Separation and Determination of Fluorescein Isothiocyanate-Labeled Amino Acids by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Abstract

Since amino acids are components of peptides and proteins, the analysis of amino acids is important from the viewpoints of biochemical, medical and clinical aspects. However, since most amino acids possess neither strong ultraviolet/visible absorption nor fluorescence, they are usually detected after fluorogenic derivatization using dansyl chloride 1 , 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole 2 , orthophthalaldehyde (OPA)/N-acetylcysteine 3 , naphthalenedialdehyde(NDA) /NaCN 4 , fluorescein isothiocyanate (FITC) 5 and so on. Of these, dansyl chloride 6-9 , OPA/N-acetylcysteine 10,11 , NDA/NaCN 12 and FITC [13][14][15][16] have been employed for the fluorescent determination of amino acids by capillary electrophoresis (CE). Although FITC derivatization seems to be suitable for the sensitive determination of amino acids by CE, because of matching of the excitation maxima of the derivatives with the emission wavelength of the argon ion laser, CE separation of the bulky FITC derivatives is not easy. [13][14][15][16] The aim of this study was to develop a method for separating and determining FITC-amino acids by capillary electrophoresis with laser-induced fluorescence detection. We reexamined the separation condition of the FITCamino acids and succeeded in the separating 20 FITCamino acids by combining two migration systems using α-cyclodextrin (CD) or 10% methanol as a modifier. Experimental ChemicalsThe amino acids used were L-amino acids, which were purchased from Takarakosan (Tokyo, Japan), Kanto Chemical (Tokyo, Japan) and Nacalai Tesque (Kyoto, Japan). FITC was purchased from Dojindo (Kumamoto, Japan). α-, β-and γ-cyclodextrins (CDs) and O-methyl α-CD were obtained from Tokyo Chemical Industry (Tokyo, Japan). Water was purified by a Milli-Q reagent system (Millipore, Bedford, MA, USA). All other reagents used were of guaranteed grade. Preparation of FITC-amino acidsEach amino acid was dissolved in 22 mM sodium phosphate buffer (pH 9.4) at a concentration of 1 mM. A FITC (2 mM) solution was prepared with acetone. Each amino acid solution (200 µl) was mixed with the FITC solution (200 µl) and incubated at 50˚C for 5 h. CE conditions for separation of FITC-amino acidsThe CE system used was the P/ACE System 5010 (Beckman Instruments Inc., Palo Alto, CA, USA). Excitation was performed by an argon-ion laser at a wavelength of 488 nm; the emission was measured at a wavelength of 520 nm, and the absorbance was mea- A method for the separation and determination of amino acids by capillary electrophoresis with laser-induced fluorescence detection has been developed. At first, the condition for the derivatization of amino acids with fluorescein isothiocyanate (FITC) was studied; it was proved to be optimal at 50˚C for 5 h. Next, the separation of 20 FITC-amino acids was attempted by using an unmodified fused silica capillary. Actually, 18 kinds of FITC-amino acids were separated with 96 mM sodium borate buffer (pH 9.5) containing either 25% acetone (separation system 1) or 10% methanol (separation system 2). The resolutio...

Characterization of amino acids in silk sericin protein from Gonometa rufobrunnae by MEKC with phenyl isothiocyanate derivatization

“…This anomaly was in contrast with earlier observations by previous workers when they used higher concentrations of borate buffer for the separation of derivatized amino acids [24,26]. A similar phenomenon was however reported by Takizawa and Nakamura [25] when they used a running buffer consisting of 80 mM sodium borate at pH 9.2 and 45 mM a-CD for separating 18 FITCamino acid derivatives. They observed a decrease in detection sensitivity of amino acids when using both the UV and LIF detection.…”

“…The effect of borate buffer concentration on the resolution of amino acids was evaluated. Previously, other researchers successfully employed borate buffer for the separation of different types of labeled amino acids [24][25][26]. Figure 1 demonstrates a separation that was achieved when 10 mM borate was used.…”

“…Characterization of proteins involves the hydrolysis of the protein into free amino acids followed by separation using highly efficient or selective techniques typified by HPLC [14][15][16][17], CZE [18][19][20], CEC [21], and MEKC [22][23][24][25].…”

HPLC with In-Capillary Optical Fiber Laser-Induced Fluorescence Detection of Picomolar Amounts of Amino Acids by Precolumn Fluorescence Derivatization with Fluorescein Isothiocyanate

“…So far, several fluorescence tagging regents including o-phthaldialdehyde [1][2][3], naphthalenedialdehyde [3], 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [4], 9-fluorenylmethylchloroformate [5], 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate [6], 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate [7] and others are commonly used for sensitive determination of amino acids in HPLC. Fluorescein isothiocyanate (FITC) is also a powerful amino acids derivatization regent which is widely used with CE system, However, as far as we know, up to now, there is few articles about FITC used in HPLC for determination of amino acids [8][9][10][11][12].…”

Determination of Amino Acids in Tobacco Samples by Capillary Electrophoresis/Indirect Absorbance Detection with Isolation of the Electrolysis Compartment and p-Aminobenzoic Acid as a Background Electrolyte

“…5,6 CE has been employed in the determination of amino acids because of its high separation efficiency, low sample consumption and fast analysis velocity. Since most amino acids have insufficient UV absorbance and no fluorescence, their spectrometric methods, such as UV-Vis absorbance, 5,6 fluorescence (FL) and laser-induced fluorescence (LIF), [7][8][9] are mainly based on pre-or post-column derivation. The derived amino acids can improve the detection sensitivity, but the deriving operations are troublesome and time-consuming.…”