is carried through a separating column by a supercritical fluid where the mixture is divided into unique bands based on the amount of interaction between the individual analytes and the stationary phase in the column. As these bands leave the column, their identities and quantities are determined by a detector ¹⁰ .

SFC is a hybrid of gas and liquid chromatography because when the mobile phase is below its critical temperature and above its critical pressure, it acts as a liquid, so the technique is liquid chromatography (LC) and when the mobile phase is above its critical temperature and below its critical pressure, it acts as a gas so the technique is gas chromatography (GC) ¹¹ . Thus SFC combines some of the best features of each, LC as well as GC. SFC is important because it permits separation and determination of group of compounds that are not conveniently handled by either GC or LC. For example, GC is inapplicable for nonvolatile or thermally unstable compounds. Similarly, LC cannot be employed for compounds with those functional groups that cannot be detected by either spectroscopic or electrochemical detectors used in LC.SFC is a relatively recent chromatographic technique and there is a large amount of research currently underway both in SFC method development and in hardware development.

SFC instrumentation ^{10, 12, 13}

The instrumentation of SFC is similar in most regards to instrumentation for HPLC because the pressure and temperature required for creating supercritical fluid from several gases or liquids lie well within the operating limits of HPLC equipment However, there are two main differences between the two. First, a thermostated oven similar to that of GC, is required to provide precise temperature control of the mobile phase and second, a restrictor or a back pressure device to maintain the pressure in the column at a desired level and to convert the eluent from SCF to a gas for transfer to detector ⁷ .

Fig. II Flow Diagram of Construction of SFC Instrument ¹⁰

In SFC, the mobile phase is initially pumped as a liquid and is brought into the supercritical region by heating it above its supercritical temperature before it enters the analytical column. It passes through an injection valve where the sample is introduced into the supercritical stream and then into the analytical column. It is maintained supercritical as it passes through the column into the detector by a pressure restrictor placed either after the detector or at the end of the column.

Pumps

In contract to HPLC pumping system, pressure rather than flow control is necessary and pulseless operation is more critical. In general, the type of high-pressure pump used in SFC is determined by the column type. For packed columns, reciprocating pumps are generally used while for capillary SFC, syringe pumps are most commonly employed. Reciprocating pumps allow easier mixing of the mobile phase or introduction of modifier fluids. Syringe pumps provide consistent pressure for a neat mobile phase.

Injector

Injection in SFC is usually achieved by switching of the content of a sample loop into the carrier fluid at the column entrance by means of a suitable valve. For packed column SFC, a conventional HPLC injection system is adequate, but for the capillary column SFC, the sample volume depends on column diameters and small sample volumes must be quickly injected into the column, therefore pneumatically driven valves are used.

Oven

A thermostated column oven is required for precise temperature control of the mobile phase. Conventional GC or LC ovens are generally used.

Columns

The strong solvating abilities of mobile phase in SFC makes the careful selection of stationary phases imperative. Basically two types of analytical columns are used in SFC, packed and capillary. Earlier work employed absorbents such as alumna, silica or polystyrene or stationary phases insoluble in SC -CO ₂ . More recent packed column work has involved bonded non-extractable stationary phases such as octadecylsilyl (C ₁₈ ) or aminopropyl bonded silica.

Restrictor or Back-Pressure Device

This is a device, which is used to maintain desired pressure in the column by a pressure-adjustable diaphragm or controlled nozzle so that the same column-outlet pressure is maintained irrespective of the mobile phase pump flow rate. It keeps the mobile phase supercritical throughout the separation and often must be heated to prevent clogging. The pressure restrictor is placed either after the detector or at the end of the column.

A typical restrictor for a 50 or 100 µm open tubular column consist of a 2-10 cm length of 5-19 capillary tubing attached to the column. Alternately the restriction may be integral part of the column formed by drawing down the end of the column in the flame.

Microprocessor

The commercial instruments for SFC are ordinarily equipped with one or more microprocessors to control such variables as pumping pressures, oven temperature and detector performance.

Detector

SFC utilizes mobile phases, which can either be liquid like or gas like. Therefore it is compatible with both HPLC and GC detectors. Conventional gas-phase detectors such as flame ionization detectors and flame photometric detectors, liquid-phase detectors like refractive index detectors, ultraviolet-visible spectrophotometric detectors and light scattering detectors have been employed for SFC. Mass spectrometry and fourier transform infrared spectrometry can also be used effectively with SFC.The choice of detectors will depend upon the mobile phase composition, column type, flow rate and ability to withstand the high pressures of SFC.

Effect of Pressure

Part of the theory of separation in SFC is based on the density of the supercritical fluid which corresponds to solvating power. As the pressure in the system is increased, the density of the supercritical fluid increases and correspondingly its solvating power increases. This in turn shortens the elution time for the eluent as pressure changes in SFC have a pronounced effect on the retention of analytes. This effect is general and similar to programmed temperature in GC or gradient elution in HPLC.

Mobile Phase

There are a number of possible fluids, which may be used in SFC as a mobile phase. However, based on its low cost, low interference with chromatographic detectors and good physical properties (nontoxic, nonflammable, low critical values) CO ₂ is the most used mobile phase for SFC. It is an excellent solvent for a variety of nonpolar organic molecules. In addition, it transmits in the UV. It permits a wide selection of temperatures and pressures without exceeding the operating limits of modern HPLC equipments.

Modifiers ^{6, 9, 14}

CO ₂ is not a very good solvent for high molecular weight, ionic and polar analytes. This can be overcome by adding a small portion of a second fluid called modifier fluid. This is generally an organic solvent, which is completely miscible with carbon dioxide (alcohols, cyclic ethers) but can almost be any liquid including water. Therefore in some applications methanol is introduced in small concentrations (1-20 mol%) to modify solvation power of CO ₂ . Including chemical additives like acids and bases in the modifier can further enhance the solubility. Modifiers can also enhance selectivity of separation and improve separation efficiency by blocking some of the highly active sites on the stationary phase. Small amount (3.5%)of methanol to CO ₂ increases solubility of cholesterol. If an analyte is only soluble in an aqueous solution, it is probably a poor candidate for SFC. Apart from methanol other solvents are also used as modifiers like acetonitrile, ethanol and1-propanol. For highly retained nonpolar solutes, modifiers increase the column efficiency. For polar solutes, they improve retention and efficiency, both.

Comparison of SFC with Other Types of Chromatography

SFC combines some of the characteristics of gas and liquid chromatography, as several physical properties of SCF are intermediate between gases and liquids. Like GC, SFC is inherently faster than LC because the lower viscosity makes use of higher flow rates. Diffusion rates in SCFs are intermediate between gases and liquids. As a consequence, band broadening is greater in SCFs but less, than in gases. Thus, the intermediate diffusivities and viscosities of SCFs result in faster separation than is achieved in LC, accompanied by lower zone broadening than is encountered in GC ¹⁰ .

The mobile phases play different role in GC, LC and SCF. In GC, the mobile phase causes the zone movement. In LC, the mobile phase transports the solute molecule and also interacts with them thus influencing the selectivity. When a molecule dissolves in supercritical medium, the process resembles volatilization but at much lower temperature than that of GC. Thus, at a given temperature the vapor pressure for a large molecule in SCF may be 10 ¹⁰ greater than in the absence of that fluid. As a consequence, high molecular weight compounds, thermally unstable spieces, polymers and large biological molecules can be eluted from a column at a reasonably low temperature. The biggest advantage that SFC holds over GC is the ability to separate thermally labile compounds. This is appreciated in the pharmaceutical fields since roughly 20% of all drugs candidates fall in this category. Unlike GC, by changing the mobile phase the selectivity can be varied in SFC ¹⁵ .

Due to the thermally unstable or non- –volatile nature of many nitrogen and / or sulfur containing compounds, they cannot be analyzed by GC. Even if HPLC is applicable to analyze these compounds, it generates a large number of organic solvents, which need to be ultimately disposed. The disposal cost of organic solvents typically ranges from $5 to $10 per gallon and is constantly rising due to the strict environmental regulations. With the desire for environmentally conscious technology, the use of organic chemicals as used in HPLC could be reduced with the use of SFC. Because SFC generally uses carbon dioxide, collected as a byproduct of other chemical reactions or is collected directly from the atmosphere, it contributes no new chemicals to the environment ¹⁶ .

Like GC, SFC is inherently faster than HPLC, because of its lower viscosity and higher diffusion rates. It is well documented that SFC provides a combination of 3-5 times increase in the speed of analysis and a decrease in the analysis cost through saving in organic solvent ¹⁷ .

Unlike GC or HPLC where the mobile phase dominates the type of detector to be used, SFC utilizes mobile phase, which can be either liquid like or gas like. Therefore both GC and HPLC detectors are applicable to SFC. This multidetector compatibility makes SFC a technique of unparallel success in the analysis of thermally liable species and/or relatively high molecular weight compounds.

Supercritical fluid chromatography has several main advantages over conventional chromatographic techniques (GC and HPLC). The biggest advantage that SFC has over HPLC lies within the differences in the mobile phases. Supercritical fluids are less viscous, possess a higher diffusivity than liquids under HPLC conditions and allow lower pressure drops along an analytical column. This provides not only the ability to increase column lengths, but also allows for faster flow rates. These factors in turn affect capacity ratios, selectivities and theoretical plate heights. It has been reported that 200,000 theoretical plates have been achieved by using eleven analytical (4.6mm i.d.) columns in series. Additionally, SFC can be set up for sub ambient temperatures, which has been key in many chiral separations ¹⁷ .

Applications

By now SFC has been applied to wide variety of materials ¹⁸ including natural products, drugs, foods, pesticides, herbicides, surfactants, polymers and polymer additives, fossils fuels, petroleum, explosives and propellants. Some of the important applications are as follows.

Natural Products

Lipophilic – amphiphilic compounds with properties between volatiles and hydrophilic compounds often create problems in connection with their isolation and analytical determination resulting in an analytical gray area, But SFC has been found to give relatively fast and simple procedures for determination of oil constituents such as chlorophyll and its derivatives ¹⁹ , carotenoids, tocopherols ^{20, 21} vitamins ^22,23 and phenolics ²⁴ which may be important for the oil quality. Thereby it gives a tool to determine the origin of oil and improved possibilities of determination of relations between oil constituents and physical as well as biochemical properties of oil.

Separation of bile salts ²⁵ and common free bile acids like ursodeoxycholic acid and chenodeoxycholic acid in pharmaceutical preparations has been reported using phenylbonded silica column and SFC-CO ₂ modified with methanol ²⁶ .

SFC has been successfully utilized for the separation of underivatized triterpene acids ²⁷ , estimation of caffeine from tea ²⁸ and conjugated bile acids ²⁹ . Capillary-SFC has been used for analysis of panaxadiol / panaxatriol in ginseng and its preparations ³⁰ , vegetable carotenoids ³¹ and pyrrolizidine alkaliods ³² .

Pesticides

Supercritical fluid extraction and chromatography has been used for the analysis of pesticide residues in canned foods, fruits and vegetables wherein pyrethroids, herbicides, fungicides and carbamates have been tested ³³ .

Surfactants

Separation of the oligomers in a sample of the nonionic surfactant Triton X100 has been reported where the detection was by measuring the total ion current produced by the chemical ionization mass spectrometer ³⁴ .

Lipids

As SFC operates at low to moderate temperature, it is most suited for the analysis of high molecular weight lipids like triacylglycerols. Even though HPLC methods give excellent resolutions, the elution times are relatively long and quantitative detection becomes a problem. With GC there is a possibility if thermal cracking of stationary phase or of the sample. Separation of paraffin wax, free fatty acid, mono-di-and tri acyl glycerol detergents like Triton X-100 has been achieved using a capillary column coated with a nonpolar stationary phase at a temperature between 60°-120° at which no thermal damage to lipids is observed ^3.

SFC has also been applied to analyze phospholipids after conversion to diacylglycerol derivatives ³⁵ . Separation of fatty acid methyl esters ³⁶ , biosynthetic polyunsaturated fatty acids (PUFA) ³⁷ , nonsaponifiable lipids ³⁸ , cholesterol and its esters in human serum ^39,40 and food samples ⁴¹ , mono-, di- and triglycerides in pharmaceutical excipients ⁴² has been carried out by SFC successfully. SFC has also been applied to analysis of archaebacterial lipids and glycosphingolipids ⁴³ . A number of review articles have appeared in recent years on this topic ^{44, 45, 46} and should be consulted for more detailed information.

Polymers

SFC provides elution of high molecular weight compounds, polymers and large biological molecules from a column at a reasonably low temperature. Separation of the series of dimethyl polysiloxane oligomers ⁴⁷ and polycyclics aromatic hydrocarbon extracted from carbon black using fluorescence detection ⁴⁸ has been reported. SFC has also been applied to analysis of polyethoxylated alkylphenols ⁴⁹ , polyolefinic antioxidants /light stabilizers ⁵⁰ and polynuclear aromatic hydrocarbons in automobile exhaust ⁵¹ .

Drugs

Modern drug substances are commonly nonvolatile and thermally or chemically labile therefore analysis by HPLC is common over GC. In SFC the conditions are mild and no volatilization is required so it is possible to handle such drug substances by SFC. Separation of various categories of drugs like antidepressants ⁵² , phenothiazine antipscychotics ⁵³ , beta blockers ⁵⁴ , felodipine ⁵⁵ , a new dihydropyridine drug-clevidipine ⁵⁶ , methylated betacyclodextrins ⁵⁷ , vasodialators ⁵⁸ like isosorbide mononitrate, isosorbide dinitrate, cyclandelate, nimodipine, amlodipine, pentifylline,pentoxifylline, lovastatin ⁵⁹ ,pyrethrins ⁶⁰ , isosorbide –5- mononitrate and related compounds in bulk substances and tablets ⁶¹ , atropine ⁶² , indol-3-yl methyl oligomers and ascorbigens ⁶³ , tolnaftate and related impurities ⁶⁴ have been carried out by SFC. Separation and trace estimation of benzidine and its macromolecular adducts ⁶⁵ , fusarium mycotoxins ⁶⁶ , oestrogens ⁶⁷ , combinations of various nonsteroidal antiinflammatory drugs ⁶⁸ like flufenamic acid, mefenamic acid, fenbufen, indomethacin mixtures, flufenamic acid, mefenamic acid, acetyl salicylic acid, ketoprofen and fenbufen mixtures and mixtures of ibuprofen,fenoprofen, naproxen, ketoprofen and fenbufen by SFC has been reported. SFC has also been applied for estimation of prostaglandins ⁶⁹ , determination of mefloquine in blood ⁷⁰ , anticancer drugs like cyclophosphamide, diaziquone, mitomycin C, thiotepa ⁷¹ , sorbitan trioleate in metered-dose inhalers ⁷² , steroids ⁷³ , [14C] propranolol ⁷⁴ , underivatized 2,4-dichlorophenoxy acetic acid ⁷⁵ , determination of urinary metabolites of styrene ⁷⁶ , high speed screening of combinatorial libraries ⁷⁷ , determination of phenylbutazone and its major metabolite oxyphenbutazone in serum, phenylbutazone in dosage forms ⁷⁸ and sulphadoxin in blood plasma ⁷⁹ , mesoprostol from tablets ⁸⁰ , simultaneous SFC of ibuprofen and methocarbomal in solid dosage form. ⁸¹

Chiral compounds

Chiral separation by SFC was first documented in 1985 ⁸² . Due to the high efficiency, fast separation, low temperature analysis and applicability to wide variety of detectors, SFC has now become an attractive alternative for chiral drug separation ⁸³ . The success of SFC in the field of bioanalytical chemistry is well documented. A real break through for SFC in the bioanalytical field has been its contribution to chiral separations and in the near future it may surpass HPLC in the ability to provide appreciable selectivity of molecular stereoisomers ⁴ . SFC has been applied to separation of a large number of enantiomers, diasterioisomers and geometrical isomers like achiral and chiral analysis of camazepam and its metabolites ⁸⁴ , diasterioisomers of Du P105- a novel oxazolidinone antibacterial agent ⁸⁵ , chiral separation of 1,3 dioxolane derivatives ^{86, 87} , diasterioisomers of 2-bromomethyl-2- [(2,4-dichlorophenyl)-1,3-dioxolan-4-yl] methyl benzoate ⁸⁸ , enantiomers of ibuprofen ⁸⁹ , chiral antifungal agents ⁹⁰ , enantiomeric separation of aminoalcohols ⁹¹ , triadimefon and triadimenol enantiomers anddiasterioisomers ⁹² , albendazole sulfoxide enantiomers ^{93, 94} , chiral separation of drugs based on macrocyclic antibiotics ⁹⁵ , separation of cis and trans beta carotene enantiomers ⁹⁶ and resolution of D- and L- alpha amino acid derivatives ⁹⁷ , enetiomeric seperation of six triazole pesticides: cyproconazole, propiconazole, diniconazole, hexaconazole, tebuconazole, and tetraconazole ⁹⁸ , enetiomeric seperation of racemic mixtures of five acidic drugs namely dichlorprop , ketoprofen ,warfarin, coumachlor and thalidomide using macrocyclic antibiotic chiral stationary phases (CSPs) ⁹⁹ .

Organometallics

Separation of metal chelates and organometals of thermally labile category, chelates of transition metals, heavy metals, lanthenides and actinides as well as organometallic compounds of lead, mercury and tin has been carried out by SFC. Determination of solubility of organometallic compounds by SFC is also reported. ¹⁰⁰ .

SFC-MS in Pharmaceuticals

In pharmaceutical industry, analyte concentrations in the picogram -per-milliliter or lower range are a commonplace. In order to detect the realistic concentration levels, a detector with highest sensitivity, broadest selectivity and best resolution must be used. Currently, the detector that fits all of these criteria is the mass spectrometer.

MS vs. Other Available Detectors for SFC ¹⁰¹

The pressure requirements for packed column supercritical fluid chromatography (pSFC) significantly limit the type of detector that can be used successfully. LC detectors need to have pressure tolerances of upto 400 bar. This upper limit is possible with special pressure-resistant absorbance detector cells, but not with the orthogonal window cell designs found in fluorimeters. GC detectors that have been used with both capillary supercritical fluid chromatography (cSFC) and pSFC include, mass spectrometers, flame ionization (FID), electron capture, nitrogen-phosphorus and phosphorus sensitive detectors, just to name a few. In all, these detectors give a wide variety of sensitive selectivity, but each in itself can be restricted in application. The only truly universal detectors available to SFC are FID and MS. FID will produce high background noise in the presence of SFC polar modifiers, concluding that the practical universal detector of choice should be MS.

Some of the applications of SFC-MS include -

Separation of avermectines, a group of potent broad spectrum antiparasitic agents by chemical ionization SFC-MS, determination of impurity profile of macrolid antibiotics using SFC–EIMS and of erythromycin-A from fermentation broth using SFC–CIMS, separation of distereoisomers of cephalosporins using SFC– EIMS, Separation of mixture of 6 pesticides namely atrazine, simazine, dimethirimol, terbacil, manazon, and ethirimol by packed column SFC-EIMS ^{102 ,} Separation of the mixture of 7 triglycerides namely trilaurin, trimyristin, tripalmitin, tristearin, triolein, trilinolien and trilinolenin, using cSFC-EIMS ¹⁰³ , identification of milk fat triacyl glycerols ¹⁰⁴ , Separation of gamma- and alpha – lenolenic acid containing triacyl glycerols in berry oils ¹⁰⁵ , analysis of beta agonists ¹⁰⁶ , characterization of triglycerides in vegetable oils ¹⁰⁷ , analysis of long chain

polyprenols ¹⁰⁸ , analysis of artemisinin ¹⁰⁹ ,determination of allicin in garlic extracts ¹¹⁰ , estimation of trichothecenes ¹¹¹ , characterization of N-linked glycans ¹¹² , characterization of glycosphingolipids by SFC-MS ^113, analysis of organic metallic containing iron, silicon, tin, chromium, arsenic, lead, mercury and antimony using plasma spectrometric detection and inductively coupled plasma mass spectrometry (ICPMS) interfacing SFC ¹¹⁴ , separation of mixture of sulphonamides ^115. A unique feature of using SFC-MS to monitor chiral synthesis is the negligible interference from achiral impurities. In addition, with SFC-MS, enantiomeric excess can be determined with much lower detection limits than in UV and much shorter analysis times compared to normal phase/reversed phase liquid chromatography ¹¹⁶ .

Conclusion

Lately SFC has found a niche in the field of pharmaceutical chemistry and has gained much support in the field of bioanalytical applications. In the overall ranking of chromatographic techniques, it has been judged that SFC falls somewhere between HPLC and GC as the chromatographic method of choice. There are too many examples in the literature testifying to the practicality of using SFC to separate specific compounds. The list of compounds separated by SFC is increasing day by day. SFC enjoys many advantages over the existing chromatographic techniques. But the most important contribution that SFC has made is towards separation of chiral compounds. SFC is enjoying great success in meeting the challenges of stereoisomer separation and may have already surpassed HPLC in the ability to provide appreciable selectivity of molecular stereoisomers. The biggest advantage that SFC holds over GC is the ability to separate thermally labile compounds, which is a very significant application in the pharmaceutical field as 20% of all drug candidates fall in this category. With the advent of SFC-MS, even picogram per milliliter concentrations can be detected easily which is not possible with other techniques. The enforcement of strict quality standards has produced a need for fast, complete and sensitive analysis of drug candidate. SFC can provide the fast and complete analysis and MS can provide universal, sensitive detection. SFC-MS shows great potential in the field of bioanalytical chemistry, but especially in chiral separation and detection. As the science advances, it would be reasonable to foresee the practicality of this analytical technique reach into mainstream of analytical chemistry.