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- Posts: 2
- Joined: Thu Aug 01, 2013 7:16 pm
Hi All,
I've been using Agilent's "Rapid, Sensitive, and Reproducible HPLC Analysis of Amino Acids" with OPA / 3-mercaptopropionic acid (without FMOC) with some success and some frustration. I'm using an Agilent 1200 HPLC, Zorbax Eclispe AAA Column (3.0 x 150 mm, 3.5 um), similar buffer conditions (altered for MS use), 17 amino acid standards at various concentrations, and norvaline as my internal standard.
For anyone that has worked with this technique or amino acid analysis before I have a few nagging questions that seem to be critical to this process:
1) I have a great calibration curve for each amino acid, BUT when I ran my first real samples (eumelanin and milk) I had pressure problems right at the front on injection. I had residual crude stuck on the column that wasn't removed until the column was rinsed with 10% IPA in the organic. I need to improve my sample workup most likely to remove the sugars from milk and a variety of components remaining after hydrolysis of melanin.
I have access to many SPE columns and a vacuum SPE manifold, but I've had some problems in the past with sample losses with SPE.
Has anyone had success using SPE without large amino acid losses? What procedure / column was used?
2) I've edited the autosampler program supplied by Agilent from the to decrease the amount of water added in the final step from 32uL to 6.5uL. I'm also using the high-resolution mass-spec with my edited buffer conditions so I know that the UV peaks I am seeing are from the amino acids expected (to a high degree of certainty). With the issues I've been seeing using my real samples I've revisited this edit. What is the benefit of using such a large amount of water in this protocol? My thought was that it dilutes the borate buffer which has a very high pH (10.2), but if the sample is in 0.1M HCl, FMOC is not being used, and the aqueous buffer is at pH 7.8 is it essential?
Thank you!
I've been using Agilent's "Rapid, Sensitive, and Reproducible HPLC Analysis of Amino Acids" with OPA / 3-mercaptopropionic acid (without FMOC) with some success and some frustration. I'm using an Agilent 1200 HPLC, Zorbax Eclispe AAA Column (3.0 x 150 mm, 3.5 um), similar buffer conditions (altered for MS use), 17 amino acid standards at various concentrations, and norvaline as my internal standard.
For anyone that has worked with this technique or amino acid analysis before I have a few nagging questions that seem to be critical to this process:
1) I have a great calibration curve for each amino acid, BUT when I ran my first real samples (eumelanin and milk) I had pressure problems right at the front on injection. I had residual crude stuck on the column that wasn't removed until the column was rinsed with 10% IPA in the organic. I need to improve my sample workup most likely to remove the sugars from milk and a variety of components remaining after hydrolysis of melanin.
I have access to many SPE columns and a vacuum SPE manifold, but I've had some problems in the past with sample losses with SPE.
Has anyone had success using SPE without large amino acid losses? What procedure / column was used?
2) I've edited the autosampler program supplied by Agilent from the to decrease the amount of water added in the final step from 32uL to 6.5uL. I'm also using the high-resolution mass-spec with my edited buffer conditions so I know that the UV peaks I am seeing are from the amino acids expected (to a high degree of certainty). With the issues I've been seeing using my real samples I've revisited this edit. What is the benefit of using such a large amount of water in this protocol? My thought was that it dilutes the borate buffer which has a very high pH (10.2), but if the sample is in 0.1M HCl, FMOC is not being used, and the aqueous buffer is at pH 7.8 is it essential?
Thank you!
