CombiSep cePRO 9600 Overview The cePRO 9600 is a multiplexed capillary electrophoresis instrument with UVabsorbance detection. The instrument can best be described by examining its major components: an exchangeable capillary array cartridge with up to 96 capillaries, a fixed optical detection platform, a high voltage power supply, a fluid pumping system, and an automatic tray handling system. Associated software provides for instrument control, data acquisition, and data processing. The capillary array cartridge houses up to 96 capillaries. The standard capillary dimensions are 75 µm inner diameter and 150 µm outer diameter. Various capillary lengths are used, depending on the application, required resolution and speed of analysis. There are 2 common lengths of capillaries: 33 cm effective length, 55 cm total length (“short” capillaries) and 55 cm effective length, 80 cm total length (“long” capillaries). The capillary array cartridge is positioned between the UV light source and the UV camera lens and detector assembly. Replacement of the capillary array cartridge is easily accomplished by simply removing the electrical and plumbing connections, placing the detection window in a transport holder, and releasing two locking tabs. A new cartridge is installed following the reverse order. The typical lifetime of a capillary array is 3-6 months, however with proper use and care it is not uncommon for the capillaries to perform well for more than six months. The optical platform consists of a UV light source, collimating lens, UV camera lens and a 1024-element linear photo diode array (PDA) detector. The UV light is provided by a line source consisting of a zinc (214 nm), cadmium (229 nm), or mercury (254 nm) lamp. Detection is performed at a fixed, single wavelength determined by the UV lamp. Light from the lamp is passed through a collimating lens, the detection windows of the capillaries, a focusing UV camera lens and onto the PDA. The capillaries are arranged in a parallel array and imaged onto the PDA, providing for the simultaneous, parallel analysis of 96 samples. The 20 kV, 6 mA total current high voltage power supply provides the appropriate voltage for electrokinetic sample injection and electrophoresis. The recommended operating limits are 16 kV, 4 mA total current (40 µA per capillary). Operation outside of these limits can damage the capillary windows and lead to increased system noise. Positive or negative power supplies are available. A syringe pump is present to provide controlled pressure or vacuum to the capillary array. The pump is connected to the outlet reservoir of the capillary array, allowing pressurization or vacuum to be performed from the outlet side of the array only. Pressure flushing is utilized for conditioning of the capillaries and introduction of buffers into the capillary array. Vacuum can be used for vacuum injection of sample, for introducing buffer solutions into the capillary array from the inlet tray, or for performing vacuum-assisted electrophoresis. An eightposition distribution valve is attached to the syringe pump. Seven positions are
utilized, allowing for the connection of five different buffer bottles and a waste bottle exterior to the instrument. Via software control, the capillaries can be rinsed in any sequence from the buffer bottles before and in between electrophoresis runs. Instruments that are configured for the analysis of synthetic oligonulceotides utilize a high-pressure pump in place of the syringe pump. This modification is because the proprietary Oligel gel matrix that is used to provide separation of oligonucleotides possesses a relatively high viscosity, requiring higher pressures to fill the capillary array. The automatic tray handler consists of a slide-out stage providing space for the simultaneous placement of four 96 well plates. The four positions on the stage are assigned to a waste tray, the inlet run buffer tray, and two sample trays, respectively. Placement of two 96 well sample trays along with the inlet buffer and waste trays allows for the uninterrupted analysis of 192 samples. The positioning of the automatic tray handler stage exterior to the instrument provides the ability to interface the cePRO 9600™ to a robotic arm device. This configuration enables unattended operation for 24-hour/7 day sample analysis. The cePRO Manager software provides instrument control and data acquisition and analysis. Methods can be edited, saved and loaded to perform capillary array conditioning and capillary electrophoresis. Conditioning methods and electrophoresis methods can be linked together to provide unattended operation. For capillary conditioning, the buffer bottle, conditioning pressure, and conditioning time can be edited. For electrophoresis, selectable parameters include an optional voltage pre run, electrokinetic or vacuum sample injection, gradient or fixed run voltage, CE run time, and vacuum-assisted CE level. Post run data analysis is accomplished with the cePRO Manager software. Data can be displayed and printed as single electropherograms or in rows of twelve. Peaks can be assigned based on user-selected criteria for peak width, minimum peak height, and maximum peak height. Peak picking parameters may be selected for all 96 capillaries or individual capillaries. Reports can be generated in a variety of file formats displaying peak migration times, peak heights, peak areas, and corrected peak areas for each capillary. Raw electropherogram data or processed data can be exported in ASCII format for integrating into third party software programs or an in house database. Companion software packages are also available for the determination of compound pKa values (pKa Estimator™), determination of compound log P values (log P Calculator™), and analysis of protein sizing separations (PRO Size™). These software programs provide seamless extraction of 96-capillary electropherogram data and automated calculations to simplify data analysis.
