COLUMN
Normal vs. Reversed Phase
PARAMETER
NORMAL PHASE
REVERSE PHASE
Polarity of packing
Medium to High
Low to Medium
Solvents Polarity
Low to medium
Medium to High
Sequence of Elution
Low polarity first
First high polarity
Increase solvent polarity
Faster elution
Slower elution
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Installation **
The direction of flow is marked in every available column in market.
Column can only be operated in the direction flow marked on the column.
Experts recommends using polyketone fittings for column up to 600 bar and the stainless steel fittings for the column up to 1200 bar column that will be operated at UHPLC pressures.
Columns have to go through the conditioning process after column connection is done.
Column conditioning
Every single has been tasted and shipped in the test eluent by the manufacturer before it has been release to the market. So rinsing with water to clean the column is not necessary for the first time use. If the mobile phase that you gone to use a buffer or ion-pair reagents, then it is better to flush the column with the mobile phase but without these additions. Flushing with 10 to 20 column volumes should help in transitioning to your mobile phase. Flow of the column 0.1ml/min will be better for the first one minute and slowly increase 0.1ml/min after 2 minutes interval to the flow that you wanted to keep for the acquisition time. It helps the column to gain the proper equilibrium with the mobile phase. This will ensure the reproducibility and help to prevent the retention time drifting.
Use guard column
If column pore size is 5 micrometer, particle greater than 5 micrometer will deposited on the inlet frit of the column. In order to solve this problem, you have to use a guard column so that column will remain safe. Guard column can be easily replaced.
Cleaning
If column pressure increases over the normal pressure, Column needs to go through a cleanup process or black flush process.
Disconnect the column from the detector and run wash into a beaker.
Start with your mobile phase without buffer salts,. Pass 20 to 30 column volumes through the column.
Use 100% organic solvent like methanol or acetonitrile.
Check the pressure, is the column pressure return to normal or not, it it is not return to normal then
Pass much stronger organic solvent like 75% acetonitrile/ 25% isopropanol through the column. If it comes to normal pressure then ok, otherwise
Use 100% isopropanol, 100% hexane or methylene chloride, if you use methylene chloride, you will need to flush the column with isopropanol in order to use
If the column particle size is less than 1.8 micrometer, do not blackflush the column just replace it.
Operational safety
Beware of leakages; all kind of connection in liquid chromatography is a possible source of leakage.so makes sure that all the connection is tight enough to lose the toxic and harmful organic chemicals to the environments.
Due to the small particle size the column particle size is breathable. Always open it to the well ventilated area.
Create a log of the column pressure so that it can be easily identify the column present conditions and unusual behaviors.
Storage
For Long term storage column packed particle have emerge in the pure organic solvent. In order to remove the mobile phase that has been used for the acquition, you have to purge the column with 20 to 30 column volumes of 50:50 mixture of methanol or acitonitrile and water, followed by 20 to 30 column volumes of pure solvent. Before storing, end fittings and the inlet fitting should be tightly capped to prevent packing from drying out. Columns may be safely stored for short periods in most mobile phases. To protect column or the equipment, it is desirable to remove salts from the column or instrument by purging the column with same mobile phase without the buffer. Because salt is corrosive to the instrument. Salt can deposit into the column and block the column forever.
Tips
Best column life can be found if it operates within 40 degree temperature.
Do not exceed the ph range that is written in the column specification of operation.
Always avoid dramatic changes in running conditions
Always degas and filter the mobile phase
Protect your column from contaminants by using guard columns
Protect your column with a saturator column
Store column in pure acetonitrile if possible
Quality test required for packing materials
Capacity factor k
Selectivity (separation factors )
Efficiency
Chemical stability
Packing stability
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Column test**
Capacity factor k
Efficiency (plate number n/m)
Asymmetry factor As
Care:
Never use immiscible solvents sequentially through the column
Make sure the solvent that pumped into the column are miscible in all portion. If they are not it can cause a damage of the column
Use HPLC or MS grade solvents as mobile phase
Highest purity solvents should be use. Must filter and degas all the solvent before use, otherwise the impurities of the solvent will decomposed in the column.
Beware of buffer precipitation upon the addition o f organic solvents
Buffers and ion pairing salts have limited solubility in organic solvents. It the percentage of organic content in your mobile phase increases it may be precipitate the salts.
Do not use mobile phase that chemically attack the bonded phase or silica
Hydrolysis of the bond of the column packing material is possible under strongly acidic conditions; therefore ph of the mobile phase should be kept above 2.0. The ph of the mobile phase below 2 is used; it will be shorten the column life time
The nature of the bonded phase determines the solubility of the packing material by affecting how easily the silica is wetted. In reversed phased materials the silica surface is protected by a waxy hydrophobic layer around the silica particles. it decreases the contact between the mobile phase and the silica.
Use guard column and saturation columns where appropriate.
Contamination from samples and mobile phases or salvation of the silica matrix will eventually destroy any column, no matter how well it is manufactured. So it is strongly recommended that Use guard column and saturation columns where appropriate, to minimize the damage.