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REACTIONS AND EFFECT OF ADDITIVES IN ORGANIZED MEDIA
ABSTRACT
' THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Bottor of PhiIo£(opl)p IN
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CHEMISTRY / I
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WASEEFA F A T M A
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DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2006
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Organized media such as micelles, microemulsions, liposomes, etc.,
are compartmentalized liquids, which may show special performance toward
reaction equilibria and reaction dynamics. Chemical and biochemical
reactions have been reported to be fairly as well as extraordinarily influenced
under compartmentalized conditions. Micelle forming surfactants or surface-
active agents are essentially chemical compounds with structural features that
impart special characteristics enabling them to accumulate at the interface,
modify interactions therein and perform various novel functions. An
understanding of the fate and behavior of reagents in this modified
environment provides valuable models for understanding the role of micro-
environmental factors, which affect the rates, products, and (in some cases)
the stereochemistry of reactions. Thus, studies in surfactant organized media
can provide mechanistic details about hydrophobic and electrostatic
influences on reactions and also on biological electron transfers which take
place on membrane surfaces or at protein-substrate interfaces
Self-organized systems constituted of amphiphilic molecules
(surfactants being one of them) have some particularities which make them
attractive not only for chemical reactivity but for physico-chemical studies as
well as for a large variety of industrial developments.'
Ninhydrin reactions using manual and automated techniques as well as
ninhydrin spray reagents are widely used to analyze and characterize amino
acids, peptides, and proteins as well as numerous other ninhydrin-positive
compounds in biomedical, clinical, food, forensic, histochemical,
microbiological, nutritional, and plant studies. The reaction is also used to
develop the latent finger prints.
It is well known that the amino acid-ninhydrin reactions proceed
through the formation of a Schiff base (unstable for isolation) and a series of
reactions, involving different intermediates, up to the formation of
Ruhemann's purple. The Schiff base undergoes decarboxylation and
hydrolysis to yield 2-amino-indanedione(A) as an intermediate (Scheme 1). A
acts as a reactant in the formation of ammonia and Ruhemann 's purple and
the two reactions (i.e., route(i) -hydrolysis and route(ii) -condensation) occur
simultaneously. These two reactions depend strongly upon the pH,
atmospheric oxygen, and temperature. A is highly sensitive to molecular
oxygen and a yellowish colored product is formed (instead of Ruhemann's
purple) in the presence of atmospheric oxygen. At low pH, the reaction has
been found to proceed chiefly by route(i) and ammonia is evolved almost
quantitatively with no Ruhemann'spurple formation. In solutions of pH > 5.0,
route(ii) predominates but the possibility of route(i) can not be ruled out
completely.
K: al R-CH-COOH , R - C H - C O O - + H^
+ NH3 +
^a2 R - C H - C O O - ^ R - C H - C O O - + H*
NH2 NH3 +
R - C H - C O O "
NH3 +
KD R - C H - C O O H
NH2
O + R - C H - C O O H
NH2
fast NH2 + CO2 + RCHO
^ H .
(C)
+ NH3
(Hydrindantin)
0]^H.O^
(Ruhemann's purple)
Scheme 1
Due to Schiff base formation between the carbonyl group of ninhydrin
and the amine functionality, the reactive species towards nucieophilic attack
on the >C=0 group of ninhydrin is RGH(NH2)C00H, which is in equiUbrium
with the zwitterionic form of amino acid.^
Due to its importance and so widely used organic reaction, a number of
researchers have modified the ninhydrin reagent by introducing organic
solvents, metal ions, reducing agents, etc., to the reaction mixture. The
objective of the modifications were in order to (i) enhance the stability of the
color; (ii) obtain reproducible results; (iii) to lower the detection limit, and
(iv) to identify the intermediates formed during the reaction.
In this direction the present work of kinetic studies on ninhydrin-amino
acid reactions in micellar organized media was undertaken with a view to find
some applications to improve contrast and visualization that may prove a step
forward from the methods already in use. For the purpose conventional as
well as gemini micellar systems were used. The latter is a special class of
surfactants which have some intriguing properties'* in comparison to
conventional surfactants. Even a cursory look at the recent literature^ will
convince that the tempo of research in the field of gemini surfactants is very
high and all signals indicate that this heightened interest will continue. ' H
NMR studies have, therefore, been carried out to investigate the micellar
morphology and location of the solubilization site of additives (sodium
anthranilate and sodium benzoate) in gemini surfactants.
The work in the thesis has been divided into five chapters, namely; (i)
Chapter-I: General Introduction; (ii) Chapter-II: Experimental; (iii)
Chapter-Ill: (A) Ninhydrin - DL-valine reaction in presence of CTAB and
effect of organic solvents, (B) Ninhydrin-DL-tryptophan reaction in presence
of TX-lOO and effect of organic solvents; (iv) Chapter-IV: Ninhydrin - DL-
tryptophan reaction in presence of gemini surfactants and ' H N M R
investigation at different concentrations of gemini surfactants; and (v)
Chapter-V: 'tl NMR study of gemini surfactants in presence of additives
(sodium anthranilate and sodium benzoate).
Chapter-I comprises of an introduction of surfactants and surfactant
organized assemblies, effect of additives, pseudophase model of micellar
catalysis, ninhydrin-amino acid reaction mechanism in aqueous medium and
statement of the problem. Chapter-I also includes pertinent literature survey
of the work performed on the kinetic and mechanistic studies of ninhydrin-
amino acid reaction in aqueous medium.
Chapter-II contains experimental details. The source and purity of various
reactants and surfactants are mentioned in this chapter. Synthesis of gemini
surfactants and their characterization by C, H, N analysis, ' H NMR, mass and
infra red (IR) spectroscopy are also given in this chapter. Procedure for the
preparation of solutions, pH measurements, kinetic measurements, viscosity
measurements, determination of cmc, etc. have been detailed.
Chapter-Ill: (A) Kinetics of the ninhydrin-DL-valine reaction has been
studied spectrophotometrically under varying conditions of [CTAB],
[ninhydrin], [DL-valine], pH, temperature and %(v/v) organic solvents
(solvents used: dimethyl sulfoxide, acetonitrile, methylcellosolve and 1-
propanol). The same mechanism (Scheme 1) has been found to be followed in
our case in the micellar media.
Application of pseudo-phase model for micelle-catalyzed reaction give
the rate constant as
where km is the second-order rate constant in micellar pseudophase. M^N
represents the mole-ratio of micellized ninhydrin, K\/ is the binding constant
of substrate with micelles and [Sn] is the total surfactant less than that of
monomer, i.e., [Sn] = [surfactant]! - cmc. Values of k^ and Ky were obtained
using a computer based program.
Leffler and Grunwald^ have pointed out that many reactions show
isokinetic relationship; AH*= C + BAS". We also obtained fairly linear AH*
vs. AS plot for the formation of Ruhemann's purple by the reaction of
ninhydrin with or-amino acids (Fig. 1). The present AH* and AS* data fit very
well on the straight line which indicates that the kinetics of the reaction of
ninhydrin with each amino acid (including the present one) follow the same
reaction mechanism in the presence of CTAB micelles.
100 -
•240 - 2 0 0 - 8 0 - 4 0 -160 -120
AS*(JK-''moC-''l
Fig. 1: Isokinetic relationship, AH* vs. AS*, for the reaction of ninhydrin with
a-amino acids. Reaction conditions: [CTAB] = 20.0 x 10" mol dm'\
[ninhydrin] = 5.0 x 10' mol dm'^ pH = 5.0, [Asp] = 1.0 x lO"'' mol dm"\ [Leu]
= 1.0 X 10"* mol dm•^ [Lys] = 1.0 x 10 ^ mol dm'^ [Phe] = 1.0 x 10 ^ mol dm•
[Glu] = 1.0 X 10- mol dm-\ [Gly] = l.O x 10"* mol dm'^ [Thr] = 2.0 x 10"* mol
dm-^ [Trp] = 1.0 x 10"* mol dm• [Ala] = 3.0 x 10"* mol dm'^ [Val] = 3.0 x
10''* mol dm' (Present work).
5)^ i^
The observations obtained in the presence of surfactant (CTAB)
suggest that the side chain of a-amino acids plays an important role: the
reaction rate increases with the hydrophobicity of the side-chain in the order
for tryptophan, phenylalanine, leucine, valine and glycine.^
%X^ > Qy^' > J„>H-CH.->^'"VcH- >H-H
As regards the ninhydrin-DL-valine reaction in different solvents, the
behavior can be interpreted in terms of solvent interaction with water and its
possible influence on solvophobic forces operating for micellization.
Effect of CTAB micelles has also been seen in the presence of
different fixed compositions of DMSO. Rate profiles obtained for the
solutions with 10-70% DMSO by volume exhibited clear maxima that shifted
to higher concentration of surfactant as a fiinction of DMSO content.
The initial increase of k^^ in k^ vs. [CTAB] profiles seems due to
combined effect of micelles as well as blocking of side reaction (arrest of
hydrolysis). As the presence of DMSO also arrests the CTAB micellization,
the above effects would compete in deciding the k^ values at a particular
DMSO volume %. This may be the reason of shifting maxima with increasing
volume % of DMSO to higher [CTAB]. However, at still higher concentration
10
of DMSO (70%), where no micelles are present, the reaction rate increase is
seemingly due to the solvent's own catalytic effect.
(B) A drawback in all the studies of ninhydrin reactions had been the
necessity of heating.^'' In presence of non-ionic TX-lOO micelles, color
development is found at a much lower temperature (40 °C). The work detailed
herein embodies these results where kinetics and mechanism of ninhydrin-
tryptophan reaction are described (the reaction does not take place at all in
pure aqueous solution at 40 °C under the conditions used). Effect of organic
solvents (MC, DMSO, AN and PrOH) on the rate of Ruhemann 's purple
formation was also studied.
Again, the pseudo-phase model was applicable. The catalytic role of
TX-lOO micelles clearly suggests the association/incorporation of ninhydrin
and tryptophan into the TX-lOO micelles. The polyoxyethylene chain of TX-
lOO micelles are polar in nature due to the presence of ether and primary
alcoholic linkages. These two functional groups ( - 0 - and -OH) play an
important role for the association of ninhydrin and tryptophan into the polar
headgroup region of TX-lOO through hydrogen bonding. Therefore, the
associated ninhydrin and tryptophan with TX-lOO micelles (through hydrogen
bonding) seem responsible of facilitating formation of Schiff base.
The observed catalytic role of solvents is due to diminished hydrolysis
of the amine. The increase in the content of solvents % (v/v) is expected to
decrease the content of water, which, in turn decreases the hydrolysis of
11
amine. As a result, the rate of the reaction is increased. Secondly, the
solubility of Ruhemann's purple is less in water in comparison to organic
solvents. Thus, blocking side reaction(s) (mainly hydrolysis) and higher
solubility of the product play important roles in presence of organic solvents.
The positive catalytic effect of organic solvents is also looked upon
from the view point of relative participation of water and organic solvent in
acid-base equilibria and hydrogen bonding. It has been established that pH of
the medium and pA[a2-values of amino acids are responsible for the rate
enhancement of amino acid-ninhydrin reactions in non-aqueous organic
o
solvents and amino acid anions are the reactive species. As the [anion] is
negligible in the vicinity of pH 5.0 (the maximum pH for ninhydrin reaction),
the neutral form of amino acid is the main reactive species under such type of
circumstances. In order to confirm the hypothesis presented by Friedman,^
i.e., rate enhancement being directly related to the pATai value of amino acid,
results of experiments performed in 65% pH 5 buffer + 35% organic solvent
are summarized in Table 1. The observations indicate that pH of the working
solution does not show any drastic change in presence of the solvents.
12
Table 1
pH and the observed pseudo-first-order rate constants (k^) for the reaction of
DL-tryptophan with ninhydrin reaction carried out in different solvents (35%
v/v).
Reaction conditions: [DL-tryptophan]n
[ninhydrinji
[TX-100]T
S.OxloVoldm-^
7.0xl0"^moldm"^
50.0xlO-^moldm"^
Temperature 40 °C
Reaction Medium pH 10';tv,(s-')
Buffer (CH3COOH + CHsCOONa)
65% pH 5.0 buffer + 35% PrOH
65% pH 5.0 buffer + 35% MC
65% pH 5.0 buffer + 35% AN
65% pH 5.0 buffer + 35% DMSO
5.0
5.48
5.58
5.89
6.08
8.3
20.0
20.7
24.6
24.6
13
Chapter-IV: No purple color developed with [ninhydrin] = 5.0 x 10" mol
dm' and [tryptophan] = 1.0 x 10'" mol dm" at pH = 5.0 and temp. = 70 °C
either in pure aqueous or [CTAB] = 20.0 x 10" mol dm"'' (however, the
reaction did occur at [CTAB] > 5.0 x 10" mol dm' and it had been studied in
detail^). With the l6-m-16 geminis the reaction occurred at a surfactant
concentration as low as 20.0 x 10" mol dm'^; therefore, detailed kinetic
investigations were made with the three geminis (m = 4, 5, 6).
Fig. 2 shows the variation of k,^ with surfactant concentrations. With
conventional surfactants, ky had been found to increase monotonically and
after the substrates completely bind the micelles, it attained constant values
(for monomolecular reactions) or passed through a maximum (for bimolecular
reactions) with increasing [surfactant].'° In the present case, however, with
the gemini surfactants only, k^ first increases with surfactant concentration
(part I), remains constant upto certain concentration (part II - parts I and II
behavior is akin to conventional surfactant micelles), and then again increases
(part III). In part I, at concentrations lower than the cmc, /r should remain
constant. The observed catalytic effect may, therefore, be due to (i) presence
of premicelles and/or (ii) preponement of micellization by reactants (as is also
confirmed by cmc decrease at reaction conditions, see Experimental).
Whereas no reaction has been observed in range II with conventional
surfactant (CTAB),^ ^^ remains constant upto 40.0 x lO''' mol dm" for gemini
surfactants. This undoubtedly shows better catalyzing power of the gemini
surfactants over the corresponding single chain surfactants. This could be due
14
o
150
Fig. 2 : Variation of k^ for the reaction of DL-tryptophan (1.0x10"* mol dm" ) with ninhydrin (5.0x10' mol dm" ) in the presence of 16-m-16 gemini micelles at 70 °C. m = 6 (3), 5 ( • ) , 4 (O).
15
to the presence of spacer in the geminis which decreases the water content in
the aggregates making the environment less polar and thus causing rate
increases (see Scheme 1 - route(i) may get suppressed).
The behavior in part II is same for all the three geminis but values of
y at all concentrations are in the order :m = 4 > 5 > 6 . Itis well known that,
to minimize its contact with water, a spacer longer than the 'equilibrium'
+
distance between two -NMe^ headgroups (the 'equilibrium' distance occurs
at m = 4 in 16-m-16 geminis) tends to loop towards the micellar interior."''^
This increased looping of the spacer (for m > 4) will progressively make the
Stem layer more wet (in comparison with the m = 4 gemini) with a resultant
decrease in k^. Thus the results are consonant with the earlier findings that
increase in the water content of the reaction environment has an inhibiting
effect. -'
The results of part III are most interesting; instead of k^ remaining
constant, it increases (though slowly) in the surfactant concentration range
40.0 X 10"''-300.0 X lO"'* mol dm"l After leveling-off, fijrther increase at
higher [gemini] is probably associated with a change of micellar structure.
That structural changes indeed occur at higher [gemini] are confirmed by
examining the ' H N M R spectra of the surfactants.
Chapter-V: Effect of addition of sodium anthranilate and sodium benzoate to
16-m-16 gemini surfactants is investigated by viscosity and ' H NMR at
16
25 °C. The solubilzation site of anthranilate and benzoate anions near tiie
micellar surface is inferred.
The results suggest that the presence of the organic anions in the micellar
solutions of 16-m-16 causes a change in micellar morphology. The
intercalation of An" ion into the micellar surface region (due to electrostatic
interactions with the oppositely charged surfactant headgroups) seems the
prime cause of inducing such morphological changes. Additionally, as NaAn
has a NH2 group attached to 2-position, it would prefer to remain in more
polar environment (than Ben" ion) due to its polar nature and the geometric
hindrance. Therefore, site of solubilization of An" has direct links to the
overall changes. In case of Ben" ion, which is solubilized deeper into the outer
core region of the micellar interior, more concentration is needed than An" to
produce the similar results.
17
References;
1. "Micelles, Topics in Current Chemistry", B. Lindman, H.
Wennerstrom and H. F. Eicke (Eds.), Springer-Verlag, Berlin, Vol.
87, 1980.
2. M. M. Joullie, T. R. Thompson and N. H. Nemeroff, Tetrahedron,
47, 8791 (1991).
3. Kabir-ud-Din, J. K. J. Salem, S. Kumar, M. Z. A. Rafiquee and Z. Khan,
J. Colloid Interface Sci., 213, 20 (1999).
4. R. Zana and Y. Talmon, Nature, 362, 228 (1993).
5. J. Yang, Curr. Opin. Colloid Interface Sci., 7, 276 (2002).
6. L. Leffler and E. Grunwald, "Rates and Equilibria of Organic
Reactions ", Wiley, New York, 1963.
7. J. Almog, in "Advances in Fingerprint Technology", H. Lee and R.
E. Gaensslen (Eds.), Elsevier Science, New York, 1991.
8. M. Friedman, J. Am. Chem. Soc, 89,4709 (1967).
9. Kabir-ud-Din, J. K. J. Salem, S. Kumar and Z. Khan, J. Colloid
Interface Sci., 215, 9 (1999).
10. C. A. Bunton, Cat. Rev. Sci. Eng., 20, 1 (1979).
11. S. De, V. K. Aswal, P. S. Goyal and S. Bhattacharya, J. Phys.
Chem.,lOQ, 11664(1996).
12. S. Karabomi, K. Esselink, P. A. J. Hilbers, B. Smit, J. Karthauser,
N. M. van Os and R. Zana, Science, 266, 254 (1994).
13. Kabir-ud-Din, U. S. Siddiqui, and Z. Khan, J. Surface Sci.
Technoi, 19, 101 (2003).
REACTIONS AND EFFECT OF ADDITIVES IN ORGANIZED MEDIA
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Boctor of Pbilo^optip
C H E M I S T R Y
OV—«.j
BY
W A S E E F A F A T M A
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DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2006
f-< (JA
5 A JAN >OtV
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T6492
rJjedicated
to
cJLovinanlemoru oj^
loLatel icjazi J4a6an ^^liaf
-.c^*^ ^ ^ s -
PROF. KABIR-UD-DIN CHAIRMAN DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (U. P.) INDIA. E-mail: [email protected]
0571-2703515 0571 - 2700920 Extn. 3353 (lab), 3351 (O)
Dated:...Ok.^^^,l.Cf\>^
Certificate
This is to certify that the thesis entitled "Reactions and Effect of
Additives in Organized Media" is the original work carried out by
Ms. Waseefa Fatma under my supervision and is suitable for submission
for the award of Ph.D. degree in Chemistry.
(Prof. Kabir-ud-Din)
ACKNOWLEDGEMENTS
In the name of Allah the most gracious, the most merciful, all praise
is for ALLAH who enables me to complete this work.
I have no words to express the gratitude, which I owe to my research guide,
Prof. Kabir-ud-Din, Chairman, Department of Chemistry, Aligarh Muslim
University, Aligarh, a legendary figure and a scientist of his own kind. He has
been a source of inspiration for me and never let me down. His commitment
to research, punctuality, integrity and probity has made him an ideal for me.
His enlightened guidance, continuous encouragement, sagacious advice and
paternal affection throughout the period of these investigations, which have
enabled me to execute this research work.
I am extremely thankful to Prof. Wajid Husain for his valuable suggestions
and all time support which can not be expressed in words.
I also thanks Dr. Zaheer Khan, and Dr. Sanjeev Kumar for their help and
cooperation at every stage.
I thankfully acknowledge the help offered to me by Dr. Mohd. Akram,
Dr. Andleeb Zehra Naqvi, Dr. Damyanti Sharma, Dr. Ziya Ahmad Khan, Mr.
S.M. Shakeel Iqubal, Miss.Nahid Perveen and Dr. Daksha Sharma.
My loving thanks and best wishes to my laboratory colleagues, Ms.Umme
Salma, Mr. Mohd. Sajid Ali, Miss Nuzhat Gull, Mr. Md. Sayem Alam, Mr.
Tanweer Ahmad, Miss Neelam Hazoor Zaidi, Ms Suraiya, Mr. Md. Altaf and
Mr. Naved Azam.
I owe my thanks to my sisters-in-law namely Dr. Shema Wasi and Dr. Samina
Wasi for their constant support and encouragement.
I will be failing in my duty if 1 will not acknowledge the sincere and
unprecedented cooperation extended to me by my husband, Mohd Shariq
Wasi. Research in any discipline unfolds untold problems for a researcher. I
shared all the problems with my husband that not only eased out my tension,
but got ready made solutions.
I specially thank my room partner, Miss Asra Kidwai, who always stood by
me whenever I needed her and friends Ms. Sabeena, Ms. Shaista, Ms.
Kahkashan and Ms. Gull Rana.
My humble gratitude must also be reserved for my mother and maternal
uncle, for their motivation and encouragement during my whole academic
period.
My sister, Faseeha Fatma and brothers namely, Qazi Abdul Nasir and Qazi
Husain Altaf who provided me all possible help and co-operation and
inculcated in me the sense of patience and appreciation of niceties of the
present competitive world where excuses no more earn laurels.
I am also thankful to Mr. Zaheer Ahmad, Limra Computers for carrying out
important type setting of the thesis.
Financial support for the work provided by UP-CST, Lucknow in the form of
Research Assistant is thankfully acknowledged and thanks are due to SIAF
for providing C, H, N analysis data, ' H N M R and mass spectra. Last, but not
the least, I have no suitable words to convey my thanks to all my teachers,
who sculptured and enabled me to achieve what I wish.
(Waseefa Fatma)
LIST OF PUBLICATIONS
1. Micelle Catalysed Reaction of Ninhydrin and DL-Tryptophan. Kabir-ud-Din and Waseefa Fatma J. Surface Sci. TechnoL, 18, 129-138 (2002).
2. Effect of Organic Solvents (Methylcellosolve, Dimethylsulfoxide, Acetonitrile and 1-Propanol) on the Tryptophan-Ninhydrin Reaction -A Kinetic Approach. Kabir-ud-Din, Waseefa Fatma and Zaheer Khan J. Indian Chem. Soc, 82, 811-813 (2005).
3. Micelle Catalyzed Reaction of Ninhydrin with DL-Valine in the Absence and Presence of Organic Solvents. Kabir-ud-Din, Waseefa Fatma and Zaheer Khan Int. J. Chem. Kinet., 38, 634-642 (2006).
4. A 'H N M R Study of 1,4-Bis(N-hexadecyl-N, N-dimethylammonium) Butane Dibromide / Sodium Anthranilate System: Spherical to Rod-Shaped Transition. Kabir-ud-Din, Waseefa Fatma and Ziya Ahmad Khan ColloidPolym. Sci., (2006) (Published online).
5. Role of Cationic Gemini Surfactants toward Enhanced Ninhydrin-Tryptophan Reaction. Kabir-ud-Din and Waseefa Fatma J. Phys. Org. Chem., (communicated).
. ^ ^ *
LIST OF PAPERS PRESENTED A T CONFERENCES
1. Micelle Catalysed Reaction of Ninhydrin with DL-Valine in the Absence and Presence of Organic Solvents. Kabir-ud-Din, Waseefa Fatma and Zaheer Khan XI Biennial National Conference on Surfactants, Emulsions and Biocolloids (NATCOSEB-XI), Mumbai, Dec. 11-13, 2003.
2. Effect of Additives on Micellization of Gemini Surfactants. Kabir-ud-Din, Waseefa Fatma and Zaheer Khan International Conference on Soft Matter, Kolkata, Nov. 18-20, 2004.
3. Structural Studies on Gemini Micelles. Kabir-ud-Din, Waseefa Fatma, Umme Salma Siddiqui and Sanjeev Kumar 7"" CRSINational Symposium in Chemistry, Kolkata, Feb. 4-6, 2005.
CONTENTS
Chapter-I General Introduction
A. Surfactants and Surfactant Organized Assemblies
B. Effect of Additives on Structural Transitions
C. Reactions in Organized Media
D. The Ninhydrin -Amino Acid Reaction Mechanism
E. Statement of the Problem
References
Chapter - II Experimental
References
Chapter - III (A) Ninhydrin-DL-Valine Reaction in Presence
of CTAB and Effect of Organic Solvents
(B) Ninhydrin-DL-Tryptophan Reaction in
Presence of TX-lOO and Effect of Organic
Solvents
References
Chapter - IV Ninhydrin-DL-Tryptophan Reaction in Presence
of Gemini Surfactants and 'H N M R Investigation
at Different Concentrations of Gemini Surfactants
References
Chapter- V H NMR Study of Gemini Surfactants in Presence
of Additives (Sodium Anthranilate and Sodium
Benzoate)
References
Page
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182
215
chapter I
QeneraC Introduction
A. Surfactants and Surfactant Organized Assemblies
The living cell contains a large number of particles composed of
aggregates of molecules. The particles associate to form subcellular bodies
such as mitochondria and chloroplasts. Thus, life processes proceed mainly
within complicated assemblages of molecules rather than in the free solution
(where control of the reactions would be difficult). Knowledge of chemical
behavior inside molecular aggregates is essential to the understanding of these
highly organized biological processes.
Recently, much effort has been directed towards the utilization of
organized media to modify reactivity and regioselectivity of products. Among
the many ordered or constrained systems utilized to organize reactants, the
notable ones are micelles, microemulsions and liquid crystals. Judicious
selection of a given organized system for a given application requires a
sufficient understanding of the properties of the organized media themselves
and those of the substrate interactions therein. Surfactants are useful because
they form aggregated structures called micelles. Individual molecules of such
materials possess hydrophobic and hydrophilic segments. At a sufficient
concentration in aqueous solution, aggregation between hydrophobic segment
is favored because it excludes water. Micelle formation thus has the effect of
creating nonpolar regions in a total structure stable in polar aqueous solution.
Because the nonpolar regions of micelles are able to solubilize nonpolar
organic materials, solutions of surfactants are able to dissolve materials that
3
remain insoluble in pure water. Increasingly, micellar solutions in various
solvents have become the focus of research due to their potential as
controllable reaction and biomimetic media.' Biochemists also have long been
interested in the micelles formed by natural compounds such as lipids, as well
as in the properties of detergents used in extractive and preparative schemes.
Of late, micelles have become a subject of great interest to the organic
chemist and the biochemist, to the former because of their unusual catalysis of
organic reactions,^ to the latter because of their similarity to biological
membranes and globular proteins.
The most important property of micelles is their ability to solubilize
substances within their distinct structured regions which are insoluble or
sparingly soluble in water, allowing their location into or at the surface of
aggregates. Moreover, ionic micelles can provide charged structure, where
attractive or repulsive interactions with ionic solutes may be present. Upon
binding to micelles, reaction kinetics, chemical equilibria and molecular
properties of solutes can be drastically altered. These changes form the basis of
number of useful analytical applications."* A fundamental understanding of the
physical chemistry of surfactant organized assemblies, their unusual properties,
and phase behavior is, therefore, essential for most industrial chemists.
A surfactant^ or surface active agent is a substance that, when present at
low concentration in the system, has the property of getting adsorbed onto the
surfaces or interfaces of systems and of altering to a marked degree the surface
4
or interfacial free energies of these surfaces. ' Many types of substances act as
surfactants but all share the property of amphipathy; the molecule is composed
of non-polar hydrophobic portion and a polar hydrophilic portion. The polar or
ionic portion of the molecule interacts strongly with water via dipole-dipole or
ion-dipole interactions. The non-polar part is hydrocarbon chain that has least
affinity for water molecules. Classifying the surfactants on the basis of
hydrophilic group, one differentiates as (i) anionic, (ii) cationic, (iii)
zwitterionic, and (iv) non-ionic surfactants:
(i) anionic, e.g., sodium dodecyl sulfate,
CH3(CH2),iOS03~Na^
(ii) cationic, e.g., cetyltrimethylammonium bromide,
CH3(CH2),5N"(CH3)3Br-
(iii) zwitterionic, e.g., N-dodecyl-N: N- dimethyl betaine,
C,2H25N (CH3)2CH2COO"
(iv) nonionic, e.g., polyoxyethylene monohexadecyl ether,
CH3(CH2)i50(CH2CH20)2iH
It is well known that surface active molecules form self-organized
aggregates known as micelles. Micelles do not exist at all concentrations and
temperatures. There is a very small concentration range below which
aggregation to micelle is absent and above which association leads to micelle
formation. This concentration is called critical micelle concentration (cmc).
A discontinuity in the physical property of the solution (techniques
Q Q
used: surface tension, conductivity, electromotive force, dye solubilization, '
water solubilization,'° NMR,"''^ etc.) can be used to identify the cmc.
Gemini Surfactants:
In recent years a new class of surfactants (known as dimeric or gemini)
has generated a lot of interest in colloidal chemistry.'"'*'* These gemini
surfactants possess two hydrophobic tails and two polar, or ionic headgroups
covalently attached through a linker or spacer (Fig. 1.1). A great deal of
O- -O Tail Head gp^^^^ Head Tail
Fig. 1.1: Schematic representation of the general structure of gemini surfactant.
variation exists in the nature of spacers, hydrophobic tails, and headgroups.''*''^
The gemini surfactants are the subject of increasing study due to their unusual
solution and inter facial properties and their enhanced performance in
applications, compared to analogous single-chain conventional surfactants.'''
These surfactants have been found to be superior to conventional surfactants on
several counts and are said to be "next generation of surfactants". '
Morphology and Aggregates
The selectivity of binding of ions to interfaces remains an important
problem in many areas of colloid and surface science. Surfactants are often
used in colloid chemistry because of their amphiphilic nature. Their self-
association in aqueous media is strongly cooperative and starts generally with
the formation of roughly spherical micelles around the cmc. When the
surfactant concentration markedly exceeds the cmc, the shape of the spherical
or ellipsoidal micelle undergoes gradual change.'*''^ Fig. 1.2 schematically
shows various structures that are formed upon increasing the concentration of
surfactant. In the beginning of structural changes spherical micelles become
cylindrical. Further increase in concentration results in a hexagonal packing of
water cylinders. It is possible to induce a transition from one structure to
another by changing the physico-chemical conditions such as temperature, pH,
addition of ionic and nonionic solutes in the surfactant solution. '' The shape
and size of these micellar aggregates can, in principle, be determined by
various methods, such as light scattering, diffusion sedimentation velocity,
sedimentation equilibrium, ultrasonic absorption, time-resolved fluorescence,
etc. The micellar sphere-to-rod transition is highly dependent upon the nature
of the counterions and it has been concluded that strong counterion binding
promotes the transition from spherical to cylindrical micelles. Transition of
spherical to larger micelles for ionic surfactants occurs upon a reduction of
inierheadgroup repulsion. It may be caused by salt or surfactant additions or
solute solubilization. Cationic surfactants in the presence of certain strongly
A,
/ \ V
Sur foc ion t Molecule? Spherical MiceHn
( a ) ( b )
Willi mmpr tiin/in)/iuffjnii iifi m
Rod - shaped Micel l ts
( C )
Lomellor Phase
( e )
Reverse Hexogonoi Phase
(f )
Hexogonoi Phose
( d )
Reverse Micelles
(g)
c ' iemra . i l l ; ' °""" °' "'''' ^ '™"" '^ f°™^^ "P"" increasing surfacan,
8
binding counterions (organic counterions) form micellar solutions with
extraordinary rheological properties. These counterions bind nearly 100% to
the micelle, suppressing the effect of electrostatic interactions in these systems.
Not only are intermicellar electrostatic interactions damped, so are the
interactions between surfactant molecules within a single micelle. This
decreases the surfactant headgroup area and causes the micelles to adopt a
cylindrical rather than a spherical morphology. These micelles grow and
become polydisperse with increasing concentration, often achieving a certain
degree of flexibility. At sufficiently high concentration the micelles overlap
and become entangled with each other, in a manner analogous to semidilute
polymer solutions.
Packing considerations constitute a factor which involves the nature of
the hydrophilic and hydrophobic groups of the surfactant. The surfactant
packing parameter, also referred to as surfactant number, surfactant parameter,
and critical packing parameter, is a dimensionless group. This group may be
expressed as Rp
Rp=Vi,/aolc (1.1)
where
V/, = the volume of the amphiphile's hydrocarbon tail,
Oo = the optimum cross- sectional area per amphiphile molecule, and
/,. = the length of the fully extended hydrocarbon tail.
9
The optimum cross-sectional area per amphiphile molecule is observed
experimentally by X-ray diffraction of bilayer systems, while the volume and
length of the hydrocarbon tail may be calculated by Tanford's fonTiulas."
Vf, = (27.4 + 26.9n) A^ (1.2)
/, = (1.5+ 1.265n)A (1.3)
(n is the number of carbon atoms in the hydrocarbon chain).
Considering the geometric dimensions, the volume and the surface area
of each association structure yield critical conditions for the formation of each
of the following shapes:
Spherical structure : Rp<\/3
Cylindrical structure: \B<Rp<l/2
Bilayer structure : 1/2 <Rp<\
Inverted structure : Rp>l
The Vh/Oglc ratio depends on the surfactant chemical structure (l^. and Vh)
and on surface repulsions between headgroups (a^). The derived curvature (and
thus type of aggregate) may be obtained upon a correct choice of the surfactant
molecule and solvent conditions (type of solvent, ionic strength, etc.) using the
Vi,/a„lr ratio as a guide. However, the ratio has to be used with caution as it
accounts only for geometrical considerations.
10
B. Effect of Additives on Structural Transitions
The properties of micellar solutions such as cmc, aggregation number,
micelle size and shape, etc., depend on the balance between "hydrophobic" and
"hydrophilic" interactions.' " '' For ionic surfactants this balance can be modified
in several ways, e.g., salt addition, counterion complexation, addition of
alcohols or other substances (that can be solubilized into the micelle), change
of the solvent, or change of the "structure" of the solvent itself. Under these
conditions it has been found that micelles undergo a change from spheres to
cylinders. This sphere-to-rod transition is important from both a theoretical and
a practical point of view. Theoretically, because (i) it implies a micellar grouch
which seems to be related to the classical Derjaguin-Landau-Verwey-Overbeek
theory (DLVO theory); (ii) the more structuralized rod micelles, as compared
to spherical micelles, can be related more closely to the formation of biological
structures (membranes, for example); (iii) if the sphere-to-rod transition can be
predicted from theoretical models it would promote a better understanding of
micellar and related organized structures.
From a practical point of view, the presence of rod shaped micelles
gives solutions a very high viscosity, which might be of importance in
industrial formulations of surfactant solutions.
11
Effect of Salts:
When salt is added to aqueous ionic surfactant solution and its
'ye TO
concentration reaches a threshold value, rod like micelles form ' because the
presence of salt ions near the polar heads of the surfactant molecules decreases
the repulsion force between the headgroups. A reduction in the repulsion makes
it possible for the surfactant molecules to approach each other more closely and
form larger aggregate, which requires much more space for the hydrophobic
chains. Because a spherical micelle has a small volume, it must change into the
rod like micelle to increase the volume/surface ratio. The existence of rod like
micelles was inferred from experiments of light scattering " and confirmed
by direct observation under the electron microscope for some systems. ' ^ In
general, in the transition from sphere to rod, micelles of course change their
aggregation number dramatically and grow linearly, keeping their radii
constant. When the salt is a simple one and is of high concentration, long
flexible rod like or thread like micelles are formed. There have been numerous
studies of dilute and moderately concentrated aqueous cationic surfactant
solutions with simple salts discussing the dynamical behavior of the solutions
in connection with network behavior of semidilute solutions of flexible
polymers.
Salts with hydrophobic counterions, such as sodium salicylate (NaSal)
and sodium tosylate (NaTos), are particularly effective in inducing micellar
growth even at low concentrations. With increasing salt content complex
12
variations occur in the rheoiogical properties. ' The classic examples are
solutions of cetyltrimethylammonium bromide (CTAB) and NaSal, '* for which
the zero-shear viscosity (;/„) goes through a maximum at low NaSal
concentrations, then a minimum and subsequently a second maximum around
IM NaSal. NMR studies on the cetyltrimethylammonium salicylate system
reveal that the ' H lines for the N(CH3) group are shifted to higher fields, and
the signals are broadened."''*' '' The salicylate anion orientates in such a way that
the negatively charged site (COO" group) stands perpendicular to the micellar
surface/^
Ikeda et al?^'^^ showed that for sodium dodecyl sulfate (SDS) and for a
series of cationic surfactants in NaCl solutions a sharp break in the apparent
micelle molecular weight is observed when the NaCl concentration reaches a
value of 0.45 M and the breakpoint correspond to the sphere-to-rod transition.
They^^ also measured light scattering from aqueous solutions of SDS in the
presence of 0.8 M NaX (X=F', CI", Br", I", or SCN") at 35 °C and found that the
molecular weight of the rod like micelles depends on the co-ion species of
added salt and changes in the order of the lyotropic series of halide ion except
for SCN" ion: NaSCN <NaF < NaCl < NaBr < Nal. The difference in micelle
size must be caused by the effect of co-ion species on hydrophobic interaction
in the micelle formation or the extent of destruction of the hydrogen-bonded
structure of water.
13
Bendedouch et al?'^ made a series of SANS measurements on micellar
solutions of lithium dodecyl sulfate (LDS) as a function of surfactant and salt
concentrations. In an electrolyte free aqueous LDS solution the micelles still
assume their smallest spherical size (n = 53) for a concentration of up to
0.037M, whereas the particle shape changes from a sphere to a prolate ellipsoid
to accommodate larger n. At low LDS (<0.3M) and salt (<0.5M) concentrations
the growth behavior of micelles was said to be somewhat similar to that of SDS
as studied by Missel et al.^^ who found that the micellar size is a very sensitive
function of [surfactant], temperature and [NaCl]. But at high salt
concentrations, there is no evidence of sphere-to-rod transition in contrast to
the SDS case. The LDS micelles grow only to a limited extent and thus the
assumption of monodispersity is probably valid.
Several reports indicate that change from Li" to Cs" induces micellar
growth, which is related to hydration of specific counterion."*' Compared to
these alkali metal counterions, symmetrical quaternary ammonium ions (R4N" )
are essentially less hydrated and, therefore, binding with the micelle will be
favorable. On the other hand, R4N^ has a low charge density and may also try
to intercalate between headgroups of anionic micelles. This will decrease the
electrostatic interactions in addition to increased hydrophobic interactions. All
these factors contribute towards micellar growth.'*^
The presence of multivalent counterions (Ca " , Al " ) in solutions of
anionic surfactant strongly enhances the formation of rod like micelles.' ''*'' Al ^
14
can bind together three surfactant headgroups at the micelle surface, thus
causing a decrease of the area per headgroup.''^ This induces a transition from
spherical to cylindrical micelles.
Effect of Organic Additives:
Micelles are well known for presenting unique structural aspects
consisting of a non-polar inner core and a polar outer surface. This structure
allows micellar aggregates to enhance the solubility of hydrophobic materials
and to modify environmental features. However, both dynamic and structural
properties of micellar solutions can be altered by the addition of a third
component in the solution. This last substance can act through two different
mechanisms: by interactions with the surfactant molecules or by changing the
solvent nature.
Surfactant solutions have a general tendency to solubilize a certain
amount of hydrocarbons. Systems with rod like micelles can actually solubilize
rather large amounts of hydrocarbons. The environment of solubilization of
different compounds in or around micellar systems can be correlated with the
structural organization of micellar aggregates and their mutual interactions.''^"^'
Interfacial partitioning of organic additives causes micellar growth while
interior solubilization produces swollen micelles. ' ^ These two types of
micelles impart different viscosity behavior to micellar solutions. The interior
(core) solubilization of organics provides swelling to the already grown micelle
and releases the requirement of the surfactant chain to reach the center of the
15
core. "* These factors may increase the smaller dimension of such anisotropic
micelles with a resultant decrease in axial ratio (more spherical). This increased
sphericity will cause micelles to flow easily with an eventual drop in viscosity.
The electrostatic repulsion terni originating from intennicellar and intramicellar
Coulombic interactions favors micelles with a higher surface area per
headgroup. On the other hand, hydrophobic interaction between the
hydrocarbon part of the micelles/ monomers tries to achieve aggregates with
closely packed monomer chains. Mukerjee^^ had proposed that an additive
which is surface active to a hydrocarbon-water interface will be mainly
solubilized at the headgroup region and will promote micellar growth. The
greater partitioning of the additive to the core was shown to retard micellar
growth by virtue of relaxing the requirement of the monomer tails to reach the
center of the aggregates which maintain the micellar shape with higher surface
area, that is spherical micelles.^ There has been considerable discussion about
the location of aromatic solutes such as benzene and toluene in ionic surfactant
micelles. Benzene solubilizes mainly in the surface region of the micelles,^^ or
primarily within the micellar interior, ' ^ or in both states.^' However,
extensive and precise solubilization studies do not indicate a strong preference
of these compounds in either the headgroup region or the interior.^^'^' The
aromatic hydrocarbons seem to be intermediate between highly polar solutes,
clearly embedded in the headgroup region, and aliphatic hydrocarbons, which
usually solubilize in the micellar interior. '*'''"
16
Kandori et al!'" studied the effect of phenol and benzene additives on
micellar struptures in aqueous solutions of dodecyltrimethylammonium
bromide by additive solubilization, tracer diffusion coefficients, electrical
conductivity, viscosit}', and ultraviolet absorbance. The solubilization of phenol
and benzene in the system causes the micelle to swell and it was observed that
phenol addition leads to a greater increase in the size of aggregates than
addition of benzene. Ultraviolet absorbance measurements revealed that the site
of solubilization within the micelles is different for the two additives. Benzene
solubilizes in the central core, while at low concentrations phenol is taken up in
the outer palisade layer.
Cerichelli and Mancini "* studied the influence of Coulombic and
specific counterions on the extent of solubilization and of the location of
benzene in aqueous solutions of cetyltrimethylammonium surfactants (CTAX,
X = Br~, Cr, NO3-, CH3SO3, and (804)1/2^! by ' H , '^C, and " 'N N M R
spectroscopy, at [CTAX] = 0.10 M. When benzene is solubilized in CTAX
aggregates, it displaces water from the clefts which characterize the micellar
surface. This displacement changes the dielectric constant inside the clefts and
has different consequences on the structure of the aggregate according to the
nature of counterion. If the counterion has an external position with respect to
the micellar surface, changing the dielectric constant between the cation
headgroups results in their repulsion and has little or no consequences on the
size of the aggregate. If the counterion is localized between the headgroups,
decreasing the medium dielectric constant results in a stronger attraction
17
between the cationic headgroups and the counterions and has the consequence
of changing the structure and the size of the aggregate. At low [benzene] the
solute displaces the water molecules which are closer to the first portion of the
surfactant aliphatic chain, once this location has been saturated the aromatic
solute displaces the deeper water.
Ruiz^^ studied the aggregation behavior of tetradecyltrimethylammonium
bromide in ethylene glycol-water mixtures across a range of temperatures by
electrical conductivity measurements. The cmc and the degree of counterion
dissociation of micelles were obtained at each temperature. Only small
differences have been observed in the standard molar Gibbs free energies of
micellization over the temperature range investigated. The enthalpy of
micellization was found to be negative in all cases, and it showed a strong
dependence on temperature in the ethylene glycol poor solvent system. An
enthalpy-entropy compensation effect was observed for all the systems, but
whereas the micellization of the surfactant in the solvent system with 20 wt%
ethylene glycol seems to occur under the same structural conditions as in pure
water, in ethylene glycol rich mixtures the results suggest that the lower
aggregation of the surfactant is due to the minor cohesive energy of the solvent
system in relation to water.
Effect of Salts + Organic Additives:
The wide attention given to the studies of size and structure of micelles
in the presence of additives is result of their importance and their complicated
18
aggregation behavior.' ' ^ For most aqueous ionic surfactant solutions just
above the cmc, the micelles are regarded as spherical in shape. Addition of
neutral additives (e.g., alcohols, amines, etc.) may affect the micellar structure.
This effect depends on the types of surfactants used, their concentration, the
salt content, and the additives.
Nguyen and Bertrand^^ used incremental calorimetric technique to study
the effect of low concentrations of alcohols on solutions of SDS with added
electrolytes at 25 °C. These measurements reveal a discontinuity in the slope of
partial molar enthalpy of solution versus concentration of alcohol curves. The
authors assert that this break corresponds to the micellar sphere-to-rod
transition.
no
Stephany et al studied the same system with varying concentration of
the electrolyte (NaCl). They varied the concentration of 1-pentanol too for each
NaCl concentration. Their data show characteristics of a continuous sphere-to-
rod transition. From static and quasielastic light scattering methods they
concluded that the micelles could be modeled as flexible wonn-like objects.
The rod-shaped micelles were formed in aqueous solution of O.IM
CTAB + 0.1 M KBr. The effects of aliphatic amines (C4, Cg, C7, or C8NH2) and
temperature on the above system show that transition of rod-shaped micelles to
larger aggregates is induced by addition of higher amines (> CeNHz) upto a
certain concentration; a further increase in concentration produced the opposite
19
effect." Addition of C6NH2 was reported to induce only a rod-to-sphere
transition.
The data had been interpreted"''^"^' in terms of solubilization/
incorporation (decrease of micellar surface charge density) of amines inside the
micelles and the nature of the effective solvent (water + amine). The latter
effect dominated the change from larger aggregates to smaller micelles at
higher concentrations of added amine. The temperature effect was similar as of
the C4NH2 addition, namely, rod-to-sphere transition.
Guerin and Bellocq^^ have shown that various phases and critical points
are present in the system SDS/Aj-pentanol/water/NaCl depending on NaCl
concentration and temperature.
C. Reactions in Organized Media
During the past few decades there has been considerable interest in
reactions which can be carried out in organized media or micelles. These
reactions often occur at the interface between the solvent, which is usually
water or an aqueous organic mixture, and the submicroscopic particle or
aggregate. Motivation for studying reactions in organized media may be
derived from three sources: first, to ftirther understanding of those factors
which influence the rates and course of organic reactions; second, and closely
related to the first, to gain additional insight into the exceptional catalysis
20
characteristic of enzymatic reactions; third, to explore the utility of micellar
systems for the purpose of organic synthesis.
The early chemical literature contains a scattering of reports concerning
reaction kinetics in aqueous-ionic or -nonionic surfactant media. However,
substantial insight into this area was first achieved in 1959 by Duynstee and
Grunwald '* in their study of the effects of cationic and anionic surfactants on
the rate of alkaline fading of cationic triphenylmethane dyes. Since that time,
related studies have been appearing at an increasing rate, and interest in still
growing. Experiments were carried out primarily in dilute aqueous solutions of
ionic micelles with some work including the effect of microemulsion , reverse
micelles, monolayers and vesicles. Progressively more sophisticated
quantitative treatments of micellar effects on reaction rates and equilibria
developed over the same time period. Chaimovich and coworkers showed that
the pseudophase ion exchange model provides a unified approach for
interpreting many of these effects.
It is the micelles, rather than individual surfactant molecules, which are
responsible for altering the rates of reactions in solutions of surfactants.
Basically, these rate effects can be attributed to electrostatic and hydrophobic
interactions between the substrate and the surfactant aggregate. However, the
fact that the experimental results are often complex, and do not bear out the
expectations based on purely electrostatic considerations, indicates that such an
explanation is an over simplification. Indeed, substrate specificity has been
21
observed in a number of micelle-catalyzed organic reactions.^^"'^ Substrate
specificity, as in the case of enzyme catalyzed reactions, arises from differences
in the nature and extent of solubilization, i.e., substrate-micelle binding, and
from differences in the reactivity of the substrate in the micellar phase and in
the bulk solvent. Though the use of surfactant structures to alter or enhance
reaction rates has been known for several decades, the use of surfactant
structures to control reaction pathways is a fairly recent development. An
example of wide interest is the use of ionic surfactants to facilitate charge
separation and retard back reactions in sensitized photochemical generation of
hydrogen from water.
Micellar effects upon reaction rates and equilibria have generally been
discussed in terms of the pseudophase model. The model aids in the
interpretation of the catalytic activity of functionalized micelles used as models
for enzymatic sites and is applicable to the effects of reverse micelles,
microemulsions, and vesicles on reaction rate and equilibria. C. A. Bunton -
"father of micellar kinetics"^^ has observed'"^: "The development of a
quantitative understanding of chemical reactivity in solution has depended on
the willingness of chemists to use models that are no more than crude
approximations. For this reason it is useful to accept the pseudophase model,
despite its imperfections, until it either fails to fit the data, or is replaced by a
better model".
22
The pseudophase description of micellar catalysis and inhibition
assumes that the relation between overall reaction rate and surfactant
concentration, for a given total concentration of reactants, can be explained in
temis of the concentrations of each reactant in water and in the micelles, and
the rate constants in the aqueous and micellar pseudophases. Provided that
equilibrium is maintained between the aqueous and micellar pseudophases
(designated by subscripts w and m) the overall reaction rate will be the sum of
rates in water and the micelles and will therefore depend upon the distribution
of reactants between each pseudophase and the appropriate rate constants in the
two pseudophases. Early studies of reactivity in aqueous micelles showed the
importance of substrate hydrophobicity in determining the extent of substrate
binding to micelles; for example, reactions of a very hydrophilic substrate
could be essentially unaffected by added surfactant, whereas large effects were
observed with chemically similar, but hydrophobic substrates. ^•^°' '^'
Scheme 1.1 describes substrate distribution and reaction in each
pseudophase.
nS „ Sp + A^ „, A ^
k'
Product
Scheme 1.1
k' A. n
23
In the scheme, S„ denotes micellized surfactant (= [(S)T] - cmc), A is
substrate, Aw and k'm are first-order rate constants, and K/^ is the binding
constant in terms of the micelhzed surfactant.
The rate equation is written as
-([Aw]+lAm]) ci[A]t dt dt
(1.4)
_ d[Product] ., rx dt
and
^ t £ ^ = 'fw[AJ.*'.(AJ (1.6) Qt
where [A]t is the stoichiometric concentration of the substrate at time t. The
observed rate constant for the product formation in micellar solution, k^, is
given by:
k^=-^^/[A],=k\¥,,+k\¥„, (1.7) dt
(Here F^ and Fm are the fractions of the unbound and bound substrate). For a
pseudo-first-order process, the micellized surfactant concentration, [Sn] »
[Am] and hence F^ is constant. The equilibrium constant, K/^, can now be
expressed as:
([A],-[AJ)[SJ [SJ(1-F,J
24
Combining Eqs. (1.7) and (1.8) and rearrangement gives:
Equation (1.9) is similar in form to the Michaelis - Menten equation of
enzyme kinetics and successfully fits the sigmoidal rate- surfactant profiles of
micellar-catalyzed unimolecular reactions, i.e., k^ increases rapidly at the cmc
and then plateaus once all of the substrate is micellar bound. The equation can
be rearranged into the reciprocal form (similar to Lineweaver-Burke equation),
which allows both A A and k^m to be estimated from the kinetic data provided
u * W • I 90,102, 103 that ATw IS known.
1 + ilc\,-k^) ik'^-k'^) (k'^-k'^)K^[SJ (1-10)
These equations have extensively been used and provided the basis for
quantitative analysis of micellar rate effects.
Another general way of treating micellar kinetic data is based on
Piszkiewicz's cooperative model.'°"* Very often, for the micelle catalyzed
reactions, the plot of rate constants versus surfactant concentration gives
sigmoid shaped curves and this observation is analogous to the positive
cooperativity (measured by Hill constant n) in the enzymatic reactions.
Considering this fact, a kinetic model analogous to the Hill model'"^ was
developed by Piszkiewicz to explain the micellar effect. It considers that the
substrate (A) and surfactant (S) molecules aggregate to form active micelle
25
(SnA). Very often, in the micelle catalyzed reaction, after attaining a rate
maximum, th? rate decreases at the higher concentrations of the surfactant. To
explain the rate retarding effect of the surfactant at its higher concentrations,
formation of kinetically inactive micelle (SnSpA) through further aggregation of
surfactant molecules has been considered. This phenomenon has been
compared with the substrate inhibition in an enzymatic reaction. The treatment
differs from that of Menger and Portnoy in that it emphasizes cooperative
effects due to substrate-micelle interactions. These interactions are probably
very important at surfactant concentrations close to the cmc because solutes
may promote micellization or bind to submicellar aggregates. Thus, when Eq.
(1.9) do not fit the data for dilute surfactant, especially when reactants are
hydrophobic and can promote micellization, the Piszkiewicz model can be
used.
The simple distribution model (Eq. (1.9)) fails for micellar catalyzed
bimolecular reactions where rate constants generally go through maxima with
increasing surfactant concentrations. The quantitative treatments of such
catalysis of reactions depend on evidence that the micelles can be treated as if
they were a separate phase, and that the distribution of ionic or nonionic
reactants between water and the micelles can be measured directly or estimated
indirectly. The pseudophase description of micellar catalysis and inhibition
assumes that the relation between overall reaction rate and surfactant
concentration, for a given total concentration of reactants, can be explained in
26
terms of the concentrations of each reactant in water and in the micelles, and
the rate constants in the aqueous and micellar pseudophases.
This can be done by extending Eq. (1.9), and a simple formalism
involves writing the first-order-rate constants A:'w and k^ in terms of second-
order-rate constants in water and micelles and reactant concentrations in each
pseudophase.'°^''°^ The problem is, however, as how to define concentration in
the micellar pseudophase. One approach is to write concentration in terms of
moles of reagent per dm'' of micelles, or to assume some volume of the micellar
pseudophase, Vm, in which reaction takes place. The problem is similar to that
of comparing second-order-rate constants, written conventionally in the
dimensions moP' dm^ s'', for a variety of solvents. The comparisons will be
completely different if concentrations are written in terms of molarities or mole
fractions.
Another approach is to define concentration in the micellar pseudophase
in terms of mole ratio. Concentration is then defined unambiguously, and the
equations take a simple form.'°^'"° Scheme 1.2 shows reaction between the
substrate A and nucleophile B (or any second reactant). The second reactant
nS . Sp - Ay ^ An
U\v ' m
Product •
Bm
Scheme 1.2
27
is generally in large excess over the substrate establishing pseudo-first-order
conditions, so that
k{,. = k,[B,^ (1.11)
k\., = kr,Ml (1.12)
where /t and km are second-order rate constants for reaction in aqueous and
micellar pseudophases, respectively, and M | is the mole ratio of micellar
bound reactive nucleophile to micellized surfactant given by
M|=[BJ/[Sn] (1.13)
Substitution of Eqs. (1.11) and (1.12) in Eq. (1.9) gives
A:,, ,= (1.14) l + ^A[Sn]
k^,[B,,] + k^K^[BJ
l + ^A[Sn] (1.15)
These, or similar, equations readily explain why first-order-rate constants of
micelle-assisted bimolecular reactions typically go through maxima with
increasing surfactant concentration if the overall reactant concentration is kept
constant. Addition of surfactant leads to binding of both reactants to micelles,
and this increased concentration increases the reaction rate. Eventually,
however, increase in surfactant concentration dilutes the reactants in the
micellar pseudophase and the rate falls. This behavior supports the original
28
assumption that substrate in one micelle does not react with reactant in another,
and that equilibrium is maintained between aqueous and micellar
pseudophases.
Equation (1.14) or (1.15) and others, which are essentially identical but
are written in different ways, can be applied to bimolecular micelle-assisted
reactions provided that the distribution of both reactants can be determined.
D. The Ninhydrin -Amino Acid Reaction Mechanism
Ninhydrin is the name given by Abderhalden and Schmidt'" to 1,2,3-
triketohydrindene (la), which, because of its peculiar color reaction with a -
amino acids and amines, was used by them as indicator in the dialysis method
for detection of the activity of specific ferments in the animals organism under
pathologic conditions. Ninhydrin is also known as Ruhemann's reagent (the
reagent was first discovered by Ruhemann in 1910 ), or more systematically,
as 2,2-dihydroxyindan-l,3-dione (because, in presence of water, ninhydrin
exists as its hydrate, I).
The application of ninhydrin for the detection and quantitative
estimation of a -amino acids has been well established since its discovery. The
interaction of ninhydrin with a -amino acids produces purple-colored product,
diketohydrindylidenediketohydrindamine (DYDA), popularly known as
Ruhemann's purple (II).
29
RCH(NH2)C00H O *-
The intensity of the color developed corresponds to the quantity of or -
amino acid present in the sample. Initially, it has been observed that the
ninhydrin-amino acid reaction does not yield reproducible results, and thus the
estimation of or-amino acids is not appropriate. To solve this problem, a
number of researchers directed their studies towards this emerging and
challenging front and tried to give a reproducible and simple method of
quantitative estimation of a -amino acids. The works were carried out in order
to (i) get reproducible results; (ii) stabilize the colored product; (iii) enhance
the intensity of color by adding other reagents (e.g., organic solvents, reducing
agents, etc.) to lower the detection limit; and (iv) identify intermediates to
achieve the above goals in a proper way. Keeping in view of the above points
many researchers carried out mechanistic studies for ninhydrin-amino acid
reaction and broadly divided the mechanism of the reaction in three steps:
(i) the initial attack of amino nitrogen of amino acid to carbonyl
group of ninhydrin;
30
(ii) oxidation and reduction steps leading to intermediates along the
pathway; and
(iii) formation of Ruhemann 's purple from these intermediates.
Mechanistic studies on the amino acid-ninhydrin reaction were first
reported by Ruhemann but the scheme of reactions proposed by him failed
to explain the formation of 2-hydroxy-l, 3-indanedione (III) and DYDA
(Scheme 1.3). He observed that both amines and a-amino acids yield the
purple-colored product but could not explain the formation of color by amines.
lOi:^:-,0
RCH(NH2)COOH ^ Q OH
H + RCHO + CO2 + NH3
(0 O o
(HI)
o o
,0 Q ,OH
OH
(HI) (I)
2NH,
(IV)
Scheme 1.3
Harding and Warneford"^ and Harding and MacLean"'* proposed a
modified mechanism based on the studies of interaction of ninhydrin with
31
amines, amides, ammonium salts and amino acids. They proposed that amino
acids decompose into the corresponding glyoxal (V) and ammonia. Glyoxal
RCH(NH2)C00H • RCOCHO + NH3
(V)
reduces ninhydrin to 2-hydroxy-l,3-indanedione (III) which, in turn, condenses
with ammonia to give 2-amino-l,3-indanedione (VI). 2-Amino-l,3-
indanedione (VI) reacts with another molecule of ninhydrin to give final
product, DYDA. The amides gave no reaction with ninhydrin. These authors
could not explain why amino acids reacted faster than amine, although a
common intermediate, ammonia, was postulated."'*
Schonberg et alV^ reported that or-amino acids are degraded to the
corresponding aldehydes or ketones containing one less C-atom by certain
carbonyl compounds (general Strecker degradation). Moubasher and Ibrahim"^
reported that the amino acid-ninhydrin reaction gives hydrindantin (IV) and
bis-l,3-diketoindanyl. The mechanism was further elaborated by McCaldin"''
and Johnson and McCaldin. The formation of hydrindantin was shown to be
a side reaction product and not involved with the colored product formation.
The proposed mechanism (Scheme 1.4) also explained the faster reactivity of
amino acids than those of amines and ammonia. In case of a -amino acids the
adjacent carboxylate (to the amino group) looses CO2, while in case of amines
and ammonia, the cleavage of C-C or C-H bonds are involved.
32
,0
OH
OH + RCH(NH2)C00H
(I) O
OH or
OH
+ Ninhydrin
(IV)
Scheme 1.4
33
MacFadyen and Fowler"^ presented the role of hydrindantin in the
purple color development for ninhydrin amino acid reaction. The rate of
disappearance of red color of hydrindantin was found to be equal to that of
appearance of purple color for hydrindantin-a-amino acid reaction. It was
postulated that hydrindantin hydrolyses to give 2-hydroxy-l,3- indanedione
(III) and its one molecule is used for the formation of each molecule of
Ruhemann 's purple (II).
To provide a model of the ninhydrin reaction mechanism, Wittmann and
coworkers observed that two moles of l,2,3-trioxo-2,3- dihydrophenalene
(VII) reacts with one mole of a-amino acid to form bis(3-hydroxy-1-
oxophenalen-2-yl)amine (VIII). This is oxidized by NaOH in methanol to yield
the final product dehydro-bis(3-hydroxy-l-oxophenalen-2-yl)amine (IX), an
analog of Ruhemann's purple (Scheme 1.5).
The reaction of 2-amino-3-hydroxy-l-oxophenalene (X) and 2,3-
dihydroxy-1-oxophenalene (XI) gave bis(3-hydroxy-l-oxophenalen-2-yl)amine
(VIII) suggesting the role of hydrindantin in the formation of 2-hydroxy-l, 3-
indanedione (III) which reacts with 2-amino-l, 3-indanedione (VI) to give the
purple-colored product.
+ RCH(NH2)C00H
(VII)
34
,0H Q .0 Q,
(X) (XI)
Scheme 1.5
Lamothe and McCormick proposed a mechanism (Scheme 1.6)
suggesting that a-amino acids react with ninhydrin to give 2-amino-l,3-
indanedione (VI) and an aldehyde with one less C-atom than the original amino
acid. On the basis of cyclic voltammetric studies the authors observed that 2-
amino-l,3-indanedione (VI) is a better reducing agent than ascorbic acid and is
also unstable. It may hydrolyse to give 2-hydroxy-l,3-indanedione (III) and
ammonia or react with ninhydrin to yield 2-imino-l,3-indanedione (XII)
alongwith 2-hydroxy-I,3-indanedione (III). The last two react to give
Ruhemann 's purple.
35
O o
(I) o o
OH
OH
OH
+ RCH(NH2)C00H OH
+ H2O NH—CHCOOH
o i
NH—CHCOOH i
O R
NH=CHR + COjf
+ Hydrolysis (^^-\-NH=CHR ^ If J NH2 + RCHO
(VI) OH
NH2 + H2O
O
NH2 + [ Q OH K,
OH
(0 O
OH
Scheme 1.6
36
Friedman and Williams'^^ modified the mechanism proposed by
Lamothe and McCormick.'"' The authors suggested that at pH > 4, excess
ninhydrin gives the best color yield for ninhydrin-amino acid reaction. At low
pH, amines are protonated and no more behave as a nucleophile. At high pH,
lone-pair of electrons of amino group attacks the carbonyl group of ninhydrin
to give a Schiff base (a condensation intermediate). The last step also differs
from that of Lamothe and McCormick (who suggested that 2-imino-l,3-
indanedione (XII) combines with 2-hydroxy-l,3- indanedione (III) to give
Ruhemann 's purple, II) in which Friedman and Williams suggested that 2-
amino-l, 3-indanedione (VI) reacts with excess ninhydrin rapidly to give the
Ruhemann's purple (II). At low concentration of ninhydrin, 2-amino-l, 3-
indanedione (VI) is not trapped by ninhydrin and is hydrolysed to 2-hydroxy-l,
3- indanedione (III) which reacts with ninhydrin to give hydrindantin (IV, a
side product), thus decreasing the color yield.
Ninhydrin
37
Bottom et al}^^ have rightly supported the mechanism proposed by
Friedman and. Williams but also did not neglect the suggestion of Lamothe and
McCormick that 2-imino-I,3- indanedione (XII) and 2-hydroxy-l, 3-indane
dione (III) are the actual reactants.
Khan and Khan' '*'' ^ carried out detailed kinetic and mechanistic studies
on the ninhydrin-amino acid reactions in aqueous medium by varying pH,
temperature, concentration of reactants and organic solvents. They proposed
mechanisms (Schemes 1.7 & 1.8) for the decarboxylation process as well as for
development of the purple color. As the amino group of amino acids is
protonated at low pH (and thus does not possess the free electron), they found
that nucleophilic attack of amino group of amino acids on carbonyl group of
ninhydrin was not possible and decarboxylation did not occur. They also
noticed that with the increase in pH, the equilibrium shifted towards
unprotonated amine which, in turn, increased the rate of decarboxylation.
38
K, + _ RCH(NH,)COOH ^. RCH(NH3)C00 + H
Kv RCH(NH3)COO
RCH(NH3)C00"
, 0
i ^ RCH(NH2X^OOH
K„ RCH(NH2)C00 + H
O
O + RCH{NH2)C00H —
(la) O
OH
NH—CHCOOH
/ o OH O
NH —CHCOOH
0 k O
o o N—CHCOOH ^
I R + H2O
O
N—CHCOOH + H2O I
R
NH, +RCHO +€0.^
G O
^ ,0
O + RCH(NH2)C00 — 1 .
O
O OH
NH —CHCOO
k\ O
O
0
R
O
OH
NH —CHCOO
N—CHCOO + H2O
R
O
*'3 =N—CHCOO ,
. i +H2O \ i
o
NH2 + RCHO + CO2 f
O (VI)
Scheme 1.7
39
RCH(NH3)C00H
RCH(NH3)G00
K, RCH(NH3)C00 + H
(III) OH
OH +
OH
( I I I )
o —
40
The decarboxylation step was fast and followed a pseudo-first-order
irreversible path. On the basis of their studies performed under varying kinetic
conditions the authors supported the mechanism proposed by Lamothe and
McCormick with a slight modification. In their mechanism 2-amino-l, 3-
indanedione (VI) was considered as a stable intermediate that behaved as
reactant for the formation of DYDA and, unlike Scheme 1.4, the conversion
was a three-step process, i.e.,
••eq NH +
H2OJ kj Y
P Q
P O
41
E. Statement of the Problem
In the preceding sections the importance of surfactants and surfactant
organized assemblies, effect of additives and reactions therein alongwith the
mechanistic aspects of amino acid-ninhydrin reaction have been discussed. As
ninhydrin reactions using manual and automated techniques as well as
ninhydrin spray reagents are widely used to analyze and characterize amino
acids, peptides, and proteins as well as numerous other ninhydrin-positive
compounds in biomedical, clinical, food, forensic, histochemical,
microbiological, nutritional, and plant studies, extensive efforts have been
made to enhance the usefulness of the method. These include sensitivity
enhancement by pretreatment with enzymes, coordination to metal salts,
change of solvent, etc. In their attempt to explore the effect of micelles on the
color development, Kabir-ud-Din and co-workers have found micellar catalysis
1 77 I " 7
of both the ninhydrin-amino acid " and ninhydrin-metal amino acid
complex' ^"'"^ reactions.
Micelle mediated reactions are similar to those occurring on lipid-
protein interfaces. Micellar pseudo-phase has been regarded as a
microenvironment having varying degree of polarity, water activit)' and
hydrophobicity with increasing distance from the interfacial region to its
core. " Micelle catalyzed reactions as models for electrostatic and hydrophobic
interactions in biological systems could provide information regarding the
mechanism of tuning of reactions occurring on biological surfaces because
42
micelles are simpler and more easily modified. There are theoretical as well a
practical reasons for the study of reactivity in the presence of micelles. On the
practical side, industrial recipes for carrying out reactions often involve the
solubilization of a reactant. Solubilization of nominally insoluble material is a
necessary process occurring in a wide range of phenomena found in
pharmaceutical delivery systems, animal digestive processes and the sensory
processes of smell and taste. Much work done on the fundamental equilibrium
aspects of solubilization has been limited to determination of the equilibrium or
saturation solubilization capacities of systems and to the effects of system
variables on these quantities. In contrast, comparatively less attention has been
focused upon the kinetics of the process with consequently little being known
definitively about its mechanism. In these areas and others such as topical drug
delivery, detergency and enhanced oil recovery, a knowledge of the kinetics of
the solubilization process is of great importance.
In this direction the present work of kinetic studies on ninhydrin-amino
acid reactions in micellar organized media was undertaken with a view to find
some applications to improve contrast and visualization that may prove a step
forward from the methods already in use. For the purpose conventional as well
as gemini micellar systems were used. The latter is a special class of surfactants
which have some intriguing properties'' '* in comparison to conventional
surfactants. These surfactant systems are popularly known as surfactants of the
twenty first century.'^ Even a cursory look at the recent literature''""'''^ will
convince that the tempo of research in the field of gemini surfactants is very
43
high and all signals indicate that this heightened interest will continue. ' H
NMR studies have, therefore, been carried out to investigate the micellar
morphology and location of the solubilization site of additives (sodium
anthranilate and sodium benzoate ) in gemini surfactants.
Lav out of the thesis
This thesis consists of the following five chapters:
Chapter - I: General introduction.
Chapter - II: Experimental.
Chapter - III: (A) Ninhydrin-DL-valine reaction in presence of CTAB and
effect of organic solvents.
(B) Ninhydrin-DL-tryptophan reaction in presence of TX-lOO
and effect of organic solvents.
Chapter - IV: Ninhydrin-DL-tryptophan reaction in presence of gemini
surfactants and 'H NMR investigation at different concentrations of gemini
surfactants.
Chapter - V: 'H NMR study of gemini surfactants in presence of additives
(sodium anthranilate and sodium benzoate )
44
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Cfiapterll
Tj(perimenta[
Materials
All the chemicals used throughout the whole stu ^ are listed in Table
2.1, which also includes their abbreviated names, chemical formulas, sources,
and purities.
Synthesis
Synthesis of bis(quaternary ammonium) surfactants:
The bis(quatemary ammonium) surfactants (2a - 2c) were synthesized
by adopting the following Scheme 2.1 and the procedure outlined in reference
(1).
Scheme 2.1: i: C16H33N (CHj)! (2.1 equiv), Dry EtOH, Reflux, 48h; 70-90% yield
i Br (CH2)„ Br ^«-C,6H33N"(CH3)2 (CH2)n.N"(CH3)2A7-C,6H33
Br" Br' (m = 4,5,6) (m = 4,5,6)
A 1:2.1 equivalent mixture of corresponding a, co - dibromoalkanes (m
= 4,5,6) with N, N-dimethylhexadecylamine in dry ethanol was refluxed (at
-80 °C) for 48 h. The progress of the reaction was monitored using TLC
technique. The solvent was removed under vacuum from the reaction mixture
and the solid thus obtained was recrystallized several times from hexane/ethyl
acetate mixture to obtain the compound in pure form. (The real difficulty was
in the purification of the raw gemini surfactant). The overall yields of the
surfactants ranged from 70 to 90%. Purity of all the gemini surfactants was
checked on the basis of C, H, N analysis and surface tension measurements
3 0-,
o ON OS
56
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59
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60
(Fig. 2.1 - absence of minima in the surface tension - log [surfactant] plots are
indicative of the surfactant purity^) which were further characterized by 'H
NMR, mass and IR spectroscopy. Pertinent details are given below.
(2a) 1,6-bis(N-hexadecyl-N,N-dimethylammonium)hexane dibromide
(16-6-16):
'H NMR, (300 MHz, CDCI3); 6 : 0.88 (t, 6H, alkylchain 2x1 C//3), 1.255-1.350
(s+br m, 48H, alkyl chain 2 x \2CH2), 1.580 - 1.715 (br m, 12H, spacer chain
1 X 2 C//2CH2lSr and alkyl chain 2 x 1 CH2CH2 CHzN^), 1.995 (br s, 4H,
spacer chain 1 x 2 CH2CH2 CHjN^), 3.355 (br s, 16H, 2 x 2 N^C/Zj and alkyl
chain 2 x 1 C//2N^), 3.431 - 3.686 (m, 4H, spacer chain 1 x 2 C/Z^N ) (Fig.
2.2).
MS-ESI; m/z (%): 701 (M^- Br", 78.52), 607 (7.97), 432 (9.82), 398 (28.22),
311(100.0), 270(27.92), 199(16.56), 128 (34.35) (Fig. 2.3).
IR: Un,ax (KBr): 1242.7 (C-N) cm"'.
C, H, N analysis: calcd. for C42H9oN2Br2: C 64.43, H 11.60, N 3.60, found: C
64.71, H 11.81, N 3.66.
(2b) l,5-b'is(N-hexadecyl-N,N-dimethylammonium)pentane dibromide
(16-5-16):
'H NMR (300 MHz, CDCI3); 5: 0.88 (t, 6H, alkyl chain 2 x 1 C//j), 1.255 -
1.352 (br m, 42H, alkyl chain 2 x 10 C//^ and spacer chain IC//2), 1.610 -
1.725 (crude t, 16H alkyl chain 2 x 4 C//^), 1.987 - 2.116 (br m, 4H spacer
chain 1 X 2 C/Z^CHjN"), 3.352 (s, 12H, 2 x 2 N^C//j), 3.45 - 3.505 (crude t,
61
4H, alkyl chain 2 x 1 CH2N*), 3.844 - 3.90 (crude t, 4H, spacer chain 1 x 2
C//2N*)(Fig.,2.4).
MS-ESI; m/z (%): 689 (M^-Br", 63.19), 418 (11.96), 384 (20.55), 304(100.0),
270 (9.50), 192 (12.27), 114 (77.91) (Fig. 2.5).
IR: u„,ax (KBr): 1230.3 - 1044.5 (C-N) cm"'.
C, H, N analysis: calcd. for C4, Hgg N2 Br2: C 64.06, H 11.45, N 3.64, found: C
64.28, H I 1.99, N 3.43.
(2c) l,4-bis(N-hexadecyl-N,N-dimethylaminonium)butane dibromide
(16-4-16):
'H N M R (300 MHz, CDCI3); 5: 0.88 (t, 6H, alkyl chain 2 x 1 CH3), 1.257 -
1.344 (br m, 44H, alkyl chain 2 x 1 1 C//^), 1.754 (m, 12H, alkyl chain 2 x 3
CH2), 2.084 (br s, 4H, spacer chain 1 x 2 CH2Cl{i^\ 3.308 (s, 12H, 2 x 2
N^C//j), 3.431 (m, 4H, alkyl chain 2 x 1 C/Z^N ), 3.811 (br s, 4H, spacer chain
2 X 1 C/Z^N ) (Fig. 2.6).
MS-ESI; m/z (%): 676 (M^- Br^ 19.63), 404 (100.0), 324 (62.27), 310 (44.78),
297 (6.75), 268 (7.97), 197 (12.88) 155 (13.50) (Fig. 2.7).
IR: Un,ax (KBr): 1043.08 (C-N) cm"'.
C, H, N analysis: calcd. for C4oH86N2Br2: C 63.62, H 11.40, N 3.71, found: C
63.49, H 11.65, N 3.40.
62
E
-6.0 -5.5 - 3 .5 -5.0 -4.5 -A.O
log Cl6-m-l63
Fig. 2.1: Surface tension vs. log [gemini] plots at 30 T . (1) 16-6-16, (2)
16-5-16,(3) 16-4-16.
63
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69
Preparation of solutions
The water used to prepare solutions was distilled twice over alkaline
KMn04 in an all-glass (Pyrex) distillation set-up. Specific conductivity of the
double-distilled water was in the range (1-2) x 10' S cm' .
Special care was observed for cleaning the glassware with chromic acid
and then by rinsing with double distilled water.
(i) Buffer solutions:
Acetate buffer was prepared by mixing appropriate volumes of 0.20 mol
dm' acetic acid and 0.20 mol dm' sodium acetate solutions.^
(ii) Surfactant solutions:
All the surfactant solutions were prepared by dissolving appropriate
amount of surfactants in the buffer solution of required pH.
(Hi) Ninhydrin and amino acid solutions:
Stock solutions of ninhydrin and amino acids were also prepared in
buffer solutions. The ninhydrin stock solution was stored in a dark bottle.
D2O of 99.9% (Aldrich) purity, for NMR experiments, was supplied by
Central Drug Research Institute (CDRI), Lucknow.
pH - measurements
pH measurements of the solutions were made with an ELICO pH-meter
type LI-I20 (ELICO, Hyderabad, India) fitted with an ELICO CH-41 glass and
calomel combination electrode.
70
Kinetic measurements
The kinetic measurements were followed spectrophotometrically by
monitoring the appearance of spectral absorption of purple color
( max = 570 nm) under pseudo-first-order conditions (ninhydrin in excess) with
a Bausch & Lomb Spectronic-20 spectrophotometer equipped with an oil-bath
thermostated at the desired temperature (±0.rC). A three-necked reaction
vessel (fitted with double-walled condenser to check evaporation) containing
required volume of amino acids with other reagents (when required) were kept
in oil bath. Required volume of thermally equilibrated ninhydrin solution was
added to start the reaction. The zero-time was recorded after half of the
addition of the ninhydrin solution. A slow stream of pure nitrogen gas (free
from CO2 and O2) vvas passed through the reaction mixture for stirring and
maintaining an inert atmosphere.
For all the kinefic runs, values of pseudo-first-order rate constants (k\^,)
were calculated from plots of log (AOO-AQ) / (A00-At) vs. time (t) by a least-
squares regression analysis of the data. The values of absorbance at infinite
time (Aoo) for each amino acid-ninhydrin system were obtained in the
following manner. At the end of each kinetic run, 10 cm^ of the solution
mixture (after taking into a standard volumetric flask) was boiled for 2 min. It
was then cooled to room temperature and after adding buffer solution to
compensate any volume loss, absorbance was recorded.
The results are recorded in Chapters III and IV. The rate constants
obtained from multiple determinations agreed within ±4%.
71
Determination of cmc by conductivity measurements
The determination of cmc by conductivity measurements were carried
out by using a conductivity bridge (ELICO, Hyderabad, India, type CM 82T).
First, tiie conductivity of the solvent was measured and then conductivity was
recorded every time after addition of small volume of the stock solution of
CTAB and ensuring complete mixing. The specific conductivity was calculated
by applying the solvent correction. The cmc values of surfactants in the
presence and absence of reactants were obtained from the break point of nearly
two straight lines of the specific conductivity vs. concentration plots.
Experiments were carried out under different conditions, i.e., solvent being
water, water + amino acid, water + ninhydrin, water + amino acid + ninhydrin.
The specific conductivity vs. concentration plots are shown in Figs. 2.8-
2.15 and estimated values of cmc are given in Tables 2.2 and 2.3.
Determination of cmc by surface tension measurements
Surface tension measurements of surfactants at the air-water interface
are the most often applied methods for determining cmc. Since pure water at
room temperature has a surface tension of about 72 mN/m and the surface
tension of the air-water interface when coated with a monolayer of amphiphile
is generally in the 20 to 40 mN/m range, one generally can observe a break in
surface tension as function of surfactant concentration. Such measurements for
pure gemini surfactant solutions are illustrated in Fig. 2.1 and their cmc values
72
are recorded in Table 2.4. A du Nouy type tensiometer (Hardson and Co.,
Kolkata) was used.
The cmc values of TX-lOO in the absence and presence of reactants
were also obtained by surface tension measurements under varying conditions,
i.e., solvent being water, water + amino acid, water + ninhydrin, water + amino
acid + ninhydrin. The surface tension vs. log [surfactant] plots are shown in
Fig. 2.16 and estimated values of cmc are given in Table 2.5.
Viscosity measurements
When a solute is added to water the flow properties of the water are
altered by the internal friction of the system being increased. Viscosity implies
resistance to flow. It is the measure of the internal friction of a liquid. It is
developed in liquids because of the shearing effect of moving one layer of
liquid past another. Viscosity is expressed as Ns/m or poise. In practice,
smaller units centipoise and millipoise are used.
There are a number of methods of different kinds for measuring
viscosity, 7. The method commonly employed is based on Poiseuille's law
which is given by,
77 = TirV / 8v/ (2.1)
where v is the volume in cm^ of the liquid flowing in t seconds through a
narrow tube of radius r cm and length / cm under a hydrostatic (driving)
pressure of P N/m^.
73
Hence viscosity of a liquid is determined with respect to another liquid,
usually water. This is called relative viscosity (^r). If ti and t2 are the times of
flow of the same volume of water and the liquid, respectively, then
'' m 8v/ •7rr^2P2 t2P2
Since the pressure is proportional to the density (p), we have
rir=^ (2.3)
Ozeki and Ikeda" found density corrections to be negligible, TJ^ value
may, therefore, be calculated using equation
r = 7 - (2.4)
Aqueous surfactant solutions containing spherical micelles are of low
viscosity.^ Addition of sahs and organic additives usually changes the
shape/size of the micelles, which is often reflected by the change in viscosity.^
Micellar growth is accompanied by a distinct rise in viscosity. In view to the
shape/size of the microscopic objects in a homogeneous suspension, one can
expect the evolution of micellar shape to be reflected in the viscosity variation.
In the present investigations viscosities of the solutions were obtained
using an Ubbelohde viscometer thermostated at particular temperature
(25 "C). The method is simple, reliable and provides information related to
micellar size. As the viscosities of certain systems were highly dependent on
the rate of flow, it was necessary to obtain values under Newtonian flow
74
conditions. For tiiis purpose, the following modification was made in the
Ubbelohde viscometer. A wide U-tube containing water was connected to one
of the limbs of the viscometer which is open to the atmospheric pressure under
normal operation conditions. Thus the pressure, P, can be varied under which
the solution flows. The viscosity values at different rates of flow can be
obtained from the slopes of the straight lines P vs. I/t. The viscosity data are
given and shown graphically at appropriate places in Chapter V.
'H N M R measurements
'H NMR spectra of the synthesized geminis were recorded on a 300
MHz Bruker Cryomagnet spectrophotometer in CDCI3 with ' H chemical shifts
relative to internal TMS.
Other ' H NMR spectra for concentration and additive effects on geminis
were also recorded on the same instrument working at 300 MHz in 5 mm thin-
walled NMR tubes with the peak for D2O as the internal standard. Stock
solutions of geminis were prepared in D2O. Sample solutions of additives were
prepared first by taking requisite amounts and making up the volumes by
freshly prepared stock solution of surfactant. To get lower concentrations, the
samples were diluted by adding appropriate volume of the surfactant stock
solution.
75
Elemental analysis
Elemental analysis was done by the Sophisticated Analytical Instrument
Facility (SAIF), Lucknow.
Mass spectroscopy
The electrospray mass spectra were recorded on a MICROMASS
QUATTRO II triple quadrupole mass spectrometer. The samples (dissolved in
suitable solvents such as methanol/acetonitrile/water) were introduced into the
ESI source through a syringe pump at the rate of 5|il per min. The ESI capillary
was set at 3.5 kV and the cone voltage was 40 V. The spectra were collected in
6s scans and the print outs are averaged spectra of 6-8 scans.
IR measurements
The IR spectra were recorded using a FT-IR Spectrometer Interspec
2020 (Spectrolab, U.K.).
76
e 00
.LL
o
10 15
lO^CCTABDCmol d rn ' ^ )
Fig. 2.8: Variation of specific conductivity (K) with CTAB concentration
at 30 °C. (I) in water, (2) in water in the presence of 3.0 x 10" mol dm'"* DL-
valine, (3) in water in the presence of 5.0 x 10' mol dm' ninhydrin, and (4) in
water in the presence of 3.0 x 10" mol dm" DL-valine and 5.0 x 10" mol dm"'
ninhydrin. The scale shown is for curve 1. Curves 2, 3, 4 have been shifted
upwards by 5, 10, 15 scale units, respectively.
- > ^ ' . M ^ ^ n '
\
r,-,.:
0 5 10 15 20 25
lO'' C C T A B D { m o l d m ~ 3 )
Fig. 2.9: Variation of specific conductivity (K) with CTAB concentration
at 80 "C. (1) in water, (2) in water in the presence of 3.0 x 10" mol dm" DL-
valine, (3) in water in the presence of 5.0 x 10' mol dm' ninhydrin, and (4) in
water in the presence of 3.0 x 10" mol dm' DL-valine and 5.0 x lO'"* mol dm"'
ninhydrin. The scale shown is for curve 1. Curves 2, 3, 4 have been shifted
upwards by 10, 20, 30 scale units, respectively.
78
Fig. 2.10: Variation of specific conductivity (K) with 16-6-16 concentration
at 70 °C. (I) in water, (2) in water in the presence of l.O x 10"* mol dm""* DL-
tryptophan, (3) in water in the presence of 5.0 x 10'" mol dm" ninhydrin, and
(4) in water in the presence of 1.0 x 10" mol dnr^ DL-try'ptophan and 5.0 x 10"'
mol dm" ninhydrin. The scale shown is for cui-ve 1. Curves 2, 3. 4 have been
shifted upwards by 2, 4, 6 scale units, respectively.
79
1 0 ^ C C i 6 - C 5 - C l 6 D ( m o l d m - ^ )
Fig. 2.11: Variation Of specific conductivity (K) with 16-5-16 concentration
at 70 °C. (1) in water, (2) in water in the presence of 1.0 x 10"* mol dm""* DL-
tryptophan, (3) in water in the presence of 5.0 x 10"' mol dm""* ninhydrin, and
(4) in water in the presence of 1.0 x 10^ mol dm' DL-tr>ptophan and 5.0 x 10'
mol dm' ninhydrin. The scale shown is for curve 1. Curves 2, 3, 4 have been
shifted upwards by 2, 4, 6 scale units, respectively.
80
3 4 5 5 7 10
Fig. 2,12: Variation of specific conductivity (K) with 16-4-16 concentration
at 70 °C. (I) in water, (2) in water in the presence of 1.0 x 10" mol dm"' DL-
tryptophan, (3) in water in the presence of 5.0 x 10" mol dm' ninhydrin, and
(4) in water in the presence of 1.0 x 10^ mol dm' DL-tryptophan and 5.0 x 10"'
mol dm' ninhydrin. The scale shown is for curve 1. Curves 2, 3, 4 have been
shifted upwards by 2, 4, 6 scale units, respectively.
81
12
10
6
1^ 6
to
(.
2
0
jt*^******
. ^ ' - ^
. * * * ^ ^
\y^j^ \X*
^^f 1 1 1
^..^^^^^^^^^^"^Z^ * ^ ^ ^ ^ ^ * < ! ^
::^0^ f^ /j^^
^
1 1 1 1 1
^ ' r l T ^ '
1 1 0 1 2 3 A 5 6 7 8 9
1 0 5 C C i 5 _ C 4 _ C ^ 5 3 ( m o l d m - ^ )
Fig. 2.13: Variation of specific conductivity (K) with 16-6-16 concentration
at 30 °C. (1) in water, (2) in water in the presence of 1.0 x 10" mol dm'* DL-
tryptophan, (3) in water in the presence of 5.0 x 10" mol dm' ninhydrm, and
(4) in water in the presence of 1.0 x 10"* mol dm" DL-tryptophan and 5.0 x lO""*
mol dm" ninhydrin. The scale shown is for curve 1. Curves 2, 3, 4 have been
shifted upwards by 2, 4, 6 scale units, respectively.
10
82
3 A 5 6 7
1 0 ^ C C i 6 - C 5 - C i 5 3 ( m o l d m - 3 )
10
Fig. 2.14: Variation of specific conductivity (K) with 16-5-16 concentration
at 30 °C. (1) in water, (2) in water in the presence of 1.0 x 10"* mol dm"'' DL-
tryptophan, (3) in water in the presence of 5.0 x 10" mol dm" ninhydrin, and
(4) in water in the presence of 1.0 x 10" mol dm' DL-tryptophan and 5.0 x 10'
mol dm" ninhydrin. The scale shown is for curve 1. Curves 2, 3, 4 have been
shifted upwards by 2, 4, 6 scale units, respectively.
83
2 3 A 5 5 7 8 9 10
1 0 ^ C C i 6 - C 4 - C i 6 3 { m o l d m - 3 )
Fig. 2.15: Variation of specific conductivity (K) with 16-4-16 concentration
at 30 °C. (1) in water, (2) in water in the presence of 1.0 x 10" mol dm""* DL-
tryptophan, (3) in water in the presence of 5.0 x 10" mol dm'" ninhydrin, and
(4) in water in the presence of 1.0 x 10" mol dm" DL-tryptophan and 5.0 x 10"
mol dm""* ninhydrin. The scale shown is for curve 1. Curves 2, 3, 4 have been
shifted upwards by 2, 4, 6 scale units, respectively.
84
Table 2.2
Critical micelle concentration (cmc) values of CTAB determined by
conductivity measurements in absence and presence of DL-valine (3.0 x 10'
mol dm" ) and ninhydrin (5.0 x 10"" mol dm" ) at 30 and 80 °C.
Solution 10 cmc (mol dm")
30 "C 80 °C
water 10.1 15.6
water + valine 10.1 15.8
water + ninhydrin 10.0 15.4
water + valine + ninhydrin 10.0 15.4
85
Table 2.3
Critical miceUe concentration (cmc) values of 16-m-16 geminis determined by
conductivity measurements in absence and presence of DL-tryptophan (1.0 x
10''' mol dm" ) and ninhydrin (5.0 x 10' mol dm" ) at 30 and 70 °C.
Solution 10 cmc (mol dm'^)
30 °C 70 °C
16-6-16
water 4.4 (4.3f 5.6
water + tryptophan 4.1 5.0
water + ninhydrin 2.6 4.9
water + tryptophan + ninhydrin 2.4 4.4
16-5-16
water 3.6 (3.6/ 5.4
water + tryptophan 3.0 3.4
water + ninhydrin
water + tryptophan + ninhydrin
16-4-16
2.5
2.0
2.8
2.4
water 2.8 (2.7)' 4.8
water + tryptophan 2.4 2.5
water + ninhydrin 1.3 1.5
water + tr}'ptophan + ninhydrin 1.0 1.3
From reference (1), determined by tluorescence measurements
86
Table 2.4
Critical micelle concentration (cmc) values of 16-m-16 geminis in water
determined by surface tension measurements at 30 ' C.
Gemini 10 cmc
(mol dm""')
16-6-16 4.5
16-5-16 3.6
16-4-16 2.6
87
Fig. 2.16: Surface tension (y) vs. log C plots for TX-lOO at 40 ''C. (I) in -4
water, (2) in water in the presence of 5.0 x 10' mol dm'"* DL-tryptophan, (3) in
water in the presence of 7.0 x 10"" mol dm""' ninhydrin, and (4) in water in the
presence of 5.0 x 10"" mol dm"" DL-tryptophan and 7.0 x 10" mol dm"'
ninhydrin.
88
Table 2.5
Critical micelle concentration (cmc) values of TX-lOO determined by surface
tension measurements in absence and presence of DL-tryptophan (5.0 x 10"''
mol dm- ) and ninhydrin (7.0 x 10' mol dm" ) at 40 °C.
Solution 10 cmc (mol dm")
water 2.5
water + tryptophan 2.5
water + ninhydrin 2.6
water + tryptophan + ninhydrin 2.5
89
References:
1. S. De, V. K. Aswal, P. S. Goyal and S. Bhattacharya, J. Phys. Chem.,
100, 11664(1996).
2. K. S. Sharma, C. Rodgers, R. M. Palepu and A. K. Rakshit, J. Colloid
Interface Sci., 268, 482 (2003).
3. H. T. S. Britton, "Hydrogen Ions", Vol. 1, Chapman and Hall, London,
1942.
4. S. Ozeki and S. Ikeda, J. Colloid Interface Sci., 77, 219 (1980).
5. H. -H. Kohler and J. Stmad, J. Phys. Chem., 94, 7628 (1990).
6. R. Gomati, J. Appell, P. Bassereau, J. Marignan and G. Porte, J. Phys.
C/zem., 91, 6203(1987).
chapter III
(fl.) !Ninfiycfrin-<DL-%^aCine ([(^action in (Presence ofCtA^
andTffect of Organic SoCvents
(<B) !NinfiycCrin-<DL-lryptopfian faction in (Presence of
TX-lOO and effect of Organic SoCvents
91
Introduction
Although the ninhydrin reaction is used daily in thousands of
laboratories throughout the world, the technique is still far from satisfaction. As
already mentioned, the amount of DYDA produced depends upon reaction
conditions, i.e., pH, temperature, reactant concentrations, presence of organic
solvents, etc. Although some qualitative information is available on the role of
organic solvents,'"^ kinetic evidence to distinguish their role is limited. For this
reason, and in the hope that introduction of organic solvents may cause the use
of low [reactants], a systematic kinetic study of ninhydrin-DL-valine reaction
has been made in the presence of cationic CTAB micelles in the absence and
presence of different fixed compositions of solvents (acetonitrile, dimethyl
sulfoxide, methylcellosolve and 1-propanol). It is pertinent to mention here that
with the concentrations of reactants used no color developed at 80 °C in the
absence of CTAB micelles. The results and probable explanations are
described below.
(A) Ninhydrin-DL-Valine Reaction in Presence of CTAB and Effect of
Organic Solvents
Results
Spectra:
In order to confirm whether the same colored product is formed in the
absence and presence of organic solvents, a series of UV-visible spectra were
recorded after completion of the reaction at 80 °C in the presence of [CTAB] =
92
20.0x10"^ mol dm" . These results are shown graphically as absorbance-
wavelength profiles in Fig. 3.1. The absorption spectra of mixtures containing
the reactants in different solvents exhibited the same maxima as that of a
solution of Ruhemann 's purple in aqueous micellar medium (see for example,
Fig. 3.1(b). The spectra consist of two broad bands with A,max - 400 and 570
nm. No variation in >.max in the presence of different solvents clearly indicates
the product of valine-ninhydrin reaction to be the same as in aqueous micellar
medium, i.e., Ruhemann's purple or diketohydrindylidenediketohydrindamine
(DYDA).''^
Effect of pH:
The solution pH plays a pivotal role in the amino acid-ninhydrin
reactions; this being the reason that the effect of pH on the rate of valine-
ninhydrin reaction was studied in the pH range 3.5 to 6.0. The results are given
in Table 3.1 and shown in Fig. 3.2. The formation of Ruhemann's purple was
known to be dependent on pH and humidity, and complete development was
known to require heating. The rate constant increased upto pH 5.0 and
thereafter became almost constant. That is why the whole study of the reaction
was undertaken at pH 5.0.
Effect offDL-ValineJ:
The ky^, values were determined at different initial concentrations of DL-
valine ranging from 3.0 x 10"'' - 4.5 x 10"'' mol dm" with a view to find the
93
order with respect to [amino acid]. The concentrations of ninhydrin, CTAB and
pH were kept constant at a fixed temperature of 80 °C. It was observed that the
values of rate constant {k^^,) were independent of the initial concentrations of
DL-valine (Table 3.2). This indicates that the order of the reaction with respect
to [amino acid] is unity. The rate law would then be
Rate = d[product] / dt = k^ [amino acidji (3.1)
Effect of [NinhydrinJ:
The dependence of the rate constants on [ninhydrin] was determined by
carrying out the kinetic experiments at different concentrations of ninhydrin
from 5.0 X 10- - 40.0 x 10" mol dm' keeping the [DL-valine], [CTAB] and
pH constant at 80 °C. The results are recorded in Table 3.2 and shown
graphically in Fig. 3.3.
The plot k^ vs. [ninhydrin] was non-linear whereas log k^^ vs. log
[ninhydrin] plot was linear with slope less than unity. This shows fractional
order in [ninhydrin].
Effect offCTABJ:
The effect of surfactant (CTAB) concentration was determined by
carrying out a series of kinetic runs at different [CTAB] from 5.0 x 10" - 70.0
X 10' mol dm"' with fixed ninhydrin and DL-valine concentrations at 80 °C.
94
The observed pseudo-first-order rate constants, ky^, increase upon
increasing concentration of the cationic CTAB surfactant and reaches a
maximum. Further increase in the surfactant concentration eventually leads to a
slow decrease. The results are given in Table 3.3 and depicted graphically in
Fig. 3.4 as rate constant (A: ,) - surfactant concentration profile.
Effect of Temperature:
A series of experiments were carried out within the temperature range of
75-90 °C at fixed [ninhydrin], [DL-valine], pH (= 5.0) and [CTAB]. The data
obtained were found to fit the Eyring equation (Table 3.4)
k^ = (kg T /h) exp (AS" / R) exp (-AH* /RT) (3.2)
(RB, h and R are, respectively, Boltzmann, Planck and gas constants).
Solvent Effect:
The effect of presence of organic solvents, viz., PrOH, MC, AN and
DMSO on the rate of purple-color formation was also seen at fixed [DL-
valine], [ninhydrin], [CTAB], pH (= 5.0) and temperature (80 °C) (Table 3.5
and Fig. 3.5).
Effect of CTAB Micelles in Presence of Different Fixed Compositions of
DMSO:
Effect of CTAB micelles has been seen for the reaction in the presence
of different fixed compositions of DMSO. Rate profiles obtained for the
95
solutions with 10-70% DMSO by volume exhibit clear maxima that shift to
higher concentration of surfactant as a function of DMSO content. Results are
recorded in Table 3.6 and graphically presented in Fig. 3.6.
Discussion
It is well known that the amino acid-ninhydrin reactions proceed
through the formation of a Schiff base (unstable for isolation) and a series of
reactions, involving different intermediates, up to the formation of Ruhemann's
purple"'^. It is, therefore, necessary to point out the main steps involved in the
mechanism of ninhydrin reaction before discussing the role of surfactant
micelles and solvents.
The reaction proceeds through the formation of a Schiff base which,
being unstable, undergoes decarboxylation and hydrolysis to yield 2-amino-
indanedione (A) as an intermediate (Scheme 3.1). A acts as a reactant in the
formation of ammonia and Ruhemann's purple and the two reactions (i.e., route
(i) -hydrolysis and route (ii) -condensation) occur simultaneously. These two
reactions depend strongly upon the pH, atmospheric oxygen, and temperature.
A is highly sensitive to molecular oxygen and a yellowish colored product is
formed (instead of Ruhemann's purple) in the presence of atmospheric oxygen.
At low pH, the reaction has been found to proceed chiefly by route (i) and
ammonia is evolved almost quantitatively with no Ruhemann's purple
formation. In solutions of pH > 5.0, route (ii) predominates but the possibility
of route (i) can not be ruled out completely.
96
U C o l_ O </)
<
360 400 600 450 500 550
Wave length ( n m )
Fig. 3.1: Absoiption spectia of the reaction product of DL-valine with
ninhydriii in aqueous micellar media at 80 "C. (a) in absence of surfactant, (b)
in presence of [CTAB] = 20.0 x 10"' tnol dm", (c) in presence of [CTAB] =
20.0 .\ 10"' mol dm"' t 20% methylcellosolve, (d) in presence of [CTAB| -
20.0 X 10"' mol dm ' - 20% 1-propanol. (e) in presence of [CTAB] - 20.0 x
10"' mol dm' - 20% dimethyl sulfoxide and (0 '" presence of [CTAB] 20.0
X 10"' mol dm"' ^ 20"o acetonitrile. Rcacilon condiiions: [DL-valine] ^ 3.0 x
10 mol dm" , [ninhydrinj ="- 5.0 x 10 mol dm"\ pH = 5.0.
97
Table 3.1
Effect of pH on pseudo-first-order rate constants (ky^) for the reaction of DL-
valine with ninhydrin.
Reaction conditions: [DL-valineJj
[ninhydrin]T
[CTABJT
Temperature
N-4 3.0 X lO'^'moldm"
5.0xlO''moldm"'
= 20.0xl0"moldm"
= 80°C
pH 10 A;.
(s-')
3.5
4.0
4.5
5.0
5.5
6.0
No reaction
4.2
4.9
6.8
7.7
8.0
98
Fig. 3.2: Effect of pH on the reaction of DL-valine with ninhydrin.
Reaction conditions: [DL-valine] = 3.0 x lO""* mol dm•^ [ninhydrin] = 5.0 x
IQ--' mol dm'-\ [CTAB] = 20.0 x 10" mol dm'\ temp. = 80 °C.
99
Table 3.2
Effect of [DLrvaline] and [ninhydrin] on pseudo-first-order rate constants (ky^,)
for the reaction of DL-valine with ninhydrin.
Reaction conditions: pH =5.0
[CTAB]T = 20.0 X 10" mol dm"
Temperature = 80 °C
10' [vaUne]T 10' [ninhydrinji Wk^
(moldm'^) (moldm'^) (s"*)
3.0 0.5 6.8
3.5 0.5 6.7
4.0 0.5 6.7
4.5 0.5 6.6
3.0 0.5 6.8
3.0 1.0 11.9
3.0 1.5 15.3
3.0 2.0 19.2
3.0 2.5 22.1
3.0 3.0 25.3
3.0 3.5 26.9
3.0 4.0 28.0
100
in o
0 10 20 30 AO
l O ^ C N i n h y c l r i n J t m o l dm"3)
Fig. 3.3: Effect of ninhydrin on the reaction of DL-valine with ninhydrin.
Reaction conditions: [DL-vahne] = 3.0 x 10" mol dm'\ pH = 5.0, [CTAB] =
20.0 .\ 10"- mol dm ^ temp. = 80 °C.
101
Table 3.3
Effect of [CTAB] on pseudo-first-order rate constants (ky^,) for the reaction of
DL-valine with ninhydrin.
Reaction conditions: pH =5.0
[DL-valine]! = 3.0 x 10""* mol dm'
[ninhydrin]T = 5.0 x 10' mol dm"
Temperature = 80 °C
10' [CTAB]T
(mol dm'" )
0.0
5.0
10.0
15.0
20.0
25.0
30.0
40.0
50.0
60.0
70.0
10'^^
(s-')
0.0
2.2
3.9
5.4
6.8
6.4
5.8
5.4
5.6
5.0
4.8
1U K^i cal
(s-')
-
2.2
3.8
5.4
6.3
6.4
5.4
5.4
5.6
5.0
4.8
Ic -k
k
-
0
+0.03
0
+0.07
0
+0.07
0
0
0
0
102
in o
20 40
10"^CCTABI|(moldm~^)
Fig. 3.4: Effect of CTAB on the reaction of DL-valine with ninhydrin.
Reaction conditions: [DL-valine] = 3.0 x 10"'' mol dm"^ [ninhydrin] = 5.0 x
10' mol dm"\ pH = 5.0, temp. = 80 °C.
103
Table 3.4
Effect of temperature on pseudo-first-order rate constants (k^^) for the reaction
of DL-valine with ninhydrin.
Reaction conditions: pH
[DL-valine]n
[ninhydrin] T
[CTAB]T
5.0
-4 -3 - 3.0 xlO"%-nol dm
= 5.0xlO-^moldm-^
2.0xl0"^moldm'^
Temperature 10'^.
(s-*)
75
80
85
90
4.8
6.8
8.4
12.7
Parameters
Ea(kJmol"')
AH^lkJmor')
-AS''(JK''mor')
A:V (mof' dm^)
Kf^ (mor' dm^)
10'^n,(S-')
65
62
152
62
65
1.3
104
Table 3.5
Effect of organic solvents on pseudo-first-order rate constants (k^^,) for the
reaction of DL-valine with ninhydrin.
Reaction conditions: pH
[DL-valine]T
[ninhydrinjx
[CTABJT
Temperature
= 5.0
S.OxlO^'moldm-^
5.0xlO"^Tioldm"^
20.0xl0'^moldm"
= 80X
% solvent
(v/v)
0
5
10
15
20
25
30
35
40
AN
6.8
6.6
15.1
24.5
44.2
55.6
72.4
78.0
83.3
DMSO
6.8
6.3
6.6
6.9
12.0
18.1
29.1
42.9
52.0
10'^^
(s-')
MC
6.8
6.4
6.7
6.8
7.2
13.9
14.5
25.0
45.2
PrOH
6.8
6.7
8.3
10.2
15.3
30.4
35.0
47.3
49.3
105
80
1 V)
^
in ^ AO
—
1
A/
1
^ b
rs/ /
1 1 0 10 /iO 20 30
% solvent (V/V)
Fig. 3.5: Effect of methylcellosolve (D), 1-propanol ( • ) , dimethyl
sulfoxide (O) and acetonitrile (A) on tiie reaction of DL-valine with ninhydrin.
Reaction conditions: [DL-valine] = 3.0 x 10""* mol dm'"', [ninhydrin] = 5.0 x
10' mol dm"\ [CTAB] = 20.0 x 10' mol dm"', pH = 5.0, temp. = 80 °C.
106
Table 3.6
Effect of [CTAB] on pseudo-first-order rate constants (k^) for the reaction of
DL-valine with ninhydrin in the presence of different fixed compositions of
DMSO.
Reaction conditions: pH =5.0
Temperature = 80 "C
10 [CTAB] "2
(mol dm")
5
10
15
20
25
30
40
50
60
70
80
90
100
% DMSO (v/v)
10
2.9
4.3
5.0
6.6
5.6
5.2
5.0
4.3
4.4
4.0
-
-
-
20
4.1
8.7
9.2
11.8
13.0
12.0
12.1
12.0
10.0
8.7
-
-
-
a
30
7.6
12.0
22.1
29.1
30.0
24.0
18.2
15.2
8.5
9.2
-
-
-
IO
CS
40
11.7
23.2
41.0
52.0
56.0
59.4
61.2
55.0
48.0
40.0
-
-
-
• ' )
50
20.6
41.4
53.1
64.1
72.0
74.0
76.0
72.6
69.8
66.2
-
-
-
% DMSO (v/v)
50
12.3
13.1
15.0
16.9
20.0
22.9
26.0
25.0
23.5
20.0
18.1
16.3
15.1
60
12.1
13.0
17.0
19.1
24.0
25.9
32.0
33.5
35.0
35.0
33.0
32.2
29.0
,"
70
26.9
34.5
38.4
48.0
51.8
60.0
64.0
72.0
73.0
74.0
76.0
72.0
69.8
-3
• b ' [ninhydrinjx = 5.0 x iO mol dm ; [DL-valine]T = 3.0 x 10 mol dm'
v3 [ninhydrin]r = 2.5 x 10" mol dm ; [DL-valinejj = 1.5 x 10 mol dm -3
107
Table 3.7
pH and the observed pseudo-first-order rate constants (k^^) for the reaction of
DL-valine with ninhydrin reaction carried out in different solvents (40% v/v).
Reaction conditions: [DL-valine]T =3.0x10"'* mol dm'
[ninhydrin]T = 5.0 xlO"^ mol dm'
[CTAB]T = 2 0 . 0 X 1 0 - ^ mol dm"
Temperature = 80 °C
Reaction Medium pH 10^A:v (s"')
Buffer (CH3COOH + CHsCOONa) 5.0 6.8
60% pH 5.0 buffer + 40% PrOH 5.51 49.3
60% pH 5.0 buffer + 40% MC 5.70 45.2
60% pH 5.0 buffer + 40% AN 5.84 83.3
60% pH 5.0 buffer + 40% DMSO 6.14 52.0
80
T w
^> >•
^ in
o
60
40
20 -
- 1 108
80 -
(B)
7 0 % O M S O
DMSO
DMSO
2 0 % DMSO
0%DMSO 10% DMSO I
100
103CCTABD( ' "o ldm~ '^ )
Fig. 3.6: (A) Effect of CTAB in the presence of various concentrations of
dimethylsulfoxide (DMSO) on the reaction of DL-valine with ninhydrin.
Reaction conditions: [DL-vaHne] ^ 3.0 x 10''' mol dm'"', [ninhydrin] = 5.0 x
10" mol dm"\ pH = 5.0, temp. = 80 °C.
(B) Effect of CTAB in the presence of various concentrations of
dimethylsulfoxide (DMSO) on the reaction of DL-valine with ninhydrin.
Reaction conditions: [DL-valine] = 1.5 x 10' mol dm'"\ [ninhydrin] ^ 2.5 x
10 ' mol dm'"', pH = 5.0, temp. = 80 ^C (Due to high absorbance, the reactant
concentrations were half of that in (A)).
109
Due to Schiff base formation between the carbonyl group of ninhydrin
and the amino group of DL-valine, cationic RCH(N^H3)C00H and
zwitterionic RCH(N"*^H3)C00~ species can not attack the middle carbonyl
group of ninhydrin and, therefore, these species are kinetically inactive (due to
the presence of positive charge, the nucleophilic character of amino nitrogen is
diminished). Species RCH(NH2)C00H and RCH(NH2)C00", on the other
hand, may be considered as the reactive ones. Under our experimental
conditions, the concentration of RCH(NH2)C00~ is negligible (because of low
K^i and high ATD values).^ Thus, towards nucleophilic attack on the >C=0 group
of ninhydrin, the reactive species is RCH(NH2)C00H, which is in equilibrium
with the zwitterionic form of DL-valine.
Though the following equilibrium states exist in aqueous solution of
ninhydrin (2,2-dihydroxyindan-l,3-dione, Nl), only the anhydride form (N)
has been found as the reactive species for the condensation.'
VH2O
(N)
DL-valine, when dissolved in water, participates in equilibria as shown in
Scheme 3.1. Under the experimental conditions of pH = 5.0, the zwitterionic
form is the major existing species. No doubt, the zwitterionic form will cause
the substrate molecules to come closer to the micellar headgroup region (due to
110
K.. al R-CH-COOH ^ R - C H - C O O - + H^ (^ai = 5.13xlO"08(«)
NH3 +
NH3 +
^a2 R - C H - C O O - ^ R - C H - C O O r + H^ (^a2 = 1.905xlO"'7(a)
NH. NHi
KD R - C H - C O O - ^ :i R - C H - COOH
I I NH3 NH2
(/:D~IOV^''>
O + R - C H - C O O H
NH2
fast NH2 + CO2 + RCHO
Kn ,
(C)
+ NH3
+ H3O
(Ruhemann's purple)
CH3
(R = CH — for valine) CH3/ '
(Hydrindantin)
Scheme 3.1
I l l
ion-pair formation between the anionic carboxylate site and the cationic
headgroups). The concentration of DL-valine thus increases within the outer
aqueous areas of the micelles. The electrostatic interactions between the
cationic micelles and the -COO", therefore, assist in localization of valine near
the micelle-water interface. On the other hand, the presence of 7i-electrons in
ninydrin' increases the possibility of partitioning between water and positively
charged micelles. Therefore, the overall increase of the reaction rate (catalysis)
is due to concentrating both the reactants in the micellar headgroup region
(Stem layer where most of the organic reactions are found to occur'^''^).
Quantitative treatment ofk^,- [surfactant] data:
The k^^ - [CTAB] profile (Fig. 3.4) shows a maximum which is
characteristic of a bimolecular reaction. Under such a situation, the reaction
of DL-valine and ninhydrin at different surfactant concentrations may easily be
explained in terms of the modified Menger and Portnoy's pseudo-phase
model.'^'"
^ v (V ah + S ^ eVa " ' / w '^n ^ V, * " ' )m
(nin)w + Sn ^ (nin)m
/:'w 'm
' rroauci -* '
Scheme 3.2
(3.3)
(3.4)
112
For the reactions of Scheme 3.2, the rate equation is written as (neglecting ^ w,
as no purple color developed in the aqueous medium'^"'^).
k^m is the first-order rate constant in micellar medium and related to the second-
order rate constant, k^ , as
k'^ = (A:n,[(ninU) /[Sn] = k^ M\ (3.6)
M N, the mole fraction of bound ninhydrin to the micellar headgroup, is given
by
M N = [(ninU] / [S„] (3.7)
Equation (3.5) can be written as Eq. (3.8) when 1^^ is substituted from Eq.
(3.6).
k„ = (k^ Ks,[S,]M\)/{l+Ky [S„]) (3.8)
Values of M N were estimated by considering the equilibrium (cf. Scheme 3.2)
[(uin) „ 1 ' " [ ( n i n ) j ( [ S J - [ ( n i n ) j ) ^'-^^
and the mass balance
[(nin)T] = [(nin),,] + [(nin)^] (3.10)
113
Upon solving Eqs. (3.9) and (3.10), a quadratic equation (3.11) results, which
was solved for [(nin)^] with the help of a computer program with K^ as an
adjustable parameter.'^'^^ M^N was then calculated with the help of Eq. (3.7).
^N [(nin)j2- (I + / N [Sn] + / N [(nin)T]) [(nin)J + K^ [S„] [(nin)T] = 0 (3.11)
In order to determine ^ and Ky kinetically we need the cmc under
kinetic conditions which were determined conductimetrically (see
Experimental). For a given value of cmc, the k^ and AV were calculated from
Eq. (3.8) using a non-linear least squares technique. Such calculations were
carried out at different presumed values ofK^. The best fit values are recorded
in Table 3.4. Validity of the rate equation is established by comparing the
observed and calculated k^^, - values with good agreement (Table 3.3).
Evolution of CO2 and aldehyde as well as dependence of rate of purple
color formation on [valine] (first order) and [ninhydrin] (fractional order) were
97 9R
found similar to those in the aqueous medium. ' These, alongwith the
spectral observations (formation of DYDA is demonstrated in Fig. 3.1),
demonstrate that the mechanism remains unchanged.
Leffler and Grunwald have pointed out that many reactions show
isokinetic relationship; AH**= C + BAS*. We also obtained fairly linear AH** vs.
AS plot for the formation of Ruhemann 's purple by the reaction of ninhydrin
with a-amino acids (Fig. 3.7). The present AH'' and AS** data fit very well on
the straight line which indicates that the kinetics of the reaction of ninhydrin
114
6
# I O
1 0 0
8 0
SO
4 0
2 0
O
—
-
-
T r p ^ - ^ "
„ 1
Thr Gly
1
Vol
Glu
1
L y s ^
1
Asp
1
- 2 4 0 - 2 0 0 •160 -120
AS*(JK-''mol~'')
- 8 0 - 4 0
Fig. 3.7: Isokinetic relationship, AH vs. AS . for the reaction of ninhydnn
with a-amino acids. Reaction comUiums: [CTAB] = 20.0 x 10"' niol dni'\
[ninhydrin] = 5.0 x 10"' mol dm"\ pH = 5.0. [.Asp] = 1.0 x 10~ niol dm"' [17].
[Leu] = 1.0 X 10" mol dnr [15], [Lys] = 1.0 x 10"" mol dm"' [18]. [Phe] = 1.0 x
lO"" mol dm'-' [15], [Glu] = 1.0 x lO"' mol dm"' [2], [Gly] = 1.0 x 10~ mol dm"'
[19], [Thr] = 2.0 x lO-" mol dm"' [4]. [Trp] = 1.0 x lO-* mol dm' [16], [Ala] =
3.0 X 10"* mol dm"' [3], [Val] = 3.0 x 10"* mol dm"' (Present work).
115
with each amino acid (including the present one) follow the same reaction
mechanism in the presence of CTAB micelles.
Effect of solvents in presence of fixed concentration of CTAB
As before, "^ addition of water-soluble organic solvents markedly
increase the rate as well as intensity of the color (Fig. 3.5).
The solvents used represent three different types which mainly affect the
properties of bulk water: (1) alcohols that are known to enhance micellization
at very low volume fractions and inhibit it at higher volume fractions ; (2) AN
that forms relatively strong H-bonds with water; and (3) DMSO that is known
for hydrate formation with water.^°'^' Although each solvent has been found to
postpone micellization, the inhibitory effect on micellization of CTAB
depends upon the nature of the solvent. The behavior can be interpreted in
terms of solvent interaction with water and its possible influence on
solvophobic forces operating for micellization. In case of PrOH or MC, the
interaction consists of the destruction of the original water's 3D structure and
the formation of new H-bonds between water and alcohols.^^ These
alcohol-water mixtures are better solvent for CTAB than pure water and
effective number of micelles thus decrease. Similarly, the decrease of micelle
number density in presence of AN can also be understood in terms of formation
of H-bonds between water and AN molecules. The effect of DMSO on CTAB
micellization has been explained on the basis of strong interaction with water
and stoichiometric hydrate (DMSO. 2H2O) formation.
116
Effect of CTAB micelles in presence of different fixed compositions of
DMSO:
Effect of CTAB micelles has been seen for the title reaction in the
presence of different fixed compositions of DMSO. Rate profiles obtained for
the solutions with 10-70% DMSO by volume exhibited clear maxima that
shifted to higher concentration of surfactant as a fiinction of DMSO content
(Fig. 3.6). The experimental results are explained in terms of specific solvent
effects and the formation of the stoichiometric hydrate DMSO.2H2O and the
inhibiting effect of dimethyl sulfoxide on the formation of micelles.^' In
confirmity with the previous studies reported in the literature, '' '*"''° addition of
dimethyl sulfoxide show an inhibitory effect on the formation of micelles of
CTAB in water.^^ An increase in the orderliness of the DMSO-H2O-CTAB
system takes place as the composition of DMSO is increased. In fact, results of
proton spin-lattice relaxation studies have shown that the increased structuring
of the H2O.DMSO liquid system overcomes the hydrophobic effect of the alkyl
chain of CTAB. Proton spin-lattice relaxation times (l/Ti) and average
rotational correlation times, x CR), for the terminal methyl, N-methyl and
methylene groups of CTAB as well as effective activation energies determined
for the various relaxation processes in water-dimethyl sulfoxide solutions
showed that the surfactant molecule became trapped in the crystalline lattice of
the stoichiometric hydrate DMS0.2H20."'''" The initial increase of k^ in k^ vs.
[CTAB] profiles seems due to combined effect of micelles as well as blocking
of side reaction (arrest of hydrolysis). As the presence of DMSO also arrests
117
the CTAB micellization, the above effects would compete in deciding the k^^
values at a particular DMSO volume %. This may be the reason of shifting
maxima with increasing volume % of BMSO to higher [CTAB]. However, at
still higher concentration of DMSO (70%), where no micelles are present, the
reaction rate increase is seemingly due to the solvent's own catalytic effect.
The observations obtained in the presence of surfactant (CTAB) suggest
that the side-chain of or-amino acids plays an important role: the reaction rate
increases with the hydrophobicity of the side chain in the order
y j ^ > (0r ' ' " ' > eH3>-="- > CH>H- > H-H
for tryptophan, phenylalanine, leucine, valine and glycine (Table 3.8 - amino
acids having -NH2, -COOH, -OH, or exceptional character, e.g., histidine, are
excluded from the sequence). As can be seen (Table 3.8), the value o^ k^ for
DL-valine is well accommodated in the hydrophobicity scale,"*' i.e., higher the
hydrophobicity of the amino acid side-chain (R), higher is the rate accelerating
effect of surfactant micelles.
118
Table 3.8
Dependence of k^ on the hydrophobicity of the amino acid side-chain (R) for
its reaction with ninhydrin in ionic micellar medium ([CTAB] = 20.0 x 10" mol
dm-^).
"R 1 0 % R^f
Or CH2-
H
QpCH.
CH3
21.4 16
9.8 15
7.5 15
H3C
^CH— 6.8 Present work
H - 5.1 19
^ [amino acid] = 1.0 x lO'"* mol dm" , [ninhydrin] = 5.0 x lO'' mol dm"\ temperature = 70 °C, except for the present case where [amino acid] = 3.0 x 10"'* mol dm'"* and temperature - 80 °C
119
(B) Ninhydrin-DL-Tryptophan Reaction in Presence of TX-lOO and
Effect of Organic Solvents
A drawback in all the studies of ninhydrin reactions had been the
necessity of heating.^ In presence of non-ionic TX-lOO micelles, we have now
found color development at a much lower temperature (40 °C). The work
detailed herein embodies these results where kinetics and mechanism of
ninhydrin-tryptophan reaction are described (the reaction does not take place at
all in pure aqueous solution at 40 °C under the conditions used). Effect of
organic solvents (MC, DMSO, AN and PrOH) on the rate of Ruhemann 's
purple formation was also studied.
Results
Spectra:
In order to choose the best kinetic conditions, experiments with varying
tryptophan and ninhydrin concentrations were tried at different temperatures,
both in aqueous and aqueous-micellar media (TX-lOO). The situation with
[tryptophan] = 5.0 x lO""* mol dm"\ [ninhydrin] = 7.0 x 10" mol dm'\ and [TX-
lOO] = 50.0 x 10- mol dm" at 40 "C was selected where no reaction occurred
in aqueous medium (Fig. 3.8, curve a) but, in contrast, the reaction did occur in
micellar medium (Fig. 3.8, curve b) with the formation of purple-colored
product (A.,„ax = 400 and 570 nm). After boiling for ca. 2 min., the same
product was formed in the aqueous medium also without TX-lOO (Fig. 3.8,
120
curve c); this confirms that the product of the reaction remains the same both in
the aqueous and TX-lOO miceiiar media. Though the reaction mixture
containing TX-lOO developed turbidity on boiling (the cloud point of TX-lOO
is 67 ^C'' and the appearance of turbidity is due to the clouding phenomenon),
it disappeared on cooling with a resultant homogeneous solution remaining
purple-colored whose spectrum was identical to that of DYDA (Fig. 3.8, curve
d). Furthermore, the absorbance was higher in the last case which shows strong
association of the product with the nonionic TX-lOO micelles.
Effect of pH:
Kinetic studies were made at different pH values with fixed [ninhydrin],
[tryptophan], [TX-lOO] and temperature. The rate of the reaction increased
sharply up to pH 5.0 and, thereafter, a slow increase occurred. Detailed studies
were, therefore, made at pH 5.0. The results are given in Table 3.9 and shown
graphically in Fig. 3.9.
Effect of [tryptophan]:
The effect of varying the [tryptophan] on the reaction rate was studied at
constant [ninhydrin] (7.0 x 10" mol dm" ), [TX-lOO] (- 50.0 x 10" mol
dm'"') and temperature (= 40 °C). The results, summarized in Table 3.10, show
independence with respect to the initial concentration of tryptophan, and thus
first-order in [tryptophan].
121
Effect of [ninhydrinj:
The concentration of ninhydrin was varied from 7.0 x 10' to 40.0 xlO"
mol dm' keeping concentrations of other reaction ingredients constant. The
pseudo-first-order rate constants {k^^, obtained at different [ninhydrin], are
shown in Fig. 3.10 and Table 3.10. The log k^ vs. log [ninhydrin] was linear
with slope less than unity; this establishes fractional-order in [ninhydrin].
Effect of [TX-IOOJ:
Dependence of the observed pseudo-first-order constant on surfactant
concentration was investigated by carrying out experiments at different [TX-
100] but keeping all other variables constant. The results are given in Table
3.11 and shown graphically in Fig. 3.11 d&Sik^- [TX-lOO] profile whose shape
is perfectly general of reactions being catalyzed by micelles.'^"''*
Effect of Temperature:
A series of kinetic runs were carried out within the temperamre range
35-55 °C at fixed [ninhydrin] = 7.0x10"^ mol dm' and [amino acid] =5.0x10'^
mol dm" in the presence of 50.0x10" mol dm' surfactant concentration. The
data obtained were found to fit the Eq. (3.2) and the activation parameters
(AH and AS) , calculated using a nonlinear least-squares technique, are
recorded in Table 3.12.
122
Solvent Effect:
The effect of the presence of organic solvents, viz. PrOH, MC, AN and
DMSO on the rate of purple-color formation was also seen. The results are
given in Tables 3.13 and 3.14 and shown graphically in Fig. 3.12.
Discussion
As the products formed in TX-lOO micellar medium at 40 °C are the
same (formation of DYDA is demonstrated in Fig. 3.8. whereas those of CO2
and aldehyde were confimied qualitatively^ ' ^), it is concluded that the
pathways outlined in Scheme 3.1 are being followed here also in the nonionic
micellar medium.
The catalytic role of TX-lOO micelles clearly suggests the
association/incorporation of ninhydrin and tryptophan into the TX-lOO
micelles. The polyoxyethylene chain of TX-lOO micelles are polar in nature
due to the presence of ether and primary alcoholic linkages.'*'' These two
functional groups ( - 0 - and -OH) play an important role for the association of
ninhydrin and tryptophan into the polar headgroup region of TX-lOO through
hydrogen bonding. Therefore, the associated ninhydrin and tryptophan with
TX-lOO micelles (through hydrogen bonding) seem responsible of facilitating
formation of Schiff base.
123
4; o c
O
o <
3 6 0 AGO 550 6 00 A50 500
Wavelength(nm)
Fig, 3.8: Absorption spectra of the reaction product of DL-tiyptophan with
ninhydrin in aqueous micellar media at 40 "C. (a) in absence of surfactant, (b)
in presence of [TX-lOO] - 50.0 \ 10"' mol dni"\ (c) in presence of [TX-lOO] =
50.0 .\ 10 ' mol dm' + 20% methylcellosolve. (d) in presence of [TX-iOO] =
50.0 \ iO' mol dm' + 20% l-propanol, (e) in presence of [TX-IOO] = 50.0 .\
lO' mol dm' + 20% dimethyl sulfoxide and (f) in presence of [TX-lOO] = 50.0
X 10"' mol dm" + 20% acetonitiile. Reaction conditions: [DL-tryptophan] = 5.0
X 10~' mol dm \ [ninhydrin] = 7.0 x 10" mol dnV , pH = 5.0.
124
Table 3.9
Effect of pH on pseudo-first-order rate constants {k^^,) for the reaction of DL-
tryptophan with ninhydrin.
Reaction conditions: [DL-tryptophan]j = 5.0 x lO""* mol dm"''
[ninhydrinjx = 7.0 xlO' mol dm'
[TX-100]T = 50.0 xlO" mol dm"
Temperature = 40 °C
pH 10'A:^(s-')
3.7 5.5
4.1 5.7
4.4 7.6
5.0 8.3
5.5 8.6
5.6 8.8
125
Fig. 3.9: Effect of pH on the reaction of DL-tryptophan with ninhydrin.
Reaction conditions: [DL-tryptophan] = 5.0 x 10 mol dm"', [ninhydrin] = 7.0
X 10"' mol dm"\ [TX-lOO] = 50.0 x 10"' mol dm"\ temp. = 40 °C.
126
Table 3.10
Effect of [DL-tryptophan] and [ninhydrin] on pseudo-first-order rate constants
(A: ,) for the reaction of DL-tryptophan with ninhydrin.
Reaction conditions: pH =5.0
[TX-1 00]T = 50.0 X 10- moi dm"
Temperature =40°C
lO'' [tryptophan]!
(mol dm" )
4.0
4.5
5.0
5.5
6.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
10" [ninhydrin]!
(mol dm'^)
0.7
0.7
0.7
0.7
0.7
0.7
1.0
1.5
2.0
2.5
3.0
3.5
4.0
10'k^
(s-')
8.0
8.1
8.3
8.2
8.1
8.3
10.8
16.3
19.9
30.3
39.9
44.0
45.0
127
0 10 20 30
lO-^CNinhydrinllCmol drn'^)
Fig. 3.10: Effect of ninhydrin on the reaction of DL-tiyptophan with
ninhydrin. Reaction conditions: [DL-tryptophan] = 5.0 x 10" inol dm"\ pH =
5.0, [TX-iOO] = 50.0 X (O' mol dnil temp. = 40 °C.
128
Table 3.11
Effect of [TX-lOO] on pseudo-first-order rate constants (k^^,) for the reaction of
DL-tryptophan with ninhydrin.
Reaction conditions: pH =5.0
[DL-tryptophanJx ==5.0x10"'* mol dm'
'
10' [TX-100]T
(mol dm'^)
2.0
5.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
[ninhydrin]T
Temperature
10'k^
0.8
1.6
2.3
3.6
5.1
7.0
8.3
7.2
7.0
10\
0.5
1.3
2.2
3.5
5.0
7.0
8.3
7.2
7.0
7.0
40'
1 cal
xlO"^
°C
mol dm"
k -k
+0.38
+0.19
+0.04
+0.03
0
0
0
0
0
129
IT) O
20 AO 60
1 0 - ^ C T X - l O O D ( r n o l d m - 3 )
Fig. 3.11: Effect of TX-lOO on the reaction of DL-tryptophan with
ninhydrin. Reaction conditions: [DL-tryptophan] = 5.0 x 10^ mol dm'\
[ninhydrin] = 7.0 x 10" mol dm"\ pH = 5.0, temp. = 40 °C.
130
Table 3.12
Effect of temperature on pseudo-first-order rate constants {k^^,) for the reaction
of DL-tryptophan with ninhydrin.
Reaction conditions: pH =5.0
[DL-tryptophan]T =5.0x10"'' mol dm"
[ninhydrinji- = 7.0 xlO'^ mol dm'
[TX-100]T = 50.0 xlO" mol dm'
Temperature 10 k^
rc) (s-')
35 5.6
40 8.3
45 11.2
50 16.5
55 25.0
Parameters
Ea (kJ mor') 62
A H'' (kJ mor') 59
-AS'^CJK-'mor') 133
^T (mol"' dm^) 82
K^ (mol"' dm^) 56
10^;fen,(s'') 1.4
131
Table 3.13
Effect of organic solvents on pseudo-first-order rate constants (k^^,) for the
reaction of DL-tryptophan with ninhydrin.
Reaction conditions:
% solvent
(v/v)
0
5
10
15
20
25
30
35
AN
8.3
11.5
9.2
14.6
16.9
18.4
21.5
24.6
pH
[DL-tryptophan]T
[ninhydrin]T
[TX-100]T
Temperature
DMSO
8.3
9.2
10.0
11.5
16.9
18.4
19.2
24.6
10
= 5.0
= 5.0
= 7.0
xlO-'
xlO-^
= 50.0x10
= 80 '
k^is')
MC
8.3
8.4
8.4
9.2
12.2
13.8
16.9
20.7
'C
mol dm"
mol dm"
• mol dm"^
PrOH
8.3
8.4
8.4
9.2
13.1
14.6
17.7
20.0
132
Table 3.14
pH and the observed pseudo-first-order rate constants (k,^,) for the reaction of
DL-tryptophan with ninhydrin reaction carried out in different solvents (35%
v/v).
Reaction conditions: [DL-tryptophan]T = 5.0 x 10'Vol dm'
[ninhydrin]T
[TX-100]T
V.OxlO'^moldm-^
= 50.0xlO-^moldm-^
Temperature = 40°C
Reaction Medium pH lO^k^is')
Buffer (CH3COOH + CHsCOONa)
65% pH 5.0 buffer + 35% PrOH
65% pH 5.0 buffer + 35% MC
65% pH 5.0 buffer + 35% AN
65% pH 5.0 buffer + 35% DMSO
5.0
5.48
5.58
5.89
6.08
8.3
20.0
20.7
24.6
24.6
133
o
10 30 40 20
% S o l v e n t ( v / v )
Fig. 3.12: Effect of methylcellosolve (a), l-propanol ( • ) , dimethyl
SLiltoxide (O) and acetonitrile (A) on the reaction of DL-valine with ninhydrin.
Reaction concUtiom: [DL-ti-yptophaii] = 5.0 x lO" mol d m ' \ [ninhydrin] = 7.0
\ 10"'' mol dm"\ [TX-lOO] = 50.0 x 10 ' mol dm"\ pH = 5.0, temp. = 40 °C.
134
Quantitative treatment of k^-fsurfactant] data:
The k^^, - [TX-lOO] profile shows a maximum which is characteristic of
a bimolecular reaction.'^ Accordingly, the catalytic effect of TX-lOO on k^^, is
explained in terms of the modified Menger and Portnoy's pseudo-phase
model'^'^ (Scheme 3.3).
(Trp)w + Sn ^ ^ (Trp)n, 3 j2)
(nin)w + Sn ^ ; ; = ^ (nin)m (3.13)
->- Product
Scheme 3.3
For the reactions of Scheme 3.3, the modified rate equation is written as
(neglecting k^, as no purple color developed in the aqueous medium)
_kMM^ (3.14)
where k^ is the second-order rate constant in the micellar pseudo-phase, M^N (-
[(nin)ni]/Sn), the mole ratio of ninhydrin bound to micellar headgroup and Sn (=
[TX-100]T - cmc) is the micellised surfactant. Values of k^ and Kj were
obtained using a computer-based program and are given in Table 3.12, As
before, the validity was established by comparing the observed and calculated
/c,|, values (Table 3.11).
135
The observed catalytic role of solvents is seemingly due to diminished
hydrolysis of the amine. The increase in the content of solvents (% v/v) is
expected to decrease the content of water, which, in turn decreases the
hydrolysis of amine. As a result, the rate of the reaction is increased. Secondly,
the solubility of Ruhemann's purple is less in water in comparison to organic
solvents.'*'' Thus, blocking side reaction(s) (mainly hydrolysis) and higher
solubility of the product play important roles in presence of organic solvents.
The positive catalytic effect of organic solvents may also be looked
upon from the view point of relative participation of water and organic solvent
in acid-base equilibria and hydrogen bonding. It has been established that pH
of the medium and pA!a2 values of amino acids are responsible for the rate
enhancement of amino acid-ninhydrin reactions in non-aqueous organic
solvents and amino acid anions are the reactive species."*^ As the [anion] is
negligible in the vicinity of pH 5.0 (the maximum pH for ninhydrin reaction),
the neutral form of amino acid is the main reactive species under such type of
circumstances.'^''^"^^ In order to confirm the hypothesis presented by
Friedman,''^ i.e., rate enhancement being directly related to the p/Tgi value of
amino acid, results of experiments performed in 65% pH 5 buffer + 35%
organic solvent are summarized in Table 3.14. The observations indicate that
pH of the working solution does not show any drastic change in presence of the
solvents (Table 3.14; similar results were obtained with DL-valine also, see
Table 3.7).
136
References;
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Cfiapterll^
!Ninliydnn-(DL-njyptopfian faction in (Presence of
gemini Surfactants and^lf^NM^Investigatton at
(Different Concentrations of Qemini Surfactants
141 Introduction
As regards the effect of surfactants we have successfully demonstrated
that both the ninhydrin-amino acid and ninhydrin-metal amino acid complex
reactions are catalyzed by the surfactant micelles.'' In the studies we used
traditional (single hydrocarbon chain/single polar head group) surfactants, the
so-called 'conventional' ones. Recently, a new class of surfactants called
'dimeric' or 'gemini', consisting of two hydrophobic alkyl tails and two polar,
or ionic, head groups covalently linked through a flexible or rigid spacer,'"'*'
has been introduced which are attracting current attention in the area of
surfactant science because of displaying a number of unique properties (e.g.,
very low cmc, high viscoelasticity, superior surface activity, better wetting,
unusual morphologies, etc.). Micellar morphologies and properties of the
gemini surfactants are found to be significantly dependent on the nature of the
hydrophobic tail, head group, and spacer. Surprisingly, despite a large body of
information being available on the physico-chemical aspects of gemini
surfactants and the assemblies they form, studies of their effects upon reaction
rates has not attracted due attention. For this reason we have performed
kinetic studies of the ninhydrin-DL-tryptophan reaction in the presence of
three dicationic gemini micelles (Fig.4.1). For comparison, the effect of the
cationic surfactant cetyltrimethylammonium bromide (CTAB), which can be
considered as "monomeric" counterpart of the above geminis, has also been
examined under similar kinetic conditions.^ The reason for choosing this
particular reaction is that the mechanism in water,''' in different solvent
142 6-9 media,' and in the presence of CTAB surfactant system is well established.
It is important to mention here that under the same reaction conditions no
color developed in the absence of gemini surfactants or in the presence of
CTAB micelles, whereas a small concentration (below cmc) of the geminis
was sufficient to accelerate the rate of the reaction. The work was undertaken
in the hope that the use of gemini surfactants may cause the use of low
reactant concentration as well as maximize the rate, thus, enhance the
sensitivity of the technique/reaction.
Br Br
MejN—(CH2)n,—NMe2
C,.H 16n33 ^16H33
Gemini surfactants (16-m-16)
(m = 4,5 ,6)
Br
MejN
C16H33
CTAB
Fig. 4.1: The surfactants used in the present study.
Results
Spectra:
In order to confirm whether the same colored product is fonmed in the
absence and presence of surfactants (CTAB, geminis), absorption spectra of
the reaction mixture, i.e., [tryptophan] = l.O x 10" mol dm" , [ninhydrin] = 5.0
v3, ,-3 -5 X 10"' mol dm"', [gemini] = 20.0 x 10"' mol dm"' and pH = 5.0 at 70 °C were
taken at the end of the reactions. These results are shown as absorbance-
wavelength profiles in Fig. 4.2. The absorption maxima were found at > ax ^
143
u c o r> (— o (/I
3 60 AOO 600 <,50 500 550
Wavele ng th(nm)
Fig. 4.2: Absorption spectra of the reaction product of DL-tiyptophan (1.0 X 0" mol dm"') with ninhydrin (5.0 .x 10" moi dm"') in aqueous micellar media at 70 °C. (a) in absence of surfactant, (b) in presence of [CTAB] = 20.0 X 10'' mol dm"\ (c) in presence of [16-6-16] = 20.0 x 10' mol dni"\ (d) m presence of [16-5-16] = 20.0 x 10" mol dm" , (e) in presence of [16-4-16] ^ 20.0 X 10" mol dm'\ (0 after boiling solution (a), (g) after boiling solution (b), (h) after boiling solution (e).
144
400 nm and 570 nm, which clearly indicate that the same purple-colored
reaction product {Ruhemann 's purple) is formed in each case due to the strong
association between purple-colored product and gemini micelles. In presence
of CTAB micelles no color developed under the similar reaction conditions;
however, at increased [CTAB] (5.0 x 10" mol dm'^), color developed at 70 °C
and pH = 5.0 and in this case also the absorption maxima were at the same
wavelengths (400 nm and 570 nm).^ No change in the absorption maxima in
the absence as well as presence of CTAB/gemini surfactants leads to the
conclusion that the same product is formed in each case (Fig. 4.2).
Effect offgeminij:
The dependence of the observed first-order rate constants on [gemini]
was studied by undertaking a number of kinetic experiments at different
gemini concentrations at constant [ninhydrin] and [amino acid] at pH = 5.0
and temp.= 70 °C. The results are given in Tables 4.1-4.3 and depicted
graphically in Fig. 4.3 as rate constant (^y)-surfactant concentration profiles.
Fig. 4.4 shows the effect of spacer (m).
'H NMR investigation at different concentrations of gemini surfactants:
'H NMR investigation was done at different concentrations of
geminis. The 'H NMR spectra for pure geminis (in D2O) at different
concentrations are shown in Fig. 4.5-4.24. Line widths at half heights (/w) of
the signal relative to N-CH3 group were also calculated and are summarized
in Table 4.4 and shown graphically in Fig.4.25.
145 Table 4.1
Effect of 16-6-16 gemini on pseudo-first-order rate constants (k^) for the
reaction of DL-tryptophan with ninhydrin.
Reaction conditions:
10"* [gemini]
(mol dm" )
0.0 0.1 0.3 0.5 0.8 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 25.0 40.0 50.0 100.0 150.0 200.0 250.0 300.0
pH
Temperature
[DL-tryptophan]
[ninhydrin]
10 A:
(s-')
0.0 5.3 6.0 7.6 10.7 11.1 15.3 15.4 15.3 15.4 15.3 15.3 15.4 15.3 15.4 15.6 15.3 21.0 28.2 32.2 41.7 49.4 55.0
z ^
1 =
=
=
1U K^^ cal
(s-')
_
-
-
7.6 10.7 10.0 15.2 15.4 15.3 15.4 15.3 -
-
-
15.4 15.7 -
21.0 28.1 32.2 41.7 49.4 55.0
5.0
70 °C
1.0 X
5.0 X
lO-"* mol dm'
lO-^moldm-^
k -k
_
-
-
0 0 +0.01 +0.01 0 0 0 0 -
-
-
0 -0.01 -
0 0 0 0 0 0
146
Table 4.2
Effect of 16-5-16 gemini on pseudo-first-order rate constants (ky^,) for the
reaction of DL-tryptophan with ninhydrin.
Reaction conditions:
lO'' [gemini]
(mol dm"'')
0.0 0.1 0.3 0.5 0.8 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 25.0 40.0 50.0 100.0 150.0 200.0 250.0 300.0
pH
Temperature
[DL-tryptophan]
[ninhydrin]
\0^k^
(s-')
0.0 5.6 9.8 11.3 12.0 13.6 16.3 16.2 16.2 16.2 16.2 16.2 16.2 16.2 16.2 16.9 16.2 24.0 30.9 38.0 45.8 50.0 59.7
—
=
=
^
1U K^i cal
(s-')
.
-
-
11.3 12.0 13.6 16.3 16.0 16.2 16.2 16.2 -
-
-
16.1 16.0 -
24.0 30.9 38.0 45.8 50.0 59.7
5.0
70 °C
1.0 X
5.0 X
10'"* mol dm-
lO'^moldm"^
k -k
-
-
-
0 0 0 0 +0.01 0 0 0 -
-
-
+0.01 +0.05 -
0 0 0 0 0 0
147
Table 4.3
Effect of 16-4-16 gemini on pseudo-first-order rate constants (k^) for the
reaction of DL-tryptophan with ninhydrin.
Reaction conditions: pH
Temperature
[DL-tryptophan]
[ninhydrin]
5.0
70 °C
l.OxlO'^moldm"^
5.0xlO"^moldm'^
lO'* [gemini]
(mol dm''')
0.0 0.1 0.3 0.5 0.8 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 25.0 40.0 50.0 100.0 150.0 200.0 250.0
10'^^
(s-')
0.0 7.0 13.5 14.8 15.1 17.1 18.3 18.2 18.3 18.7 18.9 18.3 18.0 18.7 18.5 18.4 18.9 26.9 37.2 49.4 54.9 69.3
1U /Cyj, ca!
(s-')
_
-
13.5 14.8 15.0 17.1 18.3 18.2 18.3 18.7 18.9 -
-
-
18.5 18.4 -
26.9 32.6 49.4 54.9 69.3
k -k
_
-
0 0 +0.01 0 0 0 0 0 0 -
-
-
0 0 -
0 +0.12 0 0 0
148
10 ' 'C 16-171 -1611 ( m o I d m " 3 )
Fig. 4.3: Variation of k^ for the reaction of DL-tiyptophan (1.0 x 10 mol dm" ) with ninhydrin (5.0 x 10"' mol dm"') in the presence of 16-m-16 gemini micelles at 70 °C. m = 6 (3 ) , 5 (•), 4 (O).
149
in O
Fig. 4.4: Dependence of v as a function of the spacer chain length (m-value) of 16-m-16 gemini micelles for the reaction of DL-tryptophan (1.0 x 10" moi dm') with ninhydrin (5.0 x 10' mol dm"" ) at 70 °C. [geminis] = 2.5 x 10"' mol dni'\ and [CTAB] = 5.0 x 10"- mol dm"\ For CTAB, m = 0
150
CH3 Br y j \ + 4 3 2 1
CH2
5(CH2)4
CH2 •
J^"Kl\' . 3 2 1 H.M3 g_
JU
3 ppmA 3 2 1
Fig. 4.5: 300 MHz 'H N M R spectrum of 0.5 mM 16-6-16 in D2O at 25 °C.
151
AJ
2 ppm k 3
Fig. 4.6: 300 MHz 'H N M R spectrum of 2 mM 16-6-16 in D2O at 25 "C.
152
r ppm U
T
Fig. 4.7: 300 MHz 'H N M R spectrum of 5 mM 16-6-16 in D.O at 25 T .
153
Al
ppm 4 2
Fig. 4.8: 300 MHz 'H N M R spectnim of 10 mM 16-6-16 in D.O at 25 T .
154
J^ ^ J , 1
ppm 4 3 2 1 Fig. 4.9: 300 MHz 'H NMR spectrum of 30 m.M 16-6-16 in 0 , 0 at 25 X.
155
ppm U 1 Fig. 4.10: 300 MHz ' H NMR spectrum of 50 niM 16-6-16 in D2O at 25 "C.
156
M T 2 ppm ^ 3 2 1
Fig. 4.11: 300 MHz 'H NMR spectnim of 70 inM 16-6-16 in D2O at 25 T .
157
T PPn^ ^ 3 2 1
Fig. 4.12: 300 MHz 'H N M R spectiiim of 100 mM 16-6-16 in D2O at 25 T .
158
, CH3BF yJ \"*- ^ 3 2 1
^ CH2
5(CH2)3 I
r CH3 CH2 7J ^ ' \ 6 ' 3 2 1
' C H / - - ~ ^ " ^ ~ ' ^ ' ^ 2 ) 2 - C H 2 - ( C H 2 ) I , - C H 3 Br
ppm 4
Fig. 4.13: 300 MHz ' H ^^\]R spectium of 2 mM 16-5-16 in D2O at 25 T .
159
PPm A 3 2 1 Fig. 4.14: ?00 MHz 'H N M R spectiiim of 5 mM 16-5-16 in D2O at 25 X.
160
T ppm 4 3 2 1
Fig. 4.15: 300 MHz 'H N M R spectrum of 10 mM 16-5-16 in D.O at 25 X.
161
Ppm 4 3 2 i Fig. 4.16: 300 MHz 'H N M R spectniin of 30 mN4 16-5-16 in D.O at 25 T .
162
ppm 4 3 2 1
Fig. 4.17: 300 MHz 'H N M R spectrum of 50 niM 16-5-16 in D2O at 25 "C.
163
T 2
T "I ppm 4 3 2 1
Fig. 4.18: 300 MHz 'H NMR spectrum of 70 niM 16-5-16 in DjO at 25 T .
164
ppm 4 3 2 1
Fig. 4.19: 300 MHz 'H N M R spectrum of 100 mM 16-5-16 in D.O at 25 X.
165
CH-i Br~ \ +
I C H 3 / ' j '-;,CH2-(CH2)2-CH2-(CH2)ii-CH3
CH2 I
5(CH2)2
CH3 CH2
^^ / t -CH2-(CH2)2-CH2-(CH2)i i -CH3 ^ Br
ppm ^ "T 2
Fig. 4.20: 300 MHz 'H ^ ,vlR spectrum of 0.5 niM 16-4-16 in D:0 at 25 T .
166
j\J T
ppm/. 3 ^ ^
Fig. 4.21: 300 MHz 'H N M R spectrum of 2 mM 16-4-16 in 0 , 0 at 25 T .
167
PPm u 3 2 1 '
Fig. 4.22: 300 MHz ' H N M R spectrum of 5 mM 16-4-16 in D^O at 25 T .
168
iW
ppm A T 3
T 2
Fio. 4.23: 300 MHz 'H N M R spectrum of 10 mM 16-4-16 in D.O at 25 T .
169
1 j \ 1 i PPm 4 3 2 1 0
Fig. 4.24: 300 MHz 'H NMR spectrum of 30 miM 16-4-16 in D2O at 25 X.
170
Table 4.4
Line widths (/w) of the signals from the protons of the N-methyl groups of
16-m-16 geminis obtained at different concentrations.
10 [gemini] 16-6-16 16-5-16 16-4-16
(mol dm' )
/w /w /w
(Hz) (Hz) (Hz)
0.50 30.61 - 36.01
2.0 30.61 25.21 36.01
5.0 30.61 25.21 39.61
10.0 30.61 28.21 48.62
30.0 34.21 34.21 merge
50.0 37.82 45.02
70.0 41.22 54.02
100.0 54.02 merge
171
45
35
^ 25 M X
• D
i QJ
C
^0
2 4 6 8 10 10^ri6-m-15l(moldm'^)
20 20 ^0 50 80
10^Cl6 -m - 1 6 3 (mol dm"^) Fig. 4.25: Line widths of the signals from the protons of the N-methyl groups of 16-ni-16 geminis plotted against different concentrations, m = 6 ( 3 ) , 5 ( • ) . 4 (O). Solutions o^ 16-4-16 became too viscous at higher concentrations and, therefore, results are compared at lower concentrations (see inset).
172
Discussion
As is well known, carbon dioxide, aldehyde, ammonia, hydrindantin,
and Ruhemann's purple are the products of the ninhydrin reaction ' that
proceeds through the formation of a Schiff base which is unstable and
undergoes decarboxylation and hydrolysis to yield 2-amino-indanedione (A)
as a stable intermediate (Scheme 3.1). This intermediate acts as a reactant in
the formation of ammonia and RuhemanrC% purple and the two reactions (i.e.,
hydrolysis by route (i) and condensation by route (ii) - Scheme 3.1) occur
simultaneously. Reactions of both the routes strongly depend upon conditions
like pH, presence of atmospheric oxygen, temperature, etc. A yellowish-
colored product is fonned (instead of Ruhemann''s purple) in the presence of
atmospheric oxygen, as A is highly sensitive to molecular oxygen. At low pH,
chiefly route (i) is followed and ammonia is evolved almost quantitatively
with no Ruhemann's purple formation while at pH > 5.0, route (ii)
predominates.
Relevant equilibria involving the reactant species are also shown in
Scheme 3.1. As regards the reactive species of DL-tryptophan (as a matter of
fact, any a-amino acid), it has been argued on several occasions'"^ that,
toward nucleophilic attack on the >C=0 group of ninhydrin (N), it is
RCH(NH2)C00H, which is in equilibrium with the zwitterionic form
RCH(N%)COO"(Scheme 3.1).
173
Rate-[surfactaniJ profiles for kinetics ofninhydrin with DL-tryptophan:
As already mentioned, no purple color developed with [ninhydrin] =
5.0 X 10" mol dm" and [tryptophan] = 1.0 x 10"'' mol dm" at pH = 5.0 and
temp. ^ 70 °C either in pure aqueous or [CTAB] = 20.0 x 10' mol dm"
(however, the reaction did occur at [CTAB] > 5.0 x 10" mol dm' and it had
been studied in detail ^). With the 16-m-16 geminis the reaction occurred at a
surfactant concentration as low as 20.0 x 10' mol dm'^; therefore, detailed
kinetic investigations were made with the three geminis (m = 4, 5, 6) only.
The pseudo-first-order rate constants {k^, s"') for the title reaction were
determined in micellar media at several gemini surfactant concentrations.
Fig. 4.3 shows the variation of ky with surfactant concentrations. With
conventional surfactants, A: had been found to increase monotonically and
after the substrates completely bind the micelles, it attained constant values
(for monomolecular reactions) or passed through a maximum (for bimolecular
reactions) with increasing [surfactant].'^'^^ In the present case, however, with
the gemini surfactants only, k^ first increases with surfactant concentration
(part I), remains constant upto certain concentration (part II - parts I and II
behavior is akin to conventional surfactant micelles), ' ' ° and then again
increases (part III). In part I, at concentrations lower than the cmc, k^ should
remain constant. The observed catalytic effect may, therefore, be due to (i)
presence of premicelles and/or (ii) preponement of micellization by
reactants (as is also confirmed by cmc decrease at reaction conditions, see
Experimental). Whereas no reaction has been observed in range II with
174
conventional surfactant (CTAB),"' ^ remains constant upto 40.0 x 10''* mol
dm" for gemini surfactants. This undoubtedly shows better catalyzing power
of the gemini surfactants over the corresponding single chain surfactants. This
could be due to the presence of spacer in the geminis which decreases the
water content in the aggregates making the environment less polar and thus
causing rate increases (see Scheme 3.1 - route (i) may get suppressed).
Menger et alP have already concluded that due to proximity of positive
charges in gemini micelles anion binding at surfaces is increased at the
expense of binding of H2O. The behavior in part II is same for all the three
geminis but values of k^ at all concentrations are in the order : m = 4 > 5 > 6
(Fig. 4.4). This is not for the first time but best results with 16-4-16 were
obtained earlier also. It is well known that, to minimize its contact with
+
water, a spacer longer than the 'equilibrium' distance between two -NMe^
headgroups (the 'equilibrium' distance occurs at m = 4 in 16-m-16 geminis)
tends to loop towards the micellar interior. '*' ^ This increased looping of the
spacer (for m > 4) will progressively make the Stem layer more wet (in
comparison with the m = 4 gemini) with a resultant decrease in k^. Thus the
results are consonant with the earlier findings that increase in the water
content of the reaction environment has an inhibiting effect. " ' ^
The results of part III are most interesting; instead of k^ remaining
constant, it increases (though slowly) in the surfactant concentration range
40.0 X I0'^-300.0 X 10"'* mol dm'l After leveling-off, further increase at
higher [gemini] is probably associated with a change of micellar structure.
175
That structural changes indeed occur at higher [gemini] are confirmed by
examining the ' H N M R spectra of the surfactants.
Changes of aggregates structure:
NMR is a versatile and powerful technique to investigate surfactant
aggregation. Parameters such as chemical shifts, line width, and spin-spin
coupling constant and their variations with surfactJint concentration can give
usefiil information on aggregate structures.^'
To have a better understanding of surfactant behavior at high
concentrations, we performed ' H NMR study on these surfactants (Figs. 4.4-
4.24). We calculated line width at half height (/w) of the signal relative to N-
CH3 group vs. concentration of surfactant at 25 °C as line broadening is
associated with large aggregate formation.^* No significant variation in
chemical shifts was observed; however, ' H NMR spectra show very broad
signals even at low surfactant concentrations (20-fold higher concentration
than the cmc) (Table 4.4 and Fig.4.25). Small micelles are characterized by
short correlation time, which is due to micelle rotation and difftision of
surfactant molecules within the micelle. A short correlation time means a long
NMR relaxation time and thus narrow lines in the NMR spectrum.
Broadening of signals, therefore, means that the mobility of surfactant
headgroups in a micelle is reduced. This decrease in mobility could be due to
close packing of monomers in the micelle which gives rise to large
aggregates.^^ Obviously, changes in aggregate morphology provide different
176
reaction microenvironments (less polar), hence the ^^ increases at higher
[gemini] (Fig. 4.3).
Quantitative treatment ofk^ - [gemini] data:
The observed catalytic role of gemini micelles (upto range II) can be
explained in terms of the modified pseudo-phase model'^'^'''^' (Scheme 4.1)
originally proposed by Menger and Portnoy.
(Trp)w + Sn ^ ^ ^ ^ (Trp)m ^ j
(nin)w + Sn ^ ? = ^ (nin)m (4.2)
—•^ Product •<
Scheme 4.1
w and m represent the aqueous and micellar pseudophase, respectively, and Sn
is the micellized surfactant. Neglecting ^ (as no purple color developed in
the aqueous medium' ), the first-order rate equation according to Scheme 4.1
is given by
k^ = k'^Kj[^,]l{\+Kj{S,,]) (4.3)
The first-order rate constant in micellar (A:' ) pseudophase is related to the
second-order rate constant in the micellar {k^) pseudophase by
'm = (^m[(nin)n,])/[Sn] = k^ M N (4.4)
where M \ ( = [(nin)m]/Sn) is the mole ratio of ninhydrin bound to micellar
headgroup.
177
Eq. (4.3) can be written as Eq. (4.5) when /:'„, value is substituted from Eq.
(4.4)
k^ = kJCj[S,] M\ I (l+A-TLSnl) (4.5)
Values of k^ and binding constant Ky were obtained using a computer-based
program''^ and are given in Table 4.5. The data in Table 4.5 reveal that the
binding constants (/Cj) for the substrate is larger with 16-4-16 gemini micelles
compared to that with 16-5-16, 16-6-16 and CTAB. The second-order rate
constants in the micellar pseudophase, k^, are also higher in the 16-4-16
gemini micelles. Taken together, these results confirm significantly enhanced
reaction rates in 16-m-16 gemini micelles for the ninhydrin-tryptophan
reaction.
Finally, we can conclude that unlike conventional single chain
surfactants which show a constancy in k^^, it again increases with the 16-m-16
type gemini surfactants. ' H N M R spectra show a broadening in peaks at these
higher concentrations. Both kinetic and ' H NMR results indicate that larger
aggregates are forming at these surfactant concentrations due to which a less
polar environment is available for the reaction to proceed. Hence, 4-
increases at high gemini surfactant concentrations. The ninhydrin-tryptophan
reaction is thus a simple, reliable kinetic probe in aggregate structures.
178
Table 4.5
Values of binding constants, (Kj, K^^) and k^ for the reaction of DL-
tryptophan with ninhydrin in presence of CTAB and gemini surfactants at
70 °C.
Reaction conditions: [ninhydrin] 5.0 X 10" mol dm'
[DL-tryptophan] = 1.0x10 mol dm -3
Surfactant
CTAB'
16-6-16
16-5-16
16-4-16
K.J
(mor'dm^)
59
68
70
72
Parameter
K^
(mor' dm^)
100
70
75
79
10'-t,
(s-')
0.055
6.44
6.82
7.20
Reference 3
179
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Khan, J. Colloid Interface ScL, 213, 20 (1999).
3. Kabir-ud-Din, J. K. J. Salem, S. Kumar and Z. Khan, J. Colloid
Interface Sci., 215, 9 (1999).
4. Kabir-ud-Din, J. K. J. Salem, S. Kumar and Z. Khan, Colloids Surf
v4,168,241(2000).
5. Kabir-ud-Din, U. S. Siddiqui, and Z. BChan, J. Surface Sci. Technol,
19, 101 (2003).
6. Kabir-ud-Din, M. Bano and I. A. Khan, J. Surface Sci. Technol, 18,
113(2002).
7. Kabir-ud-Din, M. Bano and I. A. Khan, Indian J. Chem., 42A, 998
(2003).
8. Kabir-ud-Din, M. Bano and I. A. Khan, Indian J. Chem., 42B, 1132
(2003).
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10. M. J. Rosen, CHEMTECH, 23, 30 (1993).
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180
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181
30. (a) C. A. Bunton, in "Surfactants in Solution", K. L. Mittal and D. O.
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CfiapterV
^!K!NM^ Study of gemini Surfactants in (Presence of
Additives (Sodium AntkraniCate and Sodium (Benzoate)
183
Introduction
Systems involving surfactants constitute a field of great interest due to
their wide-ranging applications in detergent and pharmaceutical industries,
food technology, petroleum recovery, and so forth. Surfactants are also one of
the most important constituents of cells in living systems. Therefore, physics,
chemistry, biology, and technology meet at the frontier area of interdisciplinary
research on association colloids formed by surfactants.
With increasing surfactant concentration, micelles can undergo a
structural transition under appropriate conditions of salinity, temperature, or
addition of some organic additives.^'* For most surfactants, micelles tend to
grow and, in this process, change shape when an appropriate parameter is
modified.^ In most instances, this process results in the formation of elongated
micelles that can become extremely long and referred to as giant, rod-like,
wormlike, thread-like and polymer like micelles.
In recent years, a new class of surfactants (known as dimeric or gemini
surfactants) has generated a lot of interest in colloid chemistry.'^''^ These
surfactants have been found to be superior to conventional surfactants on
several counts and are said to be the "next generation of surfactants".'^
Surfactants are generally used in the presence of additives in order to
improve their properties. Thus, it is likely that dimeric surfactants in future can
be used in mixtures with various additives (e.g., conventional surfactants,
184
organic/inorganic compounds, non-electrolytes, etc). The existence of
synergistic effects between them may render the use of such mixtures even
more attractive.'*'^°
Several reports have been published on the structural aspects and
aggregation behavior of dimeric surfactants. " However, micellization of
gemini surfactants in presence of different class of additives is scarce. To our
knowledge no report has been published on the effect of addition of salts of
aromatic acids on the structure of gemini micelles. Some of these salts,
depending on the nature of groups attached to the central benzene ring, are
known to produce viscoelasticity in conventional cationic surfactants.
Therefore, we have explored the influence of the partitioning site of two
aromatic salt counterions near the gemini micelles and their possible impact on
overall micellar structural changes. For the purpose, we have used ' H N M R to
see the effect of addition of sodium anthranilate (NaAn) and sodium benzoate
(NaBen) to three 16-m-16 geminis at 25 °C.
Results
Figs.5.1-5.3 show the ' H NMR spectra of pure 16-6-16, 16-5-16, 16-4-
16 geminis in D2O. Figs. 5.4-5.9 illustrate the spectra of 10 mM 16-6-16, 10
mM 16-5-16 and 5 mM 16-4-16 containing different concentrations of salts
(NaAn and NaBen).
185
Tables 5.1 and 5.2 record ' H chemical shift (5) data of various protons
of 10 mM 16T6-16, 10 mM 16-5-16 and 5 mM 16-4-16 using D2O as solvent.
The 6 values for such protons in presence of various concentrations of the
added salts (NaAn and NaBen) are also given in Tables 5.1 and 5.2.
The line widths at half height (/w) of the signals from the protons of the
N-methyl groups of the 16-m-16 geminis in presence of NaAn and NaBen are
given in Tables 5.3 and 5.4 and shown in Figs.5.10 and 5.11.
Viscosity data of different concentrations of NaAn and NaBen with 16-
m-16 gemini surfactants are given in Tables 5,5 and 5.6 and shown in Figs.5.12
and 5.13. ' H N M R spectra of aromatic regions of pure sodium anthranilate/
benzoate and the solutions of 10 mM 16-6-16, 10 mM 16-5-16, 5 mM 16-4-16
gemini surfactants with varying concentrations of the salts are depicted in Figs.
5.14-5.19.
Discussion
'H NMR spectra were run on aqueous solutions of 16-m-16 in the
presence and absence of added NaAn and NaBen. Because the reported
concentrations of 16-m-16 here are higher than their critical micelle
concentrations (cmc), the observed chemical shifts can be considered those of
micellized surfactants.
Tables 5.land 5.2 show the 8 values for all surfactant resonances in the
absence and presence of increasing concentrations of NaAn and NaBen.
186
7 ^ CH3 Br
\ 1 ^ N - J : H 2 - ( C H 2 ) 2 - C H 2 - ( C H 2 ) - I - , - C H 3
CH3^ I/'S CH2 I
5(CH2)4
CH2
' ] / N - C H 2 - ( C H 2 ) 2 - C H 2 - ( C H 2 ) i i - C H 3 l C H 3 ' ^ ^ -
A) —f— 3.0
—r-2.5
1.0 —7— 0.5
—T" 3.5 ppm 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Fig. 5.1: 300 MHz 'H N M R spectrum of pure 10 mM l,6-bis(N-hexadecyl-
N,N- dimethylammonium)hexane dibromide (16-6-16) in D2O.
187
CH3Br ^ 3 2 1 V - C H 2 - ( C H 2 ) 2 - C H 2 - l C H 2 h n - C H 3
C H ^ 1/6 ^ CH2
I 5!CH2)3
^ " \ l \ ^ S . 3 2 1 /N -CH2- lCH2)2 -CH2- lCH2) l l -CH3
CH3 Br
ppm 3.5 3.0 I
2.6 2.0 1.5 1.0 0.5 I
0.0
Fig. 5.2: 300 MHz 'H NMR spectrum of pure 10 mM l,5-bis(N-hexadecyl-
N,N- dimethylammomum)pentane dibromide (16-5-16) in D2O.
188
r ^ V r A 3 2 1 7^ N-CH2- (CH2)2 -CH2- {CH2) i i -CH3
CH2 I
51CH2)2 I
^ " \ l N ^ ^ 3 2 1 .N-^H2-(CH2)2-CH2-(CH2)il-CH3
• Br
1 I I I 1 1 1 1 1 — PPm 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Fig. 5.3: 300 MHz ' H N M R spectrum of pure 5 mM l,4-bis(N-hexadecyl-N,N-
dimethylammonium)butane dibromide (16-4-16) in D2O.
189
^
y\' .1,
,\i\
lOiiiM
Jl
5mM
J
3mM
W L
ImM
UL " " ' 5 1 0 M 2 0 1 5 1 0 0 5 0 0
^•JL w/L\
OmM
j ^ 3 0 ? 5 ?D 1 ^ t o 0 ^ 0 0
Fig. 5.4: 300 MHz 'H N M R spectra of 10 mM 16-6-16 containing different
concentrations of sodium anthranilate (NaAn) in D2O.
190
lOmM
5mM
3mM
a
. y ^
ImM
u »• ' » 3 0 ris Ti 1.5 I.O 0 . 5 0 . 0
0 iv.M
L Dc« 3 i ) 0 ; ^ P i " 0
Fig. 5.5: 300 MHz ' H N M R spectra of 10 mM 16-5-16 containing different
concentrations of sodium anthranilate (NaAn) in D2O.
191
J - 3 0 ? 5 J O I S 1.0 0
Fig. 5.6: 300 MHz 'H N M R spectra of 5 mM 16-4-16 containing different
concentrations of sodium anthranilate (NaAn) in D2O.
lOmM
192
5mM
_ J
3mM
.-AJ
ImM
^ — T i 7i r» j.o '-5 ' « «•» • "
w/U OmM
='• 1 i 3 0 TT To Ti To o's
Fig. 5.7: 300 MHz 'H N M R spectra of 10 mM 16-6-16 containing different
concentrations of sodium benzoate (NaBen) in D2O.
193
3mM
u
IniM
3 i 1.0 J » J.O 1.4 I t 0 1 » 0
) . l ) 0 1 i ?.0 1.5 ) 0 0 .1 0 0
Fig. 5.8: 300 MHz 'H N M R spectra of 10 mM 16-5-16 containing different
concentrations of sodium benzoate (NaBen) in D2O.
194
y\j
OmM
u "'• 3 5 3 0 J.3 JO 1.5 ^ ^ 7i W
Fig. 5.9: 300 MHz 'H N M R spectra of 5 mM 16-4-16 containing different
concentrations of sodium benzoate (NaBen) in D2O.
195
Table 5.1
' H N M R chemical shifts (5 in ppm) of geminis^ with various concentrations of
NaAn at 25 °C.
Geminis [NaAn] Chemical Shift (8, ppm)
(mM) f
16-6-16 0.0 0.929 1.345 1.431 1.518 1.814 3.200 3.416
1.0 0.911 1.328 b 1.465 1.764 3.163 3.367
3.0 0.924 1.338 b b 1.717 3.138 3.316
5.0 0.940 1.350 b b 1.685 3.125 3.281
10.0 0.946 1.353 b b Disappear 3.052 Disappear
16-5-16 0.0 0.928 1.346 1.438 1.816 1.913 3.464 3.223
1.0 0.862 1.283 b 1.727 1.813 3.370 3.140
3.0 0.920 1.337 b 1.709 1.785 3.344 3.152
5.0 0.925 1.338 b 1.677 c 3.313 3.140
10.0 0.946 1.355 b Disappear Disappear Disappear 3.092
16-4-16 0.0 0.776 1.198 1.281 1.666 1.797 3.334 3.065
1.0 0.915 1.333 b 1.726 1.866 3.413 3.174
2.0 0.920 1.335 b 1.650 1.804 3.350 3.145
3.0 0.927 1.340 b Disappear Disappear Disappear 3.123
5.0 0.928 1.318 b Disappear Disappear Disappear 3.062
•b ' [16-6-16] = 10 mM; [16-5-16] = 10 mM; [16-4-16] = 5 mM
Peak merges with 2 Peak merges with 4
196
Table 5.2
' H N M R chemical shifts (5 in ppm) of geminis^ with various concentrations of
NaBen at 25 °C.
Geminis [NaBen] Chemical Shift (8, ppm)
(mM) f
16-6-16 0.0 0.929 1.345 1.431 1.518 1.814 3.200 3.416
1.0 0.913 1.329 b 1.476 1.771 3.171 3.378
3.0 0.923 1.334 b 1.426 1.727 3.147 3.330
5.0 0.931 1.339 b b 1.685 3.125 3.287
10.0 0.943 1.352 b b 1.580 3.082 3.205
16-5-16 0.0 0.928 1.346 1.438 1.816 1.913 3.464 3.223
1.0 0.910 1.329 b 1.764 1.861 3.409 3.183
3.0 0.929 1.342 b 1.716 1.816 3.363 3.165
5.0 0.933 1.340 b 1.652 1.754 3.302 3.133
10.0 0.959 1.367 b Disappear Disappear Disappear 3.101
16-4-16 0.0 0.776 1.198 1.281 1.666 1.797 3.334 3.065
1.0 0.910 1.330 b 1.720 1.880 3.380 3.190
2.0 0.919 1.331 b 1.607 1.862 3.355 3.163
3.0 0.930 1.340 b Disappear Disappear Disappear 3.142
5.0 0.957 1.372 b Disappear Disappear Disappear 3.107
" [16-6-16] = 10 mM; [16-5-16] = 10 mM; [16-4-16] = 5 mM Peak merges with 2
197
Table 5.3
Line widths (/w) of the signals from the protons of the N-methyl groups of
geminis^ against different concentrations of added NaAn at 25 °C.
Geminis [NaAn] /w
(niM) (Hz)
16-6-16 0.0 30.61
1.0 30.61
3.0 33.41
5.0 40.09
10.0 Merge
16-5-16 0.0 28.21
1.0 28.21
3.0 33.41
5.0 40.09
10.0 Merge
16-4-16 0.0 39.61
1.0 46.77
2.0 53.46
3.0 66.82
5.0 Merge
[16-6-16] = 10 mM; [16-5-16] - 10 mM; [16-4-16] = 5 mM
198
Table 5.4
Line widths (/w) of the signals from the protons of the N-methyl groups of
geminis^ against different concentrations of added NaBen at 25 °C.
Geminis [NaBen] /w
(mM) (Hz)
16-6-16 0.0 30.61
1.0 30.61
3.0 30.61
5.0 30.61
10.0 33.41
16-5-16 0.0 28.21
1.0 28.21
3.0 28.21
5.0 33.41
10.0 Merge
16-4-16 0.0 39.61
1.0 45.00
2.0 49.65
3.0 59.12
5.0 Merge
[16-6-16] = 10 mM; [16-5-16] = 10 mM; [16-4-16] = 5 mM
199
70
N X
6 0 -
5 0 -
4 0 -
30
0
3-16-6-16 -•-16-5-16 -O-16-4-16
-j= 2 4
[NaAn] (mM) Fig. 5.10: Line widths of the signals from the protons of the N-n\ethyl groups
of 16-6-16, 16-5-16,16-4-16 plotted against different concentrations of added
NaAn.
200
70
N X
6 0 -
5 0 -
40
—3-— • -
- o -
-16-6-16 -16-5-16 -16-4-16
4 6 8
[NaBen] (mM) Fig. 5.11: Line widths of the signals from the protons of the N-methyl groups
of 16-6-16, 16-5-16,16-4-16 plotted against different concentrations of added
NaBen.
201
Table 5.5
Relative viscosities ( ri,) of geminis^ in presence of NaAn at 25 °C.
Geminis [NaAn] ij,
(mM)
16-6-16 0.0 1.05
1.0 1.05
3.0 1.05
5.0 1.05
lO.O 1.25
16-5-16 0.0 1.05
1.0 1.05
3.0 1.06
5.0 1.07
10.0 5.00
16-4-16 0.0 1.05
1.0 1.38
2.0 1.67
3.0 2.60
5.0 6.86
'"' [16-6-16] = 10 mM; [16-5-16] = 10 mM; [16-4-16] = 5 mM
202
Table 5.6
Relative viscosities (//r) of geminis^ in presence of NaBen at 25 °C.
Geminis [NaBen] T],
(mM)
16-6-16 0.0 1.05
1.0 1.05
3.0 1.05
5.0 1.05
10.0 1.06
16-5-16 0.0 1.05
1.0 1.05
3.0 1.07
5.0 1.07
10.0 4.12
16-4-16 0.0 1.05
1.0 1.28
2.0 1.41
3.0 2.00
5.0 4.77
[16-6-16] - 10 mM; [16-5-16] = 10 mM; [16-4-16] = 5 mM
203
4 -
2 -
-3-16-6-16 -e-16-5-16 -O-16-4-16
- < l
0 4 6 8
[NaAn] (mM)
to
Fig. 5.12: Plots between relative viscosity (^r) against different concentrations
of added NaAn.
204
6 -
4 -
2 -
0
- 3 - 1 6 - 6 - 1 6 - • - 1 6 - 5 - 1 6 -O-16-4 -16
O
0 4 6 8
[NaBen] (mM)
10
Fig. 5.13: Plots between relative viscosity (^r) against different concentrations
of added NaBen.
205
»»^-,-fl/»-.|.«^l**if*^>***h*JA*Wi*A** •tV/"*****^"*'
ImM
p-^V^>ii*IH'*"»'>'S*)^\*-*»^'^
pom 9.0 8.5 7^7 7.5 7.0 6.5 6.0
(A)
6-H 4-H
'1 J
3-H ,5-H
OOm 9 0 8.5 8.0 7.5 7.0 6 .5 5.0
Fig. 5.15: 300 MHz 'H N M R spectra of NaAn: (A) 10 mM pure NaAn; (B)
with lOmM 16-5-16 at different NaAn concentrations.
6^ ' "
(B)
1 1 1 ]
1 *1 wU
lOmM
5mM
206
y .1 • , i , i i . » ^ t V > ^ V ^ « p ^ ^
3mM
^^ w.
• 3 6 5
Fi.. 5.H: 300 MHz ' H N M R spectra of NaAn: (A) ,0 . M pure NaAn; with IOmM 16-6-16 at different NaAn
(B) concentrations.
207
yv*»"^-*'fT'v*'
5mM
InM
.JXJ^-^
••*,"'• >> i l l u m e s
ImM
t, ..V.'i t'liv*
(A)
6-H 4-H
ua 3-H,5-H
CDa 9.0 S.S e.O J.i 7.0 e.J 6.0
Fig. 5.16: 300 MHz ' H N M R spectra of NaAn: (A) 5 mM pure NaAn; (B) with
5 mM 16-4-16 at different NaAn concentrations.
(B)
^ ^
lOmM
L
208
5mM
3mM
- i
ImM
P°» 3.0 a.5 B.O 7.5 T'o 71 i T
2-H,6-H
(A)
4-H
VJ
3-H,5-H
CO" 9 0 8 5 8-0 ' 5 ? 0 6 5 6 0
Fig. 5.17: 300 MHz 'H N M R spectra of NaBen: (A) 10 mM pure NaBen; (B)
with 10 mM 16-6-16 at different NaBen concentrations.
209
Na O
lOmM
5mM
3iTiM
pom 9.0
(A)
•~ ' ' t
2-H,6-H
ImM
-V-
4-H
u
3-H .5-H
7 1 BT T I TT 5.5 5,0
oom 9 0 8,5 8.0 7.5 7.0 5.5 6.0
Fig. 5.18: 300 MHz 'H N M R spectra of NaBen: (A) 10 mM pure NaBen; (B)
with 10 mM 16-5-16 at different NaBen concentrations.
210
Na O ^O
h
4
(B) 5mM
3mM
2niM
ImM
M'Wi,^\ii'Hi*^'^'<l^ii^''l^^'^^Vt^^^
DOm S 0 3 5 7 .5 7 .0 5 . 5 5 . 0
2-H,6-H
(A)
DDm 9 0 8 5
4-H
3-H, 5-H
8 0 7 5 7 0 6.5 5.0
Fig. 5.19: 300 MHz 'H N M R spectra of NaBen: (A) 5 mM pure NaBen; (B)
w.tl. 5 mM 16-4-16 at different NaBen concentrations.
211
Change in environment is experienced by the different parts of the surfactant
monomers which is indicated in the chemical shift via the shielding and
deshielding effects experienced by the ' H nuclei. The non-polar part of the
surfactant (hydrophobic part) near the core of the micelle is highly shielded.
When we move towards the head group the shielding decreases. Presence of N-
atoms in the head group makes its adjacent protons more deshielded. On
increasing the salt concentration the peaks overlap and signals become broad.
This indicates the presence of grown micelles in the system. When the molar
ratio of salt to surfactant is close to unity, signals become very broad, making
evaluation difficult, as shown in Figs. 5.4-5.9.
The line widths at half height of the signals from the protons of the N-
methyl groups of the 16-m-16 geminis are shown in Figs. 5.10 and 5.11. The
data show an increase in the proton line widths with increasing [salt]. The line
width values can be used to discuss structural changes in the gemini skeleton.
Changes in the width can be inferred as the changes in the micellar
morphology.^''^^
Viscosity data of different concentrations of salts with 16-m-16 geminis
show an increase in viscosity with increase in the [salt] (Tables 5.5 and 5.6,
Figs. 5.12 and 5.13). The presence of salt ions near the polar heads of the
surfactant molecules decreases the repulsion force between the headgroups. A
reduction in the repulsion makes it possible for the surfactant molecules to
approach each other more closely and form larger aggregates. This leads to an
212
increase in the relative viscosity (rj^) indicating the formation of larger
aggregates. ' '* The viscosity remaining almost constant for the 16-6-16
indicates that salt additions to 16-4-16 and 16-5-16 causes structural changes at
fairly low concentration in comparison of 16-6-16.
This behavior of 16-m-16 geminis is understandable due to the following.
In conventional surfactants (e.g., cetyltrimethylammonium bromide; CTAB),
the headgroups are randomly distributed on the micellar surface separating the
aqueous phase and micelle core. These distances between the headgroups are
determined by the opposite forces at play in micelle formation. The reported
values of the surface area per headgroup at interface suggest that the distance is
~ 0.7 - 0.9 rmi. With gemini surfactants, the distribution distances become
bimodal.^^ Indeed, the inter-headgroup distance exhibits a maximum at the
equilibrium distance (as for conventional CTAB) and another narrow
maximum at a distance corresponding to the length of the spacer. The bimodal
distribution of headgroup distances and effect of the chemical link between
headgroup on the packing of surfactant alkyl chain in the micellar core are
expected to strongly affect the packing parameter^ or curvature of surfactant
layers, and thus to the micelle shape and the properties of the solution.
However, the morphological changes and micellar growth of gemini micelles
depend primarily on the spacer chain length. It has been found that an increase
in the spacer chain length (m value) suppresses the tendencies of micellar
growth of 16-m-16 in water. ^ The present viscosity behavior reflects the
ability of 16-4-16 to give rise to worm-like micelles at fairly low salt
213
concentrations which, however, are absent with the 16-6-16 and, to some
extent, with 16-5-16 due to packing requirements.
The spectrum of pure NaAn solution in D2O (Figs. 5.14-5.16 - A) is first-
order and consists of three multiplets. The signals for the amino protons are not
seen separately, as they are labile and thus merge with the solvent peak. The
spectra of the ring protons at different concentrations of NaAn in 16-m-16 are
also shown in Figs. 5.14-5.16 - B. It is clear that for NaAn, the signals for the
6-H protons are in a polar environment and signals for 3-H, 5-H and 4-H are in
the non-polar environment indicating that An" intercalate near the headgroups
of the dimeric surfactants. At higher sah concentrations, the peaks for the An"
shift up-field. This shows that at higher concentrations of the An" the effective
environment experienced by 3-H and 5-H protons is different (slightly polar).
This is because of the proximity of 3-H proton to the - NH2 group. Figs. 5.17-
5.19 - B show the spectra of ring protons of different concentrations of NaBen
in 10 mM 16-6-16 and 16-5-16 and 5 mM 16-4-16. An upfield shift for 3-H, 4-
H and 5-H ring protons of NaBen are observed with increasing salt
concentration. These protons give a single peak indicating they are in the same
environment. This suggests that Ben" ions are located deeper inside the micelle
(compared to An"). Again, relatively lesser broadening at the 1:1 molar ratios
(see Tables 5.3 and 5.4) also supports this proposition. Thus, due to deeper
solubilization, Ben" do not interact with the micelle as strongly as the An".
214
The data suggest that the presence of an organic anion in the micellar
solutions of 16-m-16 causes a change in micellar morphology. The
intercalation of An" ion into the micellar surface region (due to electrostatic
interactions with the oppositely charged surfactant headgroups) seems the
prime cause of inducing such morphological changes. Additionally, NaAn has
a NH2 group attached to 2-position that would prefer to remain in more polar
environment (than Ben" ion) due to its polar nature and the geometric
hindrance. Therefore, site of solubilization of An" has direct links to the overall
changes. In case of Ben" ion, which is solubilized deeper into the outer core
region of the micellar interior, one needs more concentration than An" to
produce similar results.
215
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