While performing HPLC analysis with an existing method, sometimes we will observe more base line noise.
However, we won’t observe that scenario previously. The major probability due to:
- dirty mobile phase.
- dirty flow cell.
About dirty mobile phase:
Grade of organic solvet used for analysis.
- Instead of HPLC grade if other grade (low grade) of solvet used it leads to more base line noise.
- Low grade solvents contain small amounts of miscellaneous chemicals that influence the UV background of the solvet.
Especially In case of THF is there in mobile phase we have to use HPLC grade solvent only. Otherwise in other grade THF contain anti Oxidents that can detect by UV detector and leads to more noise.
On the other hand if used old THF also leads to more noise due to formation of peroxides due to deterioration. But deterioration scenario we won’t observe in other solvents.
However, in lower UV ranges Acetonitrile gives better base line when compared to methanol. In reverse phase analysis we will use inorganic salts if low grade salts Or less quality salts used mean we will observe more noise. Low grade salts contain more contaminents those sticks on flow cell and cause more noise.
TEA, DEA or amines:
Amines deteriorates with age due to oxidation. It leads to influence the UV background. Due to this use recently opened one or recently manufactured batch to avoid high background noise.
Water used in mobile phase:
HPLC grade water need to use for mobile phase. Even after using HPLC grade water, we will observe more noise due to improper functioning of the ion exchange cartridge (saturation of cartridge)in the water purification system. Better to replace the old cartridge with a new one.
Filtration of mobile phase,
Degassing of the mobile phase and By using the ghost guard column we can avoid base line noise.
About dirty flow cell:
If problem not due to mobile phase dirty flow cell may be the reason for base line noise. Air bubbles in the flow cell can cause more base line noise. we can remove those by flushing the flow cell with more pressure (High flow rate by connecting union).
Contaminants in flow cell also cause the more base line noise. Need to clean the flow cell under supervision of service person. If issue not due to contamination of mobile phase or flow cell need to replace the lamp or detectors. It will be more useful for Quality control and validation team.
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