1. Synthesis and Reactivity in Inorganic, Metal-Organic and Nano-Metal Chemistry, 36:469–476, 2006
Copyright # 2006 Taylor & Francis Group, LLC
ISSN: 0094-5714 print/1532-2440 online
DOI: 10.1080/15533170600777960
Accurate Potentiometric Studies of Chromium-Citrate
and Ferric-Citrate Complexes in Aqueous Solutions
at Physiological and Alkaline pH Values
Yahia Z. Hamada, Nabil Bayakly, Andrew Peipho, and Brandon Carlson
Division of Natural and Mathematical Sciences, LeMoyne-Owen College, Memphis, TN, USA
an oligopeptide composed of glycine, cysteine, aspartic acid,
Speciation diagram of Fe31-citrate complexes in aqueous glutamic acid with the carboxylate comprising more than
half of the total amino acid residues.[1] Although there is a
solutions is presented. The conditions set forth for the speciation
diagram were; Fe31:citric acid in 1:1 ratio with concentration of wealth of studies available regarding the essential Cr3þ, a con-
2.5 3 1024 mol . L21, 0.4922 mol . L21 NaOH solution, and
siderable amount of experimental and theoretical calculations
pKw 5 13.781 + 0.006 taken from (J. Sol. Chem. 1974, 3,
are still needed in this area.[1 – 12] The isolation and character-
191– 214). The speciation diagram is showing that the dimeric
ization of LMMCr has not been achieved, and thus its
species is the dominant species in solutions from pH 4.0 to
pH 12.0. In addition the speciation diagram for the free precise structure is still somewhat controversial. The site and
citrate presented is in a good agreement with what had been pathway of LMMCr biosynthesis are unknown. The site and
reported in the literature (Critical Stability Constants Database,
mechanism of intestinal chromium absorption in humans
Version 6.0, NIST, 2001). In a fairly recent development
have not been determined.[1]
(Science, 2002, 295, 1715 –1719), the integral outer membrane
There is no recommended dietary allowance for chromium
cell receptor for iron citrate was crystallized. The interpretation
of the crystallographic data collected led to the conclusion that a and no reliable test for chromium deficiency exists. However,
reasonable model for the species anchored into the cleft of the the National Research Council suggests that people age 7
cell receptor is the dimer Fe31-citrate species. The data on
and older can safely meet their needs with 50 to 200 mg per
the Cr31-citrate system is also in a fairly good agreement with
day. Unfortunately, few Americans take in the 50 mg
the dimer model presented in aqueous solutions.
minimum. After absorption, Cr3þ binds to the b-globulin frac-
tions of serum proteins, specifically to transferrin. Transferrin
aqueous solutions, citrate, dimer, potentiometric titra-
Keywords
is apparently the protein responsible for chromium transport
tions, speciation diagram
into tissues; Cr3þ has about the same affinity as Fe3þ towards
transferrin.[1,4,5]
INTRODUCTION
Citrate
Citric acid is ubiquitous in nature. It is involved in the active
Chromium
sites of bacterial metalloenzymes including aconitase, a key
Cr3þ is an essential trace metal necessary for the formation
enzyme in the citric acid cycle, and dinitrogenase in
of the so called “low-molecular-mass chromium-binding”
nitrogen-fixing bacteria.[13,14] It is found in human blood
(LMMCr). The LMMCr has been suggested to be an organic
plasma at a concentration of about 0.1 mM.[15 – 17] It is con-
low-molecular mass complex that contains four Cr3þions and
sidered to be a preeminent, small molecular mass binder of a
number of essential and non-essential metal ions.[15 – 17] for
Received 1 April 2006; accepted 15 April 2006. these reasons we are testing its interaction with the essential
A. Peipho and B. Carlson conducted the initial work under the
metal ions iron and chromium.
guidance of Y. Z. Hamada when he held an assistant professor
position at Wayne State College.
Iron and Iron-Citrate System
This work was supported in part from NSF under Grant # HRD-
0411493. We also would like to thank the Faculty at the Division of Iron is the most abundant transition metal in the biosphere.
Natural and Mathematical Sciences of LeMoyne-Owen College for By far it is the most important essential metal for the growth of
reading the manuscript.
almost all bio-organisms.[13,14] However, its availability to
Address correspondence to Yahia Z. Hamada, Division of Natural
bio-organisms is limited because of its low solubility in
and Mathematical Sciences, LeMoyne-Owen College, 807 Walker
aqueous-aerobic solutions at neutral pH.[13,14,18] It has been
Ave., Memphis, TN 38126, USA. E-mail: yahia_hamada@loc.edu
469

2. 470 Y. Z. HAMADA ET AL.
known for a long time that the Fe3þ-citrate complexes are very Potentiometric Titrations
soluble.[19 – 21] Spiro et al. presented a potentiometric titration The potentiometric titration solutions were contained in a
graph of trisodium citrate with ferric nitrate that had an 250.0 mL beaker equipped with a magnetic stirring bar. The
initial inflection of 1.5 equivalents of titrant per iron. They con- beaker was covered with a custom made Teflon cover. In a
cluded from this stoichiometry that the dimer [(FeCit)2(OH)] typical titration the ligand was added first (citric acid solution
was the dominant species.[20] This dimeric species has been or trisodium citrate solution) then the metal ion solution was
isolated and characterized crystallographically by Lippard added followed by the addition of the appropriate amount of
and others.[22] The mobilization of iron in living cells water to take the total volume to 100.0 mL. The concentration
of the metal was ¼ 2.50 mmoles . L21. Before each titration,
requires complex formation to overcome the insolubility of
iron at the physiological pH. The idea that, upon binding, the titration solution mixtures were allowed to stir for 20–25
citrate and other hydroxy-carboxylates enhance the mobiliz- minutes for complete equilibrium. The NaOH titrant was
ation and bioavailability and promote absorption of dietary added in the 100 mL increments using an Eppendorf micropip-
non-heme iron has attracted our attention.[16,18,23] ette with continuous stirring. The time intervals between the
additions of the NaOH solution were set to 5 minutes, which
was sufficient to get each of the pH values stabilized and reach
complete equlibrium. The experiments were repeated in 0.1 M
EXPERIMENTAL SECTION
NaNO3 solutions as ionic strength adjustor. In these experiments
we essentially reproduced the same titration graphs. This gave
Materials
high confidence to the titration graphs presented.
All solutions were prepared using Fisher reagent grade
citric acid, C6H8O7, formula weight 192.12 g . mol21,
tri-trisodium citrate salt monohydrate, C6H5Na3O7 . H2O, RESULTS AND DISCUSSION
formula weight 258.08 g . mol21, chromium nitrate nona-
Ligand pKa Constants
hydrate, Cr(NO3)3 . 9H2O, formula weight 400.15 g . mol21,
and iron nitrate nona-hydrate, Fe(NO3)3 . 9H2O, formula Citric acid has three titratable carboxylate groups with pKa
weight 404.00 g . mol21, using doubly deionized water. values of 2.90, 4.35, and 5.65. These values are taken from
Martell and Smith.[24] We have published, essentially, an identi-
The pH values of all solutions were adjusted using
0.4922 mol . L21 sodium hydroxide NaOH, solution. The pH cal set of pKa values for this biologically important ligand. The
pKa values for citric acid published by Harris et al. were 2.92,
values were measured using Hanna Instrument HI 8314
4.35, and 5.70[17] Table 1 shows these pKa values along with
Membrane pH meter with a combination glass electrode. All
the selected stability constants taken from the literature for
experiments were repeated using the more accurate Orion pH
various metal ions with citric acid in aqueous solutions that
electrode-meter combination model 720Aþ that measures the
formed binuclear complexes. Values for the overall stability con-
pH values to three significant figures to the right of the
decimal point in 0.1 mole . L21 ionic strength using the appro- stants are given in the form of Log b220 for the equilibrium given
in Equation (1) and defined by Equation (2). In these equations,
priate amounts of NaNO3 solution.
CitH42 is the totally de-protonated form of citric acid at which
21
the three carboxylate protons as well as the alcoholic protons
are all removed. By examining the values of the stability
Preparation of the Potentiometric Titration Solutions constants of these dimers in solutions (in the range of % 11 to
In all metal-ligand potentiometric titrations, the NaOH 21 logarithm values) it is clear that the metal-citrate dimers are
solution was always the titrant. The NaOH solutions were very stable and robust complexes because they possess such
prepared from NaOH laboratory grade pellets in carbonate free high stability constants in aqueous solutions when formed.
water. The methods used to prevent the contamination of the
2Mxþ þ 2ðCitH4À Þ$½ðM CitH4À Þ2 Š½2xÀ8ÞÀ ð1Þ
titrant with atmospheric CO2 had been described elsewhere.[17] À1 À1
The NaOH solutions were standardized using primary standard
b220 ¼ ½ðM CitH4À Þ2 Šð2xÀ8ÞÀ =½Mxþ Š2 ½CitH4 – Š2 ð2Þ
À1 À1
potassium hydrogen phthalate (KHP). Both NaOH and KHP
were purchased from Fisher Chemical Co. Before any KHP titra-
Cr31-Citric Acid System
tion, the KHP was dried at 1108C, for 24 hours and stored in a
Figure 1 shows the family of titration curves for the Cr3þ-
desiccator. A stock indicator solution of about 0.2% phe-
nolphthalein in about 90% ethanol was prepared from reagent citric acid system in aqueous solutions at 258C for different
grade phenolphthalein. KHP was titrated to the phenolphthalein molar ratios. In potentiometric titrations, the presence of an
end point. Typically, fifteen runs were carried out to standardize inflection point indicates the presence of a single dominant
the NaOH solution. Standard statistical treatments of the data species. The position of the inflection point indicates the
such as the arithmetic mean, standard deviation, T-test, and number of protons released via the formation of this dominant
species present in solution.[17,25,26] Table 2 shows the detailed
Q-test were conducted using Excel software.

3. 471
DIMERIC METAL-CITRATE SPECIES
TABLE 1
Selected stability constants of binuclear citrate species form the literature
pKa/Log b Temp. 8C
Citrate pKa/metal ion Ionic strength Remarks
2.90a 2.92b
PKa1 25 0.1 M
4.35b
PKa2 4.35 25 0.1 M
5.70b
PKa3 5.65 25 0.1 M
Be2þ Log b220 13.10 25 1.0 M
UO2þ 18.87 25 0.1 M
2
Cu2þ 14.50 25 0.1 M
Pb2þ 10.70 25 1.0 M
Al3þ 12.69c 12.12b,c
37 0.16 M
Fe3þ 21.20c 20 0.1 M
Cr3þd — — —
a
Martell and Smith Ref.[24]
b
Harris et al.[17]
c
The aluminum and iron log b values given here are for the [Log b22-2], which represent the ternary binuclear com-
plexes [M2(Cit)2(OH)2]. No values have been reported in the literature for Log b220 of the simpler binuclear species
of these two metal ions.
d
Missing numbers indicate no reliable data have been reported in the literature.
account of these titration curves. 0.50 mmoles . L21 of the free maximization and decline of the four different citrate species
present in solution namely H3Cit, H2Cit2, HCit22, and Cit32.
citric acid have been used to generate the free acid curve
shown in Figure 1. From Figure 1 and Table 2 it is clear that It is clear from Figure 2 that the cross points between the plots
when the free citric acid was titrated without any metal ion, the in the graph represent the three pKa values of citric acid.
acid has been defined as an H3L ligand which meant that there The 1:1, 1:2, 1:3, and 1:4 titration curves showed well-
are three protons that can be titrated out of the three carboxylates. defined and extended buffer regions between pH % 2.0 –6.0.
The pKa values of these three protons are given in Table 1. For the 1:1 titration system beginning at pH % 6.5 –7.0 there
Figure 2 is the speciation diagram for the free citric acid in was a visible turbidity till the end of the titration. This is indi-
0.1 mol . L21 ionic strength, pKw ¼ 13.781 showing the cated by the filled and darkened data-points in this particular
titration plot. No visible precipitates were observed for the
1:2, 1:3, or 1:4 titration systems. For the 1:1 and 1:4 titration
systems the buffer regions were terminated with sharp and
well-defined inflection points at half-integers. The appearance
of these sharp inflections at half integers is good evidence
that the dominant species present in aqueous solutions is the
dimeric Cr3þ-citrate complex. We do not know why the 1:2
and the 1:3 titration ratios gave these fraction integers
perhaps due to some sort of oligomerization. The titration con-
tinued to pH % 11.5 –12.0. A detailed and careful UV-Vis titra-
tion study for the Cr3þ-citric acid system at 258C between pH
2.23 and pH 9.50 has been presented at which the characteristic
Cr3þ absorption peaks in aqueous solutions have been
observed. The detailed UV-Vis titrations for the Cr3þ-trisodium
citrate have also been shown in the same study.[16]
Fe31-Trisodium Citrate System
Figure 3 shows the family of titration curves for the Fe3þ-
trisodium citrate system in aqueous solutions at 1:2, 1:3, 1:4,
1:5, and 1:6 molar ratios. These titration curves showed
buffer regions between pH % 5.0– 6.0. For all titration curves
i.e., 1:2, 1:3, 1:4, 1:5, and 1:6 titration systems the inflection
FIG. 1. Potentiometric titration curves of free citric acid and Cr3þ -citric acid
points appeared between pH % 6.5 –9.5. There were no
in 1:1, 1:2, 1:3, and 1:4 molar ratios at 258C.

4. 472 Y. Z. HAMADA ET AL.
TABLE 2
Potentiometric titration data for [Cr3þ] ¼ 2.50 mmol . L21 with H3Cit in different molar ratios, 258C
Cr3þ/H3Cit mL of NaOH Equivalents of Proposed
mole ratio titrant NaOH titrant species Remarks
0:1a 3.00a 3.00 — Citric acid is a
tri-protic acid
(Cr CitH42)2
1:1 4.80 9.45 The dimer is
21
formed
1:2 5.20 10.24 —
1:3 6.00 11.81 —
(Cr CitH42)2
1:4 7.40 14.57 The dimer is
21
formed
0.50 mmol . L21 Citric acid was titrated to generate the free acid curve shown in Figure 1.
a
visible precipitations at all in any of these titration systems. The All previous studies were conducted within the acidic or
solubilization effect of citrate for the ferric ion in aqueous neutral pH-ranges. The researchers who observed a similar
solution is well established in the literature.[15,16,18 – 22,24] It is titration graph in aqueous solution gave us some evidence on
worth mentioning that there were no drifts for the pH- the presence of the dimer, although they were trying to
readings from the pH-meter, which indicated that the system isolate and characterize the solid ferric-citrate polymer rather
than the dimer in aqueous solutions.[20,21] These researchers
is reaching total equilibrium. For the 1:3 titration system, we
have overlaid two of the 1:3 titration plots to further ensure conducted their work on one titration graph for the 1:1 titration
data reproducibility (see Figure 3). The inflection points system which is in an agreement with our data presented for the
always appeared between 1.50 equivalents and 1.57 equivalents dimer complex. Another study has tried to build up the dimer
model but failed.[28]
regardless of the iron to trisodium citrate molar ratio. This indi-
cated that in every titration system the dimeric Fe3þ-citrate is The crystal structures of the (M CitH42)2 dimer complexes
21
have been reported for the following metal ions: Co2þ, Ni2þ,
the dominant species. The titrations were continued to
Al3þ, Fe3þ, VO2þ, Ti4þ, and V5þ.[29 – 36] Table 3 catalogues
pH % 12.0. These titration curves in Figure 3 cover wider pH
ranges than those presented previously.[20,27] references from the most current literature along with further
FIG. 3. Potentiometric titration curves of Fe3þ -Trisodium citrate in 1:2, 1:3;
1:4, 1:5, and 1:6 molar ratios. [Fe3þ] ¼ 2.5 mmol . L21 [NaOH] ¼ 0.4922
FIG. 2. Speciation diagram for the free citric acid; pK1 ¼ 2.90, pK2 ¼ 4.35,
mol . L21, 25 8C.
pK 3 ¼ 5.70, and pKw ¼ 13.781.

5. 473
DIMERIC METAL-CITRATE SPECIES
TABLE 3
Selected references from the literature for various metal ions in different oxidation states with citrate that formed
the dimer complex
Metal ion Year of
and charge Nature of the study publication Ref. # Remarks
Al3þ Aqueous solutions 2003 17
Fe3þ Crystal structure 1994 22
Co2þ Crystal structure, solid and solution EPR, 2003 29 EPR data at
and potentiometric solution studies 48C
Ni2þ Crystal structure 1997 30
Al3þ Crystal structure 2003 31
Al3þ Crystal structure 2001 32
VO2þ Crystal structure 2001 33
V5þ Crystal structure and solution studies 2003 34
Cr3þ 35a
Ion exchange in aqueous solutions 1986
Ti4þ Crystal structure 2004 36
a
In this study the authors proposed that there are 15 species present, including the dimer chromium-citrate complex.
details with the year of publication, and the nature of the study variety of metal ions in different oxidation states in both
aqueous solutions and in the solid-state.[17,22,29 – 36]
(whether in the solid-state, or in solution, and what tools have
been used) regarding the particular metal-citrate dimers. Data To further confirm that the dimer species is the dominant
in Table 3 are not intended to be a full account or a detailed species in the titration systems shown here, Figure 4 shows
review of the literature regarding the metal-citrate system, but the correlation between the first derivatives (slopes) taken for
rather a concise account of the metal-citrate dimers. The main the pH values versus the number of equivalents of titrant.
point drawn from the literature presented in Table 3 is that the This shows that the maximum slopes are appearing at 1.5
citrate ligand forms the dimer complex extensively with a equivalents. It is clear that there is an increase in the intensity
FIG. 5. Speciation diagram for Fe3þ species generated by program Htss39
FIG. 4. Correlations of slopes of the pH values vs. the equivalents of added
using the following parameter: Fe3þ: Cit. is in 1:1 molar ratio,
titrant showing that the infelction points appeared at 1.5 equivalents per ferric
0.4922 mol . L21 NaOH solution, and pKw ¼ 13.78.[40] The dimer is the
ion. This indicated that the main species present in solution is the dimeric
dominant species from pH 4 to pH 12.
species.

6. 474 Y. Z. HAMADA ET AL.
of the peaks observed going from the 1:6 titration system to the of the titrant added to the titration system and (b) the pH of the
1:2 titration system. When there were excess citrate present solution from pH 5.5 to 12.0. It is clear from Figure 6 that most
around the metal ion the chances of dimerization is less than of the titration system is in the negative voltage range,
that if there were one or two moles of citrate present. This suggesting that the major dimeric species is in its anionic
state exactly as it has been reported in the literature.[19 – 22]
further confirms the contention that when there is limited
supply of citrate, the dimerization and polymerization Figure 6 is only a representative one. The same correlation
process will be more probable as seen in Spiro et al.[20] and behavior have been observed for the 1:2, 1:4, 1:5, and
Figure 5 is the speciation graph for the 1:1 titration system for 1:6 titration systems no matter how many times the experiments
0.0025 mol . L21 total Fe3þ with 0.0025 mol . L21 total citric were repeated. It is worth mentioning that the same correlation
acid using 0.4922 mol . L21 NaOH solution, pKw ¼ 13.781. and behavior have been observed for the Cr3þ-trisodium citrate
solution in 1:3 molar ratio.[16]
This speciation diagram is clearly showing the dimeric species
is dominant in solution from pH 4.0 to pH 12.0. The best
fit was obtained by considering the following combination of
species: [Fe HCit], [Fe CitH21]2, and [(Fe CitH42)2]22 the CONCLUSION
21
Citric acid plays a major role in Fe3þ solubilization, trans-
short hand notation for these combination of species are 111,
110, and 22-2, respectively. The first index in the metal species port, and utilization in almost all forms of living
cells.[15,16,18 – 22,37] Many a –hydroxy carboxylates, particu-
notation stands for the number of ferric ions, the second index
larly citrate, form stable and soluble Fe3þ complexes in
in the metal species notation stands for the number of citrate
aqueous solution over a very wide pH range (2.5 –11.5).[18 –
ion(s), and the last index stands for the number of hydrogen ions.
Figure 6 shows the correlation of the millivolts for the Fe3þ-- 24]
The three carboxylates of citric acid have very close pKa
trisodium citrate solution in 1:3 molar ratio with both (a) the mL values in the acidic range of 2.9 –5.7. The potentiometric titra-
tion curves presented with their well-defined buffer regions and
their sharp and well-defined inflection points at half integers
suggest the presence of the dimeric complex as the dominant
species for the Cr3þ-citric acid system and the Fe3þ-trisodium
citrate system in aqueous solutions.
The structures of the [(MCitH42)2]22 dimer complexes
21
have been reported with the variety of metal ions shown in
Table 3, we are suggesting that the dimer complexes formed
for both the Cr3þ-citric acid system and the Fe3þ-citrate
system in aqueous solutions will have similar structures. We
are proposing that the dimeric species formed with these two
systems will have at least two of the carboxylates (the central
and one of the terminal carboxylates) as well as the alcohol
group participating in the metal chelation. In all cases in
which the dimer crystal structures have been
reported,[22,29,31 – 33,37] the alcohol group was coordinated to
the metal ion forming the, very-stable, fused six- and five-
membered chelating rings. In the few cases in which one of
the three carboxylates is not coordinated to the metal
ion,[30,34] it was one of the terminal carboxylates that is not
coordinated to the metal ions, but never the alcohol. The
dimer formation plays an essential role in the formation of
the unique nonairon(III) citrate complex that have been
reported.[37] In a fairly recent development, the integral outer
membrane cell receptor for iron citrate was crystallized and
the three-dimensional crystal structure was determined. The
interpretation of the crystallographic data collected led to the
suggestion-conclusion that a reasonable model for the species
anchored into the cleft of the cell receptor is the dinuclear
Fe3þ-citrate species.[38] The speciation diagram presented in
Figure 5 is identify the presence of the dimeric species
FIG. 6. (a) Correlation of millivolts measured vs mL of NaOH added to
within the physiological and the alkaline pH values.[39]
the Fe3þ -sodium citrate in 1:3 molar ratio. [Fe3þ] ¼ 2.5 . (b) Correlation of
However, there are no reliable stability constant in the
millivolts vs pH.

7. 475
DIMERIC METAL-CITRATE SPECIES
literature (thus far) for the Cr3þ-citrate dimer complex to con- 16. Hamada, Y. Z.; Carlson, B. L.; Shank, J. T. Potentiometric and
UV-Vis spectroscopy studies of citrate with the hexaquo Fe3þ
struct a successful speciation diagram similar to that of the
and Cr3þ metal ions. Syn. Reac. Inorg. Metal-Org. Chem. 2003,
Fe3þ system. Little is known about chromium transport in
33 (8), 1425– 1440.
the mammalian cells, the chromium-citrate complexes could
17. Hamada, Y. Z.; Zhepeng, W.; Harris, W. R. Competition
play a role similar to that of the role played by citrate in the
between transferrin and serum ligands citrate and phosphate
metabolism of both iron and aluminum.
for the binding of aluminum. Inorg. Chem. 2003, 42,
3262– 3273.
18. Dhungana, S.; Ratledge, C.; Crumbliss, A. L. Iron chelation prop-
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