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Anal. Chem. 1992, 6 4 , 711-714
Determination of Amino Acids by Capillary Zone Electrophoresis Based on Semiconductor Laser Fluorescence Detection Toshiyuki Higashijima, Tetsuhiro Fuchigami, Totaro Imasaka,*and Nobuhiko Ishibashi Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
Chlorophyll Is fluorescent In the deepred region and Is detennlned by semlconductor laser fluorometry after Its separation wHh capillary zone electrophoresls. The separatlon Wklency b eeveral hurbed thowand In the theoretical plate. Methylene blue Is used as a chromophore In Indirect fluorometry. The datectlon lknH achleved Is l-pmol levels. A new labeling reagent Is syntheslzed, which consists of a thlazlne chromophore for fluorescence detection and a succlnlmldyl ester lor comblnatlon wHh an amino acld. The labeled amlno ackls are clearly resolved by capillary zone electrophoresls, the detection IlmH being 10-pmol levels. Amino aclds are furlher labeled wHh a vlslMe dye such as fluorescelnlsothlocyanate (FITC) or 7-( dlethylamlno)coumarln-3tarboxyllc ackl succbrhnklyl ester (PCCS), and are detected wlth vlslble semiconductor laser fluorometry using second harmonlc emission (415 nm) of the near-lnfrared semlconductor laser. The detection limn achieved Is -100-amol levels.
A laser is currently used in analytical spectroscopy and has provided an ultrasensitive means in trace analysis.' Detection of single atoms, ions, or molecules has already been demonstrated by laser spectroscopy. However, its practical application is quite limited except for Raman spectroscopy. This is believed to be due to the cost of the laser, but lifetime and maintenance problems are more serious in commercialization of the instrument. Until now, only a few analytical instruments have been commercialized using a laser, e.g. a HeNe laser for measurement of light scattering. More recently, an air-cooled argon ion laser is used for determination of amino acids after labeling them with 7-chloro-4-nitrobenzoxadiazole (NBD) or fluoresceinisothiocyanate (FITC) and separation by high-performance liquid chromatography (HPLCh2 A semiconductor laser is developed for use in a compact disk, a laser printer, and a bar-code reader. It is less expensive than a conventional light source such as a xenon arc lamp, and the lifetime exceeds loo00 h. Then, a semiconductor laser can be used in a commercial analytical instrument without any maintenance for more than 1year. The application of the semiconductor laser to analytical spectroscopy has been reviewed el4e~here.l"~Particularly, fluorescencespectrometry is sensitive and has been used in determination of biological molecules such as metabolites, enzymes, and proteins."8 When an l-kW xenon arc lamp is used as a light source and the spectral bandwidth of a monochromator is adjusted to 20-40 nm, the radiant photon flux exceeds 10 mW. This is comparable to the output power of the semiconductor laser (3-40 mW) manufactured by mass production technology. Thus a semiconductor laser is advantageous only when good beam focusing capability and monochromaticity are successfully used. For example, a laser fluorometric detector combined with HPLC gives high sensitivity due to a small
* Author to whom correspondence should be addressed. 0003-2700/92/0384-0711$03.00/0
detector volume required and to a sharp-cut-off optical filter for efficient fluorescence collection and scattered light rej e ~ t i o n . ~However, ,~ a microliter detection volume currently used in HPLC is still too large for best use of the h e r ; since the laser beam can be easily focused down to a submicron spot, the detection volume can be minimized to nanoliter or even to picoliter levels if necessary. Recently, capillary zone electrophoresis has gained ita importance. This is due to applicability to many biological substances and ultrahigh resolution in sample separation. Unfortunately, the pass length in on-column detection is 50 Hm, so that the detection limit achieved by absorption spectrometry is restricted to 10" M levels.1° Contrarily, laser fluorometry is quite advantageous for ultrasensitive detection of samples because of good focusing capability and monochromaticity of the laser. In the extreme case, only a few thousand amino acids labeled with FITC have been detected using an argon ion laser.l'-13 Electrokinetic separation and fluorescence detection of chiral amino acids are further demonstrated with copper(II)-aspartame support ele~trolyte.'~*'~ A semiconductor laser is well-matched to capillary zone electrophoresis with respect to a price and a dimension of the instrument; an argon ion laser has a large output power in the visible region, but the dimension and the price sometimes exceed those of the instrument in capillary zone electrophoresis. Furthermore, semiconductor laser fluorometry in the near-infrared or deep-red region provides low background fluorescence, and then the sample is possibly determined at lower levels especially for a real sample containing many c o n t a " t a fluorescent in the visible region. There are many compounds fluorescent in the near-infrared and deep-red r e g i ~ n s . ~ ~However, J' no fluorescence reagent useful for capillary electrophoresis based on indirect fluorometry has been reported so far, though a few near-infrared dyes are already used in HPLC/indirect fluorometry.l8tm It is noted that adsorption of the reagent to the separation column is more serious in capillary electrophoresis due to strong negative charges at the capillary surface. Moreover, a labeling reagent of biological molecules has seldom been developed at the present ~tage.'~J'Then, a new labeling reagent is desired for application of semiconductor laser fluorometry to capillary zone electrophoresis. Though the fundamental wavelength of the semiconductor laser commercially available is limited to 660-850 nm, the frequency-doubled output of the semiconductor laser has been used as a light source in analytical spectroscopy such as an optical fiber sensor system or molecular fluorescence spectrometry.21*22However, full performance in focusing capability and monochromaticity of the laser has not yet been utilized. In this study we first demonstrate capillary zone electrophoresis based on semiconductor laser fluorometry. First, chlorophyll is separated by capillary zone electrophoresis and is detected by 670-nm semiconductor laser fluorometry. Secondly, methylene blue is used as a fluorophore in indirect spectrometry, in which a capillary with a alkylsilane-derivatized surface is used to reduce adsorption of methylene blue.
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0 1992 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1, 1992 + , ',
aIaNH + HOOC-CH2SH
Diode Laser
DCC
Condensation
(CH,)zN B
Mercaptoacetic Acid
CH3
0
0
Enclosure
;Buffer
Buffer
Flgure 1. Block diagram of experimental apparatus for Capillary zone electrophoresis combined with semiconductor laser fluorometry.
Thirdly, we synthesize a new labeling reagent with a thiazine chromophore fluorescent in the deep-red region and with a succinimidyl ester to bind with an amino acid. In fact, this reagent is applied to capillary zone electrophoresis of amino acids. Finally, a blue semiconductor laser emitting second harmonic wave (415nm) is used for excitation of the separated amino acids labeled with visible dyes. EXPERIMENTAL SECTION Apparatus. A block diagram of the experimental apparatus is shown in Figure 1. The sample was injected into a capillary (Polymicro Technology, 50-pm i.d., 375-~mo.d., 60-cm long) by a siphon method. The injection volume was determined to be 1nL, according to the method reported in ref 23. A high potential is applied between platinum electrodes immersed in the buffer solutions by a high-voltage power supply (Kansai Electric, KTS-3OKO1, 0-30 kV, 100 FA). The sample migrates from a positive to negative (ground) potential. The system is enclosed by a Plexiglass box with a safety interlock. A semiconductor laser (ILEE Laser Innovation, LDAO2, 670 nm, 2 mW) emitting at 670 nm is focused by a quartz lens (focal length, 25 mm) onto the capillary at which a polyimide coating is burned off by a flame. Fluorescence from a sample is collected by an objective lens for a microscope (Olympus, LWD MSPlan 50, X 50, NA = 0.6) and is measured by a photomultiplier (EMI, 9558QB) after passing it through interference and color filters (Melles Griot, 03FIV024 and 03FCG109). A blue semiconductor laser (Matsushita Electric Industrial Co., IMS00820-04, 50 pW, 415 nm, linewidth 3 nm, beam divergence lo, linearly polarized) consists of a near-infrared semiconductor laser (830 nm), a waveguide of LiNb03, a collimating lens, and a power supply (R&K Co., LDA-8502S, 850 MHz). The body of the laser is 8-cm long, 2-cm wide, 2-cm high. In this case, the laser beam is more tightly focused by a pair of quartz lenses (focallength, 10 and 20 mm). A color fdter (Toshiba, V-Y45) is used for isolation of fluorescence. The positions of the focusing lens and the capillary are carefully aligned by adjusting the fluorescence image to the pinhole image projected by irradiating the pinhole with a lamp placed behindeZ4The fluorescence signal is amplified 100 times by a commercial amplifier (NFCircuit Design Block, LI-75A) and is measured by a strip chart recorder (Hitachi 056). Reagents. Chlorophyll, a nonrefined mixture of chlorophyll, was purchased from Tokyo Kasei. The content of total chlorophyll is specified to be 0.5% by a manufacturer. Fluorescent dyes of methylene blue and Azur B were obtained from Wako Pure Chemical and of oxazine 750 and rhodamine 800 from Lambda Physik. For synthesis of a labeling reagent, marcaptoacetic acid (Wako Pure Chemical), N,N'-dicyclohexylcarbodiimide (DCC, emaleimidocaproy1oxy)succinimide Kishida Chemical), and N-( (EMCS, Dojindo Laboratories) were used. Labeling reagents of acid FITC (isomer-I)and 7-(diethylamino)coumarin-3-carboxylic succinimidyl ester (DCCS) fluorescent in the visible region were supplied from Dojindo Laboratories and Molecular Probes, respectively. Amino acids were obtained from Ajinomoto General Foods. Synthesis of Labeling Reagent. The procedure for synthesis of a labeling reagent is shown in Figure 2. Ethanol solutions of Azur B M), mercaptoacetic acid ( 5 X M), and DCC ( 5
Flgure 2. Scheme for organic synthesis of labeling reagent.
I
0
L--
. 5
-.-A
10
Time ( m i d
Flgure 3. Electropherogram for chlorophyll (100 fmoi). The sample
was dissolved in pure water. The pH of the carrier solution was adjusted to 6 by a phosphate buffer (60 mM). X M) were mixed with a molar ratio of 15050. The solution was reacted at room temperature during a day by mixing it with a magnetic stirrer. The product was dried by a rotary evaporator. The methanol solution of equimolar EMCS M) was added and reacted at room temperature during day. This solution was used for labeling amino acids without reagent purification. Sample Preparation. Amino acid (0.1 M, 0.1 mL) was reacted over night with the synthesized reagent (low4M, 2 mL); the time period necessary to complete the reaction may be 0.5-2 h. In order to label amino acids with a visible dye (DCCS), the sample dissolved in the carbonate buffer (pH 11,20 mM) was mixed during a day with the ethanol solution containing DCCS, their final M, reconcentrations being adjusted to 2 x W5and 2 X spectively. This sample solution was diluted stepwise before measurements.
RESULTS AND DISCUSSION Determination of Chlorophyll. Several forms of chlorophyll, e.g. chlorophyll a, are known to be fluorescent in the deep-red region.25 Then, chlorophyll is measured to check performance of the instrument developed in this study. A sharp electropherogram is observed as shown in Figure 3, giving a theoretical plate of several hundred thousand. Thus it is confirmed that the present capillary zone electrophoresis combined with semiconductor laser fluorometry is useful in direct determination of water-soluble compounds fluorescent in the deep-red region. Indirect Fluorometry. Indirect spectrometry has a potential to be used in practical analysis, since all the chemical species are measured without any labeling procedure.1° In order to find a fluorescent dye suitable for indirect fluorometry, three dyes fluorescent in the deep-red region are injected into a capillary. The electropherograms obtained are shown in Figure 4. All the signal peaks are seriously broadened. This is probably due to strong adsorption of the
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1. 1992
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Methylene Blue
Rhodamine 800
G
:
L
10
5
0
l
Methylene
Oxzine 750
0
5 Time (mid
10
Proline
Figure 4. Electropherograms for dyes (100 fmol) fluorescent in deep-red region. The pH of the carrler solution was adjusted to 6 by a phosphate buffer (60 mM). The voltage and current were 25 kV and 80 PA, respectkely. PH 5
r 5 10 15
0
Time (mid
Figure 6. Indirect fluorometry of amino acMs (10 pmol) measured by flowing methylene blue (10 PM) as fluorescence reagent. The pH of the carrier solution was adjusted to 3.8 by a citrate buffer (10 mM). The voltage and current were 20 kV and 5 PA, respectively.
pH 11
Reagent Arginine 5
Time (mtn)
0
5 Time (min)
Flgure 5. Effect of pH on electropherogram of methylene blue (100 fmol). The pH of the carrier solution was adjusted to 5 by a phosphate buffer (60 tnM) or to 11 by a carbonate buffer (20 mM).
dyes to the capillary surface, since the dye molecules are positively charged and the capillary surface is negatively charged by dissociation of the proton from the silanol group. Methylene blue gives the narrowest peak among these dyes, which might be due to a high solubility of methylene blue in an aqueous solution."j So, the effect of pH is further investigated for methylene blue, as shown in Figure 5. Adsorption is substantially reduced at higher pH, and a sharp signal peak is observed in the electropherogram though the fluorescence intensity is substantially decreased. The reason is unknown at present, but methylene blue might be negatively charged and is less fluorescent a t this pH. The detection limit was 400 am01 at pH 5 and was about 1 order of magnitude poorer a t pH 11. Unfortunately, all amino acids are almost completely dissociated to negatively-charged ions at pH 11, and it seems to be difficult to separate amino acids at such high pH. Thus the capillary conventionally used was replaced with one whose surface is alkylsilane-derivatized. In this case, most of the active sites to adsorb chemical species are end-capped, but the electroosmotic flow is still sufficient to drive the solution to a negative potential.26 Capillary electrophoresis of amino acids using methylene blue as an indirect fluorophore is demonstrated in Figure 6. Glycine and proline are clearly separated and detected. Broad bands appearing a t 4-8 min in the electropherogram are reproducible, and they probably originate from contaminants included in the sample solution. 'The detection limit in this approach is 1pmol. The variation of the fluorescence intensity for M methylene blue solution in the capillary was 8 X lo4 (time constant, 1s), the dynamic reserve being 1300. However, the variation of the output power of the semiconductor laser was 2 X when it was stabilized by feedback control. Therefore, the detection limit might be substantially improved by optimization of the
-
0
"
5
10 Time (min)
15 \
Figure 7. Electropherogram for amino acids (100 pmol) labeled with newly synthesized dye consisting of Azur B chromophore and SUCCInimidyl ester. The pH of the carrier solution was adjusted to 5 by a phosphate buffer (60 mM). The voltage and current were 20 kV and 62 PA, respectlvely.
experimental conditions in the future, e.g. by changing the concentration of the dye and the pH of the carrier solution and by using a more efficient optical filter and a vibrationally-isolated optical table. The development of a negatively-charged dye might be important to reduce adsorption of the dye at the capillary surface. Addition of a ~ u r f a c t a n t ~ . ~ ~ or an organic solvent28changes surface structure and separation mode, so that it may be possible to reduce adsorption of methylene blue to the capillary surface. Determination Using a Labeling Reagent. More sensitive detection of amino acids may be performed by labeling them with a fluorescent dye. However, no labeling reagent fluorescent in the deep-red region has been so far developed. The electropherogram obtained by labeling amino acids with a new labeling reagent synthesized in this study is shown in Figure 7. A strong peak at 6 min originates from the reagent blank, probably due to nonreacted chemicals or side-reaction products. Arginine and glycine are clearly separated and detected by semiconductor laser fluorometry. The detection limit is 10-pmol levels, at present. In this preliminary work the labeling reagent synthesized has not been purified, since it requires additional time and skill. Moreover, the reaction yield might be rather low under present nonoptimized conditions. Thus it will be necessary to synthesize a new and pure labeling reagent in the future, which is more soluble in aqueous
714
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1, 1992 6 7-Diethylammocoumarin-3-Carboxyl1~ Acid, Succinimidyl Ester (DCCS)
is already developed,30so that the sensitivity in the present method will be substantially improved in the future. ACKNOWLEDGMENT This research is supported by Grants-in-Aids for Scientific Research from the Ministry of Education of Japan, from Steel Industry Foundation for the Advancement of Environmental Protection Technology (SEPT), and from Nakatani Electronic Measuring Technology Association of Japan. REFERENCES
0 ”
2
4
6
8
Time ( m i d
Flgure 8. Electrophsrogramfor amino acids (1 fmol each) labeled with a vlsible dye (DCCS). The pH of the carrier solution was adjusted to 9 by a carbonate buffer (5 mM). The voltage and current were 20 kV and 4 PA, respectively.
solution and is more fluorescent in the deep-red region. Such an effort is now concentrated by several companies manufacturing chemical reagents. Visible Fluorometry Using a Blue Semiconductor Laser. Many efficient labeling reagents are already available in the visible region, e.g. at around 415 nm, which corresponds to the emitting wavelength of the blue semiconductor laser. The experiment was performed for two candidates, i.e. FITC and DCCS. A reagent of FITC is widely used for labeling amino acids. Then, this labeling reagent is first applied to capillary zone electrophoresis/ blue semiconductor laser fluorometry to check performance of the present analytical instrument. Complete separation of labeled amino acids, e.g. arginine, proline, serine, glycine, was readily demonstrated, as reported by detecting them with an argon ion laser.’l The detection limit was -2 fmol. However, the absorption maximum of FITC is located at around 490 nm, and the excitation efficiency at 415 nm is only several percent in comparison with that at the absorption maximum.Therefore, DCCS (absorption maximum 437 nm, excitation efficiency 66%) is applied for more sensitive detection. The electropherogram obtained is shown in Figure 8. All amino acids measured are separated in 6 min. The detection limit estimated is -100 amol, which is already much lower than the detection limit (femtomole levels) obtained by a commercial HPLC system using a combination of an air-cooled argon ion laser and a labeling reagent of NBD or FITC2 It is noted that a blue semiconductor laser with an output power of 40 mW
(1) (2) (3) (4) (5) (6) (7)
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Imasaka, T.; Ishibashi, N. Pfog. Qwnt. Nectron. 1990, 14, 131. Tosoh, Model LF-8010, reported in HLC MAILGRAM, 1991, 53(6),5. Imasaka, T.; Ishibashi, N. Anal. Chem. 1990, 62, 363A. Lawrenz, J.; Niemax, K. Spectrochim. Acta, Part B 1989, 4 4 , 155. Sauda. K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1986, 58, 2649. Imasaka, T.; Okazaki, T.; Ishibashi, N. Anal. Chim. Acta 1988, 208, 325. Imasaka, T.; Nakagawa, H.; Okazaki, T.; Ishibashi, N. Anal. Chem. 1990, 6 2 , 2404. Imasaka, T.; Fuchigami, T.; Ishibashi, N. Anal. Sci. 1991, 7, 491. Sauda, K.; Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1986, 187, 353. Yeung, E. S.; Kuhr, W. G. Anal. Chem. 1991, 6 3 , 275A. Cheng, Y. F.; Dovichi, N. J. Science 1988, 242, 562. Wu, S.; Dovichi, N. J. J . Chromatogr. 1989, 480, 141. Hernandez, L.; Escalona, J.: Joshi, N.; Guzman. N. J. Chromatogr. 1991, 559, 183. Gassmann, E.; Kuo, J. E.; Zare, R. N. Science 1985, 230, 813. Gozel, P.; Gassmann, E.; Michelsen, H.: Zare, R. N. Anal. Chem. 1987, 59,44. Imasaka, T.; Tsukamoto, A.; Ishibashi, N. Anal. Chem. 1989, 6 1 , 2285. Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 6 3 , 321A. Folestad, S.: Ahlberg, H. International Symposium on Column Liquid Chromatography, Stockholm, June 26-30, 1989. Kawazumi, H.; Nishimura, H.; Ogawa, T. AnnualMeeting of The Japan Sociefy for Analytical Chemistry,Yokohama, November 21-23, 1991. Lehotay, S. J.: Pless, A. M.; Winefordner, J. D. Anal. Sci. 1991, 7, 863. Okazaki, T.: Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1988, 209, 327. Imasaka, T.; Hiraiwa, T.; Ishibashi, N. Mikrochlm. Acta 1989, I I , 225. Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. J. Chromatogr. Sci. 1989, 2 4 , 347. Dcvichi, N. J.. Alberta University, private communication, September 1990. Yoshitake, Y.; Tanino, M.; Morishige, K.; Shigematsu, T.;Nishikawa, Y. Bunseki Kagaku I98gV3 8 , 182. Towns, J. K.; Regnler, F. E. Anal. Chem. 1991, 63, 1126. Towns, J. K.; Regnier, F. E. J. Chromatogr. 1990, 576, 69. Balchunas, A. T.; Sepaniak, M. J. Anal. Chem. 1987, 59, 1468. Roach, M. C.; Gozel, P.; Zare, R. N. J. Chromatogr. 1988, 426, 129. Kozlovsky, W. J.; Lenth, W.; Latta, E. E.; Moser, A,; Bona, G. L. Appl. Phys. Len. 1990, 56, 2291.
RECEIVED for review August 16, 1991. Accepted January 8, 1992.
