Summary:-


Iraqi J Pharm Sci , Vol.18 (1) , 2009                                   Amoxicillin and cobalt (II) complexation   

 56 

HO

H
NH2 H H

S

N

CH3

CH3

COOH

H

O

N

H

O
.   3H2O

Spectroscopic Study for Determination of Amoxicillin Using 

Cobalt(II) as Complexing Metal 
May M.J. Al-Mudhafar*

,1
 

*Department of Pharmaceutical Chemistry,College of Pharmacy,University of Baghdad,Baghdad,Iraq 

 
Abstract 
         This study includes analytical methods for the determination of the drug amoxicillin trihydrate 
(Amox.) in some pharmaceutical preparations using Cobalt ion (Co(II)) as complexing metal. The best 

conditions for complexation were: the reaction time was 20 minutes, pH=1.5 and the best temperature 

of reaction was 70 ˚C. Benzyl alcohol was the best solvent for extraction the complex. 

Keywords: Amoxicillin, Cobalt(II), Complex, Molar ratio. 

 

 الخالصة
 Amoxicillin)اتالبنما اايتضمن البحثماالحمتث لرا محلالثة ة مدا  يم قايم المم يحالبنحئم البم سلي الالثئ  مة  ا   م ا  ي م         

Trihydrate)ا)اتسذبممباكتيممثي االلممم ابةلممماناالممواليممث البيثكةمما(Co IIح ممااس مم اليضمم ااpH بلنة ممدالبتلم مم ادامم االبممداحاالضمم داا

(٥٫١pH=اسل اليض اان داححلنقابلنة دالبتلم  االاك  البلماناسلاليث البفة يائانتاكث ساا)ان داال ثيد,اسليضم امالم ابةتفادم اااا٠٧

البحا ية اهثاليض االذي ابلنة دالالحتخ ص.االاق مد,ق ا حكتاد قاالذيحاتاح ااس  اإ البيثثا٠٧ث سااييا اك

 

 
Introduction 
         

Amoxicillin is one of the important derivatives 

of semisynthetic penicillin; it is active against 

Gram positive and to less extent Gram 

negative bacteria. Its nomenclature according 

to penicillins is 6-[D(-)-α-Amino-p-

hydroxyphenyl acetamido] penicillanic acid or 

α- amino-p-hydroxy benzyl penicillin
(1)

  ,while 

its systematic (IUPAC) name is
 
7-[2-Amino-2-

(4-hydroxypHenyl)-acetyl]amino-

3,3,dimethyl-6-oxo-2-thia-5- azabicyclo  [3, 2, 

0] heptane-4-carboxylic acid, the chemical 

structure of the drug is 
(1)

 

 

 

 

  

 

 

 

 

  

 

 

         

 

The formula structure of Amoxicillin as 

trihydrate (drug) is C16H19N3O5S.3H2O, its 

molecular weight = 419.45 gm.mole
-1

. It is off 

white or almost white crystalline powder, 

slightly soluble in water and alcohol such as 

methanol and ethanol
 (2)

. It has UV max. 

(ethanol): 230,274 nm and in (0.1N HCl):
 

229,272 nm 
(1)

.Imran et al. prepared complexes 

of amoxicillin with Zn(II), Cu(II), Ni(II)and 

Ag(I), they identify these complexes by (C, H, 

N)elemental analysis and IR Spectra. These 

complexes have increased the biological 

activity of the drug 
(3)

. Jian et al. determined 

amoxicillin in tablets, they used a quick and 

simple method which is (second differential 

derivative) at λmax. 282 nm and the standard 

deviation was less than 1.2% and the standard 

recovery for the drug was 97-100.5 %
 (4)

.Denis 

et al. determined amoxicillin and clavulanic 

acid in blood plasma by HPLC supplied by UV 

detection, and they found the linearity was 

(0.62 – 20 µg.ml
-
) while the detection limit for 

amoxicillin was 0.312 µg.ml
-
 
(5)

. 

     
1 Corresponding author E-mail : may_almothaffar@yahoo.com 
  Received   : 17/11/2008 

  Accepted   :  8 /4 / 2009 
 



Iraqi J Pharm Sci , Vol.18 (1) , 2009                                   Amoxicillin and cobalt (II) complexation   

 55 

Ashry et al. detected phenolic antibiotic like 

amoxicillin by its reaction with benzocaine in 

the presence of triethylamine at λmax. = 455 nm 

the linearity was (2 – 16 µg.ml
-1

) while the 

detection limit was 0.0034 µg.ml
-1 (6)

.Co(II) 

forms blue-colored complex in the organic 

phase with Cyanex 923, a sensitive analytical 

reagent, the λmax. of the complex was 635 nm 

and the concentration that obeyed Beer’s law 

is (58.9-589.0 µg. ml
-
)

(7)
.Zayed et al. in a new 

study, prepared different complexes of 

amoxicillin with Zn (II), Ni (II), Co (II) and 

Cu (II), these complexes were studied using 

elemental analysis, IR and mass spectra. The 

molar ratio of complexes were found to be 

Metal:Drug =1:1,1:2, and the stability constant 

Kf  of these chelates was(10
7
-10

14
)

 (8)
.In a 

recent study, Alekseev et al.  prepared mixed 

complexes of β-lactam antibiotics (Ampicillin, 

Amoxicillin and Cephalexin) in solutions 

containing Co(II) and glycine anions(Gly). 

These complexes had been investigated using 

pH-metric titration at 20
o
C in alkaline medium 

as mixed ligands complex [Co Gly 

Ampicillin], [Co Gly Amoxicillin], and [Co 

Gly Cephalexin]
 (9)

. 

 

Instruments , Materials and 
Method 
A - Instruments  
1. UV-Visible Spectrophotometer (CARY 

100) wave length 200-1100nm. 

2. Shimadzu (AA-670) Flame Atomic 
Absorption Spectrophotometer (400S). 

3. Mettler, Balance Model 210S, ISO 9001. 
4. pH-meter type 60A, USA.  
5. Water Bath with Thermostat, Memmert.  

B - materials 
All the chemical stock solutions were 

prepared from analytical grade  BDH, 

SDI, and India. 

C- Method 

1.  Stock solution of Co(II) 1000 ppm is 
prepared by dissolving 0.2010 gm  

hydrated cobalt chloride (CoCl2.6H2O) in 

distilled water and complete the volume to 

50 ml. 

2. Stock solution of Amoxicillin 1000 ppm is 
prepared by dissolving 0.1 gm amoxicillin 

in 5 ml. of 1M HCl then complete to 100 

ml with distilled water. 

3. Choosing the optimum conditions for 
complex formation: The experimental 

work showed that the reaction did not 

proceed at room temperature; heating was 

needed, media must be acidic for this 

reason. We studied the effect of pH, 

temperature, reaction time, extraction 

time, and suitable solvent for the 

extraction process. 

4. Spectral study: 
a) Amoxicillin spectrum: transfer 1 ml 

from stock solution of amoxicillin to 

5 ml volumetric flask then dilute with 

distilled water the absorbance is 

measured at (200-1100 nm) using 

acidic water as blank. Table (2) shows 

the proper pH of the solution and 

Figure (1) shows UV spectrum for 

Amoxicillin. 

b) Cobalt spectrum: transfer 1 ml of Co 
(II) stock solution to 5 ml volumetric 

flask then dilute with distilled water 

the absorbance is measured at (200-

1100 nm) using distilled water as 

blank. Figure (2) shows UV spectrum 

for Cobalt. 

c) Amoxicillin-Cobalt(II) [Amox.-
Co(II)] Complex Spectrum: transfer 

(2-5 ml) from the standard of 

Amoxicillin stock solution to 5 ml 

volumetric flask then 1 ml of Cobalt 

stock solution is added. The chelating 

complex was extracted by 5 ml 

benzyl alcohol then measures the 

absorbance at (200-1100 nm) using 

benzyl alcohol as blank. Figure (3) 

Shows UV spectrum for [Amox.-Co 

(II)] Complex. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure(1): UV Spectrum of  amoxicillin 

 

 

 
 
 
 
 
 
 
 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

Figure 1 : UV Spectrum of Amoxicillin 

Figure 2 : UV Spectrum of the element Co(II) 



Iraqi J Pharm Sci , Vol.18 (1) , 2009                                   Amoxicillin and cobalt (II) complexation   

 56 

 
 
 
 
 
 
 
 

 

 

 

 

 

 

 

 

 

Figure(2) :UV Spectrum of the element 

Co(II) 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure(3): UV Spectrum of [Amox-Co(II) ] 

Complex 

 
 
Results and Discussion 
         Amoxicillin spectrum, illustrated at 
Figure (1), consist of 2 bands at λmax. (272 nm) 

and (228 nm), we depend on the band at λmax. 

(228 nm) because the other peak will disappear 

in some experiments also it may interfere with 

benzyl alcohol peak. 

 Co (II) Spectrum gives peak at λmax. (211 
nm) using distilled water as a blank, 

(Figure 2). 

 Chelating complex [Amox.-Co (II)], a 
new peak at λmax. (375 nm) as shown in 

Figure (3) which indicate the formation of 

the complex that extracted by benzyl 

alcohol. Table (1) shows the color and 

λmax. for the amoxicillin, Co (II), and the 

complex.  

 

Table (1): Color and λmax for the drug, 

metal, complex. 

 

 

 Detection the optimum conditions for 
complexation using UV-Visible 

Spectrophotometer: 

1. pH effect: Table (2) shows the 
absorbance of the [Amox.-Co (II)] 

complex using different pH (0.1-3), 

the optimum pH for the complexation 

was (1.5) where the absorbance is 

gradually increased from 0.5 to the 

maximum peak at pH 1.5 and then it 

decreases. Figure (4) shows the effect 

of pH on the absorbance of [Amox.-

Co (II)].  

Table (2): Complex absorption values at 

different pH. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure(4): The effect of  pH on the absorbance  

of the [Amox-Co(II) ] Complex 

 

Compound λmax.  

( nm) 

Color 

Amoxicillin 228 

272 

Off- white 

Co(II) 211 Bluish- white 

[Amox.-Co(II)] 375 White 

pH Absorbance 

0.5 0.69 

1.0 0.98 

1.5 1.28 

2 0.84 

2.5 0.7 

3.0 0.4 

 

 

 

 

 

 

 

  

 

  

Figure 3: UV Spectrum of [ Amox-Co(II)] Complex 

Figure 4 - The Effect of PH on the 

Absorbance of the [Amox.-Co(II)] Complex

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

PH

A
b

s
o
r
b

a
n

c
e

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

Figure 1 : UV Spectrum of Amoxicillin 

Figure 2 : UV Spectrum of the element Co(II) 

pH 



Iraqi J Pharm Sci , Vol.18 (1) , 2009                                   Amoxicillin and cobalt (II) complexation   

 56 

2. Temperature effect: the reaction of 
the metal and Amoxicillin proceeded 

slowly, to increase the reaction 

velocity we intend to increase the 

temperature of the reaction from 40 

to100 ˚C, then the complex is 

extracted and measured the 

absorbance using UV 

Spectrophotometer. Table (3) gives 

the absorbance of the complex at 

different temperatures at pH 1.5 and 

Figure (5) shows the λmax for the 

complex is at 70 ˚C. 

 

Table (3): Complex absorption values at 

different temperatures at (pH 1.5). 

 

Temp. 
o
C Absorbance 

40 0.85 

50 0..96 

60 1.11 

70 1.38 

80 1.26 

90 1.1 

100 1.1 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure(5) The effect of temperature on the 

[Amox-co(II) ] formation 

 

3. Reaction time: the complex formation 
increased as well as the absorbance 

when the reaction time is increased, 

before the extraction process takes 

place, we used different times (5-30 

min.), Table (4) and Figure (6) 

showed that 20 min. is the best time 

for the reaction and give maximum 

absorbance for the  [Amox.-Co (II)] at 

(70 ˚C and pH 1.5). 

 

 

 

Table (4): Complex absorbance value at 

different reaction time at 70 
o
C 

Time(min.) Absorbance 

5 0.75 

10 0.99 

15 1.16 

20 1.30 

25 1.22 

30 1.13 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure (6) The effect of reaction time on 

complex formation 

 

4. Suitable extraction solvent: different 
organic solvents were used like; 

methanol, ethanol, chloroform, benzyl 

alcohol, and ethyl acetate to choose 

the proper solvent that dissolves the 

complex, but can not dissolve the 

metal (Cobalt) and amoxicillin as well 

give the highest absorbance for the 

complex. Table (5) shows the 

solubility of the amoxicillin, metal 

and the complex in different organic 

solvents. 

 

Table (5): The solubility of amoxicillin, 

metal and the complex in different solvents. 

Solvent Amoxicillin 
solubility 

Metal 

solubility 

Complex 

solubility 

Methanol + + − 

Ethanol + + − 

Chloroform − − − 

Benzyl 

alcohol 
− − + 

Ethyl 

acetate 
+ − + 

(+): Soluble. 

(−) :Very slightly soluble or insoluble 

Figure 5 - The Effect of Temperature on the 

[Amox.-Co(II)] Formation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 10 20 30 40 50 60 70 80 90 100 110 120

Temperature ( 
o
C )

A
b

s
o

r
b

a
n

c
e

Figure 6 - The Effect of Reaction Time on 

Complex Formation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 5 10 15 20 25 30 35

Time (min.)

A
b

s
o

r
b

a
n

c
e



Iraqi J Pharm Sci , Vol.18 (1) , 2009                                   Amoxicillin and cobalt (II) complexation   

 56 

 

5. Extraction time: the optimum time for 
shaking during the extraction of the 

complex was 6 min. which gives 

maximum absorbance as shown in 

Table (6) and Figure (7). 

 

 

 Table (6): Effect of shaking time on the 

extraction process against the absorbance. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure(7) The effect of the extraction time 

on the absorbance of the complex 

 

6. Solvent phase ratio: it was found that 
4 ml of aqueous phase and 4 ml of 

organic phase is enough to give 

maximum absorption for the complex 

as shown in Figure (8). Table (7) 

showed that the increase in the 

aqueous phase to 6 ml with 4 ml of 

organic phase will decrease the 

absorbance of the complex.  

 

 

 

 

 

 

 

 

Table (7): Absorbance value of the [Amox.-

Co(II)] as the volume of water phase 

increased.  

Note: organic phase volume = 4 ml. 

 

Water phase 

volume (ml.) 

Absorbance 

1 0.8 

2 1.1 

3 1.32 

4 1.41 

5 1.33 

6 1.06 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure(8): The effect of water volume on the 

absorbance of the [Amox.-Co(II)] Complex 

 

 

7. Times of extraction: the first 
extraction process is enough to extract 

the major concentration of the 

complex because the second 

extraction process for the complex 

that remained in aqueous phase gives 

a very weak absorbance less than 0.1.  

 

Calculation of the ligand (Amoxicillin) to 

metal (Cobalt) ratio in the complex [Amox.-

Co (II)]: 

Molar ratio: to detect the ratio of 

complexation i.e. the molar ratio of Ligand (L) 

to the Metal (M) by taking different volumes 

of the Ligand (VL) with constant volume of 

Metal (Vm) at the same concentration for each 

(1.2 X 10 
-3

 M) at the optimum conditions for 

complexation, then drawing the relation 

between absorbance and VL/Vm as in Figure 

(9), and Table (8) shows the absorbance 

against VL/ Vm. The molar ratio about (2:1) for 

the (L: M) and the suggested chemical 

structure for the [Amox.- Co(II)] complex is:  

 

 

Extraction time 

(min) 

Absorbance 

2 0.8 

3 1.08 

4 1.27 

5 1.33 

6 1.44 

7 1.44 

8 1.43 

Figure 7 - The Effect of the Extraction Time 

on the Absorbance of the Complex

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 2 4 6 8 10
Time

A
b

s
o

r
b

a
n

c
e

Figure 8 - The effect of Water Volume on the 

Absorbance of the [Amox.-Co(II)] Complex 

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 1 2 3 4 5 6 7

Vwate r (ml)

A
b

s
o

r
b

a
n

c
e



Iraqi J Pharm Sci , Vol.18 (1) , 2009                                   Amoxicillin and cobalt (II) complexation   

 67 

HO

H NH2
H H

N
O

N

O
C

S

CH3

CH3

C

O

S

N
HC

H3C

O

N

H2N
H

H3C

O

H H

C

O

O

Co

O

H

H

++

.2Cl
-

H

OH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                        

                            

                            pH = 1.5 

Co(II)+2Amox.                      Co(II) –  2Amox. 

 

Imran
 (3)

 and his partners defined the 

Amoxicillin complex form with transition 

elements in M:L is 1:2 ratio, and this was 

identical with proportion of metal with the 

ligand (drug) that we have reached by molar 

ratio. 

Table (8): The values of the absorbance of 

the complex against VL/Vm 

 

VL/Vm Molar 

ratio 

Absorbance 

1/2 0.5 0.3 

2/2 1 0.8 

3/2 1.5 1.24 

4/2 2 1.5 

5/2 2.5 1.75 

6/2 3 1.75 

8/2 4 1.74 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure(9) : Detection the molar ratio for the 

complex formation 

Calibration curve of the UV spectrum: 

         It was carried out through taking 

different concentrations for the complex (ppm) 

and measuring the absorbance, as shown in 

Table (9). Figure (10) shows the calibration 

curve of [Amox.-Co(II)] complex that obeys 

Beer's law for the concentrations (50-500 µg. 

ml 
-
)  at 375 nm. 

 

Table (9): The value of maximum 

absorbance for the [Amox.-Co(II)] at 

different concentrations. 

 

Conc. ppm Absorbance 

50 0.6 

100 0.75 

150 0.98 

200 1.2 

300 1.6 

400 2.0 

500 2.49 

600 2.55 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure(10): The calibration curve for 

detecting  of [Amox.-Co(II)] at 375 nm 

 

Determination of the drug concentration in 

different pharmaceutical preparations: 

         This was done by taking an average 

weight of one capsule from six capsules that 

have been mixed previously then the 

absorbance of the active ingredient 

(Amoxicillin) was measured from calibration 

curve (Figure 10), after complexation process 

takes place at the optimum condition 

mentioned above. 

Note: we carried out the same procedure for 

500 mg. amoxicillin capsules of different 

trademarks (SDI, Julphar and Ajanta). Table 

(10) shows the results of these preparations. 

0 

0.5 

1 

1.5 

2 

0 1 2 3 4 5 
V L /V m 

A
b

so
r
b

a
n

c
e

 

Figure 10 - The Calibration Curve for 

Detecting of [Amox.-Co(II)] at 375 nm 

0

0.5

1

1.5

2

2.5

3

0 100 200 300 400 500 600 700

Conc. (ppm)

A
b

s
o

r
b

a
n

c
e



Iraqi J Pharm Sci , Vol.18 (1) , 2009                                   Amoxicillin and cobalt (II) complexation   

 67 

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 5 10 15 20 25

Conc. (ppm)

A
b

s
o
r
b

a
n

c
e

Table (10): The concentration of amoxicillin 

in 500 mg amoxicillin capsule of different 

trademarks. 

 

Capsule Absorbance Conc. ppm 

SDI 2.5 501 

JulpHar 2.48 499 

Ajanta 2.42 487 

 

Determination of the [Amox.-Co(II)] by  

Flame Atomic Absorption Spectrophotometer 

(FAAS): 

         The complex was prepared under the 

optimum conditions of pH, temperature, 

proper solvent etc.. and we used the FAAS  to 

detect the amoxicillin concentration by indirect 

measurement the absorbance of the Co(II) in 

the complex as shown in Figure (11), also we 

can measure the concentration of the 

amoxicillin in these pharmaceutical 

preparations using the calibration curve of  

indirect (FAAS). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  Figure(11): The calibration curve for 

detecting  amoxicillin using FAAS 

 

Conclusion 
         The developments of new analytical 

methods to determine the amount of 

amoxicillin, such methods are UV-Visible and 

flame atomic absorption spectrophotometrics, 

are very sensitive and precise. Amoxicillin 

forms chelated complex with Cobalt ions at 

70
o
C and pH: 1.5 and the molar ratio for 

complex formation, Amoxicillin:Cobalt, is 

(1:2). The results of analysis of amoxicillin 

capsules of different trademarks show the 

amount of amoxicillin in SDI and Julphar 

capsules are almost as presented by the 

package, on the other hand the Ajanta capsules 

show some differences.   

  

References 
 

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Encyclopedia of chemicals, drugs and 

Biologicals, 14
th

 edition, Merck Research 

Laboratories, USA, 2006, 92. 

2. Sean C Sweetman, Martindale; The 
Complete Drug Reference, 33

rd
 edition, 

The Pharmaceutical press, UK, 2002, 149. 

3. M. Imran, J. Iqbal and T. Mehmood, 
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Screening of Amoxicillin and its 

complexes with Ag(I), Zn(II), Cu(II) 

,Co(II), Ni(II)” , J.Bio. Sci., 2006, 5, 946 

– 949. 

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5. L. Denis, C. Frances and T. Frenque, 
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Detection”,  J. Pharm. Biomed. Anal., 

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196. 

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spectrophotometric determination of 

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Pharmaceutical Samples Using Cyanex 

923”, Analytical Sciences, 2004, 20(2), 

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8.  M. A. Zayed and S.M. Abdallah, 
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A.Molecular and Biomolecular 

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