INTERFACE FOR THE SYSTEM OF SAMPLING, PREPARATION, AND ANALYSIS OF A SAMPLE, ESPECIALLY BY CAPILLARY ELECTROPHORESIS

Abstract

The interface for the system of sampling, preparation, and analysis of a sample, especially by capillary electrophoresis, consists of a cross connector, sampling capillary, separation capillary, capillary supplying BGE, and ground capillary, wherein walls of the cross connector channels fit tightly onto at least three capillaries of an outer diameter of 300 μm to 800 μm, and the internal volume of the cross connector is less than 0.5 μL, and the capillary, onto which the cross connector channel does not fit, is sealed in the cross connector channel with an adhesive.

Claims

1. The interface for the system of sampling, preparation, and analysis of a sample by capillary electrophoresis, the interface comprising: a cross connector, sampling capillary, separation capillary, capillary supplying BGE, and ground capillary, wherein walls of the cross connector channels fit tightly onto at least three capillaries of outer diameter of 300 μm to 800 μm, and the internal volume of the cross connector is less than 0.5 μL.

2. The interface according to claim 1, wherein the internal volume of the cross connector is 0.1 μL.

3. The interface according to claim 1, wherein the capillary, onto which the cross connector channel does not fit, is sealed in the cross connector channel with an adhesive.

4. The interface according to claim 1, wherein the capillary, onto which the cross connector channel does not fit, is sealed in the cross connector channel with an adhesive.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1 depicts the connection diagram of the interface of Example 1.

[0014] FIG. 2 depicts a detail of the cross connector connected in the interface of Example 1.

[0015] FIG. 3 depicts the analysis result achieved according to Example 2, while EOF signifies electroosmotic flow, Suc signifies sucrose, Lac signifies lactose, Gal signifies galactose, Glc signifies glucose, Fru signifies fructose.

[0016] FIG. 4 depicts the analysis result achieved according to Example 3, while Lys signifies lysine, Arg signifies arginine, His signifies histidine, Gly signifies glycine, Ala signifies alanine, Val signifies valine, Ile signifies isoleucine, Leu signifies leucine, Ser signifies serine, Thr signifies threonine, Gln signifies glutamine, Glu signifies glutamic acid, Tyr signifies tyrosine, Asp signifies aspartic acid.

[0017] FIG. 5 depicts the analysis result achieved according to Example 4, while Lys signifies lysine, Arg signifies arginine, His signifies histidine, Gly signifies glycine, Ala signifies alanine, Val signifies valine, Ile signifies isoleucine, Leu signifies leucine, Ser signifies serine, Thr signifies threonine, Met signifies methionine, Gln signifies glutamine, Tyr signifies tyrosine, Asp signifies aspartic acid.

DESCRIPTION OF EMBODIMENTS

Example 1

[0018] Example 1 demonstrates a laboratory-validated interface connection between the microdialysis probe (21) and the analytical instrument (40), in this example an electrophoretic instrument.

[0019] 1) The capillary (11) of ID/OD of 100/360 μm, length of 3.0 cm, is connected to the upper inlet of the cross connector (10), at the other end, the capillary (11) is connected to the outlet of the three-way valve (30). Through this upper inlet, the carrier solution is fed to the cross connector (10) at a flow rate of 83 μL/min; the carrier solution is pumped from the 50-mL filling vessel (31), for example, a syringe, using a linear pump. When switching the valve (30) to the closed position during dosing of the sample into the analytical device (40), the carrier solution is discharged from the valve (30) by a side path directly into the waste (17).

[0020] 2) The microdialysate is introduced into the right inlet of the cross connector (10) via the capillary (12) of ID/OD of 100/150 μm; the tightness of the connection of a thinner capillary in the cross connector (10) is ensured by the UV-curable adhesive (13), which is applied around the outer surface of the capillary, 2 mm in front of its orifice. The microdialysis probe (21) itself is laboratory-made of 4.0 cm long microdialysis tubing of ID/OD of 200/216 μm, cut-off 13 kDa, into both ends of which two silica capillaries of ID/OD 75/150 μm are inserted to a depth of 1 mm, and the UV-curable adhesive is applied on the connections. The inlet capillary is connected to the 5-mL filling vessel (22) with an acceptor solution, the solution flow of 5 μL/min is ensured by a linear pump. The microdialysis probe (21) is immersed in the sample (20) and the microdialysate is fed through the outlet capillary to the cross connector (10).

[0021] 3) The 15-mm ground, preferably stainless steel, capillary (14), for example, made from an injection needle of ID/OD of 400/800 μm, is inserted into the lower inlet of the cross connector (10); and a grounding contact from the high-voltage source (41) is connected to the ground capillary (14). The other end of the capillary (14) is connected to the 5 cm long capillary of ID/OD of 100/360 μm using the UV-curable adhesive; this capillary discharges the solution from the cross connector (10) to the waste (17).

[0022] 4) The dosing end of the separation capillary (15) is inserted into the left inlet of the cross connector (10), and this end is led out of the analytical instrument (40).

[0023] 5) The cross connector (10) connected in this way has its internal (dead) volume (16) of approximately 0.1 μL.

[0024] In this case, the interface is connected to a commercially available electrophoretic instrument. The dosing end of the separation capillary (15) is pulled out of the instrument and connected to the cross connector (10). The other end of the separation capillary (15) remains in the analytical instrument (40) and is inserted into the end electrophoretic vial which is connected to a pressure pump; from this end, the separation capillary is washed with BGE between the individual analyses by applying an overpressure of 920 mbar or, conversely, a sample is dosed into its dosing end by applying a vacuum of −50 mbar. The high-voltage electrode (43) is also located in the end vial in the analytical instrument (40), and the ground electrical contact is led out of the instrument using an insulated conductor and connected to the stainless-steel capillary (14). Thus, the microdialysis moiety of the apparatus is at zero ground potential and there is no risk of injury.

[0025] The entire analysis process, including washing the capillary, dosing the sample (20), applying the separation voltage, controlling the experiment, including collecting and processing the data, is performed by a computer programme. Synchronization of the analysis with the external valve (30) is ensured using an A/D converter, which simultaneously serves to collect the analogue signal from the non-contact conductivity detector (42), which is built into the electrophoretic cassette of the analytical instrument (40), where it is thermostated.

Example 2

[0026] Example 2 demonstrates the interface connection parameters according to Example 1 to determine the carbohydrate profile in blueberry-flavoured fruit yoghurt, where the laboratory-made microdialysis probe (21) was inserted directly into untreated yoghurt and washed with 0.01 M NaOH acceptor solution, wherein the results of the analysis are shown in FIG. 2.

[0027] CE-MD determination of carbohydrates in blueberry-flavoured fruit yoghurt CE: capillary, ID/OD: 10/360 μm, length/length to detector: 35/22 cm; BGE, 50 mM NaOH, pH 12.6; separation voltage/current +10 kV/3 μA, hydrodynamic dosing with vacuum −50 mbar for 1 s; microdialysis: acceptor solution—0.01 M NaOH, flow 10 μL/min, laboratory-made microdialysis probe—length: 40 mm, OD: 216 μm, cut-off 13 kDa; the probe directly immersed in the yoghurt; FGI: carrier solution—BGE, flow 83 μL/min.

Example 3

[0028] The Example 3 demonstrates interface connection according to Example 1 for in-vitro determination of amino acid profile in human blood plasma, where a microdialysis probe intended for clinical use was directly implemented into a sample of untreated human blood plasma placed in a test tube. The microdialysis probe (21) was washed with saline, and the obtained microdialysate was analysed to determine the profile of free plasma amino acids; the results of the analysis are shown in FIG. 3.

[0029] CE-MD determination of amino acids in human blood plasma. CE: capillary, ID/OD: 25/360 μm, length/length to detector: 36/23 cm; BGE, 3.2 M acetic acid +20% v/v methanol, pH 2.1; separation voltage/current +30 kV/3.5 μA, hydrodynamic dosing with vacuum −50 mbar for 20 s; microdialysis: acceptor solution—saline, flow 2 μL/min, clinical microdialysis probe—length/diameter: 20/0.5 mm, cut-off 20 kDa; the probe directly immersed in blood plasma; FGI: carrier solution—BGE, flow 83 μL/min.

Example 4

[0030] Example 4 demonstrates interface connection parameters according to Example 1 for in-vivo determination of amino acid profile in human adipose tissue, where a microdialysis probe intended for clinical use was directly implemented into the subcutaneous tissue of human abdominal adipose tissue. The microdialysis probe (21) was washed with saline, and the obtained microdialysate was analysed to determine the profile of amino acids; the results of the analysis are shown in FIG. 4.

[0031] CE-MD real-time monitoring of the amino acid profile in human abdominal adipose tissue. CE: capillary, ID/OD: 25/360 μm, length/length to detector: 36/23 cm; BGE, 3.2 M acetic acid +20% v/v methanol, pH 2.1; separation voltage/current +30 kV/3.5 μA, hydrodynamic dosing with vacuum −50 mbar for 10 s; microdialysis: acceptor solution—saline, flow 2μL/min, clinical microdialysis probe—length/diameter 30/0.5 mm, cut-off 20 kDa; the probe directly implemented in adipose tissue; FGI: carrier solution—BGE, flow 83 μL/min.

INDUSTRIAL APPLICABILITY

[0032] The interface for the system of sampling, preparation and analysis of a sample, especially by capillary electrophoresis, is industrially applicable in medicine for the analysis of samples obtained from a human body as well as in industrial production for the analysis of biological, food and environmental materials.