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- Posts: 2
- Joined: Wed Oct 20, 2010 9:59 pm
Dear everybody
i had some problems in determining hydroxyproline by HPLC after derivatization by dabsyl chloride.
First, my samples are serum samples and i made acid hydrolysis for them, and i found their color to be black (even though i made the hydrolysis in closed ampoules) .... Did this affect hydroxyproline concentration in the samples, or just charring of other constituents and hydroxyproline wouldn't be affected???
Second, i made derivatization for primary amino groups (as a blocking) in the sample using o-phthalaldehyde, then derivatize hydroxyproline by dabsyl chloride... really i found that the medium of the sample to be basic by adding sodium carbonat and sodium hydroxide or triethylamine.... i need to know which is better for my derivatization???
Third, ethyl acetate extraction procedure was made to clean the sample by adding some ethyl acetate with shaking then centrifuging the samples and take the aqueous part... iam afraid that ethyl acetate may trap some of hydroxyproline i need to measure.... what about using this extraction???.. is it recommended or cancelled?
forth, i need to know about the stability of dabsyl chloride in acetonitrile... should i use it freshly prepared or it can be stored at certain conditions?.... also, what about the stability of my complex of hydroxyproline with dabsyl chloride?.... i.e. can i prepare my samples in a day then measure them in the next day without affecting the results?
also, i tried to get a calibration curve, but the linearity is soooooooooo poor, i don't know why? even though i prepared a series of hydroxyproline concentrations and put them under the same derivatization condtions....
finally, i tried to measure hydroxyproline standards in water and after spiking in serum and there is a great difference for the same std concentration in their peak areas in serum and water.....
so should i make it in serum?
or i can make it in water and calculate concentrations from its linear equation?
i know that i wrote so much, but really this is the only parameter remaining in my practical part to finish my thesis.... so plz i need urgnet help
best regards
i had some problems in determining hydroxyproline by HPLC after derivatization by dabsyl chloride.
First, my samples are serum samples and i made acid hydrolysis for them, and i found their color to be black (even though i made the hydrolysis in closed ampoules) .... Did this affect hydroxyproline concentration in the samples, or just charring of other constituents and hydroxyproline wouldn't be affected???
Second, i made derivatization for primary amino groups (as a blocking) in the sample using o-phthalaldehyde, then derivatize hydroxyproline by dabsyl chloride... really i found that the medium of the sample to be basic by adding sodium carbonat and sodium hydroxide or triethylamine.... i need to know which is better for my derivatization???
Third, ethyl acetate extraction procedure was made to clean the sample by adding some ethyl acetate with shaking then centrifuging the samples and take the aqueous part... iam afraid that ethyl acetate may trap some of hydroxyproline i need to measure.... what about using this extraction???.. is it recommended or cancelled?
forth, i need to know about the stability of dabsyl chloride in acetonitrile... should i use it freshly prepared or it can be stored at certain conditions?.... also, what about the stability of my complex of hydroxyproline with dabsyl chloride?.... i.e. can i prepare my samples in a day then measure them in the next day without affecting the results?
also, i tried to get a calibration curve, but the linearity is soooooooooo poor, i don't know why? even though i prepared a series of hydroxyproline concentrations and put them under the same derivatization condtions....
finally, i tried to measure hydroxyproline standards in water and after spiking in serum and there is a great difference for the same std concentration in their peak areas in serum and water.....
so should i make it in serum?
or i can make it in water and calculate concentrations from its linear equation?
i know that i wrote so much, but really this is the only parameter remaining in my practical part to finish my thesis.... so plz i need urgnet help
best regards
fatma elzahraa
