USER'S GUIDE
Contents
1. KROMATON FAST CENTRIFUGAL PARTITION CHROMATOGRAPHY FCPC USER GUIDE I E P KROMATON 45 avenue Rhin Danube 07104 Annonay France Phone 33 0 4 75 69 22 15 Fax 33 0 4 75 67 69 80 E mail kromaton kromaton com Website www kromaton com IR Type Designation Seq Rev Page KROMAIUN FCPC A200 USE KRO C 2 14 CONTENTS 1 CENTRIFUGAL PARTITION CHROMATOGRAPHY DEFINITION 3 2 INSTALLATION 4 2 1 Unpacking and inspection 4 2 2 Advice for the installation of the chromatograph 4 3 CHARACTERISTICS OF THE CHROMATOGRAPH 5 3 1 Security 5 3 2 Inappropriate uses 5 3 3 Requirement for the user 5 3 4 Precautions 5 4 USING FCPC 6 4 1 Checking before start up 6 4 2 Howto use the FCPC figure 1 6 4 3 Cleaning 7 5 CLEANING MAINTENANCE 9 5 1 After sale 9 5 2 Cleaning 9 5 3 Work with very toxic compounds or very strong bases or acids 9 5 4 Check up 9 5 5 Transport of the chromatograph 9 6 REPLACEMENT OF THE ROTOR 10 7 TROUBLESHOOTING 12 8 DIAGRAMS 13 close to the chromatograph So you can quickly refer to the instructions Before beginning to set up your FCPC please refer to this user s guide Keep this document Prices and specifications are subject of modifications without notice Without written authorization by the Kromaton Company any reproduction of this manual is forbidden All rights reserved lt jj EB Type Designation Seq Rev Page KROMATON FCPC A200 USE KRO C 32. KROMATUON FCPC A200 USE KRO C 4 14 2 INSTALLATION 2 1 Unpacking and inspection The Fast Centrifugal Partition Chromatography is delivered in a wooden crate Before unpacking the FCPC inspect the crate to be sure there is no damage After carefully unpacking the FCPC put it on the ground and inspect it to be sure there is no damage on the FCPC Keep the packing crate it would be useful if you have to file a claim for damage or in the case of future transit If the crate or the FCPC is damaged file a claim for any damage immediately with the carrier as he is responsible for damage incurred during the transit Many carriers must receive a damage claim within three days after delivery and need to inspect the shipping container Zz Advice for the installation of the chromatograph The FCPC must be handled with the maximum of caution After unpacking place it on a plane surface which can support 100 kg at least laboratory straw mattress for example The instrument must be far from heat source for example the direct daylight or a heating system corrosive or dusty atmosphere vibrations possible liquid projections Let an aeration space at least 4 inches at the back of the FCPC in order to maintain the ventilation of inlet outlet HIR Type Designation Seq Rev Page KROMAIUN FCPC A200 USE KRO C 5 14 3 CHARACTERISTICS OF THE CHROMA TOGRAPH 3 1 Security This equipment is built accordi
3. 14 1 CENTRIFUGAL PARTITION CHROMATOGRAPHY DEFINITION Centrifugal Partition Chromatography is an interesting tool for isolation and purification of different kinds of extracts This method is especially attractive for the fractionation of natural compounds from complex matrices like plant extracts which are rich sources of chemical diversities with a great potential for discovery of new drugs It is based on the principle of a liquid liquid partition between two immiscible liquid phases prepared by mixing 2 or more solvents or solutions One liquid phase is retained in the FCPC Kromaton by centrifugal forces and called stationary phase The second liquid phase is pumped through the stationary phase and is called mobile phase Main characteristics are 1 No solid support no irreversible adsorption of the metabolites no loss of compounds better decontamination of toxic residues during the purification gentle treatment of fragile natural compounds 2 Reduction of solvent consumption economic impact and better environmental protection 3 Easy and reliable scaleable from analytical to preparative scale analytical developed methods on a FCPC Kromaton with a 200 ml rotor directly up scaled to a 5 liters preparative rotor Because of the scientific and economic interests for CPC this method has increased quickly the number of applications in counter current chromatography jj E Type Designation Seq Rev Page
4. place and do not rub an obstacle gt Belt Contact us Noisy functioning gt Bearings gt Contact us gt Cabinet structure Screw unscrewed es gt Equilibrium problem Verify that the 4 feet are placed on a Vibration of the chromatograph plane surface 12 ILE Type Designation Seq Rev Page KROMATON FCPC A200 USE KRO 01 C 13 14 8 DIAGRAMS Ascending _ Descending e WI P N Ascending K AL Descending P oS e gSA IN 7 SA JI Y V 7 IN Z7 N fa d Choice of the development mode Ascending mode Descending mode ____J ___J The mobile phase is the upper phase The mobile phase is the lower phase 13 all KROMATON Type FCPC A200 Designation USE KRO Rev Page C 14 14 Rotary seal Peek tube Peek fitting Rotor Bearings Cooling inlet outlet Solvent inlet Solvent outlet j nnn Ln Et ege i f S 4 way valve d Da ob NS na Door lock Motor Belt Pulley 14
5. The sample can be injected before the equilibrium of the column sandwich injection it allows the equilibrium of the column with the sample after the equilibrium of the two phases it can modify the equilibrium CAUTION do not exceed the max pressure 60 bars otherwise rotor will leak NEVER start the rotation without solvent in rotary seals 4 3 Cleaning To avoid the eventual contamination of the sample it 1s better to clean the column with methanol if methanol is a good solvent to dissolved impurity Pump 2 column volumes Vc of methanol in ascending mode if the column is empty or in descending mode if the column if full with the stationary phase denser than the methanol It could be interesting at the end of the experiment to recover the stationary phase because some compounds could be strongly retained al Designation KROMATON USE KRO Empty column Ve l Column full of ll EEE x stationary phase sample Injection of the WEE Seel Pumping of the r mobile phase ra Vm volume of stationary phase push out by the mobile phase TT TT Vs Ve Vm U U J Ld Vom Figure 1 How to use FCPC jj EB Type Designation KROMAITUN FCPC A200 USE KRO 5 CLEANING MAINTENANCE 2 1 After sale Modifications and repairs of the FCPC instrument must be handled by KROMATON In case of technical problems please contact the company KROMATON Sarl 45 a
6. as well as the 2 small connections of the rotor with the key socket for plastic Remove the top guard by unscrewing Remove the antirotation bar by l the 7 fasteners with a male spherical unscrewing the nut with a 8 10 connection Ref P399 end 3 hex key spanner unscrew the white fitting on the rotary seal Remove the 4 screws H M8x25 with a tube spanner N 13 al E KROMATON Replace the 2 plastic connections by 2 plugs Ref P 309X Type FCPC A200 Insert 2 of the screw M8 into the extraction holes and tighten gradually until the rotor takes off of the hub Remove the rotor with the hand Extractions holes Designation USE KRO Seq Rev Page 01 C 11 14 Models shown Rotor 200 ml and rotor 50 ml Proceed to the replacement of the rotors several models are available on request Reassemble the new rotor in the reverse order of dismantling Figure 2 Replacement of the rotor 11 jj E Type Designation Seq Rev Page KROMATON FCPC A200 USE KRO I01 C 12 14 7 TROUBLESHOOTING PROBLEM POSSIBLE ORIGIN SOLUTION Leaks gt Worn out fittings or Replace fitting and ferrule and check Connections ferrule the tightness Check the back pressure Rotor gt Solvents leak between Strengthen regularly the bolts This the circular disks operation should be done after 4 or 5 uses gt Solvents leak from the Noises gt Peek piping Z Check that the pipings are in
7. d continuously until hydrodynamic equilibrium is reached when mobile phase goes out 1 kuch Inject sample dissolved in few milliliters of stationary and or mobile phase Dissolving sample in both phases can help the dissolution of the sample for example 8 ml of S P and 2 ml of M P The injected volume varies according to the quantity and the solubility of the sample up to 20 of the column volume 12 Collect mobile phase in fractions by time function 13 Runtime in general 30 90 min 14 Stop the pump and rotor lt jj EB Type Designation KROMAITUN FCPC A200 USE KRO Dual Mode 1 Change phases e g stationary phase becomes mobile phase and change flow rate ascending descending 2 Change mode switch ascending descending valve mode 3 Start pump and FCPC 4 Fractionize new mobile phase stationary phase before Remarks l Sometimes there may be a bleeding of the stationary phase It could be caused by the solvent system does not allow a good retention of the stationary phase in these experimental conditions rotation speed flow rate Parameters should be adjusted to have a good and stable retention for example increase rotation speed and decrease mobile phase flow rate Some solvent systems are known to be unstable like Aqueous Two Phase System chlorinated system a flooding of the injection volume If the sample presents a high viscosity it could push the stationary phase 2
8. fy the inlet and outlet pipings and fittings Select the elution mode by switching the 4 way valve picture 1 Turn on the power on the back of the FCPC The LCD lights up Open the rotor cabinet and check the inlet and outlet pipings and connectors of the rotor Check and or bolt the fixing arms on the high pressure rotary seals Close the cabinet door The FCPC is now ready for use 4 2 How to use the FCPC figure 1 1 Select biphasic solvent systems see list of solvent systems 2 Choose mobile phase after determination of Kd TLC and HPLC chromatograms are useful Set the pump flow direction with the mode valve diagram 2 w Upper phase mobile phase gt ascending mode Lower phase mobile phase gt descending mode Mix solvents in a funnel by shaking thoroughly Connect FCPC outlet to a 250 ml graduated cylinder Fill up FCPC with the stationary phase 20 30 ml min Change to mobile phase ON AMR Pump mobile phase with the selected flow rate Increasing the flow rate will increase the backpressure in the FCPC Be careful not to exceed maximum pressure 60 bars 9 Adjust rotor speed to stay down max pressure 60 bars as follow Flow rate 1 5 ml min 2 15 ml min 10 30 ml min 1800 to 2000 rpm 800 to 2000 rpm 800 to 2000 rpm 10 The stationary phase will be pushed out 10 50 of rotor volume depending on solvent system Wait until mobile phase breaks through Backpressure will increase
9. ng to the security standards However risks and dangers could exist e if the chromatograph is used in a different way from which it is conceived e if the chromatograph is used by unqualified person 3 2 Inappropriate uses This equipment is not adapted to injection of solids particles or other rigid products The user has to be sure that the material has no precipitation risk when injected in the FCPC The consequence would be the plugging of the rotor 3 3 Requirement for the user This chromatograph can only be used by qualified staff or experienced persons The user s guide could be a support 3 4 Precautions The system includes fuses against short circuit The door security system is on when the rotor is on rotation Thus it is impossible to open the door of the instrument during his rotation It is not necessary to lubricate the bearings It is necessary to wear protective equipment like protective glasses and laboratory clothes when you use the FCPC Modifications Modifications on the FCPC chromatograph are not allowed without written authorization by KROMATON The user has to inform the manufacturer in case of troubles and incidents that occur during the use of the instrument There is no guarantee in case of dismantling of the rotor jj EB Type Designation KROMAITUN FCPC A200 USE KRO USING FCPC 4 1 Checking before start up Plug in the electrical connection Connect and veri
10. venue Rhin Danube 07104 Annonay France Phone 33 0 4 75 69 22 15 Fax 33 0 4 75 67 69 80 E mail kromaton kromaton com 5 2 Cleaning It may be cleaned with ethanol or acetone 5 3 Work with very toxic compounds or very strong bases or acids In this case we advise to rinse out the column after each use with 2 column volumes of methanol and then with water before storing the rotors with water acetone 1 1 5 4 Check up Cleaning Monthly 1 Check the cleanliness of the valve and other parts which are in contact with solvents and samples 2 Clean the rotor cabinet when necessary with a dry tissue Maintenance Weekly 1 Check the conditions of the PEEK tubes connections fittings etc 2 Check the condition of the high pressure rotary seals 3 Check the rotor condition 4 Check if all screws are tight and well positioned Monthly 1 Check tightness and condition of the gear belt 5 5 Transport of the chromatograph Before moving the instrument the connections have to be disconnected the column has to be rinsed out with methanol or emptied with compressed air Consign the chromatograph in the original packing IL Type Designation Seq Rev Page KROMATON FCPC A200 USE KRO 01 C 10 14 6 REPLACEMENT OF THE ROTOR Antirotation bar O ae ep TCR Le Tre an r eH E ae j 1i CR Bn o Di ci Remove the rotary seal connection with the 5 16 spanner
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