I am working with hexane as mobile phase, UV detector, column c18 at 15C.
I got alot of spike air bubbles and i can't get rid of them.
Hexane as a mobile phase! it is not usual to use hexane, are your analytes very non polar? Concerning the air bubbles you should do burging at high flow rate
yes , you are right it's the first time i work with hexane >....my target is detection of vitamin E and it's offical to use hexane i try burging but it didn't solve the problem
Hexane is not suitable for C18 column and it damage your HPLC Column (C-18 column is generally used in reverse phase chromatography) and Hexane is used in normal phase chromatography, when you are using C18 column you need to use polar mobile phases, If Hexane is recommended as a mobile phase the your column should be polar in nature ( like silica column).
If your HPLC system is not converted to normal phase properly then also you found in trouble in analysis(like you are facing)
Try burging with acetonitrile for 5 minutes then allow ACN with flow rate 1 ml/min. for 15 min, then burg with methanol for 5 min. then allow methanol to flow at 1ml/min for 15 minutes. then burg with water then water at flow rate 1 ml/min for 10 minutes. the fluctuations in pressure will decrease each time and the pressure will be stable at the end
many thanks but how can i get rid off hexane residue from C18 column
and how i know that the column don't damaged
I think your C18 column can not be used for any analysis. for checking workability of column just check performance of column as mentioned in COA. if you don’t have procedure i will provide to you.
(Before starting performance check to remove all hexane from your column flush column with 100% Methanol at list for 60 minutes with 1ml/min flow rate)
Sample preparation: Prepare mixture of Acenaphthene 50mg and Acetone 600µl in 100 ml mobile phase.
Note:Acenaphthene is recommended sample but you can substitute with simple aromatic hydrocarbons like Naphthalene,P-Cymene,Xylene, Diethyl/dibutyl or dipropylphthalate, propylbenzene, Toluene, ethylbenzene
(Toluene and diehtylphthalate are least desirable substitute since they have lowest K.)
Chromatographic conditions:
Column:C18
Mobile phase: Acetonitrile/ water : 50/50 v/v
Injection volume: 5 µl or 10 µl
Detection: 254nm
Flow Rate: 1ml/min
(Equilibrate column minimum 30 column volume with mobile phase.)
Check for Theoretical pleats, symmetry factor and column back pressure.
@ Dr isamil ,i try your method and it give very good result , the base line , back pressure every thing become ok , i change the method and use mehanol water as mobile phase insted of hexane and i got good result for vitamin E
then i use column again for triglycerides . thank you alot for your help
You are welcome, Good luck, if u want any further help, Do not hesitate to contact us , God Bless U
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