1764
Anal. Chem. 1962, 5 4 , 1764-1772
(19) Blanc, G.; Gabrlelli, C.; Keddam, M. Nectrochim. Acta 1975, 2 0 , 687-689. (20) Karol, M. H.; Dlxon, C.; Brady, M.; Alarie, Y. Toxico/,~ ~ pharma~ 1 COl. 1980, 53, 260-270.
,
RECEIVEDfor review December 16, 1981. Accepted May 6,
1982. We are pleased to acknowledge the U.S. Public Health Service through Grant GM 28112, Biomedical Research Support Grant 2507-RR07084(16) through National Institutes of Health, and the support of the National Research Council of Canada.
Retention Mechanism and the Role of the Mobile Phase in Normal-Phase Separation on Amino-Bonded-Phase Columns L. R. Snyder*
’
Technicon Instruments Corporation, Tarrytown, New York 1059 1
T. C. Schunk2 Chemistty Department, Pace University, Pleasantvilie, New York 10570
Experlmental data on retention In varlous amlno-phase systems can be explained in terms of a previous model with adequate precislon. The mechanlsm of retentlon in these llquld chromatographic systems Involves monolayer coverage of the adsorbent surface by moblie phase molecules and dlsplacement of solvent molecules by adsorblng solute molecules. The solvent strength eo of any moblie phase mixture can now be predlcted for these amlno-phase systems. A practical summary of the relatlve advantages of amino-phase vs. slllca columns Is glven.
Workers using high-performance liquid chromatography (HPLC) with nonaqueous mobile phases (“normal-phase” separation) can choose among silica, alumina, or various polar-bonded-phase columns (1). There is growing interest in the use of amino-phase columns in this respect (e.g., ref 2-8 and prior references) but limited information on how such separations are best optimized. The mechanism of sample retention on amino-phase columns is still controversial, and few quantitative rules exist for predicting how change in mobile phase composition will affect sample retention. For silica and alumina, on the other hand, it is known ( I ) that systematic change in the mobile phase can have a dramatic and predictable effect on separation. Three reasonably comprehensiveand detailed studies have been reported concerningretention on amino-phase columns, i.e., using porous silica packings bonded with -Si(CH3),-RNH, groups (R equal C3 or CJ. Hammers et al. (6) conclude that amino-phase packings are similar to silica that has been partially silanized. Therefore, retention of most samples on amino-phase packings is less than on silica, but separation sequence or selectivity should be generally similar (with some exceptions). A more recent paper by Hennion et al. (7)reports major differences in relative solvent strength for different compounds as mobile phase for amino-phase packings vs. silica. This suggests that retention in these two LSC systems may not be so similar. In addition these two studies (6, 7) offer different interpretations of retention mechanism and the Present address: 2281 William Ct., Yorktown Hts., NY 10598. Present address: Chemistry Department, University of Arizona, Tucson,AZ. 0003-2700/82/0354-1764$01.25/0
role of the mobile phase in determining separations on amino-phase columns. Finally, Hurtubise and co-workers (8)have examined the effect of mobile phase composition in the amino-phase separation of various phenols. However, hydrogen bonding between mobile phase and sample molecules complicates the analysis of their system in terms of retention mechanism. In this paper we present new experimental data that bear on the questions raised above. We will propose a comprehensive theory of retention and mobile phase effects for the amino-phase packings, and we will apply this theory to a practical discussion of the relative value of these packings vs. silica in liquid-solid chromatographic (LSC) separation. Finally, the amino-phase systems offer another opportunity to examine the relative importance of adsorption vs. solution interactions in LSC. The displacement model (9-16) of retention in LSC focuses on adsorption interactions, while the Scott-Kucera “sorption”model (9,17-20)emphasizes solution interactions.
THEORY Table I summarizes some of the assumptions and conclusions of the displacement and sorption theories; for a detailed comparison, see ref 9. In terms of mobile phase effects, several practical equations can be derived from the displacement model. In this study we will be concerned mainly with solvent strength as a function of mobile phase composition, and the more important relationships are (10) log (h~/hJ= a’A,(~i- ~ =
EA
2 )
+ 1 - N B) + log (NBIOa‘nb(‘B-fA) a’nb
(1)
(2)
Equation 1 states that the ratio of capacity factors k’ for mobile phase 2 (k,) and mobile phase 1 (kl) are related to an adsorbent activity factor a’ (arbitrarily defined equal to unity for the present p Bondapak-NH, column), the relative size or area of the sample molecule (AB),and the solvent strength values (to) for mobile phase 1(e1) and 2 (eZ). Equation 2 relates the strength em of a binary mobile phase A/B to the eo values of the pure solvents A (CA) and B (eB), the mole fraction N B of B in the mobile phase, and the relative area of a molecule of B (nb). For a polar solvent B (the B solvent), the value of tB can vary with the value of NB, due to localization of B molecules when the surface coverage by B (0,) is less than 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1765
Table I. Mobile Phase Effects and Retention in LSC; Summary of Previous Theories for Silica and Amino-PhasePackings displacement model, sorption model Snyder-Soczewinskin Scott-Kucera b’ Hennion et al.c (amino phase) (silica) (silica, polar solvents) hypothesis generally one solvent molecule per amino group; three molecules for alcohol solvents displacement
1. coverage of adsorbent surface by solvent
monolayer
bilayer
2. displacement of aidsorbed solvent molecule b’y
displacement
no displacement (sorption)
not important and are ignored
very important and largely
very important
not important and can be ignored
silanol groups
adsorbed water molecules (H,O)
adsorbing solute molecule? 3. mobile phase interactions
among solvent and sample molecules important? 4. localization of solvent and sample molecules onto retention sites? 5 . nature of retention sites
(-&-OH)
somewhat important and must be taken into account in determining retention occurs; significance unclear
determine retention
amino groups (-NH,)
a See ref 9-10 for recent review (model also applies to alumina and other adsorbents, all solvents except alcohols). ref 9 and 17-20 for recent review. See ref 7 .
0.75 (10-14). This variation in tg (due to so-called restricted-access delocalization) is predictable in terms of the general displacement model, so that values of Q and tm are calculable for different LSC systernn. Similarly, polar solute molecules also localize. However, as the polarity of adjacent solvent molecules in the stationary phase increases, it appears that these solvent molecules can interact laterally with adsorption sites on which localized solute molecules reside. The net effect is a disturbance of solute localization, and a decrease in the interaction energy between solute and site as the solvent polarity increases. This leads (14) to an apparent increase in the quantity A,, by an amount AA, vs. the value calculated from the molecular size of the solute. The value of AA, can be correlated with the relative polarity (or retention) of the solute molecule, as predicted from the expected correlation of solute localization anld solute polarity. We call this phenomenon site-competition delocalization. Restricted-access delocalization (and variation in eB) is predicted to occur only for adsorbents with rigid surfaces and a high concentration of surface adsorption sit E!S. Site-competition delocalization should occur only for surfaces where the adsorption sites extend above the surfaces (e.g., silanols for silica), rather than being buried below the surface (e.g., alumina); see also ref 15. The displacement model leads to a general equation (first derived by Scott and Kucora for their sorption model (17-20)), for the prediction of retention as a function of mobile phase composition
l/k’ = A ’ + B’c,
(3)
A’and B’are constants for a given solute and B solvent, and cp refers to the concentration of B solvent in the mobile phase. Equation 3 can be shown t o be equivalent to eq 1and 2 under certain limiting conditiam and can therefore be considered as a restatement of eq 1 and 2. Hammers et al. (6) have argued that the displacement model describes their retention data for an amino-phase packing and various substituted aromatic hydrocarbon solutes. On this basis they propose that solutes adsorb on residual silanol groups which remain after the silica is bonded with aminoalkyl groups, and there is a competition among solvent, solute, and bonded amino groups for these retention sites. However, only limited data have been provided by these authors for the effect on retention of changes in mobile phase composition. Furthermore, there are some questions concerning their application of eq 1 to their data that we will examine here. Hennion et al. (7)have proposed a model for retention in amino-phase LSC that appears to be a hybrid of the dis-
See
placement and sorption models (see Table I). For solvents other than the lower alcohols, they propose that retention of solute or solvent molecules within the stationary phase occurs as
i
+
-NH2
-NH2-i
(4)
That is, a molecule (i) in solution attaches to an -NH2 group in the stationary phase. For the cases of alcohol B as solvent, they report that up to three molecules can be retained by one amino group:
3B
+
-NH2
*
-NHZ-B,
(44
Hennion et al. further postulate complexation of the solute S by polar solvent molecules P in solution
S+P+SP
(4b)
That is, their model assumes that solvent-solute interactions in the mobile phase are important in determining solute retention. For the case of all mobile phases except those containing the lower alcohols, these assumptions (eq 4-4b) lead to an equation for solute retention as a function of mobile phase composition
l/k’= A’+ E % , + C’cP2
(5)
Here, A’, B’, and C’ are constants for a given LSC system (same solute, same B solvent); these constants can be derived from equilibrium constanb corresponding to eq 4-4b. Note that the C’term of eq 5 reflects mobile phase interactions, while the A’and B’terms reflect a competition for retention sites by solute and solvent molecules (cf. eq 3). In the following discussion we will interpret experimental data for the amino-phase packings in terms of the displacement model. Some significant features of the amino-phase packings should be noted in this connection. First, we will show that the amino group constitutes the adsorption site in these packings, and this group is presumed to extend above the surface (like the silica silanols). Therefore, site-competition delocalization is predicted for the amino-phase packings, which should lead to positive values of AA, in eq 1. Second, unlike the rigid silica or alumina surfaces, the surface of an amino-phase packing is flexible, and the individual amino groups are free (to some extent) to rearrange their relative positions within the surface. Furthermore, the relative concentration of amino groups on the surface of typical packings is about 2 hmol/m2 ( 2 , 2 1 ) ,which is 4-fold smaller than the concentration of silanol groups on the surface of a chroma-
1706
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
Table 11. Experimental k' Values for Various Solutes and Tetrahydrofuran/Hexane Mobile Phases: Column, Ambient Temperature
11
12 13
14 15 16 17 18
19 20
Bondapak-NH,
log k' for indicated % THF (E') As 0.5 1.0 2.0 5.0 10.0 20.0 40.0 60.0 (0.000) (0.004) (0.008) (0,011) (0.021) (0.028) (0.045) (0.074) (0.096) exptl calcda
no.
10
fi
naphthalene acenaphthylene phenanthrene fluoranthene chrysene perylene naphthalene2,6-(COOCH,), naphthalene1&(NO,), fluorenone-2,4,7(NO,), 2-OCH3 2-SCH, 2-C0,CH3 2-acetate 2-COCH,
-0.32 -0.15 0.00
0.17 0.36 0.54
0.08
0.29 0.26
-0.55 -0.38 -0.27 -0.14 0.01 0.16 0.09
0.22
0.10
0.47
0.29
-0.5~ -0.52 -0.42 -0.33 -0.20 -0.54 -0.07 -U.43 -0.20 0.07
-0.49
2-S0,CH3
0.51
0.51 0.51
10
-0.25 -0.41 -0.22 -0.38 -0.16 -0.31 0.01 -0.17 0.05 -0.13 0.09 -0.11 0.10 -0.11 0.11 -0.08 0.88 0.97 1.29 log k ' for indicated vol % of THF 20 30 40 60
0.31
6.5 8.2 9.6 -0.72
12.1 13.2 19.1
8.1 8.6 10.2 10.7 12.3 12.8 lZ.7
-0.80
18.8
10.7
-0.15
22.9
13.9
10.8 10.7 12.9 16.3 13.5 13.7 13.7 12.9
9.3 9.8 10.4 10.4 10.1 8.7
11.1
-0.20 -0.16 -0.06 0.16 0.08 0.19 0.18 0.21 1.12
0.10
1-NO,
solute
-0.11 0.04
-0.46 -0.33 -0.19 -0.06
-0.25
1.02
1-CN
no.
-0.22 -0.08 0.09 0.27
0.90
0.27 0.57 0.57 0.51
2-SOCH, 2-CONH, 1-OH
-0.19 -0.05 0.12 0.31
exptl
10.0
9.4 9.3
calcd
octylphenol. -0.40 (ethox), (ethox), 22 0.50 0.11 -0.05 -0.27 16.7 18.9 (ethox), 0.96 23 0.46 0.09 -0.13 23.8 21.2 (ethox), 24 1.17 0.60 0.20 -.0.03 26.1 23.5 (ethox), 25 0.75 0.32 0.07 23.4 25.8 (ethox), 26 1.03 0.53 0.26 -0.49 29.9 30.4 27 0.72 0.41 -0.43 32.1 35.0 (ethox), 1 28 0.53 -0.35 39.7 39.6 (ethox1 1 3 29 42.6 43.2 0.b7 -0.27 (ethox),, 30 -0.19 (ethox),7 a Calculated from ref 15, according to molecular dimensions of molecule; no corrections for localization effects. Compounds 10-20 refer to substituted naphthalenes. 'Compounds 21-30 are fractions isolated from Triton X-100, corresponding to nonyl phenols substituted by the indicated number of ethylene oxide groups. 21
tographic silica (8 pmol/m2 (22-24)). Both of these factors work against the occurrence of restricted-access delocalization, which means that values of eg in eq 2 should be constant, and not vary with e B as hthe case of polar solvents B and alumha or silica as adsorbent. Finally, retention on an amino-phase column is much weaker than on silica or alumina. This means that the energy of interaction of solute or solvent molecules with the stationary phase will be smaller for amino-phase packings. Corresponding interaction energies between solute and solvent molecules in the mobile phase will be the same for all adsorbents, and therefore the relative importance of mobile-phase vs. stationary-phase interactions will be greater for amino-phase columns vs. silica. Since we have ignored these mobile-phase interactions in the displacement model, ita accuracy for amino-phase columns should be correspondingly reduced. EXPERIMENTAL SECTION Equipment. A modular HPLC system was assembled for these studies: Altex Model 110 pump (Altex Scientific,Berkeley, CA), a Valco Model CV-6-UHPa-N6O sample injection valve (Valco Instruments, Houston, TX), and Altex Model 152 dual-wavelength photometric detector. Materials. Columns were p Bondapak-NH2(30 X 0.39 cm) from Waters Associates (Milford, MA). This packing has a surface area of about 325 m2/g and about 2 pmol/m2of alkylaminogroups (21). Three columns were purchased at various times and gave
similar retention data; relative retention was constant at about *2%. Data reported here are for a single column. Mobile phases were formulated from HPLC-grade solvents (Fisher or Burdick & Jackson). Conditions and Procedures. All separations were carried out at ambient temperature (25 1 "C) with a flow rate of 2.0 mL/min. Solutes were made up in hexane or mobile phase; 100 pL of sample solution (10-500 ng of solute) were injected each time. Retention times, tR,were measured as in (I). Capacity factor values k'were calculated from values of t R and to (column dead time): k' = ( t R - to)/to.Values of to,determined from the first base line disturbance or injection of hexane, were constant (1.61 min) for all systems studied. Following a change in mobile phase, sufficienttime was allowed before data were collected to equilibrate the column with new mobile phase (30-60 min). This was confirmed by repetitive injection of the same sample, with resulting constant k'values. Slow column equilibration as reported by Hammers et al. (25) for more bulky polar bonded phases was observed by us also, but the effects were much less pronounced. We will comment on this elsewhere.
RESULTS AND DISCUSSION Dependence of k' Values on Mobile Phase Composition. Table I1 summarizes data for a single B solvent (tetrahydrofuran, THF)and several aromatic hydrocarbon solutes. The concentration of THF was varied over the range 0-60 vol % THF/hexane; higher concentrations of THF gave k'values
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1767
6IOQ
k'
4 -
0
14 12
p+ 10
-1 1
log k
0
2-
1
0.02
0.04
o
(C)
,lo
06
.08
.I0
Flgure 1. Verification of eq 1 for data of Table 11. Plots of log k'vs. solvent strength e'. Numbers refer to solutes of Table I1 (left-hand column).
that approached zero for d solutes. We noxt correlated these data in terms of the displacement model and eq 1. For alumina, and silica, previous work (15)has shown that the solute A, values of eq 1 can be calculated from the molecular dimensions of nonlocalizing compounds such as the aromatic hydrocarbons (no. 1-6 of Table 11). These calculated values of A, are given in the last column of Table 11. We arbitrarily define the solvent strength eA of pure hexane as zero, and a' equal one. The application of eq 1to these k'values for solutes 1-6 of Table I1 then yields average values of e' for each of the nine mobile phases of Table 11. These latter e' values are listed a t the top of Table I1 for each mobile phase. In Figure 1we test the validity of eq 1for the data of Table 11. Equation 1predicts that plots of log k'vs. eo will be linear for all solutes, and this is seen to be the case in Figure 1for some representative comlpounds (other solutes of Table I1 give comparable plots). The average scatter of the data of Figure 1 around these linear plots is only a few percent. This compares favorably with similar plots of log k'vs. e' for alumina or silica as adsorbent (16). Dependence of A , Values on Solute Molecular Structure. Values of A, for each solute in Table I1 were determined from the slopes of plots as in Figure 1 (eq l), and are listed in the next-to-last column of Table I1 (experimental A, values). For solutes that do not localize (compounds 1-6 and 21-30 of Table 111,the experimental and calculated values of A, should be the same. This is the case, within a standard deviation (std dev) of i1.8 in A,, for 8 I A, I 43. However, for more polar solutes (7-20 of Table 11), experimental A, values are expected (discussion of Theory section and ref 14) to be larger than values calculated from solute molecular dimensions, because (a11these solutes can localize and (b) site-competition delocalization is expected for amino-phase columns (exposed surface sites). Furthermore, theory predicts that the difference, AA,,between experimental and calculated A, values should increase with increasing solute polarity (and increasing solute localization). This is the case for solutes 7-20. More specifically, values of AA, for the solute should correlate with the retention of the polar (localizing) solute-group i in the molecule, as observed on silica (15). The retention of the solute group i can be defined by its well-known ARM value in a nonpolar mobile phase (hexane in Table 11) M M = log
ki = log k x - log k~
6
4
8
1.0 0
Flgure 2. Dependence of AA, values for solutes of Table I1 on group-retention factor log k,. See text.
-1 .04
, 2
log k,
26 8
.02
n 0
(6)
Here, kx refers to the k' value for a naphthalene ring substituted by the group i and kN is the k'value for naphthalene. Thus, values of AA, for the group i should be zero for small values of log ki (i nonlocalizing) and AA, should increase with log ki when the group i becomes sufficiently polar for localization of i to become significant. This relationship is tested in Figure 2 for the data of Table 11. The expected correlation is observed, and it closely resembles that for silica (ref 15, p 204). Figure 2 provides a basis for estimating values of AA, (and A 3 for polar solutes, once their ki values have been determined experimentally. This in turn allows the use of eq 1to predict k'as a function of mobile phase composition. In the case of polysubstituted aromatics, the values of AA, for each group i must be summed to obtain A, for the total solute molecule
(15). The value of log ki for an ethylene oxide group (-CH2CHzO-) was determined as 0.12 from the data for solutes 21-30 of Table 11. Thus, these compounds are not polar enough to localize, and their A, values can be calculated from their molecular size (AA, = 0). In their analysis of the amino-phase retention model, Hammers et al. (6) have taken a different approach for similar data. They arbitrarily assume values of e' for the amino-phase system that are identical with those for silica and then derive experimental values of A,. As noted by Hennion et al. (7)and further confirmed by us (see below), E' values for amino-phase columns cannot be assumed equal to e' values for silica. Dependence of Solvent Strength on Mobile Phase Composition. Values of e' vs. vol % of the B Solvent. According to eq 2, values of eo for mixtures of a B solvent and hexane can be calculated from the mole fraction of B solvent NB, the adsorbent activity function a' (equal 1.00 for Bondapak-NHJ, the B solvent molecular area &, (5.0 for THF), and the pure-solvent strengths EA (hexane, 0.00) and eB (THF, best-fit value of 0.111). The ability of the displacement model to thus predict solvent strength is tested in Figure 3, where values of 'E from Table I1 are plotted against vol % THF. The solid curve is predicted by eq 2. Table I11 provides additional retention data to test the applicability of eq 2 for solvent strength in amino-phase systems. Values of k'for several of the solutes of Table I1 are given for five additional B solvents. For four of these B solvents (all except acetonitrile), several mobile phase compositions were studied, thus allowing the fitting of these data to eq 2. This fit is summarized in Table IV where average experimental values of e' are compared with calculated values from eq 2. The standard deviations (std dev) of the fit for each B solvent are summarized at the bottom of Table IV. Two points can be made concerning Table IV. First, the accuracy of e' values calculated from eq 2 is quite acceptable. The overall standard deviation for calculated vs. experimental
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1768
/
5 1 A 0
.4
€0
04 'O:
v
h/
P
i
0
E o (Alto,)
Y
Flgure 4. Comparison of solvent strength for pure B solvents, amino phase vs. alumina (see Table V): ( 0 )present study; (0)Hennion et al. ( 7 ) .
I
20
NM
40
80
60
100
&a
Flgure 3. Dependence of solvent strength
1
on mobile phase composltlon. Tetrahydrofuran (B)/hexane(A) mixtures: (0)experimental; (-1 eq 2. to
values is f0.009 units. This compares well with a similar figure for alumina and silica as adsorbents: f0.017 in to (IO), which because a' = 0.6 for the latter adsorbents is equivalent to 0.6 X 0.017 or fO.O1 units in terms of its effect on It' (eq 1). Second, it is seen that the standard deviation values in Table IV are significantly larger for 0-100 vol % B, compared to 0-60 vol % B. That is, calculated to values are less precise for higher concentrations of B in the mobile phase. Hennion et al. (7) have observed a similar larger-than-expectedsolvent strength for mobile phases with higher concentrations of the B solvent. They attribute this (the CC ' : term of eq 5) to interactions among solvent and solute molecules in the mobile phase. The data of Table IV are in agreement with this explanation, but the practical significance of this effect seems to be minor. In terms of theory, such a deviation of experimental and calculated (eq 2) to values is not unexpected for amino-phase packings, because of the weaker interaction of solutes and solvents with the adsorbent surface, and the greater relative importance of mobile phase interactions (as discussed in the Theory section). Elsewhere we will show that the precision of calculated t o values can be significantly improved by taking this and other second-order effects into account. Dependence of the Strength of Pure B Solvents on the Structure of the Solvent. Comparison with Silica. Hennion et al. (7) note that solvent strength as determined in their study of an amino-bonded-phase column does not follow an order resembling that for polar adsorbents such as alumina. Hammers et al. (6),on the other hand, assumed that the same to values will apply for both amino-phase and silica columns. Table V summarizes experimental values of eo for various pure solvents as determined by us and by Hennion et al. This listing excludes the various alcohols studied in (7), because their behavior appears more complex than for the less polar solvents of Table V. Values of to from the study of (7) were calculated as follows. For retention of the B solvent, Hennion et al. assume the relationship
(7)
which is their eq 1with substitution of terminology used by us. If eBis arbitrarily taken as 0.5, then 1 - 0 B = 0.5 and
Since experimental values of K1 for different B solvents are
i"-
(-OH1
1.2
to
K1 = eB/(1 - eB)cp
0
a
e
/ess
basic
4l
-
0
-
4i
I
4
- 6
- 2
I
2
6
I
10
log k (SIO,)
Flgure 5. Comparison of relative sample retention on present amino-phase column (10 vol % THF/hexane) vs. silica (42 vol % CH,Cl,/hexane): (0)aromatic hydrocarbons; ( 0 )substituted naphthalenes. Data for slllca from ref 11 and 15.
reported in ref 3, we can then determine to for the mobile phase for which eB= 0.5 from an equation derived in ref 11 to
= CA
NA/~A) + log (LY'Izb
(8)
Here, NA = 1- NB and = 1- 0 ~ Finally, . the value of to obtained from eq 8 can be used in eq 2 to determine eg. Values of to from (7) determined in this fashion are listed in Table IV. Values of to for the amino-phase columns of Table IV are plotted in Figure 4 against to values on alumina for the same solvents. The dark circles from the present study fall reasonably close to the (arbitrary) solid line, except for the more basic solvents THF and ethyl acetate. Thus, an amino-phase packing appears to be less acidic tdward these various solvents than does alumina. Silica is similar to alumina in terms of relative solvent strengths, so we can say that more basic solvents will also be weaker on an amino-phase column vs. silica. The open squares of Figure 4 are the data of Hennion et al. These agree fairly well with data from the present study for CHzClz and CHC13 as solvents, but their to values for acetone (A) and nitromethane (NM) in Figure 4 are twice as large as would have been predicted from the present study. This may reflect differences in the amino-phase packings of
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1769
Table 111. Experimental h‘ Values for Aromatic Solutes and Various Polar-Solvent/Hexane Mixture as Mobile Phase, Other Conditions as in Table I1
cc1, acenaph thy!iene phenanthrene fluor ant hene chrysene perylene
10% CT -u.22 -0.09 0.06 0.22
CH,Cl, acenaphthylene phenanthrene fluoranthene chrysene perylene 2,4,7 -trinitrofluorenone Eo
CHCl, acenaphthylene phenanthrene fluoranthene chrysene perylene 2,4,7 -trinitrofluorenone
5% MC
1 U % MC
-0.47 -0.41 -0.27 -0.11 0.02
ethyl acetate acenaphthyle ne phenanthrene fluoranthene chrysene perylene
(0.081)
20% MC
40% MC
60%MC
100%MC
-0.51 -0.40 -0.29 0.76
-0.89 -0.77 -0.73 -0.62 0.07
-1.00 -0.93 -0.48
-1.51
(0.025)
(0.041)
(0.063)
(0.088)
(0.112)
(0.15)
5% CH
10% CH
2u% CH
40% CH
6070 CH
100% CH
-0.62 -0.44 -0.31 0.69
-0.65 0.03
-0.38 -0.27 -0.1 5 0.00 0.15 (0.028)
Eo
-0.63 -0.47
(0.046)
(0.026)
-0.37 -U.25 -0.11 0.05 -0.21
100% CT
-0.53 -0.44 -0.32 -0.21 -0.05
-0.35 -0.24 -0.10 0.04 0.22
(0.011)
Eo
Eo
log k’ for indicated solvent mixturea 25% CT 50% CT
2% E:A -0.24 -0.12 0.03 0.20 (0.013)
-0.52 -0.43 -0.31 -0.20 -0.Ob (0.044) 5% EA -0.33 -0.21 -0.08 0.08 (0.023)
acetonitrile acenaphth ylene phenanthrene fluoranthene chr ysene
(0.063)
(0.092)
-0.43 (0.112)
-1.7 (0.17)
10% EA
25% EA
50% EA
-0.42 -0.32 -0.20 -0.06 0.11
-0.63 -0.53 -0.44 -0.33 -0.18
-0.62 -0.47
(0.034)
(0.056)
(0.07 9)
log k’ for indicated solvent mixturea 1 vol % AC -0.29 -0.18 -0.05 0.09 (0.023)
a Indicated percent values (%) refer to vol % for CT, CCl,; MC, CH,CI, ; CH, CHC1, ; EA, ethyl acetate; AC, acetonitrile. All solvents in admixture with n-hexane.
the present study vs. ref 7, for example, residual silanols in the packing of 7 which might interact with more polar solvents. Separation Selectivity in the Present Amino-Phase System Compared w i t h O t h e r LSC Systems. A rough comparison of separation selectivity on the present aminophase packing vs. silica is offered in Figure 5. Here, log k’ values from the present study for 5 vol % THF/hexane as mobile phase are plotted vs. log k’values on silica from the study of ref 11. The latter data correspond to 42% CH2C12/hexaneas mobile phase and are supplemented with k’ values calculated from ref 15 for this LSC system. Hydrocarbon solutes in Figure 5 are shown as open circles and substituted naphthalenes by closed circles. There is considerable scatter in the corirelation of Figure 5 meaning that the selectivity offered by an amino-phase column is different from that provided by silica. As in the case of the solvent strength comparison of Figure 4, it appears in Figure 5 that more basic compounds (ethers, esters, ketones) are somewhat less strongly retained on the amino-phase vs. silica column. The solute 1-naphthol is much more strongly retained on the amino-phase column, presumably by hlydrogen bonding to amine retention sites, as suggested in ref‘ 6.
Finally, aromatic hydrocarbons of higher ring number are preferentially retained on the amino-phase column vs. silica (but not vs. alumina (15)). Inasmuch as silica and alumina provide similar retention selectivity for polar compounds, the selectivity of the amino-phase packing will also differ substantially from that provided by alumina. Solvent Selectivity in Amino-Phase Systems. Limited data were collected on the effect of the B solvent on solvent-selectivity and relative band-spacing for amino-phase packings. Table VI summarizes 12’ values for several solutes and five mobile phases of roughly equal solvent strength ( E O = 0.021). Because the slight difference in actual t o values among these mobile phases has a further effect on solvent selectivity (via eq l ) , these data were adjusted using eq 1to give k’values for exactly the same E O values as for the 5 vol % THF/hexane mobile phase of Table VI. This correction is described in Table VI. Finally, log a values were determined for adjacent bands in the chromatogram for each mobile phase (last five columns of Table VI). In the case of aromatic hydrocarbon pairs (e.g., phenanthrene/fluoroanthene), there is little effect of the mobile phase on log a; the range in log a values for a given hydrocarbon
1770
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
Table IV. Solvent Strength eo of Binary Mobile Phases A/B as a Function of vol % B, Experimental vs. Calculated Values (Equation 2 ) values of
vol % B
CCI,
THF
( E B = 0.069)
(EB = 0.111)
exptla
calcdb
0.5
exptla
calcdb
0.004
1.0
0.008 0.011
0.021 0.028 0.045
0.002 0.004 0.007 0.016 0.029 0.048
0.074
0.074
2.0 5.0 10.0
20.0 25.0 40.0 50.0 60.0 100.0
0.U11
0.013
0.026
0.028
0.046
0.046
0.081
0.069
0.096
eo
(for indicated B solvent)
ethyl acetate ( E B = 0.113) exptla calcdb
0.013 0.023 0,034
0.006 0.015 0.027
0.056
0.054
0.079
0.081
0.090
CH,CI, ( E B= 0.130)
CHCI, 0.134) exptla calcdb (CB =
exptla
calcdb
0.025 0.041 0.063
0.022 0.039 0.063
0.028 0.044 0.063
0.022 0.039 0.063
0.088
0.092
0.092
0.093
O.lla 0.150
0.110 0.130
0.112
0.111
0.170
0.134
std dev 0-60%
k0.002 t0.006 a Experimental value from Tables I1 and 111. given a best fit to experimental eo values).
t0.004
0-100%
CCI, (CT) TH F ethyl acetate (EA) CH,CI, (MC) CHCI, (CH) nitromethane ( N M ) acetone (A) actonitrileC(AC) N, N-dimethylfor mamide
exptl E' values E' for this Hennion study et al.= aluminab 0.069 0.111 0.113 0.130 0.134
0.18
0.19 0.15 0.45 0.47
0.22
t 0.003
i0.008 Calculated value, using eq 2 and indicated value of
Table V. Strengths ( E ' ) of Pure B Solvents with Amino-Phase Packing
B solvent
t 0.006
0.51 0.60 0.42 0.40 0.64 0.58 0.55
0.58
a Calculated as described in text from data of ( 7 ) . Values for delocalized solvent (e") from ref 10, except for nitromethane from ref 15. Calculated from the one mobile phase of Table I11 (for which O B = 0.17).
pair is only 0.02-0.03 units. This is expected by analogy with separation on alumina or silica (15). The case of solute pairs that involve one or two polar compounds is more interesting. For polar solutes, solvent-solute localization has been shown
e~
i0.004 i0.015 (which is chosen to
to yield large changes in a,as a result of change in mobile phase composition (12). In view of the localization of polar solute molecules onto the amino-phase surface (as indicated by positive AAs values in Figure 2), similar solvent-selectivity effects are expected for amino-phase separations as for LSC with silica or alumina. However, these changes in log a will be proportional to the extent of solvent and solute localization, which will be less for the more weakly retentive amino-phase surface (compared to silica or alumina). This is observed for the data of Table VI, where the maximum range in log a for these solutes and mobile phases is only 0.10 units (dinitronaphthalene/perylene), vs. changes in log a of as much as 0.7 units for silica and alumina (12,26),using similar solutes and solvents. Thus, we can conclude that solvent-selectivityeffects will be less important for separations on amino-phase columns vs. alumina or silica. Nevertheless, in selected cases significant changes in a can be achieved by varying mobile phase composition with amino-phase columns. Thus, for quaternarysolvent mobile phases based on methyl tert-butyl ether/ CHzClz/CHCl3/methano1, Antle (3) achieved changes in log a of as much as 0.5 units, for separation of a steroid mixture of an amino-phase column. There are insufficient data in Table VI to carry out a detailed study of solvent selectivity as in ref 12. However, a
Table VI. Solvent Selectivity for p Bondapak-NH, System, Conditions as in Table I1 unless Noted Otherwise log oi (corrected for k' for indicated mobile phasea 16% 5% 5% 8% 5% 16% 5% 5% 8% solute CT MC CH EA THF CT MC CH EA acenaphthylene X-OCH,-C,,H, phenanthrene fluoranthene chrysene l-CN-C,,H,
8-O,CCH,-C, ,H, naphthalene2,6-(C02CH3),
perylene
naphthalene-
0.63 0.79 0.85 1.20 1.74 2.05 2.30 2.81
0.53 0.62
0.69 0.93 1.30 1.40 1.61 2.07
0.45 0.55 0.58 0.82 1.12 1.2 1.33 1.51
2.70 4.47
1.86 3.07
1.71 2.60
0.42 0.50
0.55 0.74 1.06 1.16 1.06 1.28 1.52
2.01
0.47 0.63 0.65 0.87 1.26 1.55 1.44 1.82 1.95 2.95
0.14 0.06 0.02 0.06 0.05
0.06 0.05 0.13 0.15 0.03 0.06 0.11 -0.04
0.09 0.02 U.1.5 0.14 0.03 0.05 0.07 U.03
0.13 0.17 0.05 -0.02 0.10 0.04
0.16 0.09 -0.03
o.15
0.25
0.20
0.16
0.18
0.07 0.04 0.14
0.09
5% THF
0.04
0.1 3 0.01 0.1 3
0.10
0.03
i,5-(N02),
See Table I1 for solvent identification; hexane as A solvent. CY is the separation factor, equal to the ratio of k ' values for two adjacent solute bands; e.g., first entry (0.07) is for acenaphthylene and methoxynaphthalene, with CCI, as B solvent. k ' values were first corrected to same strength as 5% THF by following additions to log k ' : CT, -u.OllA,; MC. -0.OulA.: CH. +0.004A.: EA. +0.006A.: 'rHF. no correction. a
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
cursory examination of thme data suggests that the model of ref 12 can predict changeai in log CY, with an accuracy of about *0.03 units (1 std dev).
CONCLUSIONS Retention Mechanism for Amino-PhasePackings. For amino-phase LSC systems that do not involve the use of alcohols as mobile phasle solvents, we can summarize our findings as follows. First, the data reported here fully support the Snyder-Soczewinski displacement model; eq 1and 2 describe the dependence of k ’ values on mobile phase composition as accurately as for similar separations on silica or alumina. We disagree with the conclusion of ref 7 that oneto-one competition between adsorbing solute and solute molecules exists for interaction with amino sites (as in eq 4). Rather, a solute molecule can displace any number of preadsorbed solvent molecules, depending on the molecular size of the solute. Second, we agree with Hennion et al. (7) that mobile phase interactions among sample and solvent molecules probably contribute to k ’values. However, this effect is small and of little practical importance; it can be ignored in most cases. Third, it is clear that retention on the amino-phase packings differs significantly from that on alumina or silica. This can be seen in terms of differences in relative solvent strength (Figure 4) and relative retention of different solute groups (Figure 5). Basic solvents and solutes are generally less strongly held on the lesai acidic amino-phase surface. This supports the view of Herinion et al. (7) that amino groups constitute the primary adsorption sites in these systems. In view of the similarities of data reported by us and by Hammers et al. (6), their data also support this conclusion. Fourth, the solvent/solute localization effects predicted in the Theory section for retention of amino-p hase packings were observed (see also dis~u~swion of ref 9-4). Thus, restrictedaccess delocalization is abisent in amino-phase systems, because of the lower concentration of surface sites (2 pmol/m2 vs. 8 pmol/m2 for silica), and the freedom of amino sites to vary their orientation within the surface. As a result, values of EB do not vary with surface coverage eB,as in the case for alumina and silica. Site-competition delocalization is found for the amino-phase packings, however, because the amino sites are exposed to lateral solvent interactions in the adsorbed monolayer. This leads to signiificant values of hA, for polar solutes. Solvent-solute localization on amino-phase columns occurs and leads to corresponding solvent-selectivity effects. Because the amino-phase surface is less retentive to polar solutes than silica or alumina, these solvent-selectivity effects are smaller and less important for amino phases vs. alumina or silica. Practical Implications: Comparison of Amino Phase vs. Silica Packings. One reason for choosing another packing in place of silica is for M change in separation selectivity. Amino-phase columns differ significantly from silica in this respect, as summarized in Figure 5 and the related discussion. Additionally,the less acidic amino-phase surface should mean smaller changes in the retention of aromatic hydrocarbon solutes, as a result of alkyl substitution on the aromatic ring. That is, the inductive effiect of such substituents in increasing the basicity of the ring aihould be less significant (15). In agreement with this expectation, several studies ( 4 , 5 )show the lesser effect of alkyl substitution in amino-phase separations, and the better separation of aromatics by ring number (e.g., in coal liquids) that is a consequence of this. From our discussion of the flexibile configuration of aminoalkyl groups on the surface of the amino-phase packings, we can anticipate poorer resolution of different isomeric solutes, vs. separation on alumha or silica (cf. ref l, pp 356-361). Retention on the aminophase columns is generally much weaker than on silica or adumina; since it is generally observed
1771
that more polar samples present additional problems in separations on the latter adsorbents, which has been attributed to their stronger retention of polar solutes, we can expect that fewer problems will be encountered in the separation of more polar samples on amino-phase packings. Variation of mobile phase composition can be used to increase separation selectivity on the amino-phase columns, but to a lesser extent than on silica. This represents a practical advantage of silica over amino-phase separations. Equation 2 and the pure-solvent to values given in this paper allow the calculation of solvent strength for a wide range of binarysolvent mobile phase compositions. The theory of (11,14) suggests that we can similarly calculate the solvent strength of ternary or quaternary-solvent mobile phases as well. Application of this approach to the data of ref 3 confirms that this is possible, in this case with an accuracy in calculated values of to of f0.006 (1std dev) (14).For the latter system, we have derived approximate to values for two additional solvents: methyl-tert-butyl ether, 0.11; methanol, 0.24. Additional pure-solvent to values can be estimated from the correlation of Figure 4. Table IV summarizes a number of E O values for some common binary-solvent mobile phases.
ACKNOWLEDGMENT The critical comments of several workers have much improved the present manuscript: B. L. Karger, J. L. Glajch, W. E. Hammers, and M. Caude. GLOSSARY refers to a weak solvent A and strong solvent B in a mobile phase mixture A/B constants in eq 3 and 5; values are determined by a particular combination of solute and B solvent molecular area of a solute molecule; eq 1 concentration of B solvent in mobile phase A/B (mole/L) a solvent or solute molecule; also a substituent group capacity factor of a particular solute in a defined LSC system; see ref 1. values of k’ for a given solute and mobile phases 1and 2 relative retention of a solute group i; log ki = log k x - log kN values of k’ (hexane mobile phase) for naphthalene (N) and naphthalene substituted by the group i (X) equilibrium constant in eq 7 ; see ref 7 molecular area of the B solvent mole fractions of solvents A and B in the mobile phase A/B; NA = 1 - NB separation factor; ratio of k ’values for two adjacent bands in a chromatogram; see ref 1 adsorbent activity function (eq 1,2); equal 1.0 for p Bondapak-NH2,1.33 for amino-phase column of ref 6 solvent strength parameter for a given mobile phase values of to for mobile phases 1 and 2; eq 1 values of E O for pure solvents A (weak) and B (strong) value of E O for binary-solvent mobile phase A/B mole fraction of solvents A and B retained in adsorbed monolayer; e A = 1 - o~ (see ref 10, 11) increase in A, (vs. value calculated from molecular dimensions) for a group i, as result of localization of i; see Figure 2 and related discussion
LITERATURE CITED (1) Snyder, L. R.; Klrkland, J. J. “Introduction to Modern Liquid Chromatography”, 2nd ed.; Why-Interscience: New York, 1979. (2) Okamoto, M. J . Chromatcgr. 1880, 202, 55. (3) Antle, P. E. “Mobile Phase Optlmlzation for the Normal Phase Separa-
1772
Anal. Chem. 1902, 5 4 , 1772-1777
tion of Steroids", du Pont de Nemours & Co., Analytical Instr. Div.: Wiimington, DE, 1981. (4) Chmieiowiec, J.; George, A. E. Anal. Chem. 1980, 52, 1154. (5) Liphard, K.-G. Chromatographia 1980, 73,603. (6) Hammers, W. E.; Spanjer, M. C.; de Llgny, C. L. J . Chromatogr. 197g, 774. 291. (7) Hennion, M. C.; Plcard, C.; Combellas, C.; Caude. M.; Rosset, R. J . Chromatoor. 1981. 270. 211. (8) Hurtubise,"R. J.; Hussain, A.; Silver, H. F. Anal. Chem. 1981, 53,
1993. (9) Snyder, L. R.; Poppe, H. J . Chromatogr. 1980, 784, 363. (IO) Snyder, L. R.; Glajch, J. L. J . Chromatogr. 1981, 2 7 4 , 1. (11) GlaJch,J. L.; Snyder L. R. J . Chromatogr. 1981, 274,21. (12) Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. J . Chromatogr. 1981, 278, 299. (13) Snyder, L. R. J . Chromatogr.,in press. (14) Snyder, L. R.; Glajch, J. L. J . Chromatogr.,in press.
(15) Snyder, L. R. "Princlples of Adsorption Chrmatography"; Marcel Dekker: New York. 1968. (16) Snyder, L. R. Anal. Chem. 1974, 46, 1384. (17) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1978, 749, 93. (18) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1979, 777, 37. (19) Scott, R. P. W. J . Chromatogr. Scl. 1980, 18, 297. (20) Scott, R. P. W. J . Chromatogr. 1980, 196, 193. (21) MaJors,R. E. J . Chromatogr. Scl. 1980, 78, 488. (22) Iler, R. K. J . Chromatogr. 1981, 209, 341. (23) Berendsen, G. E.; de Gaian, L. J . Llq. Chromatogr. 1978, 7 , 403. (24) Unger, K. "Porous Sillca: Its Properties and Use as Support in Column Liquid Chromatography"; Elsevier: New York, 1979. (25) Hammers, W. E.; Theeuwes, A. G. M.; Brederode, W. K.; de Ligny, C. L. J . Chrornatogr. 1982, 234, 32. (26) Snyder, L. R. J . Chromatogr. 1971, 6 3 , 15.
RECEIVED for review August 21,1981. Accepted May 26,1982.
High-Throughput Microcomputer-Based Binary-Coded Search Systems for Infrared, Carbon- 13 Nuclear Magnetic Resonance, and Mass Spectral Data Alan P. Uthman," Jerry P. Koontz,' Judy Hlnderllter-Smlth,' W. Stephen Woodward, and Charles N. Reilley2 Kenan Laboratories of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27514
A rapid, microcomputer-based search system for binarycoded spectra is demonstrated for large infrared, carbon-13 NMR, and mass spectral data bases containing 91 875, 2229, and 30 478 spectra, respectively. Search throughputs of several hundred spectra per second are demonstrated on a laboratory microcomputer. The search system exhibited is entirely "free-standing" and does not depend upon the availability of any other data processing requirements for preor postsearch processing. The general performance requirements of a computer system necessary for performing exhaustive searches on large data bases are also discussed. An advantage of such exhaustlve searches as contrasted with searches resorting to I/O and computation reducing prefilter techniques is outlined. A modification of the Grotch dlstance metric which reduces the weight given extra peaks relative to missing peaks in an unknown spectrum is also presented. Thls metric accommodates the possibility of having impurity peaks or mixtures in real samples.
Various approaches to computer-assisted identification of chemical compounds have been developed over the last 2 decades. Numerous survey articles have appeared on search systems (I+), along with articles appearing in annual reviews (7-9). A number of general search systems integrating different types of chemical data bases have been previously described (10-14). The three fundamental approaches used in computer-assisted identification (6) are library searches (15-29), pattern recognition (30,31),and artificial intelligence (32). The library search is the most straight-forward method of identifying a compound from its spectrum. The unknown spectrum is compared to the spectra in a library of known compounds, and a listing of candidate compounds is given as the result of the search. If the unknown compound is contained in the Present address: Burroughs Wellcome Co., Research Triangle
Park, NC 27709. Deceased.
0003-2700/82/0354-1772$01.25/0
library, then a match should be reported between the unknown spectrum and its correspondingentry in the library, and thus the unknown would be identified. If the unknown is not contained in the library, candidate compounds reported with spectra most closely resembling the unknown spectrum would u s u d y be chemically similar to the unknown and can be used as a guide to interpreting the unknown. Much of the early work on binary spectral search systems was performed by Grotch (15-18) on mass spectral data. Binary searches have also been applied to infrared spectral searches by Dupuis (27) and Delaney (28) and to carbon-13 NMR spectral searches by Woodruff (25). More recent studies on binary libraries have involved more use of information theory by van Marlen (21-24) and Dupuis (27) to more fully characterize binary data bases and search performance. Gray (29) has discussed the different similarity measures useful for binary spectra, while Rasmussen and Isenhour (33) have recently done a study comparing the various methods of data compression and the different search algorithms using a mass spectral data base. The research that is described herein was initiated to show that searches of large binary-coded libraries could be performed on microcomputers in such a fashion as to (1) be inexpensive so that routine use on a self-contained system within the laboratory is possible, (2) be fast enough so that exhaustive searches can be completed while avoiding data backlog, (3) use an algorithm that is independent of the nature of the data, and (4) return near matches as well as exact matches. Applicability of Microcomputers to Searches of Large D a t a Bases. Most searches using large data bases have up to now been run on either main-frame computers or minicomputers. This section will address the requirements of a computer for performing binary searches and the suitability of a microcomputer for this type of study. The principal attraction for using a microcomputer to perform a search instead of a minicomputer or main-frame computer is that microcomputers are very accessible to the laboratory due to their low cost. Because the computer can be located directly in the laboratory, the person desiring to 0 1982 American Chemical Society
