METHOD AND DEVICE FOR 2D-SEPARATION OF IONIC SPECIES
Each chromatography separation technique relies on a specific separation mechanism. For this reason, the separation of complex biochemical solutions can be very challenging, because it is hard to find a separation method allowing to separate every species in the solution. To overcome this difficulty, dif- ferent techiniques can be combined together. Ion Chromatography (IC) and Capillary Electrophoresis (CE) are the most widely used techniques for the separation of ionic species. IC allows the separation of ions and protonated/deprotonated polar molecules based on their affinity to an ion exchanger, whereas CE separates ions based on their mobility.
The present invention offers a solution for the combination of IC and CE, based on the coupling of the IC- and CE-apparatus through a modulator. The separation proceeds as follows:
- First separation through IC;
- The sample is transfered from the IC to the CE-apparatus through a modulator;
- Second separation through the CE-capillary;
- The CE-capillary is led to an electrospray interface (ESI) connected to a mass spectrometer(MS). The device and method developed in the frame of the invention allow for an improved separation of ionic species, providing increased peak capacityand better cost-efficiency. Small sample volumes and short run-times ensure high throughput analysis.
This method is particularly suited for the investigation of bioanalytical samples, such as DNA, RNA and amino acids. An efficient separation of these compounds can be very important for the diagnostication and monitoring of human deseases. Additionally, this invention can be applied to the field of toxicolo- gy and forensic chemistry.
A prototype is estabilished and fully operational.
Separation of a model system containing a mixture of nucleotides (AMP, GMP, CMP) and cyclic nucleotides (cCMP, cGMP, cCMP) using separately IC and CE, and then the ICxCE system. (left, a) a separation of the mixture using capillary high performance ion chromatography (cHPIC) is shown, the traces are measured through a conductivity detector. cHPIC cannot separate the nucleotides from the corresponding cyclic species. (left, b) CE can only separate the nucleotides from the cyclic ones. (right) multidimensional illustration of the ICxCE chromatro-electropherogram as contour plot. The mass spectroscopy peak intensity (extraced ions traces, expressed in counts) are shown by me- ans of color gradients whereas each substance is assigned to a different color. The upper bar is a projection of the contour plot on the x-axis while the bar on the right represents the projection on the y-axis. Through ICxCE every species in the model system can be separated. Images adapted from (1).
(1) Anal. Chem. 87, 3134-3138 (2016).
(2) EP 3 134 725 A1; US 2017 0030860 A1.