HPLC analysis is a sophisticated method to separate and analyse components and hence require a complex system.
The HPLC parts consist of basically pump, column and detector.
Other accessories include, a computer, injector, solvent input and exhaust.
Any problems in these parts can completely hinder the HPLC process.
So it is important to procure them from a standard company and also maintain them properly.
This is the hplc part which pumps the mobile phase at a constant pressure and flow rate through the column.
Mostly piston types of pumps, also called constant displacement pumps are widely used.
They have limitations of pressure i.e one can rise the pressure of the pump only to certain limit. After that, if pressure exceeds pump gets automatically stopped.
Number of pumps are fixed to the HPLC system based on the type of flow requirement. For gradient type you use a dual pump and for isocratic flow, then single pump will do.
Video shows an automated HPLC system which loads the sample on its own and give you final results on the monitor. The more the automation, the less is the time consumed and also the chances for errors.
HPLC Injector: This is the part of HPLC used to load the sample into the system. This is done by two injections, one is external syringe which load sample into HPLC injector. This injector loads the sample into the stream of mobile phase. For more refer the article on HPLC injection.
Guard column: This is a small column placed before the actual column. This is a precautionary measure to prevent damage to the column. This column is made of stationary phase similar to the actual column. This is very short and is connected between the pump and the column. This prevents any particulate matter from entering the column. Even if the mobile phase or sample injected is not particle free, this guard column helps to filter it. Thus the actual column is saved from getting clogged.
HPLC column: This is the mandatory HPLC part on which actual separation of compounds occur. The HPLC columns have fixed lengths, diameter and also the particle size of the stationary phase to choose based on our requirement. The longer columns are used for separation of complex mixture with many components. While shorter columns are used to separate less complex mixtures. Most columns we use are of reverse phased columns made of C8 or C18 type o f non-polar stationary phase. The columns also have a specific direction for the hplc mobile phase to flow. This is indicated by an arrow on the column.
This is the critical part of HPLC. Sample separated has to be identified with the detector. If there is a problem, the entire effort goes waste. The detector mostly used are UV, Electrochemical detector, Photo-diode array detector, florescent detector etc.
Computer operated software: HPLC system is always connected to a computer having related HPLC software. This software regulates the pump, detector, sample injection etc depending on the model you purchase or the configuration you have chosen while buying it. The more the software control, the better as handling and human errors are minimized.
Guard column: This is used as a safe guard to the main column. This prevents any particulate matter from directly entering into the column and there by choking it.
Reservoir: This is the bottle having mobile phase in it. If isocratic only one is sufficient, If gradient type, more than one reservoir is used.
Syringe; This is specially designed syringe unlike locally available ones. The volume of sample injected is mostly in a few micro liters. So syringe with accurate rulings are used for the purpose. Ex: Hamilton syringe.