ANALYTICAL SCIENCES MAY 2000, VOL.

16
2000 The Japan Society for Analytical Chemistry

537

Notes

The Performance of the Benesi-Hildebrand Method in Measuring the Binding

Constants of the Cyclodextrin Complexation
Chao YANG,* Lei LIU,*,** Ting-Wei MU,* and Qing-Xiang GUO*
*Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China
**Department of Chemistry, Columbia University, New York, NY 10027, USA

(Received November 22, 1999; Accepted March 13, 2000)

Binding constants are fundamentally important to supramolecular

They are frequently measured with various
chemistry.1
spectroscopic methods2 based on the Benesi-Hildebrand (BH)
regression.3 However, the reliability of the BH method has
often been questioned,4 and the binding constants measured
with it were often controversial.5
Our previous experience of using the BH method610 prompted
us to examine in detail the sources and magnitude of its
inaccuracies in measuring binding constants, especially those of
cyclodextrin (CD) complexation. Such a study will surely
benefit the future research, though surprisingly it has not been
conducted before.

Experimental
The basic approach of the present study was computer
simulation. The computer program, written in Borland C++ 5.0,
was run on a PII400 computer. CD complexation was investigated
as a typical system, for CD was one of the most frequently
encountered host molecules in the field of molecular recognition.
In the simulation, the real concentration of the substrate was
chosen as 1.00104 M for all the solutions. The real binding
constant (K) was chosen as 50, 1000 or 10000 M1. The molar
absorption constant of the free substrate (0) was taken as 5000
m2/mol, while the molar absorption constant of the bound
substrate () was chosen as 4950, 4850, or 4750 m2/mol,
respectively. Seven different real CD concentrations were used:
0.0000, 0.0025, 0.0040, 0.0055, 0.0070, 0.0085 and 0.010 M.
Herein, the absorption of CD was presumably zero. Three
replicate solutions were prepared at every CD concentration,
and their absorption values were averaged to offer the
absorption value at the given CD concentration. It was
noteworthy that the present selections of host and guest
concentrations for simulation were reasonable, for they were
based on the practice in real experiments.
Only the random errors which arise from preparing the
solutions and from measuring the absorption values were
considered. Systematic errors, which could and should be
avoided, were assumed to be zero. Thus, the final absorption
values taken by the computer to calculate the binding constants
were not determined merely by the real host and guest
concentrations but by the influence of the random errors as well.
To whom correspondence should be addressed.
E-mail: leiliu@chem.columbia.edu

The random error from preparing the solutions was assumed to

obey the normal error curve model, i.e.
y=

(x )2
1
exp
2 2
2

Here, was the real concentration of the given solution, while x

was the concentration prepared actually. was for the standard
deviation, which was chosen as 0.003 here. y was the relative
frequency with which random sampling of the infinite
population would bring about a particular concentration x.
Similarly, the random error from measuring the absorption
value was also assumed to obey the normal error model,
wherein was chosen as 0.002. It was noteworthy that a
relative error of 0.3% from preparing the solutions and a
relative error of 0.2% from measuring the absorption values
were typical and reasonable for analytical experiments.
One million rounds of simulations were performed for every
case. In every round, when the computer generated the final
absorption values of the solutions, a test was performed to
determine whether the correlation coefficient (r) of the regression
was larger than a criterion. If it was, the round of the simulation
was accepted as a successful experiment. After one million
rounds, the relative error in the binding constants (K/K) and
the success rate (the ratio of the number of successful round (N)
to one million) were obtained. (See Chart 1)
Chart 1
Simulation Algorithm ( )
{ Summation_K=0; N=0;
For i=1 to 1000000
{ Generate the absorption values of the solutions;
Calculate r using the present absorption values;
If r > criterion
{ Calculate Ki according to the BH method;
Summation_K=abs (KiK); N=N+1;
}
}
K/K=(Summation_K/N)/K; Success_Rate=N/1000000;
}

Results and Discussion

Benesi-Hildebrand method
Generally, CD forms 1:1 complexes with the substrate.5 The

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ANALYTICAL SCIENCES MAY 2000, VOL. 16

Table 1

The relative errors of the BH method in measuring the binding constants of CD complexation

= 0
(m2/mol)
50

150

250

Table 2

K
(M1)

0.90

0.91

0.92

50
1000
10000
50
1000
10000
50
1000
10000

1.722
0.291
0.842
0.860
0.113
0.457
0.518
0.069
0.275

1.674
0.296
0.842
0.856
0.113
0.462
0.518
0.069
0.280

1.612
0.298
0.844
0.852
0.112
0.463
0.520
0.069
0.284

Criterion in correlation coefficient

0.93
0.94
0.95
0.96
1.548
0.305
0.844
0.842
0.112
0.469
0.518
0.069
0.288

1.512
0.308
0.845
0.832
0.112
0.472
0.516
0.069
0.294

1.456
0.316
0.844
0.814
0.114
0.479
0.516
0.069
0.296

1.404
0.323
0.853
0.788
0.111
0.483
0.512
0.069
0.303

0.97

0.98

0.99

1.350
0.327
0.859
0.748
0.111
0.487
0.506
0.069
0.310

1.294
0.339
0.853
0.694
0.111
0.472
0.490
0.069
0.315

1.172
0.340
0.845
0.612
0.114
0.503
0.438
0.069
0.312

The success rates of the BH method in measuring the binding constants of CD complexation

= 0
(m2/mol)

50

150

250

K
(M1)

0.90

0.91

0.92

Criterion in correlation coefficient

0.93
0.94
0.95
0.96

0.97

0.98

0.99

50
1000
10000
50
1000
10000
50
1000
10000

0.47
0.44
0.02
0.98
1.00
0.09
1.00
1.00
0.26

0.42
0.40
0.02
0.98
1.00
0.07
1.00
1.00
0.22

0.38
0.35
0.02
0.97
1.00
0.06
1.00
1.00
0.19

0.33
0.30
0.01
0.96
1.00
0.05
1.00
1.00
0.16

0.13
0.09
<0.01
0.81
0.88
0.01
0.98
1.00
0.04

0.07
0.05
<0.01
0.68
0.71
<0.01
0.94
0.99
0.02

0.02
0.01
<0.01
0.42
0.36
<0.01
0.79
0.89
0.01

binding constant is
Xi
[SCD]
K = =
(CCDCSXi)(1Xi)
[CD][S]

(1)

in which [SCD], [CD] and [S] represent the equilibrium

concentrations of the complexed substrate, free CD and free
substrate, respectively. CCD and CS stand for the analytical
concentrations of the CD and substrate, and Xi=[SCD]/CS.
Suppose that the molar absorption constants of the free and
bound substrate at certain wavelength are 0 and , respectively
(herein, the absorption of CD is usually taken as zero5). Thus,
when CCD=0, the absorption of the solution is A0=0lCS, in which
l is the length of the cell. If the analytical concentration of CD
i
, the apparent absorption of the solution is Ai=
is CCD
0lCS(1Xi)+lCSXi.
Suppose that CS is constant, then
Ai=A0+AXi, in which A=(0)lCS.
i
CS, the following equation can be obtained:
If CCD

i
A
1
(CCD
CS Xi)(1Xi)
i (1Xi)
i
1
= CCD
= CCD
=
Ai
Xi
K
Xi

(2)

where Ai=AiA0=AXi. Rearranging Eq.(2) gives

1
1
1
1
= +
i
Ai
A
AK CCD

(3)

i
Hence, plotting 1/Ai vs. 1/CCD
gives a slope of 1/(AK) and an
intercept of 1/A. The ratio of the intercept to the slope can be
taken as an estimation of the binding constant K. This is the BH
method.

Performance of the Benesi-Hildebrand method in cyclodextrin

chemistry
The relative errors and success rates of the BH method in

0.28
0.25
0.01
0.95
0.99
0.04
1.00
1.00
0.12

0.23
0.19
<0.01
0.92
0.98
0.03
1.00
1.00
0.09

0.18
0.14
<0.01
0.88
0.95
0.02
0.99
1.00
0.06

measuring the binding constants of CD complexation are

summarized in Tables 1 and 2, respectively.
From Table 1, it can be seen that the relative error of the BH
method is usually over 30% regardless of how high the
correlation coefficient can be. The only exception is when K =
1000 M1 and || 150 m2/mol. This explains why the
difference between the binding constants reported in the
literature for the same substrate is often significant, though the
reported correlation coefficients are usually excellent. This also
explains why a reproducible binding constant is often hard to
obtain with the BH method if the complexation is too weak or
too strong, or if the absorption change is too small.
Interestingly, it can be also seen that the selection of the
criterion does not significantly affect the relative error in
measuring the binding constants.
A higher correlation
coefficient does not necessarily implies a lower relative error in
the experiments, if only the random errors exist. This is
understandable, for the errors of the measured absorption values
from the real ones can possibly be correlated in such a way so
that good linearity of the BH regression is not spoiled. Thus,
the operator is not able to discern the errors and will simply
accept the calculated binding constant.
However, this
calculated binding constant is very inaccurate or even
erroneous, for it is well known that the binding constants from
the BH method are very sensitive to the intercept or slope of the
regression.11,12 Admittedly, the possibility of the occurrence of
such correlated errors is not high. However, any such
occurrence will result in a very inaccurate binding constant,
which will make the overall average relative error high.
Therefore, care should be given to the BH method regardless
of how high the correlation coefficient might be.13 Sometimes,
a very good linearity can also give an erroneous binding
constant. On the other hand, a sufficiently high correlation
coefficient is still needed in practice, for a low correlation
coefficient is more possibly due to systematic errors or due to

ANALYTICAL SCIENCES MAY 2000, VOL. 16

improper operations. Practically, a correlation coefficient of at
least 0.98 is recommended.
From Table 2, it can be seen that the selection of criterion
significantly affects the success rate of the experiment. The
higher the correlation coefficient, the lower the success rate.
When the correlation coefficient is 0.98, the success rates are
usually badly low. Only when the absorption change is
sufficiently large (i.e. || 150 m2/mol) and the complexation
is not strong (i.e. K 1000 M1), the success rate is fair. This
agrees with the experience in experiment, for an acceptably high
correlation coefficient is usually hard to obtain if the absorption
change is small or if the complexation is strong. This also
indicates that sometimes the requirement of a high correlation
coefficient is actually impractical.

Conclusion
The relative error of the BH method in measuring the binding
constants of CD complexation is usually high, while its success
rate is often low. The BH method is only recommendable when
the complexation is modest (i.e. K 1000 M1 for CD
complexation) and the absorption change is sufficiently large
(i.e. || 150 m2/mol for CD complexation). Under other
conditions, certain nonlinear methods are more desirable.1315
For other host-guest systems, similar computer simulations
were also performed. The only differences were the ranges of
the variations in the host concentrations and in the guest molar
absorptions. The results are basically the same, namely, a high
correlation coefficient does not ensure that the calculated
binding constant is accurate in the BH method; and the BH
method is only recommended when the complexation is
modestly strong and the absorption change is sufficiently large.

Acknowledgements
We are grateful to NSFC for the financial support. Encouragement

539
and insights from Professor R. Breslow of Columbia University
are highly appreciated.

References
1. K. A. Connors, Binding Constants: The Measurement of
Molecular Complex Stability, 1987, Wiley, New York.
2. H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc.,
1949, 71, 2703.
3. M. V. Rekharsky and Y. Inoue, Chem. Rev., 1998, 98, 1875.
4. S. M. Hoenigman and C. E. Evans, Anal. Chem., 1996, 68,
3274.
5. K. A. Connors, J. Pharm. Sci., 1995, 84, 843.
6. Q.-X. Guo, Z.-Z. Li, T. Ren, X.-Q. Zhu, and Y.-C. Liu, J.
Inclusion Phenom., 1994, 17, 149.
7. Q.-X. Guo, T. Ren, Y.-P. Fang, and Y.-C. Liu, J. Inclusion
Phenom., 1995, 22, 251.
8. Q.-X. Guo, X.-Q. Zheng, X.-Q. Ruan, S.-H. Luo, and Y.-C.
Liu, J. Inclusion Phenom., 1996, 26, 175.
9. Q.-X. Guo, S.-H. Luo and Y.-C. Liu, J. Inclusion Phenom.,
1998, 30, 173.
10. X.-Q. Zheng, X.-Q. Ruan, W. Wang, H.-M. Zhang, Q.-X.
Guo, and Y.-C. Liu, Bull. Chem. Soc. Jpn., 1999, 72, 253.
11. N. J. Rose and R. S. Drago, J. Am. Chem. Soc., 1959, 81,
6138.
12. B. K. Seal, H. Sil, and D. C. Mukherjee, Spectrochim. Acta,
1982, 38A, 289.
13. O. Exner, Chemom. Intell. Lab. Syst., 1997, 39, 85.
14. D. Salvatierra, C. Diez, and C. Jaime, J. Inclusion Phenom.,
1997, 27, 215.
15. G. Pistolis and A. Malliaris, Chem. Phys. Lett., 1999, 310,
501.