Anal. Chem. 1983, 55, 1009-1012
plasma emission spectrometric detector for chromatography. The detection limits and selectivities obtained with this system are comparable with the results achieved by other investigators. Further improvements in the microcomputer hardware and software should extend the applicability of this method. With two more digital output lines and some changes in the assembly language data acqusition program, potentially eight PMT channels could be monitored with an electronic amplification of any one OS eight gains. Other improvements may make it possible to increase the data acquisition rate. While the current datu acquisition rates of 1-3 ]points/s are adequate for HPLC and packed column GC separations, much faster rates (10-20 points/s) would be needed for capillary column GC. Hopefully, the characterization of this polychromator/microcomputer system will lead to further development and application of miultielement plasma emission spectrometric detections for chromatography in the near future. Particularly interesting is the possibility for elemental ratioing in addition to (quantitative determinations.
1009
LITERATURE CITED McCormack, A. J.; Tong, S. C.; Cooke, W. D. Anal. Chem. 1965, 37, 1470. Krull, I. S.; Jordan, S. J. Am. Lab. (Falrfleld, Conn.) 1980, 21. Carnahan, ,I. W.; Mulligan, K. J.; Caruso, J. A. Anal. Chlm. Acta 1981, 130, 227. Windsor, D. L.; Denton, M. E. J . Chromatogr. Scl. 1979, 17, 492. Bonnekessel, J.; Klier, M. Anal. Chlm. Acta 1978, 103,29. Brenner, K. S. J . Chromatogr. 1978, 167,365. Morita, M.: Uehiro, T.; Fuwa, K. Anal. Chem. 1980, 52, 349. Fralev. D. M.: Yates, D. A,; Manahan, S. E.; Stalling, D.: Petty, J. Appl. Spectrosc. 1981, 35,525. Hausler, D. W.; Taylor, L. T. Anal. Chem. 1981, 53, 1223. Estes, S. A,: Uden, P. C.; Barnes, R. M. Anal. Chem. 1981, 53, 1829. Muliigan, K. J.; Caruso, J. A.; Fricke, F. L. Analyst (London) 1980, 105. 1060. Skogerboe, R. K.; Lamothe, P. J.; Bastianns, G. J.; Freeland, S. J.; Coleman, G. N. Appl. Spectrosc. 1976, 30,495. Woodward, W. S.: Reilley, C. N. Pure Appl. Chem. 1978. 50, 785. Jackson, M. E. Ph.D. Dissertation, Unlverslty of Cincinnati, 1980. Eckhoff, M. A. Ph.D. Dissertation, University of Cincinnati, 1982. Quimby, E. D.; Uden, P. C.; Barnes, R. M. Anal. Chem. 1978, 50, 2112. Beenakker, C. I . M. Spectrochim. Acta, Part B 1977, 326. 173. Estes, S. A.; Uden, P. C.; Barnes, R. M. Anal. Chem. 1981, 53, 1336.
ACKNOWLEDGMENT The authors isre grateful to K. J. Mulligan for his comments and assistance. Registry No. DDT, 50-29-3; BN-2’1, 40703-79-5; BC-26, 85115-87-3;PBIB, 36355-01-8; methoxychlor, 72-43-5.
RECEIVED for review December 9,1982. Accepted February 1, 1983. The authors are grateful to the National Institute of Occupational Safety and Health for support of this work through Grant 0H-00739.
Determination of Ribonucleoside 5’-Mono-, 5’-Di-, and 5’-Triphosphates by Liquid Chromatography/Inductively Coupled Plasma Atomic Emission Spectrometry Kazuo Yoshldla, Hirokl Haraguchi, * and Keiichiro Fuwa Department of Chemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Inductlvely coupled plasma atomlc emlsslon spectrometry (ICPAES) Interfaced wlth hlgh-performance liquid chromatography (HPLC) has been applled to the determlnatlon of nucleotldes. The ICPAES was used as an element-selectlve detector for HPLC by observing the P I emission lntenslty at 213.62 nm. Twelve common 5’-rlbonucleotldes were determlned separately wlth the HPLCACPAES system uslng a concentration gradlent method. The Integrated emlsslon Intenslty of phosphorus was proportlonal to the number of phosphorus atoms In all nucleotldes. The detectlon llmll of the present HPLWICPAES system for phosphorus standard (KH,PO,) was about 0.4 pg of P/mL (wlth 200-pL sample Injectlon), and the relntlve standard dexlatlon was 4 % In the peak height measurement.
Since biological fluids or tissues contain a mixture of a large number of compounds, chemical speciation of biological substances is important for elucidation of biological metabolisms and reactions. Recent development of high-performance liquid chromatography (HPLC) has enabled rapid characterizatio’n of biiological substances. Nucleotides have been commonly separated with HPLC by using an anion exchange resin and d.etermined with a UV detector (1-3). However, identification of chromatographic peaks of biological
samples is still not easy when the detector has no element or molecular specificity. As is well-known, atomic absorption spectrometry (AAS) is an excellent method for the determination of metallic elements. Use of element-specific detection capability of AAS in addition t u the conventional nonspecific detectors, e.g., UV-visible spectrophotometry, may help to interpret the chromatograms. Actually, a number of applications of AAS to HPLC detectors have been reported (4,5),although they dealt with only metallic element detection. Recently, inductively coupled plasma atomic emission spectrometry (ICPAES) has been extensively developed as a promising spectrochemical method with versatile capability, multielement analysis, low detection limit, and element specificity (6, 7). These analytical feasibilities of the ICPAES are also suitable for its use as an element-specific detector for chromatography. The sample uptake rate of the ICPAES is about 1-2 mL/min, which is almost comparable to the flow rate of effluent from HPLC. This makes it easy to interface the ICPAES with HPLC. For these reasons, the use of the ICPAES as a chromatographic detector has been demonstrated by some workers (8-12). In the determination of nucleotides, ICPAES should be used as the detector of phosphorus, taking advantage of its high sensitivity for phosphorus. Flame emission (13) and graphite furnace atomic absorption (14) methods were applied to the
0003-2700/83/0355-1009$0~ .50/0 0 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
Table I. Instruments and Operating Conditions HPLC column column packing MC Flgure 1. Schematic diagram of the HPLWCPAES system: GP, gradient programmer: P.A, pump A: P.B, pump 8: MC, mixing coil: I , Injection valve: C, column: UV, UV monitor; P, plasma: M, monochromator; PM, photomultiplier: A, amplifier; R, recorder.
analysis of organophosphorus compounds separated with HPLC, but the ICPAES is superior in sensitivity to these methods. Morita et al. showed the separation and quantitative analysis of phosphate compounds by using a HPLC/ICPAES system (15). Heine et al. compared the UV and ICPAES detectors for the determination of nucleotides (16). They, however, separated only three nucleotides in one chromatogram, using a 5-min linear gradient method, and described the analysis of six nucleotides. In this paper, we will show the separation of 1 2 common nucleotides (5’-ribonucleotides)with a HPLC/ICPAES system using a 2-h gradient and discuss the practical value of the ICPAES detector. EXPERIMENTAL SECTION Apparatus. A HPLC/ICPAES system for the determination of nucleotides is shown in Figure 1. The HPLC system consisted of two solvent delivery pumps, an injection valve (Model 7125 from Rheodyne Co.), an associated column, and a UV absorbance detector (Model SPD-2A from Shimadzu Co.). The solvent flow rates from pump A and pump B were controlled with a gradient programmer (Model GRE-3A from Shimadzu Co.). The separation was performed by using a 4 mm i.d. X 250 mm long or 4 mm i.d. X 500 mm long stainless steel column packed with a strong anion exchanger (EX-260-SA-SIL from Toyo Soda Co.). The column temperature was maintained at 60 “C with a column oven (Model GTO-2A from Shimadzu Co.). The effluent outlet from the UV detector was fed to the cross-flow nebulizer of the ICP spectrometer, which was interfaced with small diameter Teflon tubing (0.5 mm i.d. X 300 mm long). The ICP spectrometer consisted of an ICP torch system with a RF generator (Model ICAP-50 from Nippon Jarrell Ash), a monochromator, a photomultiplier (R 787 from Hamamatsu TV Co.), and a current amplifier (Model 427 from Keithley). The monochromator was of the Czerny-Turner type (focal length 1.0 m) with a holographic grating (2400 grooves/mm). The emission signal of phosphorus was observed at 213.62 nm. The emission signal from the photomultiplier readout and the response from the chromatographic UV detector were recorded simultaneouslyon a two-pen strip chart recorder (Model R-10 from Rikadenki Co.). Chemicals. All the chemicals used were of analytical reagent grade. Formic acid, ammonium formate, and potassium dihydrogen phosphate were purchased from Wako Chemical Co., and nucleotides were from Sigma Chemical Co. These reagents were used without further purification. RESULTS AND DISCUSSION Optimization of HPLC/ICPAES System. The HPLC/ICPAES system was operated under the conditions specified in Table I. These experimental conditions were optimized while aspirating a KH2P04solution containing 10 pg of P/mL as a standard solution. The optical observation height above the coil and carrier argon gas flow rate were set so as to obtain the maximum signal-to-background ratio. Generally, phosphate solutions have been used as the buffer of the mobile phase in the determination of nucleotides with HPLC ( 2 ) . However, phosphate solution is not suitable as the buffer when an ICPAES is used as the detector of
oven mobile phase sample volume UV wavelength ICPAES monochromator RF power coolant gas auxiliary gas sample gas viewing height wavelength
Shimadzu Model LC-3A stainless steel columns (50 cm x 4 mm id., 25 cm x 4 mm i d . ) IEX-260-SA-SIL (anion exchange resin) 60 “C 0.1-0.73 M HCOONH,, pH 3.0 200 PL 260 nm Nippon Jarrell-Ash Model ICAP-50 Jobin Yvon Model HR 1000 (focal length 1000 mm, grating 2400 grooves/mm) 1.3 kW 16 L/min 1.2 L/min 0.8 L/min 1 5 mm above load coil P I 213.62 nm
phosphorus. Heine et al. used sodium acetate below 0.3 M as the buffer solution for the determination of a few nucleoside monophosphates with the ICPAES detector (16). They described the difficulty in separation of both adenosine di- and triphosphates with the concentration range used. In the present work, it was also impossible to separate diphosphates and triphosphates of nucleotides at the same concentration of sodium acetate (below 0.3 M). Then, the separation of diand triphosphates of nucleotides was examined at the higher concentration of sodium acetate, but the high concentration of sodium salts over 0.4 M resulted in a base line drift because sodium salts easily clogged both the conventional ICPAES torch and cross flow nebulizer. Therefore, in the present experiment, solutions of tris(hydroxymethy1)aminomethane (THAM), ammonium acetate, and ammonium formate were examined as buffer solutions for the mobile phase. The pH of these buffer solutions was adjusted with nitric acid, glacial acetic acid, and formic acid, respectively. The solutions of THAM and ammonium acetate a t the high concentration above 0.5 M often clogged the top of torch, causing a serious base line drift. On the other hand, ammonium formate solution provided stable emission signals and base line, and so it was employed as the mobile phase for HPLC separation. Furthermore, in the case of ammonium formate solution, it was possible to perform long-time gradient elution in the concentration range from 0.1 to 1.0 M without base line drift. The flow rate of the mobile phase was an important variable for the optimization in HPLC separation. Figure 2 illustrates the effects of the flow rate of the mobile phase on the number of theoretical plates obtained with UV and ICPAES detectors. The curve for the UV detector indicates normal pattern of HPLC, i.e., the number of theoretical plates becomes significantly smaller at the faster flow rate. On the other, the number of theoretical plates for ICPAES detection was smaller in the whole range than that for the UV detector, and it was almost constant at the flow rate above 1.4 mL/min. Thus, peak broadening was observed more remarkably with the ICP detector than with the UV detector, but it could be minimized by controlling the flow rate between 1.2 and 1.6 mL/min. The effects of the solution flow rate on phosphorus emission intensity (peak height of chromatogram) observed with ICP detection are shown in Figure 3. For UV detection, the peak height did not change at the flow rate from 1.0 to 2.0 mL/min. As can be seen in Figure 3, however, the highest peak appeared between 1.4 and 1.6 mL/min for ICPAES detection. Then, 1.4 mL/min was chosen as the flow rate of mobile phase. Quantitative Analysis of Nucleotides. The relative intensities per phosphorus atom for 5’-ribonucleotides ob-
ANALYTICAL. CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
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Table 111. Detection Limits of Various Nucleotides with the HPLC/ICPAES System (Peak Height Measurement) 1200.
8
-nm c
-m 2 1000.
nucleotide KH,PO,a AMP ADP ATP CMP; C DP CTP GMP: GDP GTP UMP UDP UTP
E
5
\ I
r
0
E
z “01 ‘r
0.39 4.4 2.9 2.5 3.8 2.4 2.2 5.4 3.5 3.2 4.3 3.0 2.7
*
2.0
1:2 1:4 1:6 1.8 Flow rate (rnL/rnin)
L-”l:O
detection limit concn as concn as nucleotides, phosphorus, &mL clg of P/mL
Flgure 2. Effect of solvent flow rate on the number of theoretical plates: column, 4 mm i.d. X 500 mm long; sample, AMP, 14 pg of P/mL; mobile phase, 0.1 M HCOONH,, pH 3.0.
0.31
0.42 0.46 0.36 0.38 0.42 0.46 0.49 0.56 0.41 0.46 0.52
Mobile a Mobile phase 0.1 M HCOONH,, pH 3.0. phase 0.64 M HCOONH,, pH 3.0. Mobile phase 0.73 M HCOONH,, pH 3.0. uv
b
-$ 1
I CP
1
0.5
q
~
-
40
.
00
80
100
120
I
Time (min)
Figure 4. Comparison of chromatograms observed with UV and an 110
112
1:4
116
1:8
210
Flow rate (mL/min) Figure 3. Effect of solvent flow rate on the peak heights of chromatograms: ICP, F’ I213.62 nm; column, 4 mm 1.d. X 500 mm long; sample, AMP, 14 pg of P/mL; mobile phase, 0.1 M HCOONH,, pH 3.0.
Table 11. Relaitive Intensities per Phosphorus Atom in Various Nucleotides with ICPAES Detection compound KH,PO, adenosine 5’-monophosphate (AMP) adenosine 5’-diphosphate (ADP) adenosine 5’4riphosphate (ATP) cytidine 5’-monophosphate (CMP) cytidine 5‘-diphosphate (CDP) cytidine 5’-triphosphate (CTP) guanosine 5‘-monophosphate (GMP) guanosine 5’-diphosphate (GDP) guanosine 5’-triphosphate (GTP) uridine 6‘-monophosphate (UMP) uridine 5‘-diphosphate (UDP) uridine 5‘-triphosphate (UTP) a Normalizedl to 1.00.
______
20
re1 intens l.Ooa 1.01
1.01 0.95 0.91 0.98 1.00
1.03 0.96 1.03 1.01
0.97 1.05
-
tained with ICPAES detection are summarized in Table 11. The relative intensities were calculated as the ratio of peak area to phosphorus atoms in each nucleotide. As can be seen from Table 11,the relative intensities of these nucelotides were
ICPAES detectors for ribonucleoside 5‘-mono-, 5’-di-, and 5‘4riphosphate: column, 4 mm i.d. X 500 mm long; (a) CMP 7.25 pg of P/mL, (b) AMP 11.7 pg of P/mL, (c) UMP 9.80 pg of P/mL, (d) GMP 7.82 pg of P/ml., (e) CDP 21.7 pg of P/mL, (f) ADP 24.1 pg of P/mL, (9) UDP 12.5 pg of P/mL, (h) CTP 45.8 pg of P/mL, (i) GDP 16.3 pg of P/mL, (j) ATP 16.6 pg of P/mL, (k) UTP 29.7 pg of P/mL, (I) GTP 16.2 pg of P/mL, (m) KH2P0, 5.0 pg of P/mL; mobile phase (linear concentration gradient method) 0.1 M HCOONH,, pH 3.0 (0-20 min), 0.37 M HCOONH,, pH 3.0 (80 min), 0.73 M HCOONH,, pH 3.0 (120 min).
almost the same within the standard deviation. This indicates that it may be possible to determine all nucleotides with ICPAES detection by use of one calibration curve obtained with a phosphorus standard such as KH2P04. The calibration curves obtained in peak areas by using phosphorus standard (KHZPO,) and some nucleotides were completely similar to each other, and showed a linear relationship with a dynamic range of 1-103 pg of P/mL. The calibration curve obtained in peak height had also a linearity with the same concentration range of phosphorus. The practical detection limits were estimated from the chromatogram by taking the peak height corresponding to twice the noise level. The detection limits of nucleotides with ICPAES detection measured in peak height are summarized in Table 111. Relative standard deviations were 5% and 4 % in pleak area and peak height measurements, respectively, with a series of 10 injections of 20 pg of P/mL of KHzPOI solution. Chromatographic Separation of Nucleotides. Typical chromatograms obtained with UV and ICPAES detections are illustrated in Figure 4, which shows the separation of the ribonucleoside 5’-mono-, W-di-, and 5’-triphosphates with the
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
uv
a
2e 0 P v)
L L
0
U
0 Q,
c Q,
0
0
5
10
Time (min) Flgure 5. Comparlson of chromatograms observed with UV and ICPAES detectors for adenoslne 5’-mOnO-, 5’di-, and 5’-triphosphate: column, 4 mm i.d. X 250 mm long; sample, (a) AMP 4.40 pg of P/mL, (b) ADP 15.2pg of P/mL, (c) ATP 19.8pg of P/mL; mobile phase, 0.73 M HCOONH,, pH 3.0.
HPLC. These chromatograms demonstrate that 12 common 5’-ribonucleotides can be determined separately with ICPAES detection using a 2-h concentration gradient method. Morita et al. described that the gradient elution caused a base line drift with the ICPAES detector. As can be seen in Figure 4, however, the change of salt concentration in the eluent did not cause any base line drift, even when the ICPAES was used as the detector. On the other hand, in the case of UV detection, the base line was shifted in accordance with the increase of salt concentration. These results indicate that the ICPAES detector can be employed even when the long-time concentration gradient elution is performed. Furthermore the chromatogram with ICPAES detection is not affected by chemical forms of samples because of its element-selective nature. Figure 5 shows the rapid separation of adenosine 5’-mono-, 5’-di-, and 5’-triphosphates without gradient elution. The rapid separations of cytidine, guanosine, and uridine nucleotides were also possible under the same conditions as shown in Figure 5. Furthermore, separation of each mono-, di-, and triphosphate of the nucleotides performed perfectly with use of following: mobile phase (monophosphate nucleotides) 0.1 M ammonium formate (pH 3.0), (diphosphate nucleotides) 0.64 M ammonium formate (pH 3.0), (triphosphate nucleotides)
0.73 M ammonium formate (pH 3.0). Comparison of UV and ICPAES Detection. As compared with a UV detector, the ICPAES detection is superior to the UV detection in the determination of nucleotides in terms of the following points, besides excellent element selectivity of the ICPAES. First, the working calibration curve of KHzPOI obtained by phosphorus emission peak area with ICPAES detection can be used for the determination of all nucleotides, because phosphorus emission intensity is proportional to the number of phosphorus atoms in nucleotides. In the case of a UV detector, since each nucleotide has a different molar extinction coefficient at 260 nm, a different calibration curve is required for each nucleotide. Second, even when the peaks of nucleotides are overlapped with other organic compounds, which is often observed with UV detection, it would be possible to find nucleotides by means of measuring phosphorus emission with an ICPAES detector. Unfortunately, the ICPAES detector is slightly inferior to the UV detector in sensitivity, when phosphorus is detected. In conclusion, it has been found out that the ICPAES and UV detectors have their own analytical figures of merits with some disadvantages. Therefore, the dual use of the UV and ICPAES detectors, which is demonstrated in this experiment, will provide the most useful information about chemical speciation of biological samples. Registry No. AMP, 61-19-8; ADP, 58-64-0; ATP, 56-65-5; CMP,63-37-6;CDP,63-38-7;CTP,65-47-4;GMP,85-32-5;GDP, 146-91-8;GTP, 86-01-1;UMP, 58-97-9; UDP, 58-98-0;UTP, 6339-8.
LITERATURE CITED (1) Horvath, C. G.; Preiss, B. A,; Llpsky, S.
R. Anal. Chem. 1967, 39, 1422- 1428. (2) Brown, P. R. J. Chromatogr. 1970, 52, 257-272. (3) Khym, J. X. J. Chromatogr. 1974, 97, 277-279. (4) Fernandez, F. J. At. Abs. News/. 1977, 10, 33-36. (5) Van Loon, J. C . Anal. Chem. 1979, 51, 1139A-1150A. ( 6 ) Boumans, P. W. J. M.; de Boer, F. J. Spectrochlm. Acta, Par? 6 1972, 276,391-414. (7) Fassel, V. A.; Knlseley, R. N. Anal. Chem. 1974, 46, IllOA-1120A. (8) Cast, C. H.; Kraat, J. C.: Poope, H.; Maessen, F. J. M. J. J. Chromat o g . 1979, 185, 549-561. (9) Fraley, D. M.: Yates, D. A.; Manahan, S. E. Anal. Chem. 1979, 51, 222522229, (10) Morita, M.; Uehlro, T.; Fuwa, K. Anal. Chem. 1980, 52, 349-351. (11) Fraley, D. M.; Yates, D. A,; Manahan, S. E.; Stalling, D.; Petty, J. Appl. SpeCtrOSC. 1981, 35, 525-531. (12) Morita, M.; Uehlro, T.; Fuwa, K. Anal. Chem. 1981, 53, 1806-1808. (13) Julin. B. G.:Vanderborn. H. W.: Kirkland. J. J. J. Chromatour. 1975. 112, 443-453. (14) Tlttarelll, P.; Mascherpa, A. Anal. Chem. 1981, 53, 1466-1469. (15) Morlta, M.; Uehiro, T. Anal. Chem. 1981, 53, 1997-2000. (16) Heine, D. R.;Denton. M. 8.; Schlabach, T. D. Anal. Chem. 1982, 54, 81-84. I
.
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RECEIVED for review January 19, 1983. Accepted March 4, 1983. The present research has been supported by the Grant-in-Aid for Environmental Science (No. 57030018) from the Ministry of Education, Science and Culture, Japan.
