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In modern analytical chemistry, chromatography has become the most widely used separation technique. In the beginning of this century, the first chromatographic experiments were carried out by Tswett, who separated plant pigments. (1) Since these first experiments, many scientists have made substantial contributions to theoretical as well as practical aspects of analytical technique and especially during last decades, chromatography reached its mature state. This development does not only stem from the growing need of many scientists for better methods to separate complex mixtures. At present, chromatography is an essential analytical tool for the determination of various substances in many fields of science and industry. (2) Separation science is an integral part of almost all analytical methods for the determination of organic, organometal and even inorganic species in complex mixtures and matrices. (3)The separation sciences have progressed continuously throughout the years but have grown exponentially in the last few decades. This is due to advances on the instrumental side by the manufacturers but also due to stationary phases in chromatography. (4)Efficient separation processes are needed to obtain high grade products in the food and pharmaceutical industries, to produce high quality water and to recover or remove valuable or toxic metal components from industrial effluents. The conventional separation methods such as distillation, crystallization, precipitation, extraction, adsorption and ion-exchange have recently been supplemented by processes that utilize semi permeable membranes as separation barriers. (5)The development of new, efficient techniques to separate different types of substances is a research area of high priority. Particularly within life sciences there is a great need for efficient and rapid separation methods on both the analytical and preparative scale in order to solve many intricate problems in basic as well as applied research. The purposes of the major categories of separation techniques were reviewed to determine the latest developments and future research needs. One of the most promising developments in separation science and technology is the realization that are relatively small numbers of underlying principles unite the field. Considerable progress has been made in understanding the mechanisms that control entire classes of separation processes. Continued development of theories of separation based on these underlying principles and mechanisms will expedite the application of advances in fundamental fields such as phase equilibrium thermodynamics, coordination chemistry, transport processes, interfacial phenomena, and chemical kinetics to the improvement of existing separation processes and the development of new ones.(6)
Multidimensional separation techniques:-
The term multidimensional separation refers to the combined use of different separation mechanisms to resolve components in complex mixtures. The possible combinations include separation methods such as simple solvent extraction, the different chromatographic mechanisms and electrophoretic techniques. This technique is referred as Coupled Column Chromatography but generally Heart Cutting is used. In multidimensional chromatography, fractions from a chromatographic system are transferred to one or more additional chromatographic separation systems to improve resolution and sensitivity or to decrease analysis time. In these techniques, a selected portion of a chromatogram is transferred to another column that operates according to a different separation mechanism.
Hyphenated separations; LC-MS, CE-MS, LC-NMR:-
The novel analytical techniques must be developed to meet the need of large scale analysis which leads to the hyphenation of multiple techniques like one dimensional and multidimensional liquid chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry, liquid chromatography-nuclear magnetic resonance spectroscopy. Chromatography is one of the most powerful separation techniques based on the different physicochemical properties of the compounds while mass spectrometry has the ability to identify and to quantify thousands of proteins from complex samples (6) Electrospray ionization mass spectrometry (ESI-MS) is the most popular method for protein identification with its powerful MS/MS ability and ease of coupling with liquid chromatography (LC). The multidimensional LC-MS is divided into different types such as Column switching multidimensional LC-MS, Integrated multidimensional LC-MS, Off-line multidimensional LC-MS. Two-dimensional column switching liquid chromatography, where the two columns were coupled with a switching valve was first reported in 1978.(7) Different modes of HPLC (reversed phase, ion exchange, size exclusion, affinity, hydrophilic interaction) have been used for the separation of peptides and other biological samples.(8-12) Integrated multidimensional LC system was first developed by Yates and coworkers.(13-15) In this system, SCX and RP columns were directly coupled or the packing materials were filled in one column. it does not need an interface between the two dimensions which effectively eliminated the valves and dead volume commonly associated with column-switching system. This system was coupled with mass spectrometry for protein identification, and it is called multidimensional protein identification technology (MudPIT).(16) Off-line multidimensional LC-MS is also convenient to perform selective fractionation to focus on a subgroup of peptides such as phosphopeptides or glycopeptides (17-23) An additional advantage of off-line coupling is that different separation columns that are not directly compatible with each other in terms of required solvents could be used, and allows the re-analysis of collected fractions. (5)
Ultra-high pressure LC:-
High Pressure Liquid Chromatography (HPLC) is used in analytical chemistry or biochemistry to separate chemical compounds in mixtures for analysis or purification. The increase in the backpressure required has led to this technique being referred to as ultrahigh-pressure liquid chromatography (UHPLC) when the column backpressure exceeds 10000 psi (l700 bar). (25)
High temperature LC:-
The use of high temperature and temperature programming in Liquid Chromatography (LC) is more popular in laboratories where high throughput is important. Mobile phase viscosity is reduced as separation temperature increases and therefore high flow rates can be utilised to achieve fast separations, without exceeding the standard operational pressure limits of the HPLC system. An additional benefit of using high temperature is that the lower mobile phase viscosity enhances the mass-transfer of the solute between the mobile and stationary phase, resulting in better chromatographic performance. (26)
High efficiency and high throughput separations:-
The high efficiency and high throughput separation methodologies such as solid phase extraction, Microchip solid phase extraction, capillary electrophoresis. The recent developments in High throughput separation methodologies employing nanomaterials such as carbon nanotubes, gold nanoparticles and magnetic NPs etc. The nanoparticles (NPs) exhibit unique physical and chemical properties (28) that have significant influence on separation science. NPs are used as stationary phases in gas chromatography (GC),(29) high performance liquid chromatography (HPLC) (29,30), capillary electrochromatography,(31) capillary electrophoresis (CE),(32) and solid-phase extraction (SPE). (27)
Electro driven separations:-
The Electrically driven separation methods including capillary zone electrophoresis (CZE),(34-36) micellar electrokinetic chromatography (37,38) and electrochromatography (39,40) are currently attracting a great deal of attention. Their common characteristic is that the flow through the "separation column" is effected by electroosmosis rather than by a pressure gradient. Many of the operating parameters (e.g., field strength, buffer concentration, column diameter, column length) affect the separation performance of all electrically driven separation techniques. An integrated instrument for electrically driven separations has been designed. The design facilitates the use of short columns and high field strengths, so that high resolutions and short analysis times can be attained. The detector cell has the required small volume (100 pl) and allows 2 .10-19 mol of fluorescein to be detected. In combination with a short capillary (4 cm), four laser dyes could be separated within 35 s. (33)
Current Practices of Separation Scinces:-
The main purpose of the review article is to offer an opportunity to young researchers to learn more about the current progress in the techniques of chromatography and other separations, hyphenation as well as sample preparation. The Review article is focused on the fundamental and practical aspects of the separation and detection methods, sample preparation as well as related or hyphenated techniques applied to the analytical, preparative and industrial purposes.
The Advances in separation science have provided the impetus for exciting new developments in the biological sciences (eg. genomics, proteomics and medicine), pharmaceutical sciences (eg. drug discovery and characterisation), environmental sciences (eg. ultra-trace residue analysis), forensic science (eg. illicit drugs, DNA fingerprinting, and explosives residues) and other areas.
Chromatography refers to a group of separation techniques that involves a retardation of molecules with respect to the solvent front that progresses through the material. The name literally means "color drawing" and was originally used to describe the separation of natural pigments on filter papers by differential retardation. The same principle is now commonly used for protein separation. Column chromatography is the most common physical configuration, in which the stationary phase is packed into a tube, a column, through which the mobile phase, the eluent is pumped. The degree to which the molecule adsorbs or interacts with the stationary phase will determine how fast it will be carried by the mobile phase. Chromatographic separation of protein mixtures has become one of the most effective and widely used means of purifying individual proteins. (41)
Traditional analysis of liquid chromatography-mass spectrometry (LC-MS) data, typically performed by reviewing chromatograms and the corresponding mass spectra, is both time-consuming and difficult. Mass spectrometry-based proteomics has the capability to identify hundreds of proteins in a single experiment, and has become an important analytical technology in modern biological and medical research. In a classical liquid chromatography-mass spectrometry (LC-MS) experiment, the resulting peptides are then separated by reversedphase micro- or nano-capillary chromatography. Peptides eluting from the LC column are usually ionized by electrospray and then introduced into the mass spectrometer.(42,43)
Capillary electrophoresis (CE) is an establishing separation technique of choice effective for a wide spectrum of analytes, ranging from small inorganic ions to DNA macromolecules as it provides reliable data, requires minimal sample preparation and offers a high degree of automation. CE is an alternative to more traditional methods such as gel electrophoresis and liquid chromatography and is employed to detect both high and low affinity molecular interactions, and separation of both charged and non-charged molecules. CE technique covers different modes that essentially rely on a high voltage electric field being applied over a solution which is held in a capillary tube. CE has proved to be an efficient and versatile approach for physicochemical characterization of bioactive molecules and resolution for charged substances such as biomolecules, low molecular weight basic or acidic drugs and ions. CE is now an established technique in several areas of analysis and is capable of detecting millimolar to nanomolar binding interactions and offers the benefits of a powerful and proven technology applied to drug discovery screening on a wide variety of targets such as enzymes, membrane receptor domains, structural proteins, nucleic acid complexes, bioactive peptides, protein-protein interactions and antibodies. CE can also determine chiral purity in pharmaceuticals and can be successfully used to support aspects of early drug discovery and drug development testing, analysis of protein-base pharmaceuticals and routine quality control of marketed pharmaceuticals. CE has found application in the analysis of complex carbohydrates and in the increased associated activities of CE-MS techniques in the biotechnology and biopharmaceutical industry. CE-frontal analysis provided a method on the interaction of drugs with plasma proteins. Various applications of hyphenated capilliary electrphoresis techniques include characterization of quantum dots and quantum dots-conjugated biological molecules. Immunoaffinity capillary electrophoresis has been reported as a versatile tool for determining protein biomarkers in inflammatory processes and for total Immunoglobulin E quantification in serum. CE technology has been valuable for the comprehensive characterization of macromolecules, used both as biologics and in proteomic study, and to analyze and characterize therapeutic proteins in biotechnology.(44)
The high resolution potential of capillary electrophoresis makes CE-techniques valuable for separations of metal species. The separation of metals with different oxidation states or of organo metal compounds are possible with CE .(46,47,48,49,50) ICPMS is an element specific multi-element detector, providing extremely low detection limits. Therefore, the combination of CE with ICP-MS promises a powerful tool for metal speciation. (46,47,49) (45)
The separation sciences have assumed an increasingly significant role in Bioanalytical chemistry.(52) Since biological samples are usually complex, separation techniques are necessary in order to isolate the components of interest before their identification and characterization can be attempted. Of the various separation techniques, high performance liquid chromatography (HPLC) and Capillary electrophoresis (CE) are usually employed. (53,54)The Electrospray ionization (ESI) interface permits the mass spectrometric analysis of large biomolecule with mass up to several hundreds thousand with significantly lower mass scan range. Thus it proves to be suitable for the coupling with HPLC,CE and even Electrochromatography (55-65). The success of coupling CE separation system to ESI-MS detection system depends on many factors including the design and operating parameters of the electrospray interface, (66) the composition and pH of the buffer solution, (67) the chemical properties of the analyte, (68) and composition of the sheath liquid (69,70-72). (51) Microscale separation techniques including high performance capillary liquid chromatography and capillary electrophoresis have a number of practical advantages over conventional scale analytical separation methods. High performance capillary electrophoresis (HPCE) is an important microseparation technique in life sciences as well as biotechnology and environmental research areas. Unlike high performance liquid chromatography (HPLC) in which separation is due to the partition of solutes between the mobile phase and stationary phase, separation by CE is based on the difference in charge to mass ratio of the analytes. One of the advantage of ESI-MS and ISP-MS with the determination of biological compounds with high molecular masses like peptides, proteins, enzymatic digests, biopolymers which lies in the multiple charging of the analytes that can occur under ESI conditions. (73)
Capillary electrophoresis (CE) also known as capillary zone electrophoresis (CZE), can be used to separate ionic species by their charge and frictional forces. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. The technique of capillary electrophoresis (CE) was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. While its use has been sporadic, CE offers unparalleled resolution and selectivity allowing for separation of analytes with very little physical difference.
Capillary zone electrophoresis (CZE) is one of the methods employed in analytical
chemistry for the separation of mixtures of chemical species by exploiting their different electrophoretic mobilities in aqueous solution. (74,75)
The development of CE methods to separate diverse analytical samples has been growing very rapidly over the past decade, (77-81) and the technique has demonstrated its efficiency in many applications, (82,83) including the determination of antibiotics. (84-86) Despite of these, CE is not very common for the determination of sulfonamides. Some papers have been published, using mainly UV (87-89) or amperometric detection (90), applying the CZE mode (91-95) or MEKC (93,96-98) in different matrices such as pharmaceutical compounds, biological fluids, or food of animal origin. (76)
Quantitative interpretation of protein and peptide mobilities obtained by capillary zone electrophoresis to this point has been based on generic relationships for their dependence on net charge.(100) This approach has seemingly allowed the net charge (valence) of a protein to be determined solely on the basis of mobility differences stemming from alteration of the protein charge either by mutation (101) or by chemical modification. (102,99)
The employment of CE for the analysis of drugs and pharmaceuticals has been demonstrated in excellent reviews. (104,105) The popularity of CE in the pharmaceutical fields has been accelerated by its simplicity, high efficiency and selectivity, and large separation capacity. Indirect UV detection is often used in CE for the analysis of chemical compounds that lack an intrinsic chromophore or fluorophore. In indirect detection, an absorbing or fluorescing ion, typically called the probe, is added to the buffer. The probe ions are displaced by analyte ions of the same charge and similar mobilities. Displacement of the probe by the analyte produces a decrease in signal. (106,107) The main advantage of indirect photometric detection is that it offers universal detection. There are only a few reports discussing the use of CE with indirect detection for drugs and pharmaceuticals. (108) The aim of this work was to explore the potential of indirect UV detection for determination of vigabatrin, a cationic model drug with poor UV absorptivity, in its pharmaceutical dosage forms. (103)
Capillary Electrochromatography (CEC) is a relatively recent electrokinetic separation technique which involves application of an electric field across a packed capillary in order to obtain separations which are achieved by the use of both electrophoretic and chromatographic mechanisms. CEC is a hybrid of both HPLC and Capillary Electrophoresis. A variety of detectors have been employed most commonly on-column UV detection, in-column fluorescence detection and mass spectrometry. Current applications of CEC also include CEC with laser induced flurescence (CEC-LIF) and electrospray ionisation MS. (109,110)
Over the last decade, monoliths or continuous beds have emerged as an alternative
to traditional packed-bed columns for use in capillary electrochromatography (CEC)
and micro-high performance liquid chromatography (Âµ-HPLC). Monolithic columns
can be divided into two categories: silica-based monolithic columns and rigid organic polymer-based monolithic columns resulting from the polymerization of acrylamide, styrene, acrylate or methacrylate monomers. In this paper, the chemistry and most recent applications of these various types of monoliths in both CEC and Âµ-HPLC are presented. (111)
This review summarizes applications of CEC for the analysis of proteins and peptides. This "hybrid" technique is useful for the analysis of a broad spectrum of proteins and peptides and is a complementary approach to liquid chromatographic and capillary electrophoretic analysis. All modes of CEC are described - granular packed columns, monolithic stationary phases as well as open-tubular CEC. Attention is also paid to pressurized CEC and the chip-based platform. (112)
The separation of basic solutes at low pH by capillary electrochromatography (CEC) has been investigated. The feasibility of separation of basic solutes by CEC was demonstrated. Influence of operational parameters, solvent composition, pH, temperature on retention and selectivity of the separation of a mixture of basic, neutral and acidic drug standards has been investigated. The observed elution behavior has been modeled to account for both chromatographic retention and differential electrophoretic mobility of the solutes. This model was verified experimentally. It is demonstrated in this work that the elution window of solutes in reversed-phase CEC is expanded to range from -1 to âˆž. (113)
High-temperature liquid chromatography (HTLC) is recognized today as a valuable technique in reversed phase high-performance liquid chromatography (RP-HPLC). Column temperature can play a role in reducing analysis time, modifying retention, controlling selectivity, changing efficiency or improving detection sensitivity. The different effects of high temperatures on reversed-phase separations, the practical limitations due to the instrumentation, the limits and the main advantages of HTLC, especially for the separation of polar and ionized compounds, are reviewed. (114)
The high temperature liquid chromatography (HTLC) reveals interesting chromatographic properties but even now, it misses some theoretical aspects concerning the influence of high temperature on thermodynamic and kinetic aspects of chromatography: such knowledge is very essential for method development. The thermodynamic properties were evaluated by using different mobile phases: acetonitrile-water, methanol-water and pure water. The type of mobile phase was found to play an important role on the retention of solutes. The kinetic aspect was studied at various temperatures ranging from ambient temperature to high temperature (typically from about 30 to 200 â-¦C) by fitting the experimental data with the Knox equation and it was shown that the efficiency is improved significantly when the temperature is increased. In this paper, we also discussed the problem of temperature control for thermo stating columns which may represent a significant source of peak broadening: by taking into account the three main parameters such as heat transfer, pressure drop and band broadening resulting from the preheating tube, suitable rules are set up for a judicious choice of the column internal diameter. (115)
Temperature changes, in particular elevated temperatures have become a valuable variable which should be considered during method development in liquid chromatography because they can lead to a reduction in analysis time, can offer an alternative selectivity and are claimed to increase column efficiency. (117-120) Previous studies have demonstrated the applicability of high-temperature separations to a range of pharmaceuticals (121), with examples such as analgesics (122), vitamins (123) and thalidomide (124) and compounds of environmental interest such as the triazine herbicides. (125) (116)