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By Anonymous on Friday, June 20, 2003 - 01:53 am:
Which RP columns and mobile phase (pH) do you advise and why to separate these components creatine NH2-C(NH)-N(CH3)-CH2-COOH, guanidino acetate NH2-C(NH)-NH-CH2-COOH, L-Arginine NH2-C(NH)-NH-CH2-CH2-CH2-C(NH2)-COOH and creatinine CO-NH-C(NH)-N(CH3)-CH2.Thanks in advance.
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By Anonymous on Friday, June 20, 2003 - 05:53 pm:
I recommend a silica HILIC column with about 75% acetonitrile, 25% water with a control of the pH, maybe acetic acid around pH 4.75. The reason for this is that you will have a hard time to get retention for these compounds in reversed-phase without trying ion-pair, which creates difficulties with MS detection. HILIC is ideal for these very polar compounds. In addition, you get much higher sensitivity in ESI MS.
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By Martine Vaglio on Friday, September 5, 2003 - 06:30 am:
Thanks for the answer.
I receive an HILIC silica,3µm,50mm,id 2.1mm,column from Waters.
I use 0.1% formic acid (A) in water and acetonitrile (B) for the mobile phase with a flow rate of 0.2 ml/min.
I try an isocratic separation, the A/B 35:65 give me the best results. Also I try a gradient separation, the A/B 30:70 to A/B 45:55 in 15 minutes give me the best results, probably I could do it in a short time.
My questions are:
is acetic acid better than formic acid and why?
is 0.1% formic acid enough, is it better with 0.2%?
which percent is necessary to wash my column after the gradient separation A/B 50:50 or 40:60?
which separation is better to use isocratic or gradient and why.
Thanks to clear my troubles.
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By Anonymous on Saturday, September 6, 2003 - 06:01 pm:
Glad you are successful! Since you are successful, there is no need to change.
I had suggested ammonium acetate to prevent potentially excessive retention due to the amine functions on your analytes. I could have suggested ammonium formate at pH 3.75 as well. But if formic acid works, leave it at that.
Higher formic acid concentrations are better if you need to inject a lot of sample, but since you are working with MS, this is not likely. If you are happy with 0.1% stick with it. I would wash the column after the runs with 50:50. Finally, I do not see a reason to prefer isocratic over gradient, unless you need to work in high speed mode and want to avoid the column reequilibration time in the gradient.
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By Jakub Krijt on Tuesday, October 21, 2003 - 05:49 am:
hello,
i plan to set up a method for creatine and guanidinoacetate in plasma and tissues. Could you please write me what kind of MS/MS detector (ion trap, Q?) do you use and in which mode (MRM,SIM?).Do you perform a SPE or other sample preparation before injecting the samples on LC?
Thanks J.
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By Anonymous on Wednesday, January 28, 2004 - 05:23 am:
Between two samples are you doing a wash (which number of column volume) then an equilibration (which number of column volume)of the HILIC column after the gradient separation of the analytes or only an equilibration and finally a wash of the column at the end of the run.
Thanks for the answer.
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By Anonymous on Tuesday, July 6, 2004 - 07:15 am:
Hello,
Do you recommend any handbook on sample preparation for LC/MS bioanalysis. We work with plsama/blood/serum samples for pharmacokinetic studies.
Regards.
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By Uwe Neue on Thursday, July 22, 2004 - 04:18 pm:
Last anomymous: if you contact me, I'll send you an article published by us in the Handbook of Analytical Separations volume 4, Bioanalytical Separations.
Which RP columns and mobile phase (pH) do you advise and why to separate these components creatine NH2-C(NH)-N(CH3)-CH2-COOH, guanidino acetate NH2-C(NH)-NH-CH2-COOH, L-Arginine NH2-C(NH)-NH-CH2-CH2-CH2-C(NH2)-COOH and creatinine CO-NH-C(NH)-N(CH3)-CH2.Thanks in advance.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 20, 2003 - 05:53 pm:
I recommend a silica HILIC column with about 75% acetonitrile, 25% water with a control of the pH, maybe acetic acid around pH 4.75. The reason for this is that you will have a hard time to get retention for these compounds in reversed-phase without trying ion-pair, which creates difficulties with MS detection. HILIC is ideal for these very polar compounds. In addition, you get much higher sensitivity in ESI MS.
-------------------------------------------------------------------------------------------------------
By Martine Vaglio on Friday, September 5, 2003 - 06:30 am:
Thanks for the answer.
I receive an HILIC silica,3µm,50mm,id 2.1mm,column from Waters.
I use 0.1% formic acid (A) in water and acetonitrile (B) for the mobile phase with a flow rate of 0.2 ml/min.
I try an isocratic separation, the A/B 35:65 give me the best results. Also I try a gradient separation, the A/B 30:70 to A/B 45:55 in 15 minutes give me the best results, probably I could do it in a short time.
My questions are:
is acetic acid better than formic acid and why?
is 0.1% formic acid enough, is it better with 0.2%?
which percent is necessary to wash my column after the gradient separation A/B 50:50 or 40:60?
which separation is better to use isocratic or gradient and why.
Thanks to clear my troubles.
-------------------------------------------------------------------------------------------------------
By Anonymous on Saturday, September 6, 2003 - 06:01 pm:
Glad you are successful! Since you are successful, there is no need to change.
I had suggested ammonium acetate to prevent potentially excessive retention due to the amine functions on your analytes. I could have suggested ammonium formate at pH 3.75 as well. But if formic acid works, leave it at that.
Higher formic acid concentrations are better if you need to inject a lot of sample, but since you are working with MS, this is not likely. If you are happy with 0.1% stick with it. I would wash the column after the runs with 50:50. Finally, I do not see a reason to prefer isocratic over gradient, unless you need to work in high speed mode and want to avoid the column reequilibration time in the gradient.
-------------------------------------------------------------------------------------------------------
By Jakub Krijt on Tuesday, October 21, 2003 - 05:49 am:
hello,
i plan to set up a method for creatine and guanidinoacetate in plasma and tissues. Could you please write me what kind of MS/MS detector (ion trap, Q?) do you use and in which mode (MRM,SIM?).Do you perform a SPE or other sample preparation before injecting the samples on LC?
Thanks J.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, January 28, 2004 - 05:23 am:
Between two samples are you doing a wash (which number of column volume) then an equilibration (which number of column volume)of the HILIC column after the gradient separation of the analytes or only an equilibration and finally a wash of the column at the end of the run.
Thanks for the answer.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, July 6, 2004 - 07:15 am:
Hello,
Do you recommend any handbook on sample preparation for LC/MS bioanalysis. We work with plsama/blood/serum samples for pharmacokinetic studies.
Regards.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, July 22, 2004 - 04:18 pm:
Last anomymous: if you contact me, I'll send you an article published by us in the Handbook of Analytical Separations volume 4, Bioanalytical Separations.
