The Center for collective use of equipment (CCUE) «High-performance liquid chromatography (HPLC)» /further – Center/ is organized on the decree of Presidium of National Academy of Sciences (NAS) of Ukraine from 03.11.2004 No.295 and on the directives of Presidium of NAS from 31.05.2005 No.337 and from 11.11.2005 No.651 with the purpose of providing of the proper conditions for the effective use of the unique imported equipment. National Botanical Garden named after M.M.Grishko (NBG) as scientific institution of NAS of Ukraine, on the base of which Center is created, undertakes obligations to provide high-qualitative and reliable functioning of scientific equipment and devices which are at disposal of center, their professional service, conditions of the best use of working hours and expendable materials in behalf of scientific community of NAS of Ukraine and other scientific organizations and institutions.
Presently CCUE «HPLC» disposes of automatic liquid chromatograph Agilent 1100 series equiped with quaternary pump and diode-array detector (Agilent Technologies). EASYpure II RF/UV system (Barnstead International (USA) is used for preparation of water.
Today the HPLC technique is widespread in practice of various analytical laboratories in the whole world. It is used mainly for analysis of polar low-volatile compounds. By this device it is possible to determine the different classes of bioactive substances, viz. secondary plant metabolites (phenolic compounds, flavonoids, anthraquinones, saponins etc.); phytohormones (auxins, gibberellins, abscisic acid and its derivates, cytokinins etc.); other bioactive substances of plant origin (alkaloids, sesquiterpene lactones, etc.).
Principal activity of CCUE «HPLC» lies in authentication and analysis of bioactive plant substances in various vegetable objects according to the scientific projects worked out in the departments of NBG.
The module design of chromatograph Agilent 1100 allows to realize various HPLC techniques from simplest isocratic elution with single wavelength detection up to fully automated complicated gradient schemes with the registration of complete UV-VIS spectrum. Autosampler substantially promotes efficiency of chromatographist work during the analysis of numerous samples. Vacuum degassing of eluents, thermostatting of columns and samples results in the improvement of producibility and authenticity of obtained results. The special software for the automatic protocolling and diagnostics of the separate modules of device allows to watch the functionability of chromatograph during work. Software does not allow operator ‘to edit’ the results of analyses in future, that corresponds the international GLP /good laboratory practice/ standards.
Basic characteristics of Agilent 1100 series chromatograph
Quaternary plunger pump (up to 400 bar) at flow rate 0.001-10 ml/min with compartment for eluents. Step of flow rate regulation: 0,001 ml/min. ðÍ range of eluents: 1-11.5. Plunger volume: 180 µl.
Vacuum degasser with 4 channels and the efficiency up to 10 ml/min.
Autosampler /Device of automatic injection of samples/ (100 units with volume up to 2 ml) with possibility of internal standard addition, dilutions of sample and conducting derivatization. Possible volume of injected samples 0.1-100 µl. Accuracy of injection 1% (relative standard error for a sample volume 0.1 µl).
Column oven /thermostat/ (temperature range from 10°C below room to +80°C with the step 1°). In the thermostat 3 columns long to 30 cm could be placed. Accuracy of temperature maintenance ±0,15°C. A thermostat is equipped with device for record and read-out information which is stored on the identification modules of columns.
Diode-array detector (1024 photodiodes) with the range of wavelengths 190-950 nm (discreteness 1 nm), with the automatically changeable programed spectral bandwidth (from 1 to 16 nm), also during the process of chromatographic analysis, with high-pressure cuvette. Detector includes an integrated filter based on holmium oxide for verification of wavelengths setting and equipped with the system of warnings about the necessity of timely prophylactic replacement of radiant. Noise level of spectrophotometric detector: no more than 6´10-6 absorbance units.
EASYpure II RF/UV ultra-pure water preparation system
The system is constructed from exceptionally inert materials to obtain ultra-pure water without organic and inorganic impurities and has a few sets of cartridges which allow to get water with specific characteristics. In the device the UV lamp with double wave-length (185 and 254 nm) is built-in. The process of water treatment is controlled by microprocessor, parameters are output on digital display, temperature compensation to 25°C. In waiting mode the system recirculates the purified water each 10 min to avoid recontamination. Purification steps: adsorption, deionization, UV-oxidation and final filtration through the 0.2 µm filter; efficiency: 0.8 l/min. Water of type 1 with parameters of: specific resistance 18,2 MOm/cm; content of total organic carbon (ÒÎÑ) < 5 ppb.
In structure NBS as the scientific division, enters the Center of collective use by devices " HPLC". The center gives to science officers an opportunity to carry out researches at a modern level a method of highly effective liquid chromatography.