Isotachophoresis: A Technique of Electrophoresis for the Separation of Charged Particles
Shobhit Shrivastava*, Ashish Jain, Sushil Mhaske and Satish Nayak
Bansal College of Pharmacy, Kokta, Anand Nagar, Bhopal-462021, M.P., India.
*Corresponding Author E-mail: shrivastava.shobhit@gmail.com
ABSTRACT
An overview mapping recent trends in the isotachophoresis technique, for separation and analysis of wide range of bioactive. Isotachophoresis utilizes electro-separation technique, which is generally based on the electro-kinetic phenomenon. In Isotachophoresis the separation occurs according to difference in ions motilities. An overview focused on technique used, instrumentation, advantages in isotachophoresis. It also comprises diverse researches that have been done by the isotachophoresis.
KEYWORDS: Isotachophoresis, Electrophoresis, Separation Technique
INTRODUCTION:
In recent years electrophoretic separation methods have become important analytical tools and have generally been used in the field of molecular biology and biochemistry where they have proved useful tool in the analysis of things such as proteins, vitamins, food additives etc. However some techniques, like capillary zone electrophoresis and isotachophoresis, can also be used for the separation of ionic substances [1]. Isotachophoresis is an electrophoresis method that van be used for the quantitative and qualitative analysis of ionic species. Isotachophoresis (Greek: iso = equal, tachos = speed, phoresis = migration) is a technique in analytical chemistry used to separate charged particles. It is a further development of electrophoresis and powerful separation technique using a discontinuous electrical field to create sharp boundaries between the sample constituents. In Isotachophoresis the separation occurs according to difference in ions mobilities. Theses mobilities cause the zones of various ions to migrate with equal velocity and just behind to each other, a steady state. Then later these zones can be detected by appropriate detection system2.
About technique:
Isotachophoresis utilizes electro-separation technique, which is generally based on the electro-kinetic phenomenon. It is simple, high resolution technical method which is utilized for the analysis of ionic substances.
The technique based on discontinuous electrolyte system which consists of a leading electrolyte, having higher mobility tan any of the ions in the sample being separated, and a trailing electrolyte, having lower mobility than any of the ions of interest in the sample. The sample is introduced between leading and trailing electrolyte, now when an electric current is applied a uniform strength electric field occurs over the sample zone. Then the separation of ions occurs due to different migration velocity of different species. The different parameters which determines movement of ionic species under an electric field can be represent as:
V= µE
Where, V= electrophoretic migration velocity
µ= electrophoretic mobility of ion
E = strength of applied electric field
Hence the decreasing electrophoretic mobility of ions species separates them in sample. In this way a situation is reached where each zone contains only one ionic species, this state is known as the steady state. At this stage migration rate of all the zones will be same; this ca be determined by migration velocity of the leading electrolyte. In the steady state the electric field strength applied across the zones increase from the leading zone to tailing zone as the mobility decrease. It’s a self correction of zone boundaries, which is a one of the significant feature of isotachophoresis system, which results in high resolution of separation. The concentration of all the separated zones can be determined by the concentration of the leading electrolyte, which is another useful feature of isotachophoresis.1
Instrumentation:
The apparatus used in isotachophoresis is comprised with a column, which arranged between two electrodes, the sample to be separated being introduced between two electrolytes a leading and trailing electrolyte respectively and detector that is attached with the column, for the detection of different zone of samples. In isotachophoresis a separation is carried out by introducing a sample , containing ions of certain polarity, between two electrodes; a leading electrolyte containing ions of polarity same as to sample but will migrate with a greater mobility than the sample ions, are introduced into the part of column which are situated between sample and electrodes towards said ions will migrate after applying voltage and a trailing electrolyte of a said polarity but with a lower mobility than sample ions, are introduced between the sample and the other electrode.
The separation of ions is achieved in form of separated and sharp boundaries that will be obtained between the zones formed by different ions. In these zones the concentration of ions depends on the mobility of each ions and their concentration in initial sample mixture. Finally the detection of resolved ions is done either by conductivity or UV/VIS detection. The higher resolution of sample zone is obtained by means of a thermal or optical detector arranged in the column.3
The commercially available system has power supply which can provide 0-15 kV and current of 0-500 µA. It has valve injection system which includes a 30 µl sample loop. Two fluorinated ethylene-propylene copolymer (FEP) made separating column are used. A pre-separation column with 0.8 mm internal diameter, 90 mm long and a 160 mm long, fe0.3 mm internal diameter, analytical column. Each column is fitted with an on column conductivity detector.
Isotachogram:
for the individual zones the different signal intensities recoded by the detector. In isotachophoresis, the identification parameter of a substance is the signal height, which is characteristic for each of the different zones and is a specific parameter for a respective zone. The quantification of the analytes is done by recording the zones length for the individual zones.
Advantages:
· Simple design: An advantage with the isotsachophoersis is that it requires a more simple design as compared to chromatographic methods.
· Fast development: The optimum separation condition easily and effectively attained by variation in the pH, this gives a rapid separation of the analytes.
· Low chemicals required: IT generally require aqueos solution only so low consumption of chemicals saves te cost of many expensive solvents.
· Minimum sample preparation: In isotachophoresis it is generally possible to define condition such that the sample matrix is not transported through the analytical separation path. the advantage of this is that practically all samples can be injected either directly or require only a simple dilution step before analysis, whereas with chromatographic techniques the flow of the mobile phase usually cause the sample matrix to also transported through the analytical column. This technique is expensive and time consuming and require sample preparation steps before the analysis.
· Operating cost: The above description also describe the economic consideration and generally lead to lower cost per analysis as compared with conventional methods.
Recent development:
1. Jozef Polonsky et al., (2003) has been separated and quantified nine organic acids and phosphate in tobacco by capillary isotachophoresis. he found Two operating systems for the separation : hydrochloric acid (10 mmol l-1 ) adjusted with -alanine to pH 2.9 plus 0.1% poly(vinylpyrrolidone) was used as the leading electrolyte and 10 mol l-1 nicotinic acid as the terminating electrolyte and hydrochloric acid (10 mmol l-1) including 5.5 mmol l-1 1,3-bis[tris (hydroxy methyl)methyl amino]propane plus 0.1% poly(vinyl pyrrolidone) was used as the leading electrolyte and 5 mmol l-1 2-morpholino ethanesulfonic acid as the terminating electrolyte. he was observed linearity from 0.008 to 0.100 mmol l-1 with a coefficient of determination of 0.999. The separation of anions was achieved in less than 16 min. The minimal sample pretreatment and relatively low running cost make isotachophoresis a good alternative to existing methods.[4]
2. Milton L. Lee et. al.(2003) successfully coupled capillary isotachophoresis (ITP) and comprehensive isotachophoresis–capillary electrophoresis (ITP–CE) to electrospray ionization (ESI) orthogonal acceleration time-of-flight mass spectrometry (TOF-MS) using angiotensin peptides as model analytes. ITP–TOF-MS and ITP–CE–TOF-MS utilize of for the analysis of samples containing analyte amounts sufficient to form flat-top ITP zones (30 mM) as well as for samples with trace analyte amounts (0.3 mM) was studied. The fused-silica columns were coated with poly (vinyl alcohol) to suppress electroosmotic flow that can disrupt ITP zone profiles. The sample loading capacity in both ITP and comprehensive ITP–CE was greatly enhanced (up to 10 ml) compared with typical nano liter-sized injection volumes in CE. He was concluded that ITP–TOF-MS alone was adequate for the separation and detection of high concentration samples. At lower concentrations of analyte the outcome was different, where mixed zones or very sharp peaks formed. With formation of mixed zones, ion suppression and discrimination could occur, complicating quantitative determination of the analytes. This problem was effectively overcome by inserting a CE capillary between the ITP and TOF-MS. In such an arrangement, samples were pre-concentrated in the high load ITP capillary and then injected into a CE capillary where they were separated into non-overlapping peaks prior to their detection by TOF-MS. The advantage of this comprehensive arrangement is that there is no need to discard portions of the sample in order to avoid overloading of the CE capillary. The whole sample is analyzed by multiple injections from ITP to CE. Thus, this method can be used for the analysis of complex samples with wide ranges of component concentrations.[5]
3. Jeff E. Prest et al. (2004) has been developed a method, the miniaturised isotachophoretic analysis of amino acids. The problems of carbonate contamination which occur when performing separations at alkaline pH levels was overcome by using glycolate as the leading ion. The method has been used on a poly (methyl methacrylate)microdevice with integrated on-column conductivity detectors. The behaviour of a range of common amino acids was investigated andsuccessful separations of up to seven amino acids were made. [6]
4. S.J. Baldock et. al (2004) designed a micro-device furnished with a novel sample injector, capable of delivering variable volume samples, for miniaturised isotachophoretic separations . Micromachining by direct milling was used to realize two flow channel network designs on poly(methyl methacrylate) chips. Both designs comprised a wide bore sample channel interfaced, via a short connection channel, to a narrow bore separation channel. Superior injection performance was observed with a connection channel angled at 45◦ to the separation channel compared to a device using a channel angled at 90◦. Automated delivery of electrolytes to the microdevice was demonstrated with both hydrostatic pumping and syringe pumps; both gave reproducible sample injection. Isotachophoretic separations of model analytes (metal ions and an anionic dye) demonstrated the potential of the device. Separations of ten metal cations were achieved in under 475 s.[7]
5. Yongwon Jeong et al. (2004) worked on Transient isotachophoresis (TITP) of a highly saline solution on a polydimethylsiloxane chip of 18mm × 12 mm. The micro-channel dimensions were 30m in width and 30m in depth. The injection plug length was varied from 12 to 28mm to inject samples of large volume while the total channel length was fixed at 75 mm. Chloride ions abundant in the saline sample and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonate ions in the background electrolyte were used respectively as the leading and terminating electrolytes for TITP of anionic analytes fluorescein and 2,7-dichlorofluorescein.[8]
6. Takeshi Hirokawa et al. (2004) found that concentrations of inorganic anions, both as individual species and biotransformation products, in physiological fluids are of strong concern in clinical studies. The analytical methodologies have either required different analytical procedures to determine these analytes in plasma and urine, or extensive sample preparation, or unconventional and often expensive detection schemes, or both. A simple and sensitive capillary electrophoresis (CE) method with direct UV detection was developed for the simultaneous determination of iodide, bromide and nitrate in human plasma and urine, with a special focus on reliable quantification of the trace serum iodide. With the latter objective, he incorporated a method transient isotachophoresis (tITP) procedure enabling an efficient on-line pre-concentration of iodide (limit of detection, 1.4g l−1) as well as other moderately mobile analytes that fall into the tITP range.[9]
7. Shin-ichiro Miura et al (2005) described a method for the charge isolation of lipoproteins using capillary isotachophoresis (cITP), and suggested that this method may be clinically useful throughut the acute phase of myocardial infarction(MI). cITP was performed according to the method of Bottcher et al. [10] with some modifications [11]. this is the first report of the use of cITP in a patient with acute IM (AMI). HDL and LDL can be separated into electronegative HDL or LDL and major LDL with the oxidation of LDL by cITP.[12]
8. Duˇsan Kaniansky et al (2005) determine the total sulfite content in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP–ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite.[13]
9. Marie Pospısilova et al (2005) developed isotachophoersis (ITP) separation of acebutolol{N-{3-acetyl-4-[(2-hydroxy-3-(isopropylamino)propoxy] phenyl butanamide]} which is a cardioselective -blocker with a potent antihypertensive and antiarrhythmic effect. The optimised operational system of electrolytes consisted of 10mM potassium acetate +10mM acetic acid (pH 4.65) as the leading electrolyte and 10mM -alanine with pH ≈4 (adjusted with acetic acid) as the terminating electrolyte. The driving and detection currents were 75 and 20A, respectively and the analysis took ≈13 min. Under these conditions the effective mobility of acebutolol was determined as 20.7×10−9 m2 V−1 s−1. The method was applied to the assay of acebutolol in Sectral tablets, Acecor tablets, Apo-acebutol tablets (nominal content 400 mg of acebutolol per tablet) and Acebirex tablets (nominal content 200 mg of acebutolol per tablet). No interference from any excipients present in the tablets was observed. [14]
10. J. Sa´decka et al (2005) was investigated modified capillary isotachophoresis for the separation of 2-arylpropionic acids (fenoprofen, flurbiprofen, ibuprofen, ketoprofen and naproxen) and benzoic acid and its derivatives (salicylic, acetylsalicylic and gallic acids). The relative step height (RSH) values of analytes were found to be dependent on the type and concentration of the surfactant {Non-ionic surfactant (Brij 35, Tween 20, Tween 80 and Tergitol NPX)}. The strength of the affinity of the 2-arylpropionic acids to the non-ionic micelles was found to be as follows: flurbiprofen > fenoprofen > ibuprofen > naproxen > ketoprofen. Separation of all acids was obtained with the Tween 20 (1.5%, w/v) in the leading electrolyte 10 mmol L−1 hydrochloric acid/l-histidine (pH 6.0). [15]
11. Pavel Jac et al (2006) presented an overview mapping recent trends in the determination of polyphenols of natural origin (mostly flavonoids) and their synthetic derivatives by electromigration methods. The overview is focused on capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with various detection methods. Techniques comprisingon-line pre-separation such as isotachophoresis (ITP)-CZE and flow-injection-CZE, chiral separations and CZE evaluation of antioxidation activity are also discussed.[16]
12. Jeff E. Prest et al (2007) has studied the behaviour of DNA under conditions of miniaturised isotachophoresis. An electrolyte system comprising a leading electrolyte of 5mMperchloric acid at pH 6.0 and a terminating electrolyte of 10mMgallic acid was used to perform isotachophoresis of DNA containing samples on a miniaturised poly(methyl methacrylate) device. Under such conditions it was found that no separation of DNA fragments was observed with the substance migrating instead as a single isotachophoretic zone.The result showed the method is unsuitable for analysis DNA, it offers significant potential as a means of sample preparation for subsequent analysis using another method. This is because the single zone of DNA formed is preconcentrated to a constant concentration governed by the leading ion and is separated from all species with different effective electrophoretic mobilities.[17]
13. Jeff E. Prest (2007) has been developed a new method for the determination of small inorganic anions using isotachophoresis.This method makes use of indium(III) as a counter ion to manipulate the effective mobilities of inorganic anion species by means of complexation reactions. This new procedure successfully allowed the simultaneous determination of nitrate, chloride and sulphate to be realized on a capillary scale instrument and in a chip-based separation device. The electrolyte system developed to allow the separation to be achieved employed a 10 mM bromide-based leading electrolyte containing 1.25 mM indium(III) at pH 3.15 and a terminating electrolyte of cyanoacetic acid [18]
14. S.J. Baldock (2008) designed a variable-volume sample injector and evaluated it for performing sample introduction protocols for carrying out miniaturised isotachophoresis (ITP) separations. The microdevice design features a wide bore sample loop channel connected to a narrower bore separation channel via a short injection channel angled at 45°. An additional side arm channel located at the injection point enabled a range of injection strategies, using a gravity-fed system, to be implemented and assessed. A model analyte was used to demonstrate the versatility of the injector design for low and high sample loading regimes suitable for concentrated and dilute sample solutions, respectively.[19]
15. Tianlin Wang (2008) was first time developed a transient isotachophoresis–capillary zone electrophoresis (tITP–CZE) method for determining iodate in table salt. Sensitivity enhancement was accomplished by coupling on-capillary tITP with CZE. The interference of sample matrix was overcome by using electrophoretic buffer containing high concentration of sodium chloride. The optimal terminating electrolyte for tITP was 1500 mmol/L phosphate and the separation buffer of capillary zone electrophoresis was 10 g/L sodium chloride (pH 8.0) containing 20 mmol/L cetyltrimethylammonium chloride (CTAC). Calibration graphs based on peak height and peak area showed good linearity. The quantitative results of iodate in table salt measured by the tITP–CZE method were compared with those measured by the redox titration and there was no significant difference between the two means. The method developed was sensitive, fast and simple.[20]
16. Jeff E. Prest (2008) has been developed a new method for the determination of the chlorate, chloride and perchlorate anions in inorganic explosive residues by using isotachophoresis(ITP).Indium(III) is used to allow the determination of chloride using nitrate as the leading ion and cyclodextrin is used to allow the separation of chlorate and perchlorate. Separations were carried out using a miniaturised poly(methyl methacrylate) (PMMA) separation device. Successful determinations of these samples were achieved with no interference from other anions typically found in inorganic explosive residues. [21]
17. Ying Chen (2008) was developed a new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration . The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT8–13), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. The detection limits based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT8–13, NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT8–13 in human cerebrospinal fluid sample.[22]
18. Peter Mikus (2008) worked on the possibilities of column-coupling capillary electrophoresis (CE-CE) combined with chiral selector (2-hydroxypropyl-β-cyclodextrin, HP-β-CD) and fiber-based diode array detection (DAD) for the direct quantitative enantioselective determination of trace drug (amlodipine, AML) in biological multicomponent ionic matrices (human urine). Capillary isotachophoresis (ITP) served as an ideal injection technique in CE-CE. Moreover, the ITP provided an effective on-line sample pretreatment prior to the capillary zone electrophoresis (CZE) separation.The proposed ITP–CZE–DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied to an enantio-selective pharmacokinetic study of AML.[23]
CONCLUSION:
Isotachophoresis shows its superiority to conventional separation techniques when the maximum resolution is achieved with the latter. The choice of the experimental parameters remains complex but is very rewarding when successful. The isotachophoresis was proved to be a simple and inexpensive technique in sample analysis. Good analytical performance, short analysis time, no sample pretreatment and low running cost makes isotachophoretic separation of charged particles in a very promising manner.
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Received on 20.07.2009 Modified on 23.09.2009
Accepted on 30.10.2009 © RJPT All right reserved
Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 642-647