The quality of medicine will decided based on analytical results on pre defined analytical parameters.
For every analysis, the validated testing procedures are available and analyst follow the respective written procedures. No any departure will accept from written procedures.
Some of analysis are of chemical analysis and some of are Instrumental analysis. The precautions should be taken at every steps of analytical activities. Here we are going to discuss about Instrumental analysis i.e. analysis on HPLC Instrument.
The abbreviation of HPLC is High performance liquid chromatography. In HPLC, liquid mobile phase and liquid sample being used. Earlier, manual sampler were widely used but now a days, auto-samplers are introduced and in almost laboratory, auto sampler HPLCs are available.
The HPLC can be handled without software control and with software control but for analytical purpose and data storage, the software control is must.
There are different make HPLCs available in laboratory but most commonly are as Shimadzu, Waters, Agilent, Dionex, Perkin Elmer etc. The mechanism of all make HPLCS are almost common. But the operating procedures are little bit different. We can easily understood by reading the user manuals or Standard Operating Procedures.
There are different configurations of every make HPLC i.e. Isocratic, quaternary gradient, binary etc. and based on our analytical requirements, we have to select specific HPLC Instrument.
Figure:-1. Waters make HPLC
In every HPLC there are different modules i.e. Pump module, Sampler module, Column compartment and detector module.
Pump module:- The purpose of pump module is to deliver solvent i.e. mobile phase from mobile phase bottle to detector via sampler column compartment. In pump module having delivery system i.e. vacuum pump based on delivery piston, degasser, MCGV (multi composition gradient valve), solvent mixing unit, Active inlet valve, Outlet ball valve, purge valve.
The delivery system pumps the mobile phase from mobile phase bottle and degasser remove the dissolve gases from mobile phase. The MCGV, make the correct composition of mobile A, B, C, D as required and send to mixing unit for proper mixing of same. If purge valve is properly close, then mobile phase will deliver in next compartment i.e. sampler compartment. If purge valve is open then mobile phase will go in waste tube. To remove the earlier mobile phase from mobile phase port, we need to purge with high flow i.e. about 5- 8 ml/ minutes by open the purge valve. After removal of previous mobile phase or any bubble, keep down the flow gradually as required and close the purge valve.
Note:- If we give high flow and purge valve is closed, then over pressure message will occur and may be any leakage observed. Hence make sure in purge valve close condition, give the low flow as mentioned in testing procedures.
Sampler compartment:- The sampler may be two type one is manual where in manually load the injection after every runs and second is autosampler which automatically load the injection from vials which was already placed in racks at different vial position. Autosampler automatically inject the injection at defined time interval.
Some of sampler inbuilt with cooler which maintains the temperature of solutions in vial which was sampler compartment.
The pump outlet is connected with 6 port valve. The 6 port valve is connected with measuring device, needle seat, column compartment.
The measuring device is connected with sample loop, sample loop connected with needle. The needle is momentary part but mostly it seat on needle seat. And in above paragraph said as needle seat is connected with 6 port valve. Hence a cycle created in sample compartment and flow move towards this cycle and reach to column.
When needle move to vial for collection of sample, the mobile phase flows from 6 port valve to column directly and when after sample collection needle move and inject the sample in needle seat, the mobile phase immediately move and getting flow through needle and injected sample solutions getting floated with mobile phase. Sample solutions and mobile phase reach to mixture (mixture is available in 6 port valve) where sample mixed with mobile phase then reach to column.
Here precautions to be taken as needle should be wash before and after injection with appropriate was solvent to avoid carry over.
Figure 3:- Open view of sample compartment.
Column compartment:- The column compartment is connected with outlet of sampler compartment. The oven is inbuilt in column compartment which maintains column oven temperature. As per analytical requirements, specific column to be selected. Make sure for analysis, column to be installed in forward direction otherwise the stationary phase will disturb and after some time column life will decrease. Also ensure as give the column temperature above 25°C, only when mobile phase running in column otherwise stationary phase will damage.
Abhishek Singh is the author of Pharma technical support. He is pharmaceutical professionals, having experience of more than 12 years in Quality.