Anal. Chem. 1992, 6 4 , 166-170
166
Role of Electron-DonatingIWithdrawing Character, pH, and Stoichiometry on the Chemiluminescent Reaction of Tris(2,2’-bipyridyl)ruthenium( I I I) with Amino Acids Stephen N. B r u n e and Donald R. Bobbitt* Department of Chemistry a n d Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701
A postcolumn Chemiluminescent technique for the detection of underlvatized amino acids using eiectrogenerated tris(2,2’-bipyrldyl)ruthenium( I I I ) is described. The reaction chemistry has been investigated, and lt has been shown that the electron-withdrawing/donating character of the R group attached to the a-carbon of the amine influences the chemiluminescent efficiency of the reaction. Stoichiometric studies conducted on four amines produced a mole ratio of 2:1, ruthenium:amine. At optlmum reaction conditions the reiatlve intendties of the primary amino acids tested varied by a factor of 55, wlth serine yielding the lowest chemiluminescence and leucine yielding the highest chemiluminescence. Detection limits for serine and leucine have been calculated to be 135 and 3 pmol, respectively, at a S / N of 3. Linearity has been demonstrated over 2 orders of magnitude for Leu. The abillty to implement thls system for postcolumn chemiluminescence detection of amino acids following a protein digest has been demonstrated.
INTRO DUCT10N Current chromatographic techniques are limited by the sensitivity of the detectors available. This is especially true for the detection of amino acids. Current methods of detecting amino acids rely heavily on either pre- or postcolum derivatization of the amino acid with a compound that makes it more easily detectable. These derivatizing reagents generally involve the addition of a fluorophor through the reaction of the amino acid with either dansyl chloride (1,2) or o-phthalaldehyde (3, 4). Recently, chemiluminescent-based systems have been reported that use of either 4-(isocyanato)phthalhydrazide(5) or bis(2,4,6-trichlorophenyl)oxalate/hydrogen peroxide/ dansyl chloride (6) to generate chemiluminescence. Other methods involve phenylthiohydantoin derivatization followed as by UV detection (7) or naphthalene-2,3-dicarboxaldehyde a precolumn derivatizing agent which has been used for postcolumn amperometric detection in open tubular liquid chromatography (8). The methods cited above each have limitations. For example, when dansyl chloride is used as a precolumn derivatizing reagent it can react with some amino acids twice, producing extra peaks. To minimize this problem, reaction time and the ratio of derivatizing reagent to amino acid must be controlled. o-Phthalaldehyde does not react with all amino acids, and its derivatives are unstable. The 4(isocyanato)phthalhydrazide derivatives are light sensitive and must be refrigerated when not used. The bis(2,4,6-trichlorophenyl) oxalate system is itself unstable and also suffers from the same problems stated earlier for the dansyl chloride system. The phenylthiohydantion derivatization method can produce artifact peaks. The naphthalene-2,3-dicarboxaldehyde method requires control of the reaction stoichiometry and does not detect all amino acids. These problems can add
* To whom correspondence should be addressed.
both time and complexity to the analysis and in some cases affect the reproducibility of the data. There are a few techniques available for the postcolumn detection of underivatized amino acids. They involve the chemiluminescent quenching of the luminol/ hydrogen peroxide reaction by the complexation of either cobalt(I1) or copper(I1) ions with the amino acids (9-ll),amperometric detection with a copper electrode (12), reaction of L-amino acids with amino acid oxidase followed by either conductance detection (13) or chemiluminescence detection (14), and chemiluminescent detection of amino acids using electrogenerated tris(2,2’-bipyridyl)ruthenium(III) (15). Both the amperometric and luminol methods rely on the complexation of the amino acid with metal ions. The rates at which amino acids complex with metal ions can therefore affect the sensitivity and speed of the technique. The amino acid oxidase method suffers because the system does not react with all amino acids and is not stable over long periods of time. The ruthenium-based system under the conditions reported earlier showed poor sensitivity. The development of a chemiluminescent detection method for underivatized amino acids has implications in protein sequencing. As smaller and smaller amounts of protein are isolated for sequencing, new and improved methods for separating and detecting the amino acids must be found. As stated above, the current methods of detecting amino acids are inadequate. It has been shown that a wide range of primary, secondary, and tertiary amines will react with tris(Z,Z’-bipyridyl)ruthenium(III)to yield chemiluminescence (CL) centered around 600 nm (16). Later it was shown that amino acids can also react with tris(2,2’-bipyridyl)ruthenium(II1) to yield chemiluminescence (15). The reaction conditions under which this reaction occurs can significantly affect the efficiency of the reaction and thus the intensity of the resulting chemiluminescence (17). We present here the implementation of a chemiluminescent postcolumn reaction detector for the detection of underivatized amino acids using electrogenerated tris(2,2’-bipyridyl)ruthenium(III). Detection limits significantly lower than those previously observed will be demonstrated. A discussion of the reaction conditions which yield optimum chemiluminescence will be presented. Implementation of this technique for postcolumn chemiluminescence detection of underivatized amino acids following a protein digest will be demonstrated.
EXPERIMENTAL SECTION Materials. Tris(2,2’-bipyridyl)ruthenium(II)chloride was purchased from Aldrich Chemical Co. (Milwaukee, WI) and converted to tris(2,2’-bipyridyl)ruthenium(III)perchlorate [Ru( b ~ y ) ~ ~prior ’ ] to use. The conversion of the tris(2,2’-bipyridyl)ruthenium(II) chloride to the tris(2,2’-bipyridyl)ruthenium(II1) perchlorate is necessary to prevent the oxidation of chloride ion to chlorine gas, which readily occurs at the potential required to keep the tris(2,2’-bipyridyl)ruthenium(III) perchlorate in the 3+ oxidation state. This can cause problems with the analysis in that the chlorine gas can collect in the detection cell
0003-2700/92/0364-0166$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
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f
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minimum of three times. In a previous study it had been determined that maximum chemiluminescence occurs for the amino acids when they are buffered at a pH of 10 (17). Therefore, both the eluent and amino acid standards were prepared in a 0.05 M boric acid buffer at pH 10. In the above study, the flow rate of the amino acids were 0.5 mL/min. and the R ~ ( b p y )flow ~ ~ +rate was 0.5 mL/min. The ability to utilize this technique in the postcolumn detection I of underivatized amino acids following a digest was demonstrated by digesting gramicidin D in 3 N p-toluenesulfonic acid for 24 h at 110 "C followed by separation on a Whatman PartisillO SCX column. The column eluent was 1mM Na2S04adjusted to pH 2.5 with 12 M HC1. The column flow rate was 1mL/min. The column eluent was postcolumn adjusted to pH 10 with a 0.05 M solution of boric acid buffered at a pH of 12.5 & 0.1. The POstcolumn buffer was delivered via the peristaltic pump at a flow rate of 0.32 mL/min. The R ~ ( b p y )reagent ~ ~ + flow rate was also set at 0.32 mL/min. The amino acid standards were made up to have approximatelythe same composition as that expected from the Gramicidin digest.
Figure 1. Experimental configuration for the Chemiluminescent detection of underivattvedamino acids: (SP) syringe pump; (IN) injectlon system; (AC) analytical column; (LDS) liiht detection system (detection cell coupled to a photomultipliertube); (HV) high-vottage power supply; (PA) picoammeter; (DA) data acquisition and display; (PP) peristaltic pump; (RR) Ru(bpy),*+ reservoir; (MS) magnetic stirrer; (EC) eiectrochemical cell; (PS) potentiostat; (W) waste.
causing system instability (18). All ruthenium solutions used in this study were 1mM in tris(2,2'-bipyridyl)ruthenium(III) perchlorate and 0.2 M in Na2S04. DL-Amino acids were obtained from Sigma Chemical Co. (St. Louis, MO) and used as received. a-Methylglutamic acid, 2-aminoisobutyric acid, and the phenylamines were obtained from Aldrich. All other chemicals were reagent grade unless otherwise specified. Deionized water was used in the preparation of all solutions. Instrumentation. A schematic of the experimental setup is ~ ~ +generated by using a PAR shown in Figure 1. R ~ ( b p y ) was (Princeton, NJ) Model 363 potentiastat/galvanostat with a standard three-electrode arrangement consisting of a platinumgauze working electrode, a silver-wire quasi reference electrode, and a platinum-wire auxiliary electrode. Since the reaction is reversible, the auxiliary electrode was isolated from the Ru(bpy)33+ solution by a glas frit to prevent the Ru(bpy)? from being ~ ~the + auxiliary electrode. The potential reduced to R ~ ( b p y ) at at the working electrode was set to 0.89 V vs the Ag wire. Electrolysis was typically run for 30 min/50 mL of ruthenium solution. An ISCO (Lincoln, NE)LC-5OOO syringe pump delivers the amino acid from a Rheodyne (Cotati, CA) 7125 injector fitted with a 20-pL loop to a 4.6 mm X 25 cm Whatman PartisillO SCX column (P. J. Colbert Associates, St. Louis, MO). The column was connected to the detection cell via a mixing tee which was used to introduce the postcolumn buffer. The detection cell consisted of a small coil of 0.5 mm i.d. glass tubing positioned directly in front of a Hamamastu (Hamilton, NJ) R928 photomultiplier tube (PMT). The detection cell volume was determined ~ ~ +delivered to the detection coil via to be 80 rL. R ~ ( b p y ) was a four-channel Gilson (Middleton, WI) MiniPuls 3 peristaltic pump. It should be noted that the electrolysis cell was continually refilled with Ru(bpy)?+ at the same rate as the Ru(bpy)?+ was being removed. This was done via the peristaltic pump and allowed for continuous operation for periods exceeding 2 h. The postcolumn buffer was also delivered to the mixing tee vie. the peristaltic pump. The resultant CL was detected by the PMT. The PMT current was monitored with a Keithley (Cleveland,OH) 485 picoammeter, the output of which was sent to a personal computer via an IEEE488 interface (Model PC-W; National Instruments, Austin, TX). A Hewlett Packard 8452A diode array spectrophotometer was used to monitor the reaction for the production of the Ru(bpy),+ species. Analysis. To determine the relative CL intensity of 15 physiologically important amino acids, the column and mixing tee were removed from the apparatus shown in Figure 1 and 20 WLof standard solutions of the amino acids were injected a
RESULTS AND DISCUSSION A sensitive chemiluminescent detection method for amino acids that requires neither pre- nor postcolumn derivatization would be highly desirable for purposes of protein sequencing. The CL derived from such a technique should be directly proportional to the quantity of amino acid present, the method should have a wide dynamic working range and it should be sensitive to the detection of all amino acids. There are currently only a few chemiluminescent methods available for the detection of amino acids as previously mentioned. In order to evaluate the potential of the reaction of tris(2,2'-bipyridyl)ruthenium(III) for the CL detection of amino acids, the experimental conditions affecting the R ~ ( b p y ) , ~chem+ iluminescence were investigated. Properly optimized, the technique will be applied to the postcolumn detection of amino acids following a protein digest. The chemiluminescent reaction between R ~ ( b p y ) and ~~+ amino acids was studied using a flow injection analysis (FIA) system to reproducibly introduce amino acid samples into the CL detection system. A variety of experimental conditions were studied in this manner. As previously reported, the CL intensity of the reaction is very dependent on the pH at which the reaction is conducted, with optimum signal to noise ratio being obtained when the amino acid is buffered at pH 10 using a 0.05 M boric acid buffer (17). This pH dependence has been observed for all amino acids tested (Val, Thr, Phe, Met, Lys, Ala, Glu, a-methylglutamic acid, and 2-aminoisobutyric acid) except proline, a secondary amino acid, which exhibited a maxima at pH 9. This is very similar to the pH dependence found for the oxidation of amino acids by alkaline hexacyanoferrate(II1) ions (19). Since both ruthenium and iron are in the same group, it is conceivable, in light of the similar reaction p H dependence, that the two reactions proceed by a similar mechanism. The following mechanism is therefore proposed to account for the observed pH dependence of the Ru(bpy)?+/amino acid chemiluminescence reaction:
H,N+CHRCOO-
+ OH-
-
H,NCHRCOO-
H2NCHRCOO- + Ru(bpy)S3+ H2N'+CHRCOO- + Ru(bpy),2+ H2N'+CHRCOO-
+ R~(bpy),~+ -+
+ R ~ ( b p y ) ~ +~ +2H+ * Ru(bpy),'+* Ru(bpy),'+ + HN=CRCOO- + H2O RCOCOO- + NH3 NH=CRCOO-
-.
-
Note that in the proposed reaction sequence 2 equiv of Ru( b ~ y ) are ~ ~consumed + per equivalent of amino acid.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
168
i a0
0.41
r
162
0.40
44
0.38
-
7
.$ 1 2 6 Y
E
108
C a,
90
%
72
LL
.-3 m E c
0.36 0.34
Glu
-
0.32 > ._
5 -
54
0.31
-
0.29
-
Ser
36
ia 0
"
'
"
"
'
~
'
"
"
"
"
'
'
000 0 1 3 0 2 6 0 3 9 0 5 2 0 6 5 0 7 8 091 1 0 4 1 1 7 1 3 0
0
130
260
390
520
Leu mM
Figure 2. Stoichiometric study of the reaction of R ~ ( b p y ) , ~ +with leucine. Leucine was buffered at pH 10. The concentration of Ru(bpy):' is 1 mM with 0.2 M Na,SO,. Flow rates for both the Ru(bpy):+ and amino acid solutions were 0.32 mL/min.
Table I. Relative Chemiluminescence Intensities of 15 Amino Acids amino acid"
re1 CLh
amino acidn
re1 CL"
PRO LEU
1.000 0.222 0.160
CYS TYR ASN MET
0.012 0.011 0.010 0.009
GLY
0.009
THR SER
0.007 0.004
VAL GLU
PHE ALA ARG
HIS
0.133 0.100 0.062 0.044 0.013
650
780
910 1040 ' 1 7 0 1300
Time (sec)
"All solutions buffered a t pH 10 with 0.05 M boric acid buffer. "Intensities normalized to Pro.
Flgure 3. Representative detection limi-ts for serine and glutamic acid. Concentration of Ru(bpy):+ is 1.0 mM with 0.2 M Na,SO,. Flow rates . are 0.75 mL/min for amino acid and 0.5 mL/min for R ~ ( b p y ) , ~ +Key: (B) four injections of a blank buffer solution: (Glu) six injections of 10 pM glutamic acid: (Ser) six injections of 40 pM serine.
Table 11. Effect of Electron-Donating/Withdrawing Groups on the Chemiluminescence of Ethylamine amine"
CL signalh
2-chloroethylamine 2-hydroxyethylamine 2-methoxyethylamine ethylenediamine phenethylamine
0.266
0.46
0.347
0.27
0.402 0.974 1.00
0.27
UII
0.12
0.10
"Amines were buffered at pH 10, flow rates were 0.32 mL/min for 1 mM Ru(bpy),33+and 0.5 mL/min of amine. CL signals normalized to phenethylamine. ' uI values taken from ref 20.
given in Table I. System stability was found to be better than In support of this mechanism a stoichiometry study was 5%, as determined by the repetitive injection of the same standard over a 2-h period when operated in the FIA mode. conducted in which steady-state concentrations of Ru(bpy),,' and amino acid were mixed in front of the PMT at equivalent The system was found to be linear over 2 orders of magnitude for valine, with a correlation coefficient of 0.998 (5 points). flow rates (0.32 mL/min). The Ru(bpy)?+ concentration was checked spectrophotometrically by converting it to Ru(bpy)? Detection limits of 135 pmol for serine and 6 pmol for glutamic acid at a signal to noise ratio of 3 have been obtained. The and comparing its absorbance with a standard Ru(bpy)32+ solution prepared from commercially available Rudata from which these detection limits were calculated are shown in Figure 3. These values represent concentration (bpy),C12.6H20. In the stoichiometric study, the amine or amino acid concentration was varied while the R ~ ( b p y ) , ~ + limits of detection of 6.8 and 0.3 pM, respectively. For the concentration was held constant. This resulted in the most part, the new detection limits presented here represent "titration" of the R ~ ( b p y ) , ~reagent + with amine. When the an increase in detectability of 2 orders of magnitude over those previously cited, where the mass limit of detection was 50 nmol correct stoichiometric amount of amine was added to comfor serine and 2.5 nmol for glutamic acid (15). pletely react with Ru(bpy):+, the CL signal should reach a From Table I it can be seen that not all amino acids react plateau. This titration was repeated for four compounds: to yield CL with the same efficiency. It has been previously leucine, glutamic acid, valine, and triethylamine. A repreobserved that CL efficiency for amines proceeds from tertiary sentative titration curve is presented in Figure 2. For the > secondary > primary, with tertiary amines having the lowest four compounds studied a stoichiometric ratio of 2:1, Ru(bpy),"+:amine was obtained within experimental error. To detection limits (16) (i.e. greatest CL efficiency). This would our knowledge this is the first experimental data published account for the difference in CL intensity observed between showing the 2:l stoichiometry of the reaction. We were iniproline, a secondary amine, and leucine, a primary amine. One tially concerned about the efficient transport of R ~ ( b p y ) ~ ~ +possible explanation for the different CL intensities of the to the detection cell. Since the residence time of Ru(bpy)3J+ primary amino acids, which vary by a factor of about 55, is based on the R group attached to the a-carbon atom of the in the tube from the electrolysis cell to the detection cell was 10 s, the peristaltic pump was turned off for 10 s, and turned amino acid. To study the effect of varying the electronback on and the chemiluminescence intensity then noted. withdrawing character of the R group attached to the aGenerally, there was less than a 5% decrease in the signal carbon, various derivatives of ethylamine were tested using which supports the idea that there is efficient transport of the FIA/CL system. The results of this study are presented Ru(bpy)gStto the detection cell. in Table 11. A plot of u,! which is a measure of the elecThe relative CL of amino acids using the Ru(bpy)$+ reagent tron-withdrawing character of the R group (ZO),versus the has been reported previously (15). However, the pH at which log CL signal yields a straight line with an R2 coefficient of the reactions were conducted was not optimized for maximum 0.82. Although this study is a preliminary finding, it appears chemiluminescence. A comparative study was initiated at the that electron-withdrawing R groups tend to decrease chemoptimum pH in order to assess the relative CL efficiencies of iluminescence (Le. alcohols, serine, and threonine) while the various amino acids. The relative CL for 15 amino acids, electron-donating R groups tend to enhance chemiluminesbuffered at pH 10, as determined by triplicate injections are cence (i.e. alkyl side chains, leucine, and valine). Therefore,
-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
160
Table 111. Effect of a-Hydrogen on Chemiluminescence of Amino Acids amino acid'
re1 CL"
amino acid"
re1 CL'
GLY ALA a-methyl ALA
142 824 1702
GLU a-methyl GLU
1750 5473
Amino acids buffered at DH 10. Intensity in arbitrary units.
it would be expected that if the a-hydrogen of the amino acid was replaced with an electron-donating group, such as a methyl group, the chemiluminescencewould be expected to increase. To test this hypothesis, the relative CL of glycine, alanine, a-methylalanine, glutamic acid, and a-methylglutamic acid were compared. The results of this study are presented in Table 111. From this study, the data suggest that substitution at the a-carbon of the amino acid with an electron-donating group enhances the chemiluminescent signal. These results support the conclusions from the previous study of electrondonatinglwithdrawing character at the a-carbon. Although the generality of these observations are still under investigation, it is possible that the ability of the a-carbon and the substituents attached to it to stabilize the radical initially formed, either through hyperconjugation or electron-donating effects, may play an important role in determining the CL efficiency for that species. Studies are in progress to determine the predictive value of the above observations in determining which analytes are suitable for detection using this system and which analytes can be made detectable through derivatization with an appropriate functionality. It has also been observed that aromatic groups such as benzene tend to quench chemiluminescence when attached directly to the amine group, i.e. aniline. This is surprising because phenyl groups are only weakly electron withdrawing and would therefore be expected to decrease the chemiluminescence. In order to determine the effect of proximity of phenyl groups to the amine, a series of phenylamines were studied in which the phenyl group was progressively removed one methylene group from the amine. The relative chemiluminescence was monitored for each compound starting with aniline and proceeding to 3-phenyl-1-propylamine. Aniline quenched the CL reaction while benzylamine, phenethylamine, and 3-phenyl-1-propylamineall produced chemiluminescence varying in intensity by a factor of -4. These data suggest that the ability of an R group to delocalize the radical formed through an extended r-system could be important in determining whether or not an amine will be detected in the above system. Chromatography. To determine if the R~(bpy),~+-based chemiluminescent detection of underivatized amino acids could be implemented as a postcolumn detector, a separation scheme for five amino acids (Gly, Ala, Val, Trp, and Leu) using a Whatman Partisil 10 SCX column was devised. These amino acids were chosen because they are the same amino acids present in gramicidin D, a small 15 amino acid peptide with antibiotic properties. To show the applicability of this technique in the detection of amino acids from a protein digest, gramicidin D was digested in 3 N p-toluenesulfonic acid (pTS) for 24 h at 110 O C (21). pTS was neutralized with sodium hydroxide to the pH range 5-7 and diluted 1:lO with water. A 20-pL aliquot of this digest was subsequently chromatographed and detected using the Ru(bpy),,+ reagent. The conditions for separation are given in Figure 4, which shows the separation of a five-component amino acid standard at two different concentrations. Figure 5 shows the separation of the gramicidin digest, using the same conditions as in Figure 4. Since glycine is present in such a small quantity, it was necessary to inject higher concentrations of the digested
0.12 0.00 0 ~
250
500
750 1000 1250 1500 1750 2000 2250 2500 Time (sec)
Flgure 4. Chromatogram of amino acid standards. Separation was conducted on a 4.6 mm X 25 cm Whatman Particil 10 SCX column at ambient temperature. The eluent was 1 mM Na,SO, adjusted to pH 2.5 with 12 M HCI. The column flow rate was 1 mL/min. The first set of peaks represent an amino acid standard 4 times as concentrated as the second set. Key: (Glu) glycine; (Ala) alanine; (Val) valine; (Trp) tryptophan; (Leu) leucine. See Experimental Section for details of postcolumn buffering and detection. 0.64
c
Val
Leu
0.59 0.53
,x .m
6 c
> .-;j
0.48 0.42 0.37
- 0.32
d
0.26 0.21 0.15 0.1 0
~
0
108 216
324
432
540
648
756
864
972 1080
Time (sec)
Flgwe 5. Chromatogram of gramicidin digest. Condiiis are the same as in Figure 4.
Table IV. Summary of Amino Acid Standard Chromatogram amino acid
nmol injected
peak areao
peak vol, mL
peak width, s
GLYb ALAb ALA* VAL* TRP* LEU*
1.65 3.20 0.800 1.20 1.20 1.20
0.463 8.02 2.00 5.31 2.33 10.2
0.90 1.4 1.3 1.6 2.4 2.7
33 52 48 61 89 95
Arbitrary units. Numbers presented were obtained from the injection of an amino acid standard 4 times more concentrated than those denoted bv an asterisk. Table V. Summary of Gramicidin Digest Chromatogram mol ratio foundb expected
amino acid
peak area'
nmol found
GLY' ALA' ALA VAL
1.02 18.7 2.99 10.6 4.17 20.4
3.63
1.00
7.46 1.20 2.40 2.15 2.41
2.06 1.98 3.97 3.55 3.98
TRP
1 2 2
4 4
LEU 4 "Arbitrary units. bDetermined by dividing all amino acid nanomole amounts found by the smallest nanomole amount found, i.e. Gly. Numbers presented were obtained from the injection of a digest 6 times more concentrated than that shown in Figure 7.
170
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
Table VI. Solvent Effects on the CL of Ru(bpy):;'+ O/o
solvent citrate methanol 2-propanol acetone acetonitrile
5%
300 800 400 35 (-5) 20 (+O)
10%
U" 800 450 60 (-5) 30 (+17)
50 70
20%
U U U 75 (-30) 46 (+17)
U U U U -12 (-66)
Numbers outside parentheses represent an increase in background CL over that obtained without any additive. Numbers in parentheses represent an increase in signal above the background increase. i. Numbers inside parentheses represent the effect of solvent on the CL of Glu when compared to a Glu solution having no solvent. ' Value for citrate was obtained from a 1 mM sodium citrate solution. U = solvent was not tested.
protein in order to obtain quantitative data. This overloaded the column, resulting in the coelution of valine and tryptophan (not shown). The results of this study, based on peak areas, are presented in Tables IV and V. The slightly low value for tryptophan can be attributed to its partial air oxidation/decomposition. From the data obtained it is evident that the Ru(bpy),"+-based CL detection method is applicable for use as a postcolumn detector for all underivatized amino acids. We are currently limited by the quality of the cation-exchange column being used. This is evident by consideration of the peak volumes given in Table IV, where it can be seen that the chromatographic peak widths are much larger than the detection volume. Further improvements could be expected if a more efficient chromatographic system were used. The present system is not confined to being used with strictly water-based systems. Although the separations presented in this paper used a 1mM Na2SO4solution buffered at pH 2.5, other eluents have been tested. Among those tested were sodium citrate, methanol, 2-propanol, acetone, and acetonitrile. The results of this study are presented in Table VI. From these data it appears that sodium citrate, methanol, and 2-propanol are all unsatisfactory eluents due to their large background signals. However, acetone can be used without significant detrimental effects at concentrations less than 10% v/v. The use of acetonitrile actually increased the chemiluminescent intensity of amino acids by 17% when used at concentrations less than 20% v/v. The behavior of citrate is not unexpected since organic acids have been known to produce CL upon reaction with Ru(bpy),,+. The CL of the alcohols was, however, unexpected. Therefore, in order to determine if the alcohols were affecting the pH dependence of the reaction, a pH study was conducted in which both amino acid (Phe, 0.05 mM) and blank buffer solutions contained 20% by volume methanol. The results of this study showed that the maximum S I N with MeOH added occurred at pH 9. When the Phe/MeOH signal at pH 9 was compared with that of Phe with no MeOH at pH 10, the ratio of the two signals was approximately 3:1, Phe/no MeOH:Phe/MeOH. This was due to the fact that the background CL signal for the pH 9 buffer/MeOH blank was 2.5 times higher than the pH 10 buffer blank with no MeOH. It is believed that the high background signal is caused by the deprotonated form of the alcohol (ie. methoxy ion, -OCH,) reacting with Ru(bpy), i+at elevated pHs. Acetone and acetonitrile, although completely miscible with water, impart an organic nature to the solution which could affect the pH a t concentrations greater than 20%. Separations have also been successfully
conducted on a CI8 column using 0.1% H3P04with 5% acetonitrile as the eluent using Ru(bpy),3+ CL detection.
CONCLUSION The ability to implement the reaction of Ru(bpy),3+with underivatized amino acids as a postcolumn chemiluminescent detection system has been demonstrated. Amino acids are generally not well suited for detection without prior derivatization due to the absence of a strong chromophore. The Ru(bpy),,+ reagent has the potential to be generated in line prior to the detection cell, which can make the system more rugged and easy to use. Research is currently under way to miniaturize the present system by using an experimental arrangement that allows for the generation of Ru(bpy)?+ at the site of detection. It is believed that several orders of magnitude improvement in the mass limit of detection can be gained through such a miniaturization. In an effort to understand and utilize the R ~ ( b p y ) , ~reaction + for the detection of other analytes, the structural requirements for chemiluminescence were investigated. In general, analytes with electron-donating groups will generate chemiluminescence more efficiently than analytes with electron-withdrawing groups. Phenyl groups or groups capable of delocalizing the radical when attached directly to the amine result in decreased chemiluminescence via a quenching mechanism. The characteristics of the Ru(bpy),3+/amino acid reaction make it directly amenable to the postcolumn CL detection of amino acids following a protein digest. Results determined by the Ru(bpy),3+CL scheme for gramicidin were in exact agreement with the actual composition of the peptide. With the reaction characteristics now well defined, work can progress toward miniaturization of the system. The technique is currently being investigated as a post-column detection system for microbore HPLC and CZE separations.
ACKNOWLEDGMENT We thank an anonymous reviewer for helpful comments.
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RECEIVED for review July 22,1991. Accepted October 8,1991. D.R.B. acknowledges the support of the Camille and Henry Dreyfus Foundation through a teacher-scholar fellowship.
