Navigation Links
ANALYSIS OF AMINO ACIDS BY REVERSED PHASE CHROMATOGRAPHY WITH PRECOLUMN DERIVATISATION AND UV/VISIBLE DETECTION


Key words:Amino Acids, RP-HPLC, Precolumn derivatisation, o-Phthalaldehyde (OPA), 2-Mercaptopropionic acid, Ultraviolet detection.


Introduction:
Underivatised amino acids do not significantly absorb light in the UV/visible spectrum. In order to increase sensitivity, their analysis normally includes a derivatisation step in which the amino acids react with a precursor to yield a strong UV/Visible chromophore or a fluorescent compound. Derivatisation can be performed before the chromatographic separation or after the separation step itself.

In this Application Note we describe a procedure which consists in pre-column derivatisation with O-Phthalaldehyde (OPA) in a borate buffer, giving UV absorbing iso-indoles that can be separated by reversed phase chromatography. The derivatisation is performed automatically by the autosampler before injection, and detection is carried out using a UV detector set at λ=338nm.


Sample treatment:
Samples are digested in acid medium to the complete hydrolysis of the protein fraction. After extraction and washing, the hydrolised amino acids are filtered and vialed, ready for analysis.

Derivatisation of the amino acids is performed automatically by the autosampler. This instrument is able to draw reagents from different vials and mix them according to a program.


Chromatographic method:
10L of a standard mix (equivalent to 10pmol of each amino acid) are mixed with the OPA reagent and given sufficient time to react.

The subsequent iso-indole derivatives are injected onto a C18 column and are eluted with a phosphate AcN/MeOH/H2O gradient.


Reagents:
Amino acid standards, OPA (o-Phthalaldehyde), 3-MPA (3-mercaptopropanoic acid), Sodium tetraborate, Sodium hydroxide were obtained from Sigma-Aldrich Company, Ltd. (Dorset, England)

De-ionised water was produced in site using a Model 6C Houseman Hegro de-ioniser, with mixed bed ion exchange resins from Helga. Acetonitrile, Methanol Gradient Quality (Romil SpS), from Romil Ltd (Waterbeach, Cambs, England.)


Typical chromatogram:


Instrumentation:

Basic Instrument:
Adept System 6T Automatic Binary Gradient HPLC consisting of:
Two CE 4100 HPLC pumps
CE 4120 dynamic mixer
CE 4200 UV-Vis detector
PowerStream chromatography control software.

CE 4800 Triathlon automatic sample injector.
With the addition of a CE 4601 column oven


Optional Modules:
CE 4020 solvent degasser
CE 4300 WaveQuest scanning UV-Vis detector
DataStream scanning control software and spectral library manager.


Chromatographic conditions:

Analytical column:

Hypersil GOLD C18 3m 150x4.6mm

Guard column:

Hypersil GOLD C18 3m

Col. temperature:

40 C

Inj. volume:

10L

Flow:

2.0mL/min

Mobile phase:

A: 40mM NaH2PO4/Na2HPO4 (1:1) buffer pH=7.8
B: AcN/MeOH/H2O (45/45/10 v/v/v)

Detection:

UV absorbance @ λ=338nm


Precolumn reaction and analysis conditions:


5L borate buffer + 5L sample ◊ mix 30s
+ 1L OPA reagent ◊ mix 90s
+ 64L H2O ◊ mix 30s ◊ inject.


Gradient program:


We supply Analytical Columns and Guard Column kits upon request.


References:
Bartolomeo MP, Maisano F. Validation of a Reversed-Phase HPLC Method for Quatitative Amino Acid Analysis. J Biol Tech 2006; 17(2):131-137


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Using THERMAL DESORPTION GAS CHROMATOGRAPHY for the identification of fragrance and other components in cosmetic products
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:3/28/2017)... ... 2017 , ... Executive search firm, Slone Partners, announces the ... is a distinguished life sciences expert with a proven track record in strategic ... wide range of services related to laboratory testing and analysis for the pharmaceutical, ...
(Date:3/27/2017)... , March 27, 2017 Cousins Properties (NYSE: ... the world,s leading biotechnology companies, has signed a 10-year, approximately ... Class A office asset located in the Westshore submarket of ... are thrilled that Amgen has chosen Corporate Center for their ... Larry Gellerstedt , president and chief executive officer of Cousins ...
(Date:3/27/2017)... , March 27, 2017  The global market ... 2016, according to a new report from Kalorama ... testing is performed to evaluate disease progression, monitor ... among other reasons.  The healthcare market research firm,s ... , provides an overview of the medical laboratory ...
(Date:3/27/2017)... ... March 27, 2017 , ... PMG Research is pleased to announce ... by The Conference Forum in Boston on April 3-4, 2017. The CTC conference focuses ... trial outcomes and bring them closer to the patient. Clinical Trial Collaborations also will ...
Breaking Biology Technology:
(Date:2/27/2017)... 27, 2017   Strategic Cyber Ventures , the ... led a $3.5 million investment in  Polarity , the ... Ventures is DC based and is led by cybersecurity ... . Ron Gula , also a longtime cybersecurity ... in this series A round of funding. This new ...
(Date:2/21/2017)... ARMONK, N.Y. and PORTLAND, Ore. ... IBM ) and the Avamere Family of Companies (Avamere ... Care) today announced a six-month research study that will ... caregivers improve eldercare at senior living and health centers. ... facilities, Avamere hopes to gain insights into physical and ...
(Date:2/13/2017)... , Feb. 13, 2017  RSA Conference -- RSA, ... that is designed to enhance fraud detection and ... in the RSA Fraud & Risk Intelligence Suite. ... to leverage additional insights from internal and external ... better protect their customers from targeted cybercrime attacks. ...
Breaking Biology News(10 mins):