Anal. Chem. 1001, 63, 1222-1227
1222
Split-Peak Phenomenon in Nonlinear Chromatography. 2. Characterization of the Adsorption Kinetics of Proteins on Reversed-Phase Supports H616ne Place, Bernard S6bille, and Claire Vidal-Madjar* Laboratoire de Physico-Chimie des Biopolymbes, CNRS, Universit6 de Paris Val de Marne, UM 27, 2 rue Henry Dunant, 94320 Thiais, France
The kinetics of adrorptlon d human serum albunin (HSA) on a reversed-phase support was studied by analyzing the “rpllt-peak” effoct in mcrrcoverload conditions. ThIs effect occurs when a fraction of solute is eluted as a nonretained peak. I t can be convonhtty characterized by cdarlrrthg the fraction of unretained solute obtainod from cumulated InJectionr. The experimental data were fitted with a madel that assumes irreverdble adsorption and Langmuk secondorder klnetks. Its analytical expression, relating the nonretained fraction to the sample dze and the flow rate, is a function of both the number of transfer units (related to the apparent adsorption rate constant k , ) and the maximum loading capactty of the cohnnn 0,. From zonal duHon experiments the method permits the determination of the parameters k , and Ox, characterizing the adsorption of HSA on the reversedphase support. The influences of acetonitrile concentration and temperature on the spllt-peak effect were examined. I n pure buffer, the number of transfer units is equal to 16 at 25 O C . I t Is about 5 thm as large as when acetonltriie is added to the duent. An hrpoltanl ckcrease of the maxlmun loadhg capaclty (0,) is observed in the presence of acetonitrile in the mobile phase. This study gives information about the interaction d free protein with both the support and the solvent.
INTRODUCTION One of the major problems in the chromatography of proteins is the slow adsorption-desorption kinetics that leads to important band broadening and even to incomplete recovery of proteins with ghost or split peaks. If some of these effects may be due to a modification of the protein structure after adsorption, others originate in the slow adsorption kinetics which characterizes the interactions of free protein with the stationary phase. It thus appeared useful to carry out a few fundamental studies to elucidate these effects. In the present work we focus attention on the appearance of the “split-peak”phenomenon in mass-overload conditions. This effect occurs with slow adsorption kinetics (1): a fraction of solute elutes immediately as a nonretained peak, while the rest of the solute is retained. This last fraction may elute as a long trailing band or may remain irreversibly adsorbed on the support and can be eventually eluted with a different mobile phase. Sportsman et al. ( 2 , 3 )were the first to observe this effect when proteins are eluted on immobilized antibody supports. An apparent equilibrium constant depending on the flow rate was found, which reveals a kinetic effect. Hage et al. ( 4 , 5 ) gave an analytical expression that connects the nonretained fraction to the adsorption rate constant. The model, valid for linear chromatography, is applied to the analysis by
high-performance liquid chromatography (HPLC) of two experimental systems: the retention of immunoglobulin G on immobilized protein A and that of hemoglobin A on reversed-phase columns. Our goal is to study, by HPLC, the adsorption of proteins on reversed-phase supports in mass-overload conditions with the split-peak model developed in the previous paper (6) and based on the same hypothesis as those used to analyze breakthrough curves ( 7 , 8 ) . A simple expression was given in the case of irreversible adsorption and a Langmuir second-order kinetic law. It relates the unretained solute fraction to the column loading capacity and to the number of transfer units characterizing mass-transfer kinetics. This number includes two terms: the effective second-order adsorption rate constant and a global first-order one relative to the transfer in the stagnant fluid. Differentiating between the two ratelimiting steps is experimentally difficult. Larew and Walters (9) designed an experimental syatem where the mass transfer for chemical adsorption is negligible: they used a reversedphase support of large pore size and therefore of high loading capacity. In this work we select an experimental syatem where the magnitude of the global fiit-order rate constant is minimized: the studied protein, human serum albumin (HSA), is excluded from the small pores of the support (80A). This implies a low loading capacity and an increase of the role of the kinetic mechanism for the adsorptive exchange on the support. One of the criteria to apply the model is a second-order Langmuir kinetic law. This is the case with albumin adsorption (IO), which is often found to follow a Langmuir type isotherm: the surface area for protein adsorption is generally evaluated from the maximum albumin loading capacity. THEORY In the previous work (6) we established an equation that connects the sample size Qi with the fraction f of compound eluted as a nonretained compound in the case of irreversible adsorption:
Here Q, is the maximum loading capacity of the column and a is the number of transfer units characterizing the kinetic process. Characteristic curves are obtained that relate the fraction f or the adsorption yield, p = 1- f to the ratio QJQ, and to a = k,Q,/6. In this last expression, k, is the apparent second-order adsorption rate constant and 6 is the flow rate. If the kinetic process can be described simultaneously by a transfer between the mobile and stagnant fluid and by an adsorptive exchange of comparable rates, Arnold and Blanch (8)showed that an additive law may be applied to the number of transfer units, which is expressed in the case of irreversible adsorption as
To whom correspondence should be sent. 0003-2700/91/0363-1222802.50/0
0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991
where k,* is the effective second-order rate constant for the adsorption process, k,, is a global first-order rate constant for the transfer in the stagnant fluid (at the particle boundary and inside the pores), and Vois the interstitial fluid volume or excluded volume. The expression for k,, can be derived from the plate height model (11-13):
(3) where the first mass-transfer term is for totally porous spherical particles and does not depend on flow rate. The second one, for the transfer at the particle boundary, is flow rate dependent as the fluid film mass-transfer coefficient, kt. In this expression d, is the particle diameter, Di is the effective particle diffusion coefficient with Di = DV,/(Voe). D is the solute diffusion coefficient in the bulk liquid, V , is the pore volume accessible to the solute, and 8 is a tortuosity factor. For totally nonporous particles only the second term must be considered and for high Peclet number kf = QDu’fa/d,(111, where u is the reduced velocity ( u = ud,/D), u is the liquid linear velocity, and Q is a constant whose value lies between 3.2 and 3.6, depending on the column interparticle porosity. Equation 3 shows that k,, is flow rate dependent: the importance of the first-order kinetic contribution increases at lower flow rates, especially with totally nonporous particles, for which the first term is not to be considered. The limiting form of eq 1, for small Qi values, gives the split-peak expression at infinite dilution: fo = e-* = e-kaQJ6 (4) Considering two rate-limiting steps for the adsorption process, diffusion through the stagnant mobile phase and adsorption on the support, Hage et al. (4) found a similar expression in the linear case and for irreversible adsorption. The asymptotic expression off (jJ for large amounts injected or for zero flow rate may be written by using the obvious equation relating f to Qi and Q, for batch experiments: fm = 0 for Qi < Q, Three parameters define the model given by eq 1: the apparent adsorption rate constant k,, the maximum loading capacity of the column Q,, and the flow rate 6. The occurrence of the split-peak phenomenon in mass-overload conditions depends on the ratio a and on the sample size Qi.The larger the flow rate and the shorter the column, the lower will be the amount injected necessary to give rise to a split-peak. Because of given experimental conditions, it may even happen that no first peak is observed at low solute concentration. When increasing amounts of solute are injected, the fraction f of nonretained compound increases and the first peak can be detected. The adjustment of eq 1 to the experimental f and Qi values permits the determination of the apparent adsorption rate constant k , and the maximum loading capacity Q,, for the studied protein. Until now, the kinetic adsorption constant could be determined from the value of fo (4),measured at infinite dilution in the linear domain of the adsorption isotherm (eq 4), but three important drawbacks arise from this approach. First, the split-peak effect has to be analyzed for low sample sizes and this seriously restricts the possibilities for applying the method: one must use short columns, high flow rates, and low Q, values. With fast kinetics (high k , values) split-peak measurements in the linear elution range are impossible. Second, the experimental value of Q, has to be found from independent experiments. Third, measurements imply a good precision: this is extremely difficult to achieve at low con-
1223
centration, and experimental results have to be extrapolated at infinite dilution. And last, it is important to account carefully for impurities that interfere in the determination of the unretained fraction. The results of numerical simulation have previously shown (6) that the variation off with the amount injected is independent of the shape of the injection signal. Simulations with irreversible adsorption give characteristic curves identical with those obtained from eq 1, even if successive injections are carried out. Therefore it is valid to determine the nonretained fraction experimentally from the ratio of the cumulated values of the amounts of solute injected to the eluted ones. EXPERIMENTAL SECTION The HPLC system consisted of a Waters Model 6000A pump (Milford, MA) and a Rheodyne 7125 sampling valve (Berkeley, CA) with a 20-pL sample loop. A UV multiwavelength detector (Shimadzu SPD-6A, Tokyo) was set at 280 nm for the HSA detection. The analog outputs of the detector were connected to a digital voltmeter (data acquisition unit 3497, Hewlett-Packard, Palo Alto,CA). With four digits precision, the maximum sampling rate is 50 readings/s. A reversed-phase support (Spherisorb RPC6, particle size 10 pm, pore six 80 A) was obtained from Phase Separation (Norwalk, CO), and 0.5 g was packed into a 50 X 4.6 mm stainless steel column, using the slurry technique. The specific surface area of the support was of 220 m2/g. The temperature of the column and that of the eluent were kept constant within 0.1 OC by using a thermostated-cryostated water bath. The eluent was a 0.067 M potassium phosphate buffer (pH = 7.4) to which various concentrations of acetonitrile (HPLC grade) were added. HSA was obtained from Sigma (A1887,St. Louis, MO). The solutions of HSA were prepared by diluting the protein in the same solvent as that pumped through the column. Split-peak studies are based on peak area measurements, and therefore we applied the usual protocol for quantitative analysis: a blank injection of the eluent to check for the zero response of the UV detector; calibration of the detector and check of the linearity of its response by injecting increasing sample sizes of HSA on the column in cases when the protein is totally eluted as a first peak, i.e. on a column saturated with the protein; acquisition rate selected for defining the elution peak with a minimum of 15 data per peak width measured at half peak height. The data were collected and treated for peak integration with a microcomputer IBM PC AT2 (Greenock, Scotland). An integrator could as well be used for this type of study. Prior to performing the initial measurements, the column was saturated with HSA in order to eliminate the most active sites on which the protein is definitivelyadsorbed: these sites cannot be regenerated, even with a solvent containing large concentrations of acetonitrile. The split-peak experiments were performed by injecting increasing concentrations of HSA into the column. The solute was split into two fractions, and their relative importance was a function of various experimental conditions: flow rate, organic solvent concentration, and temperature. The f i t fraction was eluted at the exclusion volume ( Vo= 0.37 mL), and the second one is irreversibly adsorbed onto the support. This last fraction can be desorbed by changing the nature of the mobile phase, i.e. by increasing the amount of organic solvent in the mobile phase. Figure 1 illustrates the increase of the split-peak effect when successive injections of HSA solution are performed (2 g/L in the eluent). The saturation of the support was reached when the protein was totally eluted as a first peak and the increase of the amount eluted is related to the acetonitrile content in the mobile phase. The column was regenerated after saturation, by eluting the protein with a buffer containing 40% acetonitrile. The maximum loading capacity of the column QSwas measured from the area of the peak eluted after performing the desorption step. Within 10% relative error, it is in good agreement with the amount adsorbed calculated from the difference between the cumulated values injected and the cumulated nonretained values. The unretained fraction f was determined from the ratio between the cumulated amounts eluted as nonretained solutes and calculated from the first peak area and the cumulated amounts injected. Figure 2 shows that an impurity, slightly resolved from
1224
ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991
Table I. Influence of the Flow Rate on the "Split-Peak"Effect (HSA Adsorption at 25 OC with 30% Acetonitrile in the Eluent) 6, mL/min
8 x 3 mg/g
k,, M-'*s-'
ff
ffa/ v,, s-1
V
0.55 1.00 1.45
2.7 f 0.1 1.5 f 0.05 1.8 f 0.04
1500 f 100 3500 f 300 3100 f 200
3.3 2.3 1.6
0.082 0.104 0.105
200 370 540
12kf/d,,
8-l
148 180 204
1.
SO0
LMM
;SO0
2000
I
2500
.
8.
FigUe 1. Successive injections of HSA on a reversed-phase support. Conditions: eluent, 0.067 M phosphate buffer pH 7.4 20% v/v acetonitrile; injected volume, 20 pL; [HSA] = 2 g/L; data acquisition rate, 5 readingsls; t = 25 O C ; L = 5 cm; V , = 0.37 mL; 6 = 1
+
mL/min.
H)
io0
I
.
a. 50
io0
I
a.
!lo
100
Chromatogram of first injections of HSA eluted on a reversed-phase support: (a) [HSA] = 1.0 g/L; (b) [HSA] = 1.5 g/L; (c) [HSA] = 2.0 g/L. Experimental conditions are the same as in Figure Flgure 2. 1.
HSA peak, is eluted at a larger retention volume larger than the exclusion volume. The presence of impuritiesat a low level (about 2%)in the protein sample is not so critical for measurements in mass-overload conditions. One can notice that an enrichment of the protein occurs in the first peak eluted when the amount irreversibly adsorbed increases, while the fraction of impurity relative to the amount injected is not changed. If noticed during the experiments, one can easily correct for the impurity amount in the calculation of the nonretained fraction. This is true for impurities that are not irreversiblyretained and therefore do not interfere with split-peak measurements.
RESULTS The kinetics of HSA adsorption was studied as a function of various experimental conditions (flow rate, temperature, organic solvent concentrations) in relation to the theoretical model previously established (6). The data were analyzed to determine the apparent adsorption rate constant k, and the column loading capacity 8,. The determination of the parameters was performed by using a program for nonlinear least-squares fitting written in Fortran language. The intervals on the parameter estimation at 95% confidence level are given. Except for experiments with fast adsorption kinetics (in pure buffer), the fit of the model was restricted to injected values corresponding to the formation of the first adsorbed layer (Qi < 8,). On the other hand, for low sample sizes, in order to avoid the lack of precision due to impurities interference, the fittings were performed for injected amounts larger than 0.04 mg or l / f values lower than 100. Influence of the Flow Rate. A kinetic effect could be clearly demonstrated from studies of elution behaviors as a function of the flow rate. With reversible elution and in the absence of the split-peak effect, one usually measures the band broadening or plate height as a function of mobile-phase velocity. In linear chromatography and when the split-peak phenomenon occurs, one measures the variation of the nonretained fraction fo with the flow rate. Similarly, in mass-overload conditions, we can observe (Figure 3) a decrease of the adsorption yield p with an increase of the flow rate. The theoretical model given by eq 1 was fitted
1
2
3
Q i mg
Figure 3. Variation of the adsorption yield p with the amount of HSA injected (Influence of Row rate). conditkns: ehmnt, 0.067 M phosphate buffer + 30% acetonitrile; t = 25 O C ; L = 5 cm; V , = 0.37 mL; 6 = (0)0.55, (0) 1, (A)1.45 mL/min.
to the experimental data, and the results are given in Table I for 30% of acetonitrile in the buffer at 25 "C.As expected from the model, the number of transfer units a decreases with an increase of the flow rate. If the adsorption process were the only rate-limiting step, one would expect that the quantity a6/Vobe a function independent of the flow rate. This is true for 1.00 and 1.45mL/min flow rates but not for 0.55 mL/min. This shows that the mass transfer in the stagnant fluid cannot be neglected because the apparent adsorption rate constant increases with a flow rate increase. In the same table we compare the experimental ratio a6/Vo with the theoretical contribution for the stagnant fluid transfer at the particle boundary 12kf/dp(eq 3). We assumed that the diffusion coefficient of HSA in the mobile phase is 6.1 X lo-' cm2/s (9). The results of Table I show that 12kf/d, is about IO3 times larger and its contribution is negligible. A larger value for the diffusion coefficient should be used in this calculation, since the viscosity of the eluent used with an
ANALYTICAL CHEMISTRY, VOL.
63,NO. 13, JULY 1, 1991 1225
Table 11. Influence of Eluent Composition on HSA Adsorption at 25 OC
Saturation amt of acetonitrile, vol %
Qs,mg/g of support
Q,, mg/g of support
0 20 25 30 40
3.6 3.5 3.7 3.6 0
2.2 f 0.1 2.0 f 0.05 1.7 f 0.05 1.5 f 0.05
split-peak effect k,, M%-' 15000 f 5000 5100 f 300 3300 f 300 3500 f 300
aa/
a
16 4.4 2.7 2.3
v,, s-1
0.720 0.198 0.122 0.104
Table 111. Influence of Temperature on HSA Adsorption
amt of acetonitrile, vol % 0
30
t,
"C
10 25 10 15 20 25 35
saturation Qs, mg/g of support
split-peak effect
Q,, mg/g of support
3.4 3.6 0.4 0.7 1.6 3.6 7.4
organic modifier is lower. This correction would increase the calculated mass-transfer rate at the particle boundary. From these experiments one cannot exclude a partial diffusion in the stagnant fluid present in the pores or located between the particles, using a term similar to the first one in eq 3 which assumes a partial diffusion in a stagnant fluid volume (9). This preliminary study does not permit the differentiation between transfer kinetics limited by a diffusion mechanism or by an adsorption chemical step, and more detailed experiments should be performed to study the influence of the flow rate, porosity, and particle diameter. Therefore the adsorption rate constants given in this work must be considered as apparent ones, measured at a given flow rate. The parameters found for 1.00 mL/min (Figure 3) were used to predict the variation of the adsorption yield at other flow rates (0.55and 1.45 mL/min). As previously noticed from the results in Table I, the split-peak variations at the higher flow rates are in good agreement with the model, but the divergence with the experiments performed at the lower flow rates shows that the mass transfer due to the diffusion in the stagnant fluid cannot be neglected. For sample sizes larger than the maximum loading capacity, an important deviation between the model and the experimental results may be observed: the amount of protein adsorbed is consistently larger than the predicted one (Figure 3). This reveals another kinetic mechanism occurring for amounts of adsorbed protein larger than the amount necessary to form the first adsorbed layer. Influence of the Solvent Composition. The influence of the amount of acetonitrile added to the buffer is studied at a flow rate of 1.00 mL/min and a temperature of 25 "C. The variation of l/f as a function of the amount injected is shown in Figure 4,a more convenient display for the nonlinear regression. The importance of the split-peak effect depends on the acetonitrile concentration added to the eluent. In pure buffer, this effect is not observed at low sample sizes but appears suddenly at a value close to saturation. A solute splitting is noticed between 20 and 30% of acetonitrile in the buffer, even for low injected amounts of HSA. With 40% of acetonitrile, the protein is totally eluted in the first peak and the phenomenon cannot then be studied further. Table I1 gives the k, and Q, values determined from fitting of eq 1 to the experimental data. For increasing concentrations of acetonitrile, a decrease of the number of transfer units is observed, which explains the increase of the split-peak effect.
loo00 15000
2.2 f 0.1 2.2 f 0.1
b
* 5000 5000
3000 f 1000 2000 300 3500 300
0.27 f 0.01 0.89 f 0.03 1.50 f 0.05
I
k,, M%-'
a
12 16 0.3 0.8 2.3
I
1
2
3
Q i mg
Flguro 4. Varletion of the nonretained fractlon f with the amount of HSA Injected (influence of solvent COmpOSitlOn). ConditlonS: t = 25 "C; L = 5 cm; V , = 0.37 mL; 6 = 1-00 mL/min; eluent, 0.067 M phosphate buffer (+) 0 % , ( 0 )20%, (A)25%, (0)30% acetonlblle.
+
The apparent adsorption rate constant k, measured in pure buffer is 4-7 times as large as when acetonitrile is added to the buffer. For comparison with Q, values determined from model fitting, the capacity of the column, Qs, measured after column saturation is listed in Table 11. Qs is always larger than Q,. A possible explanation for the increase of the loading capacity could be protein aggregation, which could lead to the reorganization of the adsorbed layer or multiIayers formation. Influence of Temperature. The influence of temperature was studied with 30% of acetonitrile in the buffer and a flow rate of 1.00mL/min. A decrease in the split-peak effect was observed with increasing temperatures: the higher the temperature, the lower the fraction of nonadsorbed protein. At 10 O C the protein is totally eluted as a first peak (fal),while at 35 "C the protein is almost completely adsorbed (f = 0) and saturation is difficult to achieve. Therefore, at low or high temperatures, it is almost impossible to observe a splitting of the eluted protein and no analysis of the effect can be performed. From the plot of l/f vs Qi (Figure 5),the value of the number of transfer units a and the adsorption capacity Q. were determined at 15,20,and 25 "C (Table 111). For comparison, the values measured in pure buffer at 10 and 25 "C are listed in the same table. In pure buffer, the loading capacities (Q,and Qs)do not change with temperature, but the
1226
':
ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991 l/f
8
4 1
3 i
\
o
O
\",%-
1
2
3
Q i mg
Flgwo 5. Variation of the nonretalned fraction f wlth the amount of HSA InJected (Influence of temperature). Condltlons: eluent, 0.067 mM phosphate buffer i-30% acetonitrile; L = 5 cm; V o = 0.37 mL; 6 = 1.00 mL/min; r = (0)25, (A)20, ( 0 )15 "C.
number of transfer units slightly increases. With buffer + acetonitrile, a marked increase of the number of transfer units is noticed with temperature, which is related to the increase of the maximum loading capacity Q,. The same trend may be observed for Q s values, measured after desorption from saturation experiments, but these values are higher than Q,, as already noticed.
DISCUSSION Kinetic measurements from the split-peak effect are possible with experiments carried out in overload conditions, if saturation of the column can be obtained. Even with pure buffer, when the nonretained fraction does not elute at first injection, the apparent adsorption rate constant can be determined. In zonal elution and linear chromatography, the kinetic measurements are performed from the plate height variation with the flow rates (12,131. In mass-overload conditions, the adjustment of the theoretical profile to an experimental one eluted from the column (14) enables the determination of the parameters of the adsorption isotherm (for a Langmuirian type: the adsorption equilibrium constant and the maximum loading capacity) plus the number of transfer units. In the present experiments, irreversible retention occurs, and it is of course impossible to analyze the total elution peak and determine k'or kd: for the given set of experimental conditions used (nature of the support and of the eluent), the only observable phenomenon is the split-peak effect. When exploiting the split-peak effect in linear elution (at low sample sizes), one can only determine the number of transfer units. In the present work, the fit of the theoretical law (eq 1)to the experimental data makes it possible to determine a and Q, in the same experiment. From these values the apparent adsorption rate constant k, can be determined. This measurement is possible in a range of a values between 16 and 0.3. Because of its Stokes radius of 36 A (15), one can assume that the protein is excluded from the pores of an 80-A support (16) and is adsorbed onto the external surface of the beads (0.6 m2/g of packing, calculated for lO-pm spherical nonporous beads). When a monolayer of HSA is assumed, the amount of protein adsorbed is between 2.5 mg/m2 ("side on") and 6 mg/m2 ('end on") (17). The maximum loading capacity Q, calculated from the model and measured in pure buffer is 2.2 mg/g of packing (Table 11) and correspondsto an amount of protein adsorbed equal to 3.6 mg/m2. This is in good agreement with the adsorption of HSA as a monolayer. In all experiments the value of QSobtained after column satu-
ration is about twice as large: it is the upper limit for the assumption of a monolayer formation. The apparent adsorption kinetic constant is about 4-7 times as large in pure buffer as in the presence of acetonitrile (Table 11). For comparison with a first-order rate constant we give in the same table the ratio a,(/ Vo). In a weak mobile-phase eluent (0.1 M ammonium acetate containing 0.05% morpholine) Larew and Walters (9) studied the adsorption of bovine serum albumin on a reversed-phasesupport of 500 A, using the split-peak method for linear elution. For bovine serum albumin a fit-order adsorption rate constant of 0.104 s-* was found. This result is of the same magnitude as the apparent first-order rate constant (ah/V,) measured in this work with the solvent of higher eluent strength. The nature of the eluent used in their work is probably closer to the phosphate buffer used in these experiments, and the kinetic first-order rate constant for diffusion in a porous 500-Asupport is 7 times as low as the apparent first-order rate constant found with the 80-1%support the reversed-phase support. Since the 500-A support has a much larger adsorption capacity, one can conclude that the effective adsorption of HSA on the 80-A support contributes largely to the overall kinetic process. An important decrease of the apparent adsorption rate constant is observed when an organic solvent is added to the buffer (Table II). This indicates the solvatation of the protein by the organic solvent, and this is in agreement with the displacement retention model for proteins in reversed-phase chromatography described by Geng and Regnier (18): one of the equilibria considered in their model is the association of the organic solvent with the free protein. In fact, mixtures of water with low-energy organic solvents such as acetonitrile cause the decrease of the interfacial or hydrophobic interaction energies compared to such interactions in water. This phenomenon is fundamental to the elution step in reversed-phase liquid chromatography (19) and could explain the decrease of the apparent adsorption rate constant in the presence of acetonitrile. It is noticed that the loading capacity does not change with temperature in pure buffer. It increases with temperature, in the presence of acetonitrile. This is true both with capacities obtained from saturation and from model adjustments. A possible explanation is the adsorption of acetonitrile on the support that modifies the structure of the alkyl chains of the reversed phase and therefore the surface available for adsorption: Slaats et al. (20) measured the equilibrium sorption isotherm of acetonitrile from aqueous solutions onto reversed-phase supports and show that there is a maximum in the amount of acetonitrile adsorbed for 40% (v/v). Moreover, the results of Zhu (21) show that acetonitrile adsorption is larger at lower temperatures, but the variation between 10 and 20 "C does not exceed 10%. The variations of 8, observed with temperature or with organic solvent additions could be due to the modification of the reversed-phase support characteristics in the presence of acetonitrile, but the important increase of the support capacity for HSA observed with temperature (Table 111) cannot be totally explained by this effect. More likely, hydration of the protein decreases at higher temperatures in acetonitrile aqueous solutions. This mechanism is described by Van Oss (22) to explain the protein precipitation in water-ethanol mixtures. This effect leaves the protein surface more hydrophobic and increases the protein-protein interactions. Therefore, because of protein self-aasociations,the maximum loading capacity increases with temperature and is much larger at 35 "C than at lower temperatures. The effect of increasing temperature on the apparent adsorption rate constant (Table 111)is not clear, and the values
ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991
lie in the range of experimental errors. Moreover, in the presence of the organic modifier, important variations of the loading capacity as a function of temperature reveal modificationsof the adsorption mechanism. The activation energy that could then be deduced from an Arrhenius plot is not a significative quantity of the kinetic behavior. The study of the split-peak peak effect, with irreversible adsorption, gives information about the state of the protein in solution, but not about its adsorbed state and its possible alteration after desorption. The denaturation of proteins by reversed-phase liquid chromatographywas demonstrated by Karger et al. (23,241. They showed that, under certain experimental conditions, some proteins are eluted in two peaks: the first eluted peak is active and the last one is inactive, because irreversible denaturation can occur after adsorption on the bonded surface. This chromatographic behavior, which shows a splitting of peaks in gradient elution, could have its origin in slow adsorption kinetic effects similar to those described in this work. The activity of the protein after desorption was not checked, but the analysis of the split-peak effect gives information about the association step of free protein with the support.
CONCLUSION The method developed in this work permits the measurement of the number of transfer units and the maximum loading capacities in mass-overload conditions, even if no split-peak effect occurs at low amounts injected. It is easy to perform by successive pulse injections and easy to exploit because the method is based on peak area measurements. Similar experiments can be achieved from the analysis of breakthrough curves (7,8) with models based on the same hypothesis as those used to derive the split-peak expression. This last method accounts for band-broadening effects other than the kinetic ones, such as axial diffusion, eddy dispersion, or extracolumn contributions. This is not the case in frontal analysis, but it is not a serious drawback since the contribution due to axial dispersion is negligible with proteins and the others can usually be neglected in HPLC experiments. Even when using nonporous particles, one cannot exclude the importance of the mass transfers in the stagnant fluid. However the method permits the measurement of the loading capacity of the column relative to the Langmuir adsorption mechanism. Both this parameter and the apparent adsorption
1227
rate constant are useful for a better understanding of the behavior of the protein in solution. This type of experiment in mass-overload conditions can be performed with systems leading to Langmuir type isotherms and therefore to support saturation but cannot be applied to study the adsorption of self-associated proteins. New perspectives can be expected from the application of the method, not only to obtain information about the interaction of free protein with both the support and the solvent but also to understand better the mechanisms of elution of proteins in reversed-phase chromatography.
ACKNOWLEDGMENT We thank C. J. Van Oss for our helpful discussions. LITERATURE CITED (1) Giddlngs, J. C.; Eyrlng, H. J . phvs. Chem. 1955, 59. 416. (2) Sportsman, J. R.; Wilson, 0. S. Anal. Chem. 1980, 5 2 , 2013. (3) Sportsman, J. R.; Lkldll. J. D.; Wilson, G. S. AMI. Chem. 1983, 5 5 ,
771. (4) Hage, D. S.;Welters, R. R.; Hethcote, H. W. Anal. Chem. 1988, 58, 274. (5) Hage, D. S.; Welters, R. R. J . Chrometogr. 1988, 436, 111. (6) Jaulmes. A.; VidaCMedjar, C. AM/. Chem. 1991, 63,1165. (7) Chase, H. A. J . chrometog.1984, 297, 179. (8) Arnold, F. H.; Blench, H. W. J . Ch”?togv. 1988, 355, 13. (9) Larew, L. A.; Waiters. R. R. Anal. Blochem. 1987, 164. 537. (IO) Boisspn, C.; Jozefonvicr, J.; Brash, J. L. Bkmeterlels 1988, 9 , 47. (11) Horvath, C.; Lln, H. J. J . C I K 0 I I y ) t o g . 1978, 126. 401. (12) Horvith, C.; Lin, H. J. J . chrometogr. 1978, 149, 43. (13) Amdd. F. H.; Blanch, H. W.; Wke. C. R. J . Chrmrogr. 1985, 330, 159. (14) Wade, J. L.; Bergold, A. F.; Can, P. W. Anal. Chem. 1987. 5 9 , 1286. (15) Frk” R. P.: LevDddt. J. K.: bell. . . S.: Henderson. L. W. Anal. Chem. l o b , 55. 1349:’ (16) Schmkk D. E.; Qleuese. R. W.; Conron, D.; Karger, B. L. AM/. Chem.
inno. - - - - , 52 - - , 177 .. . .
(17) Soderqulst, M. E.; Waiton, A. G. J . CdW Interface Scl. 1980, 75, 386. (18) a n g , X.; Regnier. F. E. J . chrometugr. 1984, 296, 111. (19) Van O s , C. J. Israel J . Chem. 1990, 30, 251. (20) slaats, E. H.; Markovskl, W.; Fekete, J.; Poppe, H. J . Chromatog. 1981, 207, 99. (21) Zhu, P. L. Chromefogrephie 1985, 7 , 425. (22) Van Oss, C. J. J . Rotein Chem. 1989, 8 . 661. (23) &hen. S.A.; Benedek, K. P.;Dong, S.; Tapuhl, Y.; Karger, B. L. Anal. Chem. 1984. 5 6 , 217. (24) Benedek, K.; Dong, S.; Karger, B. L. J . chromerogr. 1984, 317, 227.
RE”
for review August 7,1990. Accepted March 25,1991. One of the authors was supported by a fellowship from the Soci6t6 FranGaise Chromato Colonne (Neuilly-Plaisance, France).
