Determination of T ris( 2,2'- bipyridyl)Rut henium(I I) Derivatives by Reverse Phase Paired-Ion High Performance Liquid Chromatography Steven J. Valenty" and Paul E. Behnken General Electric Corporate Research and Development, Schenectady, New York
Reverse-phase paired-ion high performance liquid chromatography (HPLC) has been used to determine diester, monoester-monocarboxylate and dicarboxylate derivatives of tris( 2,2'-bipyridyl)ruthenium(11) ( Ru"( bpy),*+) which can be distinguished on the basis of water solubility and molecular charge. Positively charged surfactant ruthenium derivatives have been ion-paired with methanesulfonate anions in the aqueous tetrahydrofuran mobile phase while their water soluble analogues have been paired with n-heptanesulfonate anions to provide suitable retention for separation on a CI8 bonded column. Using optical detection at 280 nm, the lower limit of sensitivity is 1 X mol with f2% precision at the 2.5 X mol level. Monitoring hydrolysis reactions of diester derivatives of Ru'1(bpy),2+ by HPLC has proven superior to absorption and emission spectrometry.
T h e chemistry of tris(2,2'-bipyridyl)ruthenium(II)(Rurl(bpy),'+) and its derivatives is of current interest especially when these compounds are employed as photopromoted electron transfer reagents (1-3). Characterization of these materials and monitoring the course of chemical reactions have been mainly accomplished by elemental analysis and optical spectroscopy. While ion-exchange chromatography has been utilized in several instances (4-6), there has been no routine application of chromatography for purity assay, reaction monitoring, or isolation of reaction products. We wish to report the first use of reverse-phase paired-ion high performance liquid chromatography (HPLC) (7-10) to separate and quantitate Rull(bpy)gz+derivatives-which can be distinguished on the basis of water solubility and molecular charge-observed in the hydrolysis of I and 11.
I, R E, R
i
R 'CieH37
'R'C2H5
E,R - C l a n 3 7 P, R ' C z H 5
m
EXPERIMENTAL Materials. The synthesis and characterization of compounds I-IV is described elsewhere (11). Methanesulfonic acid (puriss, Fluka), glacial acetic acid (Baker Analyzed), PIC Reagent B-7 (1-heptanesulfonic acid buffered to ca. pH 3.5, Waters Associates), tetrahydrofuran (THF) (Burdick and Jackson), and triply distilled water were used in the preparation of the mobile phase. The hydrolysis reaction buffer (1.0 x M, pH 10.0) was prepared from boric acid (Baker Analyzed, 50 mL, 0.1 M), potassium chloride (Mallinckrodt AR, 50 mL, 0.10 M), and standarized sodium hydroxide (Fisher, 43.7 mL, 0.10 N) diluted to 500 mL with water. Chromatography. A Model ALC/GPC 244 Liquid Chromatograph equipped with two Model 6000A pumps, Model 660 Programmer, Model U6K Injector, Model 440 Dual Channel
1230 1
Absorbance Detector and a 30 cm X 3.9 mm i.d. pBondapak/C18 column (Waters Associates, Milford, Mass.) was used for these analyses. Components in the eluent were detected by their absorbancies a t 254 and 280 nm. The analysis for I, 111, and I V employed a 20-min linear solvent gradient, 5 0 7 ~THF in HzOto 100% THF (both 0.015 M MeSO,H, 0.5% ( v / v ) HOAc), with a 2.0 mL/min flow rate. The analysis for 11, 111, and V employed a 10-min linear solvent gradient, 1070 THF in HzO to 40% THF in H20 (both 0.005 M n-heptanesulfonic acid, HOAc, pH 3.5, PIC B-7 reagent), with a 2.0 mL/min flow rate. The column equilibration time between analyses was 6-7 min. Optical Spectrometry. Absorption spectra (10 nm/min, 60 or 120 nm/min, 1.0-nm effective band width) were recorded with a Perkin-Elmer Model 575 spectrophotometer. The emission spectra (Aex = 475 nm, 8.5-nm band pass for both excitation and emission monochromators, Oriel long pass 475 emission filter, 30 nm/min) were recorded at 22 O C using right angle geometry on an apparatus constructed in this laboratory (11) and are not corrected for variation in detector/emission monochromator sensitivity with wavelength. Hydrolysis Reaction Procedure (11). A 0.90 mL, 1.11X M solution of I1 in HzO was transferred to a 10-mL Erlenmeyer flask containing 9.10 mL 1.0 X M borate buffer (pH 10) thermostated at 20 f 1 "C and magnetically stirred; 25-pL aliquots were removed at time intervals and analyzed by HPLC. The detector response at 280 nm was calibrated by 25-pL, 2.5-nmol injections of standard solutions of I1 and I11 and found to be identical ( f 2 7 0 ) . Due to the tailing of component 111, the areas of 11,111, and V were obtained by copying the trace, cutting out the areas under each band, and weighing. The procedure for monitoring the course of the reaction by optical spectrometry M I1 and 36.0 mL 1.0 X varied slightly: 4.0 mL 1.0 X M borate buffer (pH 10) were mixed and stirred at 20 f 1 "C. Aliquots (2.5 mL) were withdrawn at intervals and the absorption or emission spectra recorded. Note that the aliquots taken for spectroscopic analysis were not acid quenched and the reaction continued as the spectra were recorded. The aliquot times indicate the start of the scan; total scan times for absorption and emission M are 1.7 min and 8.3 min, respectively. Spectra of 1.0 X solutions of I1 in H20 and I11 in 1.0 X lo-' M borate buffer (pH 10) were recorded t o provide calibrations at t = 0 and t = m, respectively. Hydrolysis Reaction Procedure (I). A 1.0 mL, 1.0 X M solution of I in THF and 4.0 mL fresh THF equilibrated to 20 "C were transferred to 5.0 mL 1.0 X M borate buffer (pH 10) stirred at 20 f 1 "C; 25-pL aliquots were removed at time intervals and analyzed by HPLC. The detector response at 280 nm was calibrated by 25 pL, 2.5 nmol injections of I and 111. The areas of I, 111, and IV were obtained by weighing the trace cutouts. Calculations of the amount of IV used the same calibration factor as for I based on their similar peak shapes. Hydrolysis solutions, analyzed in 2.5-mL aliquots by optical spectroscopy, were prepared by mixing 4.0 mL 1.00 X lo-, M I in THF, 16.0 mL THF, and 20 mL 1.0 X lo-' M borate buffer (pH 10) with magnetic stirring M solutions of at 20 f 1 "C. Calibration spectra of 1.0 x I in 50% THF in H20and I11 in 50% THF in 1.0 X M borate buffer (pH 10)were also recorded. RESULTS A N D DISCUSSION HPLC Analysis. Initial attempts to determine the purity of I by conventional normal or reverse phase chromatography
0003-2700/78/0350-0834$01.00/00 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL 50, NO. 7, JUNE 1978
Compound IIIe IVe Iae Ie Ibe IIIf Vf 11,
Retention time, min" 1.1 2.4 6.8 7.3 8.0 4.9 7.7 10.4
O9
-
--ABSORPTION
r---
Table I. Chromatographic Data
~
-~~
EMISSION
08 -
PH2s b
PH280 0.54 i. 0.54 t 0.54 t 0.54 i 0.54 * 0.54 t 0.54 t 0.54 *
0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
835
-
130
-
I10 100 90
I15
__
Recovery,
E 280
%d
60
.. . ... ...
50
€zsc
0.54 0.54 0.53 0.53 0.53 0.54 0.52 0.51
96
a Solvent flow rate 2.0 mL/min, time required t o displace one column volume is 0.9 min. Ratio of recorder peak heights (PH) as each component is detected at 254 and 280 nm. Ratio of the molar extinction coefficients ( e ) obtained from a spectrum of the known compound (in the HPLC solvent mixture passing through the detector % Recovery = O.D.hLC of a at the retention time). component collected off the column + O.D.hSta*d of the component a t the same concentration in the solvent system at the retention time but not injected onto the column x 100. e THF/H,O with methanesulfonic acid. f THF/H,O with n-heptanesulfonic acid.
failed. The adsorption of the dication onto the bare or partially coated silica surfaces of the stationary phases used was irreversible. Upon addition of methanesulfonic acid (MeS03H)to both reservoirs of the H20/THF solvent system, I eluted from a CI8-bondedcolumn with increasing T H F as a sharp, symmetrical peak at an optimum MeS03H concentration of 0.015M. As shown in Table I, the resolution is sufficient to separate and quantify compounds differing by only two methylene units in the two long hydrocarbon tails (I, R1 = R2 = C18H37;Ia, R1 = C16H33, Rz = C18H37; Ib, R1 = C20H41, Rz = C18H37). The lower limit of detection sensitivity mol at S / N = 2 (etrn = 5 X lo4). for I a t 280 nm was 1 X The detector response was linear over the range 5 X to 2X mol I with a precision of f.2% a t the 2.5 X mol level. The data in Table I are based upon analyses of independently synthesized and characterized samples (11)with the exception of V whose structure was assumed on the similarity of its absorption spectrum (obtained on a sample collected in a fraction of column eluent) to IV and its intermediacy in the hydrolysis of I1 to 111. Note that comparing the ratios of the absorbancies observed a t 254 and 280 nm to those obtained from the full absorption spectrum (recorded in the HPLC solvent system) allows an additional diagnostic tool for identification of a compound as a Ru11(bpy)32+ derivative. To determine whether material was being irreversibly bound to the stationary phase, 5.4 x mol (50 pL) of I, 11, and I11 were injected separately, ca. 4 mL fraction of eluent collected about the retention volume of that component, diluted to 5 mL with additional solvent of the same composition and an absorption spectrum was recorded (200-600 nm). The optical densities of the absorption maxima were then compared to those of standard solutions of 5.4 x mol of I, 11, and I11 in 5.0 mL of the same eluting solvent composition. The ratio of these values shows a 96-97% recovery of the originally injected material, indicating little or no irreversible adsorption onto the column. Reverse-phase ion-pair chromatography has been described by Wittmer, Nuessle, and Haney (7), Waters Associates, Inc. (8),and Knox and Laird (9). In this report, the positively charged organometallic molecules are thought to form neutral ion-pairs with one or two alkyl sulfonate counterions present in the mobile phase. The hydrophobic interaction of the
5
80 I 70
40
Y
30 20 IC
96
... 97 ...
~
2
0
350 400
450
550
500
600
650
700
750
800
WAVELENGTH i n m )
Figure 1. Absorption and uncorrected emission spectra of the hydrolysis M pH 10 borate buffer. Curves reaction of I1 in aqueous 1.0 X are marked in hydrolysis reaction times (min). The initial concentration of I1 is 1.0 X M and spectra were recorded in 1.0-cm path length cells (right angle geometry for emission spectra)at 21 f 2 O C . Excltation wavelength is 475 nm (bp 8.5 nm) I
035 E
-
I
I
-
I
03OC
c 0
E
025-
I
49
0
I
2
3
10 4
77
4 5 6 7 8 RETENTION T I M E (rnin.)
9
IO
I1
Figure 2. Chromatograms of the hydrolysis reaction of I1 in aqueous 1.0 X M pH 10 borate buffer. Traces shown at 0, 2.5,21, 90, and m min reaction times. The initial concentration of I1 is 1 .O X IO-' mol) aliquots are injected for each analysis. M and 25 I.LL(2.5X
The diagonal line across the figure shows the composition of mobile phase wlth 0.005 M n-heptane-sulfonic acid buffered to pH -3.5 hydrocarbon chains of this ion-pair with the bonded octadecylhydrocarbon surface of the stationary phase provides the basis for separation. Alternatively, the alkyl sulfonate additives might be thought of as initially adsorbing onto the hydrophobic surface of the substrate to change it into a sulfonate based ion-exchange column (10). Hydrolysis of I1 in Aqueous Solution. At a concenM, I1 forms a golden-hued, homogeneous tration of 1.0 X solution in M borate buffer (pH 10). The results of monitoring the absorption spectrum of this solution as a function of time are shown in Figure 1. A more dramatic change appears upon observing the emission from this solution (also Figure 1) when excited a t 475 nm where the absorption spectra indicate only a 5-7% variation in optical density. The slight enhancement in emission intensity seen during the latter stages of reaction relative to that finally observed for I11 has been seen on several occasions and is not understood. Both the trends in absorption and emission spectral shifts have been noted previously in the pH titration of I11 (12) with specific differences presumably due to having ethyl substitutents rather than protons bonded t o the carboxyl groups. Following the course of the reaction by HPLC provides a V quantitative description of the interconversions, I1 111, as shown in Figure 2 and Table 11. The separation depends upon adjusting the pH of the mobile phase to -3.5 such that all carboxylic acid groups are ionized as expected from the titration of I11 (12). At this pH, the formation of a neutral ion-pair in the mobile phase with I1 requires two +
+
836
ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
Table 11. Hydrolysis of I1 Followed by HPLC Hydrolysis time, min 0.0 1.0
2.5 5.0 21 36 53 70 90 120 142
V
I1 No. nmola,b
Mol %'
2.5 2.2 2.0 1.5 0.56
100 88 80 60 22 4
0.10
0.03 0 0 0 0 0
No. nmolb 0
0.24 0.55 0.85 1.6 1.3 0.97 0.76 0.51 0.28 0.19 0
1
0 0 0 0 0
a Initial concentration of I1 is 1.0 x ponent in 25-bL aliquot withdrawn at t
Mol %'
=
I11 No. nmolb Mol %'
0 10
0 0
0 0
22 34 64 52 39 30 20
0.03 0.07 0.38
1
3 15 44 60 72 84 92 96
1.1
1.5 1.8 2.1 2.3 2.4 2.5
11 8 0
100
I1 + I11 + v Mol %c
NO. nmolb
2.50 2.44 2.58 2.42 2.54 2.50 2.50 2.56 2.61 2.58 2.59 2.50
M in aqueous 1.0 x M borate buffer pH 10, 20 + 1"C. t. mol x 100. Mol % = No. mo1,,,/2.50 x
100 98 103 97 101 100
100 102 104 103 104 100 Amount of com-
Table 111. Hydrolysis of I Followed by HPLC I Hydrolysis time, min No. nmolasb 0.0
0.5 1.25 5.0 13 25 45 61 96 m
2.50 1.40 0.59 0.04
