High-performance liquid chromatography has the following basic HPLC parts like
- An injector
These are basic parts of the HPLC instrument to analyze components. But with the advancement of technology, there are many additions to it.
- a computer
- Guard column
- solvent input (reservoir)
Any problems in these parts can hinder the HPLC process. So it is important to procure them from a standard company and also maintain them properly.
HPLC parts in detail
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 raise the pressure of the pump only to a certain limit. After that, if the pressure exceeds the pump gets automatically stopped.
A 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 a single pump will do.
The video shows an automated HPLC system that loads the sample on its own and gives you final results on the monitor. The more automation, the less is the time consumed and also the chances for errors.
This is the part of HPLC used to load the sample into the system. This is done by two injections; one is an external syringe that loads the sample into the HPLC injector. This injector loads the sample into the stream of the mobile phase. For more refer the article on HPLC injection.
Autosampler is a device fixed to the advanced HPLC systems. It finds its use to analyze multiple samples.
The autosampler has a plate with multiple channels into which vials containing different samples are placed. The injector draws the sample in a series from the vials into the syringe and injects it into the column automatically after each HPLC run is completed.
This device is not needed if one wishes to just run a couple of samples. But when there are tens or hundreds of samples, autosampler is of great benefit.
It saves time of the experimenter and also minimizes handling error due to manual injection.
This is the mandatory HPLC part on which the actual separation of compounds occurs. 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 the separation of the 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 of 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 a small column placed before the actual column. This is a precautionary measure to prevent damage to the column.
This column is made of a 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.
This is a critical part of HPLC. The sample separated has to be identified with the detector. If there is a problem, the entire effort goes to waste. The detector mostly used are UV-Vis detector, Electrochemical detector, Photo-diode array detector, fluorescent 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.
This is mostly a glass bottle having a mobile phase in it. It is placed over the HPLC system. The tubes emptying the mobile into the column are immersed into the reservoir at one end.
If the HPLC method depends on the isocratic procedure, only one solvent is used hence on the reservoir is sufficient.
If the procedure employs a gradient method, multiple solvents and mixtures are used. So more than one reservoir is used.
This is a specially designed syringe, unlike those available for medicine. This syringe is used to load a specific quantity of sample into the column.
The volume of the sample injected is mostly in a few microliters.
So syringe with accurate rulings is used for the purpose. Ex: Hamilton syringe.
However, due to autosampler technology, these injections are mostly not employed