I answered Ting,in detail,but did not send the message to the community,but
I have a couple of comments.I found out,very quickly,
that larger volumes caused havoc to the earlier part of the
chromatogram.This is obvious when there is very little sample to go
around.In my case,I compounded the problem by using more total
acetonitrile(I had synthesized my own reagent,but 20 microliters
worked ok for a week at the most.So,I started using more reagent,up
to 35 microliters in acetonitrile.I solved the problem by vacuum
drying the sample,and redissolving in A buffer.After centrifuging
some insoluble mess,I could inject the entire sample.So,basically,
I went the opposite way,but came up with the same conclusions,i.e.
too much acetonitrile=too much volume cause loss of resolution.
FYI:I am using a Waters Nova-Pak C18,which works as well as the special
and more expensive column.Only these columns give excellent separations
of the dozen tried.I hope that this may be of some usefulness.
schegg@unr.edu@aecom.yu.edu> on 06/22/2000 12:58:54 PM
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Subject: Re: AAA on HP1100
On Wed, 21 Jun 2000 16:24:40 -0600 TWong@biomira.com (Ting Wong) wrote:
>To all those AAA experts out there:
>
>I am trying to run the AAA (amino acid analysis) using the Waters'
>AccQTag method on the HP1100 HPLC system according to a paper Liu et al.
>in Journal of AOAC international, 78 (1995). Unfortunately, the Asp and
>the Glu peaks are eluting with a shoulder and tend to split into two
>peaks. Anybody know how to fix that?
>
>Thanks in advance.
>
>Ting Wong
Hi Ting,
I have been running the AccQ-Tag method for years on an HP1090. I
initially
encountered the same problem with split Asp peaks, and discovered, as Len
Packman did, that the splitting of peaks was dependent on the sample volume
injected. Optimum sensitivity is very important to me. My solution was to
decrease the total reaction volume from 100 ul to 20 ul and to inject 8 ul
onto
the column. My samples are hydrolyzed by gas phase hydrolysis in 100 ul
hydrolysis vials. Following hydrolysis, the samples are redissolved in 5
ul 20
mM HCl, then 15 ul borate buffer and 5 ul AQC solution is added. Injection
volumes of 5 and 8 ul produced lovely chromatograms, but an injection of 10
ul
produced split Asp peaks.
Kathy Schegg
University of Nevada, Reno
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