The capillary format of the tITP was described and used for the first time in 1992 by Foret et al. [54]. In 2003 Kurnik et al. used computer simulations to study the
separation of electrokinetically injected fluorescein inside a chip with a double-T cross geometry using an electrokinetic sample injection and consequently tITP-ZE separation [116]. The main aim of this work was to investigate the effects of pinch-and-pull-back current and floating electrodes while using tITP for sample stacking. The predicted better sensitivity for a method with floating electrodes was confirmed using practical tITP experiments and could be explained by the sample loss when the pinch-and-pull- back methods was used. Practical experiments were done with fluorescein, a tyrosine kinase assay using a FITC labelled peptide substrate, and twelve small fluorescent molecules eTagTM. The device was cast in acrylic resin with channels 30 µm deep and 85 µm wide. In the same year Vreeland et al. described the tITP-ZE separation of fluorescent eTagTM reporters in a commercial microfluidic PMMA chip with a double-T cross geometry [117].
In 2003 Xu et al. used the commercial instrument Shimadzu MCE-2010 with a silica glass single channel chip for separation of six SDS denatured proteins followed by UV detection [118]. Prior the analysis the channel was filled with LE and sample was loaded in TE reservoir and electrokinetically injected. The sample was then manually
26 replaced with TE and TE was electrokinetically injected, forming an ITP system. The voltage was switched off and the TE was manually replaced with BGE (same
composition as LE without dextran), creating a tITP system when the voltages were re- applied. The analytes were separated by GE. The same method was applied for
separation of a standard DNA ladder marker consisting of 16 DNA fragments in a range of 50 to 800 bp [119], and for analysing DNA fragments obtained after 30 PCR cycles [120]. In the last mentioned work, a simple cross geometry chip was used as a single channel chip by blocking non-used channels using a small pullback current. This work was later followed by Hirokawa et al. [109] as described in section 1.1.2
Preconcentration ITP.
Jeong et al. described a sensitive method for tITP-ZE of fluorescein and
2.7-dichlorofluorescein in highly saline samples (250 mM NaCl). A LOD of 3 pM was obtained for both analytes using a PDMS chip of double T cross geometry treated with fluorocarbon neutral surfactant FC-PN (EOF suppressor) [121]. The chip was filled with BGE containing N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) as a terminating anion. Depending on the geometry, a plug (12, 20 or 28 mm long) of high salinity sample was electrokinetically transported from the reservoir with sample into the waste reservoir through the spiralled part of the chip by applying constant voltage. The injection time was judged by the current as a steady current indicates steady state. The tITP separation was initiated by applying voltage between reservoirs at either end of the main separation channel, each filled with with BGE. The chloride from the high salinity sample acts as a leading anion until it dissipates,
resulting in tITP.
A number of examples in the literature used tITP-GE, including for the analysis of fluorescently labelled human serum albumin and its monoclonal antibody
27 immunocomplex [122], and the immunocomplex of FITC labelled bovine serum
albumin with specific mouse antibody [123].
In 2008 Lin et al. presented a chip for separation of dsDNA from PCR reaction [124]. Authors described the method as ITP-GE, but based on the procedure of electrolyte loading and voltage application, the method is classified here as tITP-GE. All the channels in the PMMA chip were 20 µm deep and the functionalities can be divided in three parts. First, the “injection” part contained four reservoirs (TE, sample, LE and vacuum) joined with a 500 µm wide and 10 mm long channel. Second the “stacking” part contained 140 µm wide and 20 mm long channel ending at the junction with separation channel and the first waste reservoir. Third, the GE separation channel which had the same width as stacking channel and a length of 25 mm. Prior to the separation the chip was hydrodynamicaly filled with LE and the sample and TE reservoirs were filled with sample and TE, respectively. Then negative pressure was applied to vacuum reservoir and zones of TE and sample were introduced between zones of LE in injection channel. The tITP separation was initiated by applying high voltage between LE reservoir and waste reservoir 1, followed by GE by switching the separation voltage from waste reservoir 1 to waste reservoir 2. Because the separation voltage was applied between reservoirs filled with LE, this work is classified as tITP not ITP.
As summarized in Table 1.III, a number of groups used tITP GE for DNA analysis using relatively simple cross or double T cross geometry devices [125-127]. In 2009
Liu et al. showed more than a hundredfold improvement in sensitivity using tITP CGE
in comparison with CGE [128]. The chip they used consisted of two parallel channels joined at reservoir 2 (R2); the first one joined reservoirs R1 and R2 and the second one reservoirs R3 and R4. The fluorescent detector was placed close to R1. Reservoir 1
28 (R1) contained electrolyte with sieving matrix (SE1), reservoir 2 (R2) was for waste (W), reservoir 3 (R3) for electrolyte without sieving matrix (SE2) and reservoir 4 (R4) for the DNA sample. Prior the analysis R1 was filed with SE1 and negative pressure was applied to R2. Than R3 and R4 were filed with SE2 and the negative pressure was applied again to R2 to fill the channels with SE2. After filling of the channels, SE2 in R4 was replaced with the DNA sample, which was injected by applying a voltage between R2 and R4. The tITP-GE was initiated by applying a voltage between R1 and R3, applying pull back voltage to R4 during the separation. The chloride worked as a leading anion to focus the DNA in the separation electrolyte without HPMC. When the tITP zones reached the separation electrolyte with HPMC, the DNA fractions separated by GE according to their length.