1464
Anal. Chem. 1985, 57, 1464-1469
pounds with DCC and DMBA are proposed as eq 2.
(3) Williams, A.; Ibrahim, I. T. Cbem. Rev. 1981, 8 1 , 589-636. (4) Kasai, Y.; Tanimura, T.; Tamura, 2. Anal. Chem. 1975, 47, 34-37. ACKNOWLEDGMENT (5) Kasai, Y.; Tanimura, T.; Tamura, 2.; Ozawa, Y. Anal. Cbem. 1977, 49, 655-656. The author thanks Shu-Jen Chang and Chia-Li Wu, Tam(6) Chen, S.-C. J . Chromatogr. 1982, 238, 480-482. kang University, for the mass spectra measurements. (7) Chen, S.-C. Anal. Cbem. 1982, 5 4 , 2587-2590. (8) Chen, S.-C. Anal. Biocbem. 1983, 132, 272-275. Registry No. 11, 78902-42-8; 111, 78902-50-8;DCC, 538-75-0; (9) Chen, S.-C. Anal. Biocbem. 1984, 140, 196-199. DMBA, 769-42-6;pyridine, 110-86-1;pyrimidine, 289-95-2; py(IO) Wilchek, M.; Miron, T.; Kohn, J. Anal. Biocbem. 1981, 114, 419-421. (11) Kohn, J.; Wilchek, M. Biocbem. Biopbys. Res. Commun. 1978, 84, ridazine, 289-80-5; 2-aminopyrimidine, 109-12-6; 2-amino-47-14. methylpyrimidine, 108-52-1;sulfadiazine,68-35-9;sulfamerazine, (12) Chaudhuri, D. K. Indian J . Med. Res. 1951, 39, 491-505. 127-79-7; thiamine HCl, 67-03-8; chlorpheniramine maleate, (13) Asmus, E.; Garschagen, H. 2.Anal. Chem. 1953, 139, 81-89. 113-92-8;isoniazid, 54-85-3; niacin, 59-67-6; quinoline, 91-22-5; (14) Anger, V.; Ofri, S. Talanta 1983, IO, 1302-1303. K. Sitzungsber. Naturforscb. Ges. Rostock 1916, 6 , 33; isoquinoline, 119-65-3;1-(1,3-dimethyl-2,4,6-pyrimidinetrione-5- (15) Fujiwara, Cbem. Abstr. 1917, 1 1 , 3201. y1)-3-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-ylidene)methyl1(16) Friedman, P. J.; Cooper, J. R. Anal. Cbem. 1958, 30, 1674-1676. propene, 95798-81-5; 1-(1,3-dimethyl-2,4,6-pyrimidinetrione-5- (17) Leibman, K. C.; Hindman, J. D. Anal. Cbem. 1964, 36, 348-351. (18) Uno, T.; Okumura, K.: Kuroda, Y. Chem. Pharm. Bull. 1982, 30, y1)-3-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-ylidene)-2-( 3-thia1876-1 879, zolyliummethyl)propenamine, 95798-82-6; [3,7-bis(1,3-di(19) Sasagi, T. "Heterocyclic Chemistry", 1st ed. (Japanese);Tokyo Kagamethyl-2,4,6-pyrimidinetrione-5-yl)heptyl] dimethylamine, ku Dojin: Tokyo, 1972; p 106. 95798-84-8; 1-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-y1)-5-(di- (20) Barnes, R. A. "Pyridine and Its Derivatives, Part one", Interscience Publishers, Wiley: New York, 1960; pp 70-74. methyl-2,4,6-pyrimidinetrione-5-ylidene)-3-aminocarbamoyl-l,3J. N. T.; Millard, B. J.; Powell, J. W. J . Pharm. Pbarmac. 1970, pentadiene, 95841-20-6;54 1,3-dimethy1-2,4,6-pyrimidinetrione- (21) 2Gilbert, 2 , 897-901. 5-y1)-2-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-ylmethyl)penta- (22) Skinner, R. F.: Gallaher. E. G.; Predmore, D. B. Anal. Cbem. 1973, 45, 574-576. dienoic acid, 95798-86-0.
LITERATURE CITED (1) Khorana, H. G. Chem. Rev. 1953, 53, 145-166. (2) Mikolajczyk, M.; Kielbasinski, P. Tetrahedron 1981, 37, 233-264.
RECEIVED for review February 5 , 1985. Accepted February 26, 1985.
Reverse-Phase High-Performance Liquid Chromatography/Nuclear Magnetic Resonance Spectrometry Separations of Biomolecules with 1-1 Hard Pulse Solvent Suppression D. A. Laude, Jr., R. W.-K. Lee, and C. L. Wilkins*
Department of Chemistry, University of California, Riverside, California 92521
Recently developed solvent suppresslon methods that rely upon appllcatlon of rf excitation wlth zero spectral denslty at solvent resonances are demonstrated to provide slgnlflcant advantages for the reverse-phase LC/NMR experiment. I n particular, with mlnimal delays between scans and the abillty to suppress multiple solvent resonances, these suppresslon techniques are clearly superior to presaturation methods previously employed. I n the present work, parameters for the 1-1 hard pulse suppression technique are optimized for continuous-flow LC/NMR. Applicatlon of the method Is demonstrated wlth reverse-phase separations of amino acid, vltamln, and nucleoside mixtures.
Modern reverse-phase high-performance liquid chromatography (LC) has developed into the preeminent tool for biochemical analysis. Unfortunately, the coupling of reverse-phase methods with NMR detection is made significantly more difficult by solvent limitations; protonated HzO, acetonitrile, and methanol are not as readily substituted for as their normal phase solvent counterparts. Protonated solvents present two major difficulties for LC/NMR including the potential for spectral interference with the analyte, and constraints upon detection limits imposed by the dynamic
range limitations of the analog to digital converter (ADC). Attempts to solve these critical problems have included the use of larger ADCs (I),deuterated or halogenated solvents (2),or selective saturation solvent suppression pulse sequences ( I , 3 , 4 ) . Although all three approaches improve the dynamic range of LC/NMR, only the use of nonprotonated solvents effectively eliminates solvent spectral interferences. Especially for normal-phase methods, nonprotonated solvents such as Freon-113, carbon tetrachloride, and deuteriochloroform have been utilized. Although the application of deuterated solvents to reverse-phase separations on a preparative scale would be cost prohibitive, the development of analytical scale LC/NMR ( 1 , 5 )reduces solvent volumes with typical separations requiring 10 to 20 mL of solvent. At these levels, DzO becomes a viable solvent and several chromatographic applications (aqueous size exclusion and ion exchange) are amenable to LC/NMR analysis. Deuterated acetonitrile and methanol are several orders of magnitude more costly and, except for use as modifiers, are not feasible. The dynamic range of the ADC determines a ratio of the largest to smallest observable NMR signal equal to
R = 2b where b is the number of bits in the ADC. FT-NMR spectrometers are often equipped with 12-bit ADCs which limit
0003-2700/85/0357-1464$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
the dynamic range to 3 orders of magnitude, insufficient for the reverse-phase experiment. (In a 20-pL cell with HzO as the solvent, ca. 100 pg of analyte would be required to produce a signal above the noise, far above the minimum detection limits established (1, 5).) The problem may be partially circumvented through the use of commercially available 16-bit ADCs and Bayer has reported that solvent suppression is unnecessary with the larger ADC (1). However, larger ADCs will not provide the ultimate solution as LC/NMR detection limits continue to decrease. Solvent suppression techniques, widely used in NMR for the suppression of the HzO signal in aqueous biological samples, fall into two categories dependent upon differences in chemical shift or Tl values for the solvent and analyte. Among the methods that rely upon chemical shift differences, selective presaturation (6) and the binomial suppression techniques (7-10) are prominent. Methods that employ Tl differences include progressive saturation and spin echo techniques (11). Unfortunately, the strict requirements of on-line continuous flow LC/NMR preclude the use of many of the suppression techniques listed above. For example, because of the finite lifetime of the solute in the observe cell, the sensitivity afforded by the suppression technique is critical. Not only must the sensitivity for a single scan not be affected, but delays between scans must be minimized. The technique should also operate over a narrow chemical shift or Tl range specific for the solvent and be applicable to the suppression of multiple solvent resonances that occur in reverse-phase separations. None of the suppression techniques listed above conform to all of these requirements; the presaturation methods, for example, are inefficient because of the lengthy delays required between scans, and Tl methods have limited applicability. Bayer ( I ) and Herzog (3, 4 ) have previously employed suppression techniques in the continuous-flow experiment. Both used selective presaturation homo-decoupling pulse sequences in which, during a delay between scans, the solvent resonance was saturated. Bayer extended the method to the suppression of two solvent resonances through the use of a homogated-decoupling technique in which rf excitation was alternately applied to each solvent resonance every 0.04 s. The major disadvantage of the method, which limits its application on an analytical scale, is the large cycle time per scan, T,; both workers used cycle times of as much as 3 s to permit efficient saturation with a minimum perturbation of the base line. SIN was thereby decreased by as much as (T,/Ta)'I2where T, is the acquisition time. With the introduction of the Redfield pulse sequence in 1971 (12), a new class of solvent suppression methods that relies upon the application of rf excitation with zero spectral density at the solvent resonances has emerged. Null points in an FT-power spectrum are generated by a series of pulses with appropriate delays; binomial-suppression techniques, based upon the nth series of binomial coefficients, produce excitation pulses with nulls at cosn 0 (7). Examples include the 1-1 hard pulse (8)and the 1-2-1 (9) and 1-3-3-1 soft pulse sequences (10). The principal advantages of these pulse sequences for LC/NMR are the imposition of minimal delays (a few milliseconds) prior to data acquisition and multiple solvent suppression capabilities. In addition, although not selective for solvent resonances, windows in the excitation spectrum can be limited to less than 100 Hz. The 1-1 hard pulse (1-1 HP), simplest of the binomial suppression methods, consists of a Ox-r-O, sequence with 28, generally equal to 90'. If the carrier is spaced 4 u from the solvent resonance, then following a 45' pulse, a delay, 7,equal to 1 / ( 2 4 v ) allows precession of the solvent magnetization exactly 180'. Application of an equivalent 45' pulse nulls the solvent magnetization along the z axis. All other resonances
1465
Figure 1. Vector representation of 1-1 hard pulse yz magnetization.
Table I. Mixtures Separated by Isocratic Reverse-Phase HPLC/NMR
mol. separation
wt
elution amt time, injected (rg/50 CLL) min
Mixture 1. Amino Acids histidine glycyl-alanine methionine
174
400
2.9
146 149
300 400
3.6 6.3
Mixture 2. Vitamins vitamin C (ascorbic acid) vitamin B1 (thiamine) niacin vitamin B, (pyridoxine)
176 337
300 300
123
300
169
300
3.2 4.4 4.8 7.0
Mixture 3. Nucleosides uridine cytidine adenosine
244 243 267
500 500 500
4.1 4.7
5.6
+
not equal to (2n l ) 4 u retain Mxyand are detected. A vector representation of the magnetization in the yz plane is presented in Figure 1. EXPERIMENTAL SECTION The experimental work is divided into two sections: characterization and optimization of the 1-1 hard pulse sequence for flow LC/NMR conditions; the reverse-phase separation of biomolecules with optimized 1-1 hard pulse parameters. The LC/NMR interface and system configuration described in detail elsewhere (5) employs a 300-MHz spectrometer and unmodified commercial 5-mm 'H probe. 1-1 Hard Pulse Characterization. Various amounts of ethylbenzene in acetonitrile were allowed to flow through a 50-kL cell at a rate of 1.0 mL/min; 'H NMR spectra with 4K data collected over a spectral width of 12000 Hz were acquired ( T , = 0.507 s). The 1-1 HP sequence was optimized for the suppression of the acetonitrile resonance as the carrier frequency and delay were varied. In all cases a Ox value of 6.5 ks (45") was used. LC/NMR Separations. The 50-kL capillary glass observe cell was coupled to a 300 A wide pore C-18 bonded phase column by 6 cm of 0.25 mm i.d. Teflon tubing (a transfer volume of 3 kL). The solvent system used was a low-grade D20-protonated acetonitrile mixture. For the particular compounds separated, the maximum percentage of acetonitrile required to permit elution of all compounds with a k'of 3 or less was 3%. All separations were performed isocratically at flow rates of 1.0 mL/min. The 1-1 HP sequence was used for solvent suppression with 0, equal to 45" and the carrier positioned for optimum suppression of either single or dual solvent resonances. For all experiments, four coadded scans of 4K data collected over a bandwidth of 12000 to 12500 Hz (delays between scans were 0.3 s), provided a chromatographic time resolution per file of about 3.5 s. Table I lists pertinent information for the three separations. The spectra were base-line corrected,subjected to a 0.5-Hz line broadening, and transformed. Because of the often severe phase distortions produced by the 1-1 HP sequence, linear phase correction over the entire spectral region was difficult and magnitude calculations were performed to produce positive resonances. Selected windows of the frequency domain spectrum were integrated to provide intensity information for the reconstructions. DISCUSSION Application of 1-1 H a r d P u l s e t o LC/NMR. The reported suppression efficiencies for the 1-1 hard pulse have
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ANALYTICAL CHEMISTRY, VOL.
57, NO. 7, JUNE 1985
Table 11. Comparison S I N Values for Ethylbenzene in Acetonitrile Using 1-1 Hard Pulse Solvent Suppression
experiment
solvent
flow 1-pulse flow 1-pulse static 1-1 HP flow 1-1 HP
freon acetonitrile
flow 1-pulse flow 1-pulse static 1-1 HP flow 1-1 HP
freon
acetonitrile
ethylbenzene concn, Wid50 WL
aromatic
ethylbenzene S I N quartet
triplet
solvent S j N
100
45
31
70
100 100
46
135
100
677
46
15 22
136
307
20 20 20
16
11
21
9
5
20
8
25 33
>2000
a)
>2000 249 154
a)
I
. ACN
SF 12
8
0 PPm
4
b)
,
1p 0
3A;7;[Au,AcN
-5AQ-
~
0
12
2000-
8
4
oppm
Figure 3. Suggested placement of the carrier to permit single and dual 1- 1 hard pulse solvent suppression with minimal analyte interferences: (a) single suppression; (b) dual solvent suppression. >
z!
(0
w z
f
1000-
-1 W E
0,
7
1000
ranged from 100 to 1000, sufficient for the requirements of the LC/NMR experiment (8). Table I1 presents the results for a series of experiments in which the SIN for ethylbenzene a t decreased solute concentrations is contrasted for various solvents and pulse sequences. The S I N for ethylbenzene in the nonprotonated solvent, Freon-113, is indicative of the results that should be obtained with proper application of the suppression technique to protonated solvent resonances. Even at the high concentration of 1 mg of ethylbenzene in acetonitrile, in the absence of solvent suppression, the limited dynamic range of the ADC decreases solute S I N . As seen in Table 11,at the lower solute levels in the cell which correspond to chromatographic analyte levels, no ethylbenzene signal is observed. Application of the 1-1 hard pulse sequence is satisfactory for suppression of the solvent signal as analyte S I N values improve by a comparable amount for both the static and flow cases to approach the S I N values reported for the Freon-113 spectra.
Despite its efficiency, a major shortcoming of the Redfield pulse sequence is the difficulty of optimizing suppression parameters. In contrast, the simplicity of the 1-1 hard pulse sequence recommends it for LC/NMR; the hard nature of the pulse (high rf power and short pulse width), however, requires accuracy in the determination of delay and pulse lengths. Ideally, the width of the suppression window is narrow to avoid the unwanted suppression of analyte resonances but large enough to allow tolerances for variations in sequence delay and pulse times. A study of the suppression window for the 1-1 hard pulse and its effect on the LC/NMR experiment was conducted. As a 100 pg/50 pL sample of ethylbenzene in acetonitrile was allowed to flow at 1.0 mL/min, the delay time that permitted precession of the solvent resonance in the 1-1 H P sequence was incremented (the carrier frequency, set 570 Hz from the solvent resonance, was held constant). Figure 2a presents the acetonitrile SIN as a function of delay. The curve exhibits a fairly flat minimum with S I N between 100 to 200 over a 15-ps range (delays between 878 and 893 ys) that corresponds to a 10 Hz spectral region. Partial suppression occurs over a 125-Hz region (800 to 1000 p s delays). As Figure 2b demonstrates for the ethylbenzene triplet, complete solvent suppression is not required to maximize solute peak intensity. A somewhat larger region than that determined in Figure 2a for optimum suppression (a spectral region of 25 Hz corresponding to delays of 870-910 p s ) produces an unattenuated analyte signal. This broader spectral region encompasses a solvent S I N of about 600 on either side of the minimum and indicates that for the 50-pL flow cell, solvent suppression need not exceed an order of magnitude to be effective. The nulled spectral regions produced by the binomial suppression techniques have a cyclic nature which must be considered in the design of the experiment. An advantage
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
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Table 111. Chemical Shift for 100% H 2 0 to 100% Acetonitrile LC Gradient chemical shift, PPm % HzO
% CHaCN
HzO
CH&N
99.9 95 85 75 65 55 45 35 25 15 5 0.1
0.1 5 15 25 35 45 55 65 75 85 95 99.9
4.03 4.03 4.02 3.97 3.94 3.88 3.82 3.66 3.36 2.80 2.40 2.32
1.38 1.42 1.48 1.60 1.67 1.77 1.84 1.93 1.96 2.02 2.09 2.11
solvent HzO acetonitrile
gradient 100% 25 % 100% 100% 25% 100%
75% 0% 0% 75% 0% 0%
A PPm
Hz
A
0.058 1.04 1.71 0.15 0.244 0.73
17 310 512 45 73 219
s
OPPm
I
to 100YOacetonitrile reverse-phase gradient. Chemical shift data are listed in Table 111.
!
a)
is the potential for suppression of multiple solvent resonances; a disadvantage is that suppression of analyte resonances may also occur. For the 1-1 hard pulse, nulls occur at spacings of (2n 1)Av offset by AV from the carrier. Figure 3 presents typical carrier and delay conditions for both the single and dual solvent system. If only a single resonance is to be suppressed, Figure 3a, the carrier should be set so that no other region of the spectrum is perturbed; unfortunately, the position of the HzO resonance in the center of the 'H spectrum forces the carrier to be placed so that an increased spectral window is usually required. Dual solvent suppression is demonstrated in Figure 3b with the carrier equally spaced, Au, between the two resonances. However, this approach risks the suppression of analyte resonances as, for example, with the H20-acetonitrile solvent system for which a null falls close to the aromatic region of the 'H NMR spectrum. The general discussion of solvent suppression has been limited to the fixed solvent concentrations used in isocratic separations. Reverse-phase LC/NMR is made considerably more versatile through the use of solvent gradients. As with most detection methods, however, the NMB spectrum changes markedly as solvent concentration is varied, especially for polar reverse phase solvents. Figure 4 and Table I11 illustrate the changes in solvent resonances for the 100% acetonitrile gradient; the shifts in HzO and acetonitrile peaks are 512 and 219 Hz, respectively. The chemical shift change imposes another constraint upon the solvent suppression method; either the suppression window must be broad enough to encompass changes in chemical shifts or the course of changing solvent resonances must be tracked by variation of pulse sequence parameters. As Table I11 indicates, however, gradients that span the regions of high HzO or acetonitrile concentration do not produce significant changes in chemical shifts and are accommodated by the 25 Hz 1-1 hard pulse suppression window. For example, provided D 2 0 is used, a gradient from 100% to 75% acetonitrile produces a chemical shift of only 45 Hz permitting use of the 1-1 hard pulse sequence. If larger gradients are used, broader and flatter windows for suppression are required. In that case, higher-order binomial suppression methods such as the 1-3-3-1 sequence which permit windows several hundred hertz wide are more effective.
2
3
4
Flgure 4. 100 % HO ,
+
LBO
420
300 t, (s)
b)
C)
,
L-LL
-L I ' _ , _-
8
6
4
2
ppm
(a) LClNMR chromatographic reconstruction for amino acid separation. Integration windows were 2.2-4.5 and 6.5-8 ppm. (b) Spectrum of histidine with 1-1 hard pulse single solvent suppression of the residual H,O. (c) Spectrum of glycyl-alanine. (d) Spectrum of methionine. All spectra were obtained from the nonweighted summation of FIDs over each chromatographic peak. Figure 5.
Isocratic Reverse-Phase LC/NMR Separations. Several examples of reverse-phase separations of biomolecules are presented in Table I with the components from amino acid, vitamin, and nucleoside separations and their elution times listed. In all cases D20 (with residual H20 at about 1-2%) and protonated acetonitrile were used as mobile phase solvents. The 1-1 hard pulse sequence was employed for solvent
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
Table IV. Effectiveness of 1-1 Hard Pulse Solvent Suppression for Reverse-Phase Separations % DzO
separation
(1-270 residual H 2 0 )
% acetonitrile
amino acid
99.9
0.1
ref Figure 5
vitamin
99
1
nucleoside
97
3
Figures 6, 7 Figures 8. 9
1
2
,
I
IBC
4 za
300
I , (SI
Reconstruction of the four-component vitamin separation with 300 pg injected per component. Integration over both the 2.5-4 and 6-8 ppm windows was used. Figure 8.
suppression with placement of the carrier to permit the particular single and multiple suppression procedures illustrated in Figure 3. Table IV presents comparative solvent S I N values from each separation to demonstrate the effectiveness of the suppression.
pulse sequence
SIN (HzO)
no suppression single 1-1 H P no suppression dual 1-1 H P no suppression dual 1-1 H P
357 11 271 38 158 44
SIN (acetonitrile) 60 716 35 1606 95
The separation of histidine, the dipeptide glycyl-alanine, and methionine in 99.5% D20-0.5% acetonitrile is displayed in the chromatographic reconstruction, Figure 5a. All three components are base line separated; a rough estimate of number of theoretical plates is 6000 for the second peak. However, this value is substantially reduced from the 18000 plates calculated from the UV trace of the separation because of the larger observe volume used (50 pL) and column overload. LC/NMR separation efficiencies similar to UV and NMR detector efficiencies only have been demonstrated for a 20-pL observe cell and maximum injections limits of about 200 pg (5). Figure 5b-d presents the 'H NMR spectra of several amino acids with residual H 2 0 suppressed. Figure 6 presents the reconstruction of the four-component separation of vitamin C, vitamin B6, niacin, and vitamin B1 in 99% D20-1 % acetonitrile with 1-1 HP suppression of both solvent resonances. Improved chromatographic efficiency was observed with 200-300 pg per component injected; peaks 2 and 3 which were not resolved at injections of 500 and 800 pg per component are separated for the lower amount injected. Figure 7 presents NMR spectra of the four vitamins obtained from a single file (Figure 7a-d) and coadded across the entire chromatographic peak (Figure 7e-h). S I N is substantially improved, especially at larger h', by coaddition of files. The danger of this approach is demonstrated in Figure 7g, however, as the two resonances at ca. 3.0 ppm, although imperceptible in the individual files, are actually a contaminant from the vitamin B,, Figure 7f. Finally, the intensities of the two e)
d)
I
1 1
h)
Figure 7. LCINMR vitamin separation spectra. Spectra a-d are extracted from a single maximum intensity file of four coadded scans with: spectrum a, vitamin C; spectrum b, vitamin B,; spectrum c, niacin; and spectrum d, vitamin Be. Spectra e-h, which correspond to spectra a-d, result from the nonweighted coaddition of files on the eluting peak.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE
1985
1469
< I
IO
6
PPM
Flgure 9, Stacked plot of nucleoside separation extracted from the
aromatic region, 6-8 ppm. Time resolution per file of four coadded scans is 3.5 s.
I ' " ' " ' ,
9
~
8
1
7
"
'
"
"
'
6
'
I " ' I
1
5
4 wm
Flgure 8. (a) Reconstruction of LC/NMR separation of nucleosides from integration of 6-8 ppm window. (b-d) Indivdual spectra for the nucleoside separation coadded over eluting peak with (a) uridine, (b)
cytidine, and (c) adenosine. doublets in Figure 7c,f are significantly different despite an equivalent number of protons and similar TI values. The explanation, a partial suppression of the resonance at ca. 8.0 ppm by the 1-1 HP sequence, demonstrates the danger in placement of the carrier between two closely spaced solvent resonances; despite greater suppression efficiency, the potential for suppression of analyte peaks is substantially increased. A reconstruction for the separation of the nucleosides uridine, cytidine, and adenosine is presented in Figure 8a with the 1-1 hard pulse again applied effectively for dual suppression of a 97% D20-3% acetonitrile solvent system. The calculated efficiency for the base line resolved second peak, about 4000, was substantially less than the value of 15000 calculated from the UV trace, again because of a larger cell volume and slight column overload. For all three reconstructions, several peak profiles exhibited significant tailing which is not explained by column overload or an increased observe volume. For example, both adenosine in the nucleoside separation and methionine in the amino acid separation yield calculated efficiencies of less than 1000. One possible explanation is solute interaction with active sites on the untreated glass in the observation region.
The NMR spectra of the nucleosides are presented in Figure 8b-d with differences in purine and pyrimidine ring structure providing sufficient information for identification. The aromatic region that contains the ring information is shown in a stacked plot of the separation in Figure 9. The plot illustrates the potential of LC/NMR as a tool for a mixture analysis with the separation power of HPLC coupled with the unparalleled diagnostic capabilities of NMR. These data also demonstrate successful application of LC/NMR to significantly more complex biomolecules that contain relatively few protons, while operating within the constraints of an analytical-scale separation. Registry No. Histidine, 71-00-1; glycyl-alanine, 3695-73-6; methionine, 63-68-3; vitamin C, 50-81-7; vitamin B1, 59-43-8; niacin, 59-67-6;vitamin B,, 8059-24-3; uridine, 58-96-8; cytidine, 65-46-3; adenosine, 58-61-7; ethylbenzene, 100-41-4;acetonitrile, 75-05-8; water, 7732-18-5.
LITERATURE CITED (1) Bayer, E.; Albert, K.; Nieder, M.;Grom, E.; Wolff, G.; Rindlisbacher, M. Anal. Chem. 1982, 54, 1747-1750. (2) Dorn, H. C. Anal. Chem. 1984, 56, 747A-758A. (3) Buddrus, J.; Herzog, H.; Cooper, J. W. J. Magn. Reson. 1981, 42, 453-459. (4) Buddrus, J.; Herzog, H. Anal. Chem. 1983, 55, 1611-1614. (5) Laude, D. A.; Wilkins, C. L. Anal. Chem. 1984, 56, 2471-2475. (6) Schaefer, J. J. Magn. Reson. 1972, 6 , 670-671. (7) Hore, P. J. J . Magn. Reson. 1983, 55, 283-300. (8) Clore, G. M.; Kimber, B. J ; Gronenborn, A. M.J . Magn. Reson. 1983, 54, 170-173. (9) Sklengr, V.; Starcuk, 2 . J . Magn. Reson. 1982, 50, 495-501. (10) Turner, D. L. J. Magn. Reson. 1983, 5 4 , 146-148. (11) Patt, S.L.; Sykes, B. D. J. Chem. Phys. 1972, 56, 3182-3184. (12) Redfield, A. G.; Gupta, R. K. J . Chem. Phys. 1971, 5 4 , 1418-1419.
RECEIVED for review January 16,1985. Accepted March 12, 1985. Support of the National Science Foundation through Grant CHE-82-08073and a Department Research Instrument Grant, CHE-82-03497, is gratefully acknowledged. Partial support was also provided by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the National Institutes of Health, through Grant RR01857-01.
