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:
10µL 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 3µm 150x4.6mm
Guard column:
Hypersil GOLD C18 3µm
Col. temperature:
40 °C
Inj. volume:
10µL
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:
5µL borate buffer + 5µL sample ◊ mix 30s
+ 1µL OPA reagent ◊ mix 90s
+ 64µL 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
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