Date: Fri, 16 Jun 1995 09:48:52 -0500
From: ggrant@pharmdec.wustl.edu (Gregory Grant)
Subject: AAA: alternatives
In response to Antonio de Miranda about alternative AAA to Beckman 6300.
In the last 6 months we have shut down our 6300 because it was costing us
too much to run. We have switched to AQC pre column chemistry (Waters) and
it seems to be doing well and it runs on an ordinary HPLC. Dan Crimmins
will be presenting a poster at the protein Society meeting in Boston which
presents the results of a rigorous test of the method.
Greg Grant
Washington University School of Medicine
Department of Molecular Biology and Pharmacology
St. Louis, Mo 63110
314-362-3367
FAX 314-362-4698
ggrant@pharmdec.wustl.edu
(I (David Schooley) sent message praising AQC with these references)
Cohen S. A. and Michaud D. P. (1993) Synthesis of a Fluorescent
Derivatizing Reagent, 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate, and
Its Application for the Analysis of Hydrolysate Amino Acids via
High-Performance Liquid Chromatography. Anal. Biochem. 211, 279-287,
(no abstract in database)
Liu H. J. (1994) Determination of amino acids by precolumn derivatization
with 6 aminoquinolyl-N-hydroxysuccinimidyl carbamate and high-performance
liquid chromatography with ultraviolet detection. J. Chromatogr. A 670,
59-66,
A precolumn derivatization method for the determination of amino
acids using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed
by high-performance liquid chromatography is described. Ultraviolet
detection was used for the assay of AQC derivatives of amino acids with the
detection wavelength set at 248 nm. The reagent peak interference was
minimized by optimizing the pH of the eluent and the gradient elution
profile to improve the resolution between the reagent peak and amino acid
derivatives. All nineteen amino acids were separated in 35 min with
resolutions greater than or equal to 1.6. The correlation coefficients of
the calibration graphs for seventeen amino acids were fairly good (r
greater than or equal to 0.9999) at concentrations of 25-500 mu M. The
detection limits for all common amino acids including cystine and
tryptophan were at the range 0.07-0.3 pmol. Good reproducibility and
accuracy of the method were demonstrated by the determination of amino
acids in three typical kinds of samples (protein, peptide and feed). The
average relative standard deviations for bovine serum albumin (BSA) and
neuromedin were 0.86% and 1.36, respectively, and the average relative
errors were 3.2% and 2.3%, respectively. The results of the analysis of
feed hydrolysates agreed with those obtained by an ion-exchange method and
the average recovery of the method for feed hydrolysates was 98%.
Strydom D. J. and Cohen S. A. (1994) Comparison of amino acid analyses by
phenylisothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
precolumn derivatization. Anal. Biochem. 222, 19-28,
An extensive retrospective comparison was conducted of the
long-term repeatability and consistency of amino acid analyses using
phenylisothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC) precolumn derivatization. Amino acid standards were analyzed more
than 130 times on more than 60 independent occasions by each of these two
precolumn derivatization methodologies, during routine amino acid analysis
procedures. Similar coefficients of variation were seen only when very
freshly prepared derivatives were analyzed. When realistic aging for <20 h
was taken into account, the extreme stability of the AQC derivatives stood
out. Chromatography of AQC derivatives using HPLC solvents prepared on a
large scale provided the lowest coefficients of variation. The superiority
of AQC over PTC methodology was clearly apparent. (C) 1994 Academic Press,
Inc.
Date: Tue, 26 Nov 96 13:43:50 -0500
From: Steve Cohen <cohen_steven@waters.com>
Subject: AccQTag by Capillary Electrophoresis
Hello all,
Responding to a question from Deb McMillen regarding sensitivity of
AccQ-Tag derivatives at 254nm by CE:
Swartz et al. (Anal. Biochem.) v 231 (1995, pp 65-71) report that LOD for
their CE system is approximately 0.1 microgram/ml for derivatized Trp. LOQ
is 3x higher. Although they scaled up the volume 10x for derivatization,
this would correspond to LOD of 1 ng of Trp derivatized using the standard
protocol (10 microliters of sample) - corresponds to ~ 6 pmol of Trp in 10
microliters. You can easily derivatize 20-40 microliters of sample and cut
the borate derivatization buffer volume accordingly (if the final sample pH
is between 8.2 and 10.0) to decrease the detection limits by up to 4x.
Although you might think the Trp response is much greater than other amino
acids, this is not true, and the response is only ~10 - 20% higher.
In unpublished work we have separated free amino acids in an achiral manner
(the Swartz paper is for enantiomeric separations) using SDS with borate
buffer at pH 7.5 - 10.0 with [SDS] ranging from 25 - 100mM. No system
resolved all hydrolysate amino acids. Adding MeOH at 5-20% can improve the
separation.
Steve Cohen
>Hello all,
> We are planning to analyze the release of amino-terminal amino acids
>from pentapeptides
>by aminopeptidases via derivitization with AccQ-TAG and analysis using
>MEKC on an HP capillary electrophoresis system with detection at 254nm.
>(We don't have a fluorescence detector). Before purchasing the reagents,
>we would like to have some idea of the sensitivity of the method at
>254nm. Also, any suggestions as to detergent additives for the MEKC
>run would also be
>helpful: we have seen papers for the use of SDS, CTAB and deoxycholate.
>Thanks for your help,
>Deb McMillen
>Institute of Molecular Biology
>University of Oregon
Steven A. Cohen, PhD
Principal Research Chemist
Waters Corporation
PH # 508-482-2501
FAX# 508-482-3625
email cohen_steven@waters.com
David A. Schooley
Dept. of Biochemistry/330
Univ. of Nevada
Reno, NV 89557
schooley@med.unr.edu
tel: (702) 784-4136; fax (702) 784-1419