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ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
Plasma Chromatography with Ammonium Reactant Ions S. H. Kim and F. W. Karasek" Guelph- Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo, Ontario NZL 3G1, Canada
Souji Rokushika Department of Chemistry, Faculty of Science, Kyoto University, Kyoto, 606, Japan
By addition of NH, vapors to the N2 carrier gas in plasma chromatography (PC), a single positive reactant ion mobility peak is formed containing ions of the type (H,0),(N2)mNH,+. These reactant ions give product ions only with compounds having a higher proton affinity. This permits the introduction of dilute solutions directly into the plasma chromatograph, when the solvent has a lower proton affinity (primarily nonpolar hydrocarbons) and the solute has a higher proton affinity, to produce mobility spectra containing prominent MH' ions for the solute. The solution mobility spectra obtained for a series of amines correspond to those produced for the pure solute vapors alone using the reactant ions of N2 carrier gas. Use of the ammonium reactant ions makes feasible the direct introduction of liquid chromatographic effluent for detection and identification of high proton affinity solute compounds.
When using nitrogen carrier gas in plasma chromatography, three reactant ion peaks are observed in the positive ion mobility spectrum. These ion mobility peaks have been identified by use of mass spectrometry as (H20),NH4+, (H20),NO+ ( n = 0, 1, 2), and (H20),H+ ions (n= 2, 3, 4) ( 1 , 2 ) . Each of these mobility peaks is a mixture of ions undergoing rapid equilibrium reactions having a mobility which corresponds to the average velocity of the individual clusters. Among these ions the dominant ones are those of the type (H20),H+, whose low proton affinity results in protonation of most organic compounds through proton transfer reactions. When an appropriate amount of NH3 vapors is added to the nitrogen carrier gas, only a single reactant ion mobility peak containing ions of the type (H20),(N2),NH4+ (n,m = 0, 1, 2, 3) is observed in the positive ion mobility spectrum. Some workers in the field feel that the value of ( m )for N2 is zero during transit of the drift spectrometer and that clustering occurs during the adiabatic expansion of the gas into the mass spectrometer through the interfacing orifice ( 3 ) . When ammonia is used as a n ionizing gas in chemical ionization mass spectrometry (CIMS), it is known to protonate only those compounds having a higher proton affinity than NH3, and quite specifically those containing the basic amine group (4-9). Since a number of studies have established a relationship between the type of ions formed in plasma chromatography with those formed in CIMS, this work was undertaken to explore the type of P C mobility spectra obtained for amines in solution with low proton affinity solvents using reactant ions produced by NH3. It was found that the amine solutes could be selectively detected, giving mobility spectra with simple MH+ ions with little effect from the solvent.
EXPERIMENTAL The plasma chromatograph used in this study was the Beta-VI Model. Details of instrumentation have been reported previously 0003-2700/78/0350-0152$01.OO/O
(20). To introduce ammonia a small flask, removable via a ground glass joint, was installed in the carrier gas line immediately before the inlet to the reaction section. The carrier gas flow mixes with NH3 vapors by passing over the headspace of 1 mL of 0.003 to 0.03% ammonium hydroxide solution in the flask. Unless otherwise indicated in figure captions, the operation conditions of the PC are: carrier gas flow rate, 65 mL/min, drift gas flow rate, 350 mL/min; electric field gradient 121 V/cm; drift tube temperature, 207 f 3 "C; sample inlet temperature, 205 "C; ion injection pulse 0.2 ms; scan gating pulse 0.2 ms; 20-ms scan was recorded in 2 min; typical pressure was 729-739 Torr. Dry nitrogen gas (Linde high purity 99.99670)was used both for carrier gas and drift gas after passing through traps of Linde molecular sieve 13X to remove traces of impurities. Three different types of samples were introduced syringe injection of headspace vapors from sample vials, syringe injection of dilute solutions, and insertion of a Pt wire on which sample solutions had been deposited and the solvent allowed to evaporate prior to insertion. Reagents. Ethylamine was obtained from Dow Chemical, n-amylamine (pentylamine) and benzene from MCB Co., Norwood, Ohio. Other amines and pyridine were from Poly Science Analytical Standard Kit of 31A, 32A, and 81A from Poly Science Co. Evanston Ill. The other chemicals used were from J. T. Baker Chemical Co., reagent grade.
RESULTS AND DISCUSSION The sequence of reaction steps producing the positive reactant ions of (H,O),NO+ and (H20),H+when using dry nitrogen reactant gas in plasma chromatography have been reported previously by several authors (11, 12) based on the reaction steps originally reported by Good (13). The type of reactant ions present greatly affects the type of product ions formed. An increased abundance of (H20),NO+ gives more of the M(N0)' ions for organic compounds and increases the sensitivity of detection (12). The reactant ion peak with the greatest mobility of the three observed has recently been identified using PC/MS instrumentation as (H20),NH4+with n = 0, 1, 2 depending upon the amounts of NH3 and H 2 0 in the nitrogen gas and its temperature. The formation of (H20),NH4' from reaction between NH3 and (H20),Hf is dependent on the relative concentration of NH3 and H 2 0 as reported by Kebarle (14, 25). Data obtained with a coupled P C / M S instrument show that ions of the type (HzO),(Nz),NH4' are observed a t the mass spectrometer detector. The order of increasing proton affinity (PA) determined by Munson ( 1 6 ) ,and Kebarle (17) by observing proton exchange reactions for polar molecules is given as H20 < CH30H < Benzene < CH3CHO < CH3COOH3 < C2HSOH < CzH5OC2H5< NHS < CH3NH2 < (CHJ2NH < (CH3I3N. This order suggests that all the amines can be selectively detected when the amine is introduced into plasma chromatograph as a solution in a solvent having a lower proton affinity than "3. To produce (H20),(N2),NH4+ reactant ions, a 1-mL flask containing N H 4 0 H solution is installed in the carrier gas line. The estimation of the N H 4 0 H concentration needed t o produce these reactant ions is based on concentration limits G 1977 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
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Figure 1. Comparison of positive reactant ion mobility spectra. (a) Reactant ions produced by mixing of ammonia gas with N, carrier gas. (b) Conventional reactant ions produced by dry nitrogen carrier gas
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DRIFT TiME Figure 2. Positive ion mobility spectra produced by the injection of 0.5 pL each of nonreactive compounds. Arrows indicate the positions of product ion peaks when N, gas reactant ions were employed. (a)Blank, (b) pentane, (c) hexane, (d) cyclohexane
Table I. Ionic Species of Cluster Ions Identified by PC/MS in Reactant Ion Peak at Mobility of K O= 2.93 Relative m/e Ion intensity
of NH3 to HzO reported by Kebarle for proton transfer reaction (14). Using 0.03% solution gives a ratio of [NH,]: [HzO]:[Nz]of 1:847:3 X lo4 mol when the nitrogen carrier gas passes over the N H 4 0 H solution. This amount of NH3 is sufficient to obtain a single reactant ion peak whose mobility coincides with that of the NH4+ reactant ion ( K O= 3.08 cm2/V.s) produced by pure nitrogen reactant gas at 200 "C as shown in Figure 1. The amount of NH3 from solutions ranging between 0.003% to 0.03% NHIOH only changes the relative amounts of the individual cluster ions, but does not affect the type of product ion formed. The ions associated with this mobility peak have been mass identified using a directly coupled plasma chromatograph/ mass spectrometer system under slightly different conditions (18) [PHEMTO-CHEM MMS-160 Ion Mobility Spectrometer-Mass Spectrometer, P C P Inc., 2155 Indian Rd. West, Palm Beach, Fla. 334091. The mass spectral data for reactant ions using Nz carrier gas show 15 ions in the m / e range of 18 to 111 a t 155 "C. Among these ions those of m / e 18, 36,46, 64,74, and 102 have the same mobility (KO= 2.93) at 155 "C. The peak a t a mobility of 2.93 is an equilibrium mixture of many species of cluster ions and differs slightly from that of the essentially single NH4+observed in the Nz carrier gas at 200 "C. The relative intensities and identities of these ions are given in Table I. These relative intensities are those observed for these experimental conditions and can be expected to vary depending upon moisture content of the N2 gas, temperature, and size of the interfacing orifice. All of these ionic species react with the amine compounds to produce predominately MH+ ions. A t this high temperature some M(N2)H+ions were observed a t a relative abundance of 5%. while no M(H20)H' ions were observed. The lack of response of the reactant ions to solvents of low proton affinity is shown in Figure 2, where 0.5-pL liquid samples of n-pentane, n-hexane, and cyclohexane were injected. The slight indication of product ions appearing can
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Figure 3. Comparison of ion mobility spectre of pyridine dissolved in different solvents and pure compound. (a) Blank, (b) 0.1 pL pyridine solution in cyclohexane in the concentration of lO-*g/pL, (c) 0.1 p L pyridine solution in benzene in the same concentration as (b), (d) 0.01 pL of head space vapor of pyridine
he attributed t o trace impurities in the solvents. However, injection of such large quantities of the s,olventsdoes decrease the total intensity of the reactant ion peak by about 1&15%. The probable reason for this is the decrease of reactant ion density inside the tube by dilution w i t h the solvent vapors. Figure 3 shows the selective detection of pyridine obtained when solutions containing lo-' g of pyridine in 0.1 pL cyclohexane and benzene are introduced. The formation of a single product ion peak is observed at a mobility KOof 2.20 cm'/V.s. This peak has the same mobility as that formed when pure pyridine vapors are introduced into the PC containing either the NHI+ reactant ions or those formed from nitrogen carrier gas alone. To be useful as a qualitative detector for either GC or LC, the PC instrument must have a reasonakily fast response time. Although the design of the PC tube used in these experiments is far from optimum to achieve fast response times, conditions can be chosen to demonstrate the effect of this parameter. Figure 4 shows the data for five sequential injections at the time scale shown of 0.1-pL solutions of lo-" g of pyridine in cyclohexane. By tuning the detector response to the drift time of the pyridine product ion, and using a temperature of 207 "C with a very high 308 mL/min carrier gas flow rate, a reasonably reproducible response peak with a response time
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
I II
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Figure 4. Product ion mobility spectrum by repeated injection of 0.1 ng of pyridine in 0.1 WLcyclohexane. Carrier as flow rate, 308 mLlmin. Drift gas flow rate, 560 mLlmin
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Figure 6. Comparison of ion mobility spectrum of n-amylamine using both N, carrier gas and NH3 mixed with N2carrier gas. (a, b) Ammonium reactant ion spectra. (c, d) Conventional reactant ion spectra. (b, d) 0.02 pL of head space vapor of n-amylamine was injected bMlhES
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Flgure 5. Ion mobility spectra of ethylamine. (a) Ammonium reactant ion spectrum. (b) Ethylamine product ion using NH,+ reactant ion. (c) Reactant ions using N, carrier gas. (d) Ethyl amine product ion spectrum using N2 carrier gas with 0.02 yL of head space vapor taken from ethylamine reagent bottle
of less than 30 s is observed in Figure 4. The results of blank injection under these conditions are shown in Figure 2. When the three reactant ion mobility peaks formed from a pure nitrogen carrier gas are converted t o the single, high mobility ammonium peak, low molecular weight product ions of high mobility can be observed more easily. The region of the mobility spectrum normally occupied by the (H20),NO+, and (HzO),H+ ions is free for observing product ions, as illustrated in Figure 5 for the ethylamine product ions. With dry nitrogen reactant ions, the ethylamine product ion peak overlaps with the (H*O),H+ reactant ion peak as shown. However, with ammonium reactant ions, a clear single peak was obtained as seen in trace a and b in Figure 5. Most primary amines gave some minor product ion peaks in addition to the prominent MH+ ones when using a pure nitrogen carrier gas. These minor peaks were not observed when ammonium reactant ions are used. Figure 6 shows such a result for n-amylamine. The composite results obtained for the alkylamines with the ammonium reactant ion are shown in Figure 7. The reduced mobilities of these amine product ions coincided exactly with those formed from nitrogen gas reactant ions. The product ions for these peaks were assumed to be MH' ions from the results of PC/MS analysis of methylamine a t temperatures greater than 150 "C and from the results of APIMS using ammonium ions (19). Table I1 shows the reduced mobilities of alkylamines a t 200 "C. A t this temperature the ions associated with the mobility peaks
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Figure 7. Normalized mobility spectra of primary alkyl amines from nitrogen carrier gas mixed with ammonia
Table 11. Reduced Mobilities of the Reactant Ion,the Product Ions, and Ionic Mass of the n-Alkyl Amines Compound
Ionic mass M H
NH,+ reactant ion n-Methyl amine n-Ethyl amine n-Propyl amine n-Amyl amine
18 32 45 57 69
Reduced mobility, K O (cm2/V.s) a t 200 " C MH 3.08 2.63 2.46 2.16 1.83
between 95 and 100% are the MH+ ions. This technique using NH4+reactant ions suggests the direct application of the PC as a sensitive and selective detector for primary, secondary, and tertiary amines eluted from a liquid chromatographic column. To be a practical LC detector, the PC tube must be redesigned to minimize surface areas and adsorption.
LITERATURE CITED (1) D. 1. Carroi, I . Dzidic, R. N. Stillwell, and E. C. Horning, Anal. Chem., 47, 1956 (1975). (2) F. W. Karasek, S. H. Kim, and H. H. Hill. Jr., Anal. Chem.. 48, 1133 (1976). (3) David J. Carroll, private communication, August 1977. (4) D. F. Hunt, C. N. McEwen, and R. A. Upham, Tetrahedron Lett., 47, 4539 (1971).
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(15) J. D. Payzant, A . J. Cunningham, and P. Kebarle, Can. J . Chem., 3242 (19731. (16) M S. '8. Munson, J . Am. Chem. SOC.,87, 2332 (1965). (17) P. Kebarle, R. Yamdagni, K. Hiraoka, and T. E . McMahom, Int. J . Mass Spectrom. Ion Phys., 19, 71 (1977). (18) S. H. Kim and F. W.Karasek, "The Study on the Mobilities of Positive Reactant Ions using N, Carrier Gas", in preparation (1977). (19) I. Dzidic, D. I. Carrol, R. N. Stillwell, and E. C. Horning, Anal. Chem., 48, 1763 (1976)
(5) M. S. Wilson, I. Dzidic, and J. McClosky, Siochim. Siophys. Acta, 240, 623 (1971). (6) J. I. Brauman. J. M. Riveros, and L. K. Blair, J . Am. Chem. SOC.,93, 3914 (1971). (7) I. Dzidic, J . Am. Chem. SOC., 94, 8333 (1972). (8) R. Yamadagni and P. Kebarle, J . Am. Chem. SOC., 95, 3504 (1975). (9) I. Dzidic and J. A. McClosky, Org. Mass Spectrom., 6, 939 (1972). (10) F. W.Karasek and D. M. Kane, Anal. Chem., 45, 576 (1973). (11) E. C. Horning, M. G. Horning, D. I. Carrol, I . Dzidic, and R. N. Stillwell, Anal. Chem., 45, 936 (1973). (12) F. W.Karasek and D. W. Denney, Anal. Chem., 46, 633 (1974). (13) A. Good, D. A. Durden, and P. Kebarle, J Chem. Phys., 5 2 , 212 (1970). (14) P. Kebarle, Adv. Chem. Ser. 72, 24-47 (1968).
RECEIVED for review June 21, 1977. Accepted October 25, 1977.
Test for Racemization in Model Peptide Synthesis by Direct Chromatographic Separation of Diastereomers of the Tetrapeptide Leucylalanylglycylvaline S. B. H. Kent,* A. R. Mitchell,' G. Barany, and R. B. Merrifield The Rockefeller University, New York, New York 10021
model system that provides a stringent test for racemization in peptide synthesis and have used this to investigate the occurrence of racemization in stepwise solid phase peptide synthesis. The peptide Leu-Ala-Gly-Val has been used as a model for synthetic methods (11-18). Standard analytical conditions on the automatic amino acid analyzer (19) exist for the separation of most of the known peptide by-products (20). As a result of extensive work with the chromatographic separation of diastereomeric dipeptides according to Manning and Moore (21),it seemed likely to us that it would be possible to separate the diastereomers of the tetrapeptide Leu-Ala-Gly-Val. A preliminary investigation with L-Leu-D-Ala-Gly-L-Val showed that it was possible to achieve a large separation of diastereomeric tetrapeptides under standard chromatographic conditions. We therefore prepared diastereomeric tetrapeptide standards and studied their separation as a function of p H to determine the optimal separation. The color yields of the individual peptides were determined and the sensitivity and precision of the determination of D-amino acid-containing peptides were evaluated. Use of the method was illustrated by analyses of stepwise solid phase syntheses of the tetrapeptide.
The peptide Leu-Ala-Gly-Val has been developed as a test for racemization in model peptide synthesis. The single D-amino acid diastereomers, L-Leu-D-Ala-Gly-L-Val and D-Leu-L-AlaGly-L-Val, were separated from one another and from the all L-amino acid tetrapeptide on the standard amino acid analyzer 0.9 X 58 cm column of sulfonated polystyrene resin with 0.2 N sodium citrate buffers. Chromatography at pH 3.49 was chosen as the standard condition for separating the diastereomers. The color yields of the diastereomers were similar to that of valine. The determination of the D-amino acidcontaining diastereomers was accurate above 0.1 % for a standard load of 4 pmol of the tetrapeptide. The limit of detection was less than 0.01% for a 12-pmol load. The analysis was applied to the crude products of stepwise solid phase syntheses; no D-amino acid-containing diastereomers were detected (
