Analyst, January 1996, Vol. 121 (37-41) 37 Study of the Chemiluminescent Characteristics of Ninhydrin and its Application Guo Nan Chen, Xue Qin Xu and Fan Zhang Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350002, China Ninhydrin was found to exhibit chemiluminescence (CL) when it was oxidized with hydrogen peroxide in neutral or weakly acidic solution. Copper(n) and Con can catalyse the CL reaction, on the basis of which ppb levels of CU" and Co" can be determined. In the presence of Cu", some amino acids enhanced the CL reaction, whereas others inhibited the CL reaction in the presence of Co". A flow-injection system with CL detection was developed for the determination of some amino acids. The mechanism of the CL reaction is discussed. Keywords: Ninhydrin; chemiluminescence; amino acid; flow injection Introduction Ninhydrin has been widely used as a reagent for the determina- tion of amino acids by spectrophotometry;1,2 however, its chemiluminescent characteristics have not been studied. It was found that when ninhydrin was oxidized with hydrogen peroxide in neutral or weakly acidic solution, chemilumines- cence (CL) was observed with a peak at 436 nm.This CL reaction proceeded very rapidly and its intensity was pH- dependent. An Na2HP04-KH2P04 buffer solution was used in this work, and the CL intensity was found to be constant between pH 6.7 and 7.0. In this pH range, the effects of more than 30 metal ions on the CL reaction were investigated; it was found that only Cu" and Co" could catalyse the CL reaction, and that the catalytic CL intensity was proportional to the concentra- tion of Cu" and Co".A CL method could, therefore, be established for the determination of Cull and Co" at the ppb level. Chemiluminescence is one of the most sensitive methods for the determination of amino acids, and many CL systems have been used for this purpose, the peroxyoxalate and luminol systems being the most common. As ninhydrin reacts with amino acids quantitatively under suitable conditions, proce- dures for the determination of amino acids were developed based on the CL reaction of ninhydrin. In addition, a flow- injection (FI) system with CL detection was developed for the determination of some amino acids. Experimental Apparatus A Lambda 9 spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) and an RF-540 fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) were used. The FI system for the determination of amino acids consisted of an HFC-1 chem- iluminescent detector, an LZ-2000 Flow Injection Processor and a recorder. Details of the HFC- 1 chemiluminescent detector and LZ-2000 Flow Injection Processor can be found in refs.3 and 4, respectively. The FI manifold is shown in Fig. 1. One of the dual peristaltic pumps (A) was used to pump the reagents (only three channels were used), while the sample solution was delivered to the injection valve by pump B. Reagents All reagents were of analytical-reagent grade or better and water doubly distilled in a fused-silica apparatus was used through- out. Ninhydrin solution. A stock solution (1 X 10-2 moll-l) was prepared by dissolving 1.7814 g of ninhydrin (AR, Shanghai Chemical) in 100 ml of water and diluting to 1000 ml with water.This solution was diluted further as required. The amino acids used were Asp, Asn, Thr, Glu, Gly, Val, Met, Ile, Leu, Tyr, Phe, His, Lys, Trp, Pro, Gln, Hyp and Arg (Shanghai Chemical). These amino acids were dissolved in water and diluted to 1 X 10-2 mol 1-1 to give a stock solution. Procedure Procedure for investigating the CL characteristics of ninhydrin Ninhydrin, buffer (or NaOH), Cu" (or Co") solutions and water were added to the reaction cell in turn by pipette to make the total volume up to 2.05 ml and mixed. The cell was then placed in the detector chamber. The shutter was opened and the zero point of the recorder was adjusted.Then, 0.3 ml of H202 solution was injected into the reaction cell and the CL signal was recorded. Procedure for determination of amino acids The procedure consisted of three steps; step 1: buffer + Cut' (or Co") (Rl), H202 (I) (R2) and H202 (11) (R3) were continuously pumped into the manifold by pump A at flow rates of 4.2, 5.1 and 3.75 ml min-1, respectively, for 20 s (during this step pump B was stopped); step 2: pump A was stopped, and pump B was used to load ninhydrin + amino acid (R4) into the injection valve at a flow rate of 3.75 ml min-1 for 15 s; step 3: pump B was stopped, and R1, R2 were pumped into the CL detector by pump A for 20 s, at the same time R3 was pumped into the injection A Fig. 1 injection valve; R, recorder; W, waste The FI manifold.A, Pump A; B, pump B; CD, CL detector; V,38 Analyst, January 1996, Vol. 121 valve as the carrier to take & in the injection valve loop to the CL detector, and the CL signal was then recorded. Results and Discussion Initial Investigation of the Ninhydrin-HzOz CL Reaction The initial investigation showed that when ninhydrin was oxidized by H202 in a suitable medium, CL was observed. The reaction was instantaneous; the maximum CL intensity was reached in 4 s. The effect of pH on the CL reaction of ninhydrin was examined, and it was found that the reaction was pH- dependent. H3B03-KC1-Na2CO3 buffer solution (pH 7.4-1 1 .O), Na2HP04-KH2P04 buffer solution (pH 5.2-8.3) and 0.5 moll-1 NaOH solution were used to examine the variation in the CL of the reaction.It was found that CL could be observed over a wide pH range; however, the CL intensity was strongest under neutral or weakly acidic conditions. The Na2HP04- KH2P04 buffer solution was therefore used as the reaction medium and the pH was fixed at 6.1 as this gave the strongest CL intensity. The experiment also showed that the optimum concentration of H202 was 0.223 mol 1-l (concentration in cell). The effects of more than 30 metal ions on the CL reaction were examined under the above-mentioned conditions. The results are shown in Table 1 from which it can be seen that only Cu" and Coil can catalyse the CL reaction effectively; therefore, a detailed investigation of the effects of Curl and CO" on the CL reaction was carried out and the results are presented below.Catalytic Effect of Cu" and Co" on the Ninhydrin-H202 CL Reaction As mentioned above, the reaction between ninhydrin and H202 gave CL over a wide pH range, which was called the Table 1 Effect of metal ions on the ninhydrin-H202 CL reaction* Ion c/mg 1-1 h/mm h/ho Ion c/mg 1-I hlmm h/ho Bim Tall Au"I Pd" Al"' Rh" u022+ VV Fellr SnIv Pt" Hg" AsV TeIV Smrrr YbUi CU" Pb" ym 0.37 1.72 0.138 0.357 3.75 0.25 0.357 0.357 0.212 0.106 0.212 0.106 0.212 0.212 1.06 0.021 0.212 35.7 31.7 37.5 0.75 54.5 1.09 37.5 0.75 39.5 0.79 37.0 0.74 39.0 0.78 46.5 0.93 4.10 0.82 4.10 0.82 45.9 0.92 28.0 0.56 48.5 0.97 43.0 0.86 46.5 0.93 51.0 1.02 59.0 1.18 167.8 3.35 56.0 1.12 30.5 0.61 562 0.69 3.73 1.78 0.357 0.212 0.357 0.357 0.2 12 1.06 0.106 0.212 0.2 12 0.2 12 0.02 1 0.212 0.25 16.3 33.5 0.67 34.5 0.69 31.0 0.62 37.5 0.75 36.5 0.73 36.5 0.73 51.0 1.02 34.0 0.68 36.0 0.72 38.5 0.77 20.5 0.41 44.0 0.88 31.0 0.62 52.5 1.05 52.0 1.04 73.0 1.46 39.0 0.78 616.0 12.3 * c: concentration of metal ions; h: CL peak height; ho: background CL peak height, ho = 50.0 mm in this experiment.background CL. When the catalytic effect of metal ions on the CL reaction is to be examined, it is advantageous to decrease the background CL in order to emphasize the catalytic effect of the metal ions. Therefore, the conditions for catalytic CL will be different from the optimum conditions mentioned above for the initial investigation. Three main factors, viz., pH, concentration of H202 and ninhydrin, were examined at this stage. In consideration of the interdependence of these factors, they could not easily be optimized by traditional methods.The modified simplex method, however, can readily be adapted to solve such problems, and was therefore used for this purpose. The optimum conditions found for the Cu" and Corl systems using the modified simplex method are listed in Table 2. Mechanism of the Chemiluminescent Reaction Treatment of ninhydrin by HMO method In order to probe the mechanism of CL, ninhydrin was treated by the Huckel Molecular Orbital (HMO) method. For conven- ience in the discussion, the carbon and oxygen atoms of ninhydrin are numbered as follows: lo The n-electron density distribution and the bond strength are shown in Table 3, from which it can be seen that the x-electron density at C-2 is the lowest; therefore, the radical anion 0 2 - * would attack the C-2 position first.Moreover, the calculation showed that the bond strength between C-1 and C-2 was the smallest; thus after 02-• attacks C-2, it is easier to form a peroxide ring between C-1 and C-2. In general, the occurrence of CL from organic compounds is due to decomposition of a peroxide ring. CL, fluorescence and UV spectra Fig. 2 shows the CL spectra of ninhydrin with the maximum at 436 nm [see Fig. 2, A]; after the addition of Cu" or Co", the CL Table 2 Optimum conditions for Cu" and Co", linear response range and detection limits* 410-3 CJ Ion pH moll-' moll-' LRR/gml-I DL/g ml-l Co" 6.9 3.45 0.388 1.0 X 10-9-1.0 X 10-8 5.0 X 10-10 Cull 6.7 3.0 0.378 5.0 x 10-9-1.0 x 10-6 1.0 x 10-9 * cl: concentration of ninhydrin; c2: concentration of H205 LRR: linear response range; and DL: detection limit.Table 3 n-electron density distribution and bond strength of ninhydrin* Atom No. 1 2 3 4 5 6 7 8 9 10 11 12 x-edd 0.6290 0.5626 0.6290 0.9248 0.9099 0.9100 0.9100 0.9099 0.9247 1.5936 1.5025 1.5936 Atom No. 1-2 1-9 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9 4 10-1 11-2 12-3 Bond strength 0.2432 0.4314 0.2432 0.4314 0.6148 0.6612 0.6362 0.6612 0.6148 0.4854 0.7087 0.7931 0.7087 * x-edd: n-electron density distribution. Parameters: ho = 2; h, = 0.2; K, = 1.4.Analyst, January 1996, Vol. 121 39 intensity increased, but there was no change in the shape and peak position of the spectra [see Fig. 2, B and C]. The fluorescence emission spectra of the product are shown in Fig.3. Fig. 3, A, shows the fluorescence spectrum of ninhydrin, its peak maximum is at 438 nm; Fig. 3, B, is the fluorescence spectrum of the product after adding Hz02; the fluorescence intensity is greatly increased in the presence of Co'I or Cu" [see Fig. 3, C]. From a comparison of Figs. 2 and 3, it was found that the CL spectrum of ninhydrin was very similar to that of the product. 14 10 c .- C c 2 p tu % a 6 a, C v c .- c - 2 C \ 360 400 440 480 520 560 Wavelength/nm Fig. 2 Chemiluminescent spectra of ninhydrin. A, Ninhydrin (3.45 X 10-3 mol 1-')-buffer solution (pH 6.90); and B, A + H202 (0.382 moll-'); and C, B + Col* (20 mg 1-I). C 80 h c .- 5 60 P s c 2 v .- 40 c C - 20 0 400 450 500 550 600 Wavelength/nm Fig. 3 Fig. 2. Fluorescent emission spectra of the product he, = 350 nm, A-C as The UV spectra of ninhydrin and its oxidation product were measured and are shown in Fig.4. There are two characteristic absorption bands at 232 and 255 nm before the addition of H202 [see Fig. 4, A], which are mainly due to the presence of a five- membered ring with ketone groups. However, the characteristic absorption bands at 232 and 255 nm gradually disappear after the addition of H202, and only the absorption band of the benzene ring can be observed [see Fig. 4, B], which indicates that the five-membered ring is oxidized by H202. Mechanism Based on the results of the treatment by the HMO method and the investigation of the CL and UV spectra, a possible mechanism for the CL reaction of ninhydrin can be proposed. The radical anion 0 2 - * attacks C-2 first, a peroxide ring between C-1 and C-2 is then formed.The peroxide is further decomposed by H202 to produce an excited intermediate, which will emit light when it returns to the ground state. The mechanism can be shown as follows: a t 8-H i- c@ ---+ + hv b Application to the Determination of Amino Acids Ninhydrin can react with amino acids under heating to give a blue-violet product, Ruhemann Violet, which has a maximum absorption at 570 nm; therefore, ninhydrin has been widely used as a reagent for the determination of amino acids. We found that the product, Ruhemann Violet, did not give CL when it was oxidized by H202; hence, in the presence of excess of ninhydrin, amino acids can be determined indirectly by examining the A 0.48 h c v) .- 5 0.32 c $ g) 5 a 2 m v f! 0.16 200 240 280 320 Fig.4 The UV spectra of ninhydrin and its oxidation product. A, Ninhydrin (5.0 X 10-3 mol l-')-buffer solution (pH 6.90); and B, A + H202 (0.382 mol 1-I).40 Analyst, January 1996, Vol. 121 variation in the CL intensity of the reaction between ninhydrin and the amino acids. Moreover, we also found some interesting phenomena, which are described below. 1. In the presence of Cu", when some amino acids were added to and heated with ninhydrin, and the resulting solution was reacted with H202, it was found that the CL intensity of the system was greatly enhanced, and that the enhancement was proportional to the concentration of amino acids. 2. In the presence of Co", when some amino acids were added to and heated with ninhydrin, and the resulting solution was reacted with H202, it was found that the CL intensity of the system was greatly inhibited, and that the inhibition was proportional to the concentration of amino acids.Based on these properties of ninhydrin, the possibility of the determination of amino acids by using these CL reactions was investigated, and procedures for the determination of amino acids by FI with CL detection were developed. Ninhydrin-H202-Cu11 system for determination of some amino acids Treatment of sample. A 1 ml volume of amino acid solution was placed in a 15 ml test-tube, and 1 ml of 1 X moll-' ninhydrin solution was then added and mixed. The test-tube was plugged with cotton, and placed in a boiling-water bath for 15 min; it was then cooled with water and set aside for 5-10 min, after which the contents were diluted to 5 ml with water.The solution obtained was used as the sample solution for FI with CL detection. Selection of conditions. It was found that Trp, Gly, Glu, Phe, Gln, Pro, Asp, Leu, Thr, Ile, Val and Lys exhibited an enhancement for this CL system, and that the enhancement was dependent on the pH of the buffer solution, and on the concentrations of H202, Cu" and ninhydrin. Trp is taken as an example for selection of the optimum conditions. The effect of pH on the determination of Trp is shown in Fig. 5, from which it can be seen that the enhancement of the amino acid CL is pH-dependent, and that the optimum enhancement is obtained at pH 6.70. The optimum concentration of Cu" is 20 mg 1-1 (see Fig.6). As regards the concentration of H202, good results were obtained between 0.29 and 0.59 mol 1-l; 0.44 mol 1-1 H202 was chosen subsequently (see Fig. 7). Fig. 8 shows that the enhancement of amino acid CL is proportional to the concentration of ninhydrin; therefore, the concentration of ninhydrin can be selected based on the concentration of amino acid in the sample. In general, ninhydrin should be in excess; a 10-fold excess would be suitable. Relative standard deviation, linear response range and detection limit. Under the selected conditions, the s, (n = 10) for 4.0 X 10-6 moll-1 of Trp was 1.7%. The linear response range and the detection limit of 12 amino acids were examined and the results are shown in Table 4. 6 6:3 6.5 6.7 6.9 7.2 7.5 PH Fig.5 Effect of pH on determination of Trp. 10, background CL of ninhydrin; I, enhanced CL, A I = I - I. [Cu"] = 20 mg 1-1; [ninhydrin] = 3.33 X 10-3 moll-1; [Trp] = 2.0 X 10-4 moll-'; and [H202] = 0.294 mol 1-1. Ninhydrin-H202-Co1[ system for determination of some amino acids For the ninhydrin-H2O2-CoI1 system, His, Arg, Tyr, Asn, Met and Hyp were found to inhibit the CL reaction; the inhibition was dependent on the pH of the buffer solution, and on the concentrations of H202, Co" and ninhydrin. The treatment of the sample solution was the same as that for the ninhydrin-H202-Cu11 system. Taking His as an example for - 0 0 2i 5 14 20 25 30 35 2 [Cu"]/mg I-' Fig. 6 Effect of Cu" on determination of Trp. Io, background CL of ninhydrin; I, enhanced CL; AI = I - I,; pH 6.70; [ninhydrin] = 3.33 X 10-3 moll-1; [Trp] = 2.0 x 10-4 moll-l; [H202] = 0.294 moll-'.l o r - o 0.144 0.3 0.384 0.408 0.6 7 [H,O,]/mol I-' Fig. 7 Effect of H202 on determination of Trp. 10, background CL of ninhydrin; I, enhanced CL; A I = I - IO [Cu"] = 20 mg 1-l; [ninhydrin] = 3.33 x moll-l; [Trp] = 2.0 x moll-I; pH 6.70. 12 r & In 10 .- ? * c S 6 = 4 2 0 I A1 0 1 2 3 4 5 6 7 [Ninhydrin]/mmol 1-l Fig. 8 Effect of ninhydrin on determination of Trp. lo, background CL of ninhydrin; I, enhanced CL; A I = I - IO [Cu'l] = 20 mg 1-I; pH 6.70; [Trp] = 2.0 x 10-4 mol 1-1; [H202] = 0.441 mol 1-1. Table 4 Linear response range and detection limit of 12 amino acids Amino acid Trp GlY ASP Phe Gln Leu Glu Pro Ile Thr Val LYS Linear range/mol 1- 1.0 x 10-6-1.0 x 10-4 1.0 x 10-5-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 1.0 x 10-5-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 2.0 x 10-5-2.0 x 10-4 1.0 x 10-5-1.0 x 10-4 2.0 x 10-6-1.0 x 10-3 1.0 x 10-6-1.0 x 10-3 1.0 x 10-5-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 Detection limit/mol I-' 2.7 X 6.8 x 10-6 2.1 x 10-6 5.4 x 10-6 4.8 x 10-7 5.9 x 10-7 - 4.5 x 10-7 4.2 x 10-7 4.2 x 10-7 5.1 x 10-7 2.9 xAnalyst, January 1996, Vol.121 41 Table 5 Linear response range and detection limit of six amino acids Amino acid Linear range/mol1-1 Detection limit/moll-l His 2.0 x 10-6-1.0 x 10-3 6.1 x 10-7 A% 1.0 x 10-54.0 x 10-4 4.0 x 10-6 Asn 1.0 x 10-6-4.0 x 10-4 - Met 2.0 x 10-5-1.0 x 10-4 9.2 x 10-6 TYr 1.0 x 10-5-1.0 x 10-4 4.2 x 10-6 2.0 x 10-6-8.0 x 10-4 4.5 x 10-7 HYP selecting the optimum conditions, the results showed that the optimum conditions for the inhibition of the CL reaction were: pH 6.9; [HZ021 = 0.29 mol 1-1; [ninhydrin] = 3 X 10-3 moll-1; and [Cotl] = 14 mg 1-1. Under the optimum conditions, the s, found (ten replicates) for 6 X 10-6 mol 1-1 of His was 1.7%. The linear response range and the detection limit of six amino acids were examined and the results are shown in Table 5. This work was financially supported by the State Education Commission, China, and the Natural Sciences Foundation of Fujian Province, China. References 1 Zarkadas, C. G., Can. J . Biochem., 1975,53, 96. 2 Zhong, H. S., and Guang, J. S., Biochemistry, Gaodeng Jiaoyu Press, Beijing, 1990, p. 64. 3 Chen, G. N., Duan, J. P., and Hu, Q. F., Anal. Chim. Acta, 1994,292, 159. 4 Chen, G. N., Duan, J. P., and Hu, Q. F., Mikrochim. Acta, 1994, 116, 227. Paper 5l047.586 Received July 19, 1995 Accepted September 7, I995