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:5/24/2017)... 2017 As Ebola resurfaces in the ... 20 suspected cases now reported, a new analysis of the ... a correlation between the 2014 and 2017 outbreaks of the ... in 2012-13, which preceded the 2014 outbreak. An analysis of ... Replikin counts in 2014-15, which again precedes the current outbreak ...
(Date:5/23/2017)... ... May 23, 2017 , ... A recent survey conducted by the Weed Science ... control weed in 12 categories of broadleaf crops, fruits and vegetables, while common lambsquarters ... U.S. and Canada participated in the 2016 survey, the second conducted by WSSA. ...
(Date:5/23/2017)... , ... May 23, 2017 , ... ... this month its 20th anniversary, marking the occasion with a strong presence at ... meeting’s Welcome Reception and further extends an invitation to all attendees to view ...
(Date:5/23/2017)... ... May 22, 2017 , ... A new Technology Hot Topics ... this August will feature high-level speakers on quantum devices, graphene electronic tattoo sensors, ... Photonics, the largest multidisciplinary optical sciences meeting in North America, will run 6-10 ...
Breaking Biology Technology:
(Date:4/19/2017)... 19, 2017 The global military ... is marked by the presence of several large global ... by five major players - 3M Cogent, NEC Corporation, ... for nearly 61% of the global military biometric market ... the global military biometrics market boast global presence, which ...
(Date:4/17/2017)... 2017 NXT-ID, Inc. (NASDAQ: NXTD ) ... of its 2016 Annual Report on Form 10-K on Thursday April ... ... in the Investor Relations section of the Company,s website at ... at http://www.sec.gov . 2016 Year Highlights: ...
(Date:4/13/2017)... 13, 2017 According to a new market research ... Analytics, Identity Administration, and Authorization), Service, Authentication Type, Deployment Mode, Vertical, and ... is expected to grow from USD 14.30 Billion in 2017 to USD ... 17.3%. ... MarketsandMarkets Logo ...
Breaking Biology News(10 mins):