What is HPLC GRADE & its importance in HPLC ?
High pressure liquid chromatography is one of the widely used analytical technique.
But it very sensitive to operate at constant pressure.
Further the mobile phase passes through a dense column so all the material used in the procedure should be of high quality.
While performing analysis by HPLC chromatography, one is advised to use HPLC grade components.
This is intended for an effective, uninterrupted performance and also obtain precise results on analysis.
This HPLC grade mostly implies to solvents, water, chemicals used to make mobile phase, standard substances etc.
Manufacturers in general make chemicals of different grades based on the requirement.
These grades vary based on the extent of purity of the said substance.
They are termed as analytical grade, HPLC grade, reagent grade etc.
HPLC is very sensitive procedure and use of less pure compounds can produce unnecessary peaks in data.
So most of the time, one is advised to use HPLC grade materials and they quite expensive.
HPLC Grade material include
HPLC grade water:
This is water which is devoid of even fine particles and is filtered using 0.02 micron filter paper. It is also devoid of air bubbles and also free from any minerals, pyrogens and any other substances which can interfere in the data. So HPLC grade water is particle free up to 0.02 micron size and also air and substance free. This can be made in lab by obtaining triple distilled water and then degassing to remove air bubbles in them. Also there are commercially available as 1 liter packs. Even there are instruments which can yield readily HPLC grade water from normal tap water.
HPLC grade buffers:
Most of the solvents of organic nature like methanol, acetonitrile etc. used are to be of HPLC grade . They have the same qualities as above like particle free up to 0.02microns, other substance free. These solvents are of highest purity without any second substance or solvent. Their percentage level of concentration exceeds 99%.
HPLC garde chemicals:
Chemicals used like buffer, available in solid powder form from the market are chosen to possess highest quality or purity. They are termed HPLC grade as their purity exceeds 97 to 99% range. Moreover on dissolution in solvents they are mostly particle free. Some companies specialize to provide chemicals of different grade for the purpose like chemical of analytical grade, extra pure or reagent grade. Mostly grades with highest purity on analysis are used. once these chemicals are made into solutions by used of solvents, they are to be filter through 0.02 microns filter for safety.
HPLC grade standards:
Standards are the substances used for comparison and identification of test substance. They are also analysed by HPLC along with sample. They help in standardization of the instrument and see if the instrument is performing well. These standards used are to be of HPLC grade i.e. of highest purity like above 97% and above 99% are the best. As per my experience, chemicals from Sigma aldrich are especially used as they comply with these requirements.
If one does not find HPLC grade material, one should refrain from performing any analysis by HPLC method. This is because, the result will not be precise and also the chances of damage to the column. If one cannot find such high grade chemicals, they can try to filter the them and use. But for water and other solvents they need HPLC grade.