2496
Anal. Chem. 1987, 5 9 , 2496-2501
Reversed-Phase High-Performance Liquid Chromatography of Substituted Anilines Utilizing Molecular-Recognizing Ability of Crown Ether: Comparison with Ion-Pair Chromatography Akimasa Shibukawa, Terumichi Nakagawa,* Atsunori Kaihara, Kumiko Yagi, and Hisashi Tanaka Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto-shi 606, Japan Host-guest interaction between crown ether and protonated amino group has been applled to the specific Separation of podtlonai isomers of mono- and disubstituted anlllnes by reversed-phase hlgh-performance liquid chromatography with mobile phase containlng 18-crown-6. The retention of these amlnes on a hydrophobic statlonary llgand was enhanced by association with 18-crown-6, and the degree of the enhancement reflected the molecular structure of the guest. The host-guest assoclatlon constant and the degree of maxlmum enhancement were evaluated from the experimental values of capacity factor by the use of an equatlon derlved from the equlllbrla involved In this particular system. The retention and separation of the Isomers were characterized In terms of number, posltion, and functionality of the substituent groups on the anlllne rlng in comparison wlth those obtained by conventlonal ion-palr reversed-phase chromatography.
The selectivity in high-performance liquid chromatography (HPLC) separation can be improved by modification of the interaction between solute and mobile phase and/or stationary phase, especially by incorporation of a second equilibrium system (1)that involves host-guest interaction (2-17). In the previous papers (2-B), the authors have clarified the effect of host-guest interaction between crown ether and amino compounds in reversed-phase HPLC where crown ether was involved as an additive to the aqueous mobile phase (CERPLC). It has been known that 18-membered crown ethers associate with protonated primary amino groups by hydrogen bonding and ion-dipole interaction to form a relatively stable cationic complex in solution (19). Hydration of amino groups in the mobile phase may be weakened by the formation of the complex, so that the retention of the guest on the hydrophobic stationary ligand (e.g. ODS) can be enhanced depending on the stability of the complex and the hydrophobicity of crown ether. According to the model for this particular chromatographic system; we derived the equation that expressed capacity factor of a guest as a function of crown ether and proton concentrations in the mobile phase. The experimental results allowed discussion of the retention behavior, in good agreement with the equation in terms of class, number, and location of amino groups and the chemical structure around amino groups in the guest molecule. Some practical methods for the separation of catecholamines (4), p-lactam antibiotics (3),and amino acids and peptides (7,8) have been developed by the use of crown ethers. The present paper deals with the effects of substituent groups on the retention of aniline derivatives in CERPLC, putting emphasis on the molecular recognizing separation of positional isomers of mono- and disubstituted anilines. The discussion is also focused on the comparison between CERPLC and conventional ion-pair reversed-phase liquid chromatography (IPRPLC). EXPERIMENTAL SECTION Reagents. 18-Crown-6was a product of Merck (Darmstadt, FRG). Sodium pentanesulfonate used for ion-pair chromatog0003-2700/87/0359-2496$01.50/0
raphy and triethylamine of analytical reagent grade were purchased from Nakarai Chemicals Co. (Kyoto, Japan). All the guest compounds of analytical reagent grade were obtained from Nakarai Chemicals and Wako Pure Chemical Co. (Osaka, Japan). These reagents were used without further purification. Glassdistilled water and methanol were used to prepare the mobile phase after degassing. Hydrochloric acid of analytical reagent grade was used to adjust the pH of the mobile phase. The analytical column packed with 5-wm C18 silica (Chemcosorb 50DSH) was purchased from Chemco (Osaka, Japan). Measurements of Capacity Factors. A liquid chromatograph (LC-GA,Shimadzu,Japan) equipped with a W detector (PAD-GA, Shimadzu) was used for the measurements of capacity factors. The operating conditions are given in Table I. The guest materials were dissolved in a small volume of mobile phase or methanol, and the minimum amount required for UV detection was used in order to maintain linearity of the chromatographic system. The capacity factors (k’) were calculated from the equation,k ’ = (tR- to)/to,where tRis the retention time of a guest compound averaged over repeated measurements at the top of the elution curve and to is that of a nonabsorbed substance (NaN03). Data Analysis. Nonlinear least-squares fittings were carried out with a PC-9801F microcomputer (NEC, Tokyo, Japan) specially programmed in BASIC (18).
THEORY In the reversed-phase liquid chromatography where crown ether is contained in the aqueous mobile phase, a sample compound with an amino group may undergo the equilibria shown in Figure 1,where the pH of the mobile phase is low enough to allow complete protonation of amino group and the host-guest interaction is assumed to take place with 1:l stoichiometry. SH, C, and L represent a protonated guest molecule, host compound (crown ether), and stationary ligand, respectively. The K’s are the association constants specified in Figure 1. By introducing the equilibria in Figure 1to the capacity factor (k’) equation defined as [LSH], + [LCSH],
k’= @
[ W m + [CSHlm
we obtain
where 0 designates phase ratio (stationary phase/mobile phase), the subscripts “s” and “m” specify stationary phase and mobile phase, and B represents KLCSHKCSH,KLc~HKLc, OF KLCSHKLSH. Since the stationary phase occupied by the guest accounts for a very small part of the total ([LT]) Therefore (3)
From eq 2 and 3
0 1987 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 20, OCTOBER 15, 1987
2497
Table I. HPLC Conditions" experiment
mobile phase
stationary phase
(A) k'vs. pH profile
H20/CH30H = 8/2(v/v), pH 2.2-5.4 adjusted by HC1, containing 50 pM triethylamine* (B)dependence of k'on [18-crown-6] H20/CHsOH = 8 / 2 (v/v), pH 2.5 adjusted by HC1, containing 0-20 mM 18-crown-6 (C) dependence of k'on [C6Hl,S03Na] H20/CH30H = 8 / 2 (v/v), pH 2.5 adjusted by HC1, containing 0-10 mM sodium pentanesulfonate (D) separation of methoxyaniline isomers H20/CH30H = 8 / 2 (v/v), pH 2.5 adjusted by HCl, containing 10 mM sodium pentanesulfonate or 1 mM 18-crown-6. (E) separation of aminocresol isomers H,O/CHsOH = 8/2 (v/v), pH 2.5 adjusted by HC1, containing 10 mM sodium pentanesulfonate or 2 mM 18-crown-6.
Chemcosorb 5DSH (15 cm
X
4.6-mm i.d.)
Chemcosorb 5DSH (15 cm
X
4.6-mm i.d.)
Chemcosorb 5DSH (15 cm
X
4.6-mm i.d.)
Chemcosorb 5DSH (15 cm
X
4.6-mm i.d.)
Chemcosorb 5DSH (15 cm x 4.6-mm i.d.)
"Flow rate, 0.9 mL/min; detection, UV 220 nm: column temperature, 40 OC. *Triethylamine was added to avoid silanophilic effect. SH
C W CSH-'
''
SH
J'
C
10 I
SH
I I/
c 3 LSH
v
LC 7 L ' CSH KLCSH
KLCSH
Flgure 1. Equilibria involved in reversed-phase llquld chromatography with a crown ether containing moblle phase: SH, protonated amine; C, crown ether; L, hydrophobic stationary phase; K , association constant; K,, dissociation constant.
where kd is the capacity factor of free guest measured without using crown ether. The capacity factor of the host-guest complex (k:) is expressed by
k,' = @ [ L T I B / K C S H
-
8 ,
V
I/,
L 0
' 0
15
10
5
(C),
20
0
I 10
20
30
40
[CIm ImMI
ImMI
Figure 2. Simulated curves of capaclty factor against the concentration of crown ether in the mobile phase at various values of k,' and KCSH.The simulation Is based on eq 4. (A) k,' = 1; K,, = 50 M-'; KCsH= 200 M-l; k,' = 10 (line a), 15 (line b), 20 (line c). (B) k,' = 1; K,, = 20 h4-l; k,' = 10; KCSH = 50 M-' (line a), 100 M-' (line b), 200 M-' (line c).
(5)
When an anionic ion-pairing agent such as hydrophobic sulfonate is involved instead of crowd ether, the following equations in place of eq 4 and 5 are valid:
k' =
k,' + @.[LTIBT1lm (1 + K L I [ I l m ) ( l + K I S H [ I l m )
kI' = @[LTIB'/KISH
(6)
(7)
where subscript "I" designates the ion-pairing agent and k{ is the capacity factor of a 1:1 ion pair formed between guest and sulfonate. These equations are conformable to those previously derived according to ion-pair partition theory (20), because of the similarity in modeling of the chromatographic processes. However, the role of crown ether is different from that of the ion-pairing reagent in that the former is a hydrophobic nonelectrolyte, which can participate in stereospecific interactions with organic cations. Equations 4 and 6 predict that the additive exerts opposite effects on the retention of a guest; one is to increase the capacity factor by the complex formation, and the other is to decrease it by competing with the guest in binding to the stationary ligand. Thus, the k'value changes depending on the balance of these effects. The degree of the former effect (retention enhancement effect) depends on the K C ~or HKBHand k,' or k{ values. Figure 2 shows the curves of eq 4, where three different values were arbitrarily substituted in k,' (Figure 2A) and K a H(Figure 2B), while the other parameters were kept constant. As shown in Figure 2, a larger k,' value causes a larger increase in k', and a larger KCsH value causes a more rapid increase in k', reaching maximum level a t a lower concentration of crown ether. This indicates that the solutes, which are hardly separated from each other in the usual reversed-phasemode, can be separated because of the difference in K C ~and/or H k,' value(s). On the other hand, when the additive does not make a complex with the guest but competes with it in binding to
0
10
20
(18-crown-6) ImM i
Figure 3. Effect of 18-crown-6 concentration in the mobiie phase on the capacity factors of benzylamine (A),m-toluidine (W), N-methyland N,Ndimethylbenzylamine m-toluidine (O),N,Ndimethylaniiine (V), (A).For HPLC conditions, see part B in Table I.
the stationary phase (Le. K C ~ H = KIsH = 0), the eq 4 and 6 can be simplified to eq 8 and 9, respectively. Apparently, a larger K L C or KLI value causes a larger decrease in k'. k' = ko'/(l + K ~ c [ C l m ) (8)
k' = k,'/(l
+ KLI[Ilm)
(9)
RESULTS AND DISCUSSION Estimation of KLCand KLIValues. Figure 3 shows the relationship between capacity factor and concentration of 18-crown-6. The capacity factors of primary amines (benzylamine, m-toluidine) were markedly increased by addition of a low concentration (I30 000 plrtw/m with good permeablltty and peak symmetry. *par a t b of multkompnent peptide and protein mixtures were petfofmed in less than a minute wtth good recoveries of such
solutes. Permanent address: Department of Chemistry, Miami University, Oxford, OH 45056. 0003-2700/87/0359-2501$01.50/0
The theoretical desirability of using very small particles (e.g.,
