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Spectrophotometric determination of iminodiacetic acid in presence of primary amino-acids
Summary-Alkgl-l~biguanides are determined by titration with copper(H), or excess of copper(H) is added and the surplus determined by titration with EDTA, a liquid-state copper-sensitive electrode being used in both cs~ses far potentiometric detection of the end-point.
~~5photume~~c and &her meth5ds-3 have been reportd for the ~t~~~~t~on of amino-a&s. None of &em is. however, appliible to ~rn~n~~a~e~~ acidid,IDA, in presence of gIycine and other primary amino-acids. The method proposed here is based on complex formation between IDA and ferric ions. Clycine also complexes ferric ion, but its interference can be eliminated by proper choice of pH and wavelength. Other primary amino-acids do not interfere. Several
EXPERlMEWTAC
Reagenfs
mixing the requisite amounts of Ferric afum and IDA, precipitated with alcohol and then recrystallized twice from doubly distilled water. PR?&&Wt? To an nliquot of an unknown IDA solution, add a measured amount of ferric alum solution and dilute the solution to give a ferric alum concentration of -2%3X) x 10s4M, and an IDA concentration of _ l-9 x 10VSM at pH 3.c3.5. Measure the absorbance at 250 and 300 nm. The concentrations of iron and IDA are chosen to give absorbances in the range 0%0.7.
Ferric alum was reagent grade- IDA was recrystallized from doubly distilled water. Fe(III jXDA was prepared bv Figure L shows the absorption spectra of ferric alum ~~~~I~~~A and F~III~gly~~ne complexes at pH 3-o-3-5. The ferric alum spectrum is characterized by a maximum at 300 nm and an identical absorbance at 2.50 nm. Fe(W)glycme (1 :J) also gives a similar, slightly more intense absorption spectrum. The spectrum of the Fe(KII)-IDA (1: 1) complex has no maximum but the absorbance at 250 nm is much higher than that at 300 nm. All three systems obey Beer’s law at the two selected wavelengths. Hence, if 6%and E* are the molar absorptivities of l?e(EII)-IDA at 250 and 300 nm respectively and Azso and AJno are the absorhnces at 250 and 3OOnm,
A,,, - &a
= AA = (+ - E,)@+IDA] = K DDA].
'%US a @ut of ~$4 KS.[IDA]
serves as a ~l~b~~ion found to be T.Gi x IO3 1.mole".",cm-'. & IDA concentration as low as _ IO- ‘M can be determined with a fair degree of accuracy. The pH has an effect in the system, being optimum over the range 34-3~5. At pH greater than 3.5, the excess of iron(III) hydrolyses and at pH less than 3.0, AA for Fe(III)IDA diminishes slightly. The accuracy of the method is further dependent upon the correct selection of the shorter wavelength such that there is automatic correction for the curve. K was
220
1
I
240
260 2t10 300 Wawelbngth, nm
1
I
320
:
Fig. 1. Absorption spectra of (I), ferric alum; (II), (1:i) Fe(III)-IDA; and (III), (I ~3) Fe(III)-GCY. pH = 3G3.5; [Fe’3] =: 1 x’ 10”4niI.
(I1
SHORT COMMUNICATIONS
332 Table
1. Effects of primary
amino-acids
Amino-acid added
Molar ratio, amino-acid : IDA
IDA taken, 10-6M
IDA found, 10-6M
Glycine
1:4 1:2 3:4 1:l I:4 1:2 3:4 1:l
80.0 80.0 80.0 80.0 80.0 80.0 80.0 80.0
80.0 80.5 80.6 82.0 81.0 82.0 80.8 81.0
Alanine
Table
2. Effects of various
* 0.0 +O6 +@8 + 2.5 +1.3 + 2.5 + 1.0 + 1.3
metal ions
Ion added
Molar ratio, metal ion : IDA
IDA taken, lo-‘jM
IDA found, 10-6M
Ca(I1) Ca(I1) Ba(I1) Ba(I1) Cu(I1) Cu(I1) Co(I1) Co(I1) Ni(I1) Ni(I1) Cr(II1) Cr(II1)
1:3 1:2 1:3 1:2 1:3 1:2 1:3 1:2 1:3 1:2 1:3 1:2
90.0 80.0 90.0 80.0 90.0 80.0 90.0 80.0 90.0 80.0 90.0 80.0
90.0 82.0 92.0 81.0 92.0 82.0 87.0 82.0 88.0 81.0 91.5 80.5
absorbance of the excess of iron(II1) and of the iron(IIIt glycine complex. This shorter wavelength was found to lie in the range 250 + 2 nm. It should therefore be separately determined for each experimental arrangement. Table 1 shows the effect of primary amino-acids. e.g., glycine and alanine. The amino-acid:IDA mole ratio was varied from 1:4 to 1:l. It is evident from the results that the method is very promising in these cases too. Probably the metal complexes of the primary amino-acids are not stable at pH 3-3.5 or else AA then is negligible. It is sometimes necessary to determine IDA in the presence of metal ions, so the effect of some chosen metal ions was also studied. Metal ion complexes of IDA which are not stable at the pH range used or from which the metal ion can be easily displaced by ferric ion, do not
Summary-Iminodiacetic acid down to lo-‘M of primary amino-acids by complex-formation fairly accurate, rapid and free from interference
Error, %
Error, %
rf:0.0 +25 +2.2 +1.3 + 2.2 + 2.5 -3.6 + 2.5 -2.2 +1.3 +1.7 + 0.6
interfere. Table 2 shows some typical results; it is evident that the metal ions under study do not interfere seriously. It can be claimed that the method is very simple, reproducible and is easily adaptable. Acknowledgement-The authors are grateful A. K. Saha for his kind encouragement.
to Professor
REFERENCES
1. N. Furman, H. Morrison and F. Wagner, Anal. Chem., 1950, 22, 1561. 2. S. Moore and W. H. Stein, J. Biol. Chem., 1948, 176, 367. 3. E. Critchfield and B. Johnson, Ad. Gem., 1956, 28, 436.
is determined spectrophotometrically in the presence with iron(II1) at pH 3.s3.5. The method is simple, from most common metal ions.
~~5photume~~c and &her meth5ds-3 have been reportd for the ~t~~~~t~on of amino-a&s. None of &em is. however, appliible to ~rn~n~~a~e~~ acidid,IDA, in presence of gIycine and other primary amino-acids. The method proposed here is based on complex formation between IDA and ferric ions. Clycine also complexes ferric ion, but its interference can be eliminated by proper choice of pH and wavelength. Other primary amino-acids do not interfere. Several
EXPERlMEWTAC
Reagenfs
mixing the requisite amounts of Ferric afum and IDA, precipitated with alcohol and then recrystallized twice from doubly distilled water. PR?&&Wt? To an nliquot of an unknown IDA solution, add a measured amount of ferric alum solution and dilute the solution to give a ferric alum concentration of -2%3X) x 10s4M, and an IDA concentration of _ l-9 x 10VSM at pH 3.c3.5. Measure the absorbance at 250 and 300 nm. The concentrations of iron and IDA are chosen to give absorbances in the range 0%0.7.
Ferric alum was reagent grade- IDA was recrystallized from doubly distilled water. Fe(III jXDA was prepared bv Figure L shows the absorption spectra of ferric alum ~~~~I~~~A and F~III~gly~~ne complexes at pH 3-o-3-5. The ferric alum spectrum is characterized by a maximum at 300 nm and an identical absorbance at 2.50 nm. Fe(W)glycme (1 :J) also gives a similar, slightly more intense absorption spectrum. The spectrum of the Fe(KII)-IDA (1: 1) complex has no maximum but the absorbance at 250 nm is much higher than that at 300 nm. All three systems obey Beer’s law at the two selected wavelengths. Hence, if 6%and E* are the molar absorptivities of l?e(EII)-IDA at 250 and 300 nm respectively and Azso and AJno are the absorhnces at 250 and 3OOnm,
A,,, - &a
= AA = (+ - E,)@+IDA] = K DDA].
'%US a @ut of ~$4 KS.[IDA]
serves as a ~l~b~~ion found to be T.Gi x IO3 1.mole".",cm-'. & IDA concentration as low as _ IO- ‘M can be determined with a fair degree of accuracy. The pH has an effect in the system, being optimum over the range 34-3~5. At pH greater than 3.5, the excess of iron(III) hydrolyses and at pH less than 3.0, AA for Fe(III)IDA diminishes slightly. The accuracy of the method is further dependent upon the correct selection of the shorter wavelength such that there is automatic correction for the curve. K was
220
1
I
240
260 2t10 300 Wawelbngth, nm
1
I
320
:
Fig. 1. Absorption spectra of (I), ferric alum; (II), (1:i) Fe(III)-IDA; and (III), (I ~3) Fe(III)-GCY. pH = 3G3.5; [Fe’3] =: 1 x’ 10”4niI.
(I1
SHORT COMMUNICATIONS
332 Table
1. Effects of primary
amino-acids
Amino-acid added
Molar ratio, amino-acid : IDA
IDA taken, 10-6M
IDA found, 10-6M
Glycine
1:4 1:2 3:4 1:l I:4 1:2 3:4 1:l
80.0 80.0 80.0 80.0 80.0 80.0 80.0 80.0
80.0 80.5 80.6 82.0 81.0 82.0 80.8 81.0
Alanine
Table
2. Effects of various
* 0.0 +O6 +@8 + 2.5 +1.3 + 2.5 + 1.0 + 1.3
metal ions
Ion added
Molar ratio, metal ion : IDA
IDA taken, lo-‘jM
IDA found, 10-6M
Ca(I1) Ca(I1) Ba(I1) Ba(I1) Cu(I1) Cu(I1) Co(I1) Co(I1) Ni(I1) Ni(I1) Cr(II1) Cr(II1)
1:3 1:2 1:3 1:2 1:3 1:2 1:3 1:2 1:3 1:2 1:3 1:2
90.0 80.0 90.0 80.0 90.0 80.0 90.0 80.0 90.0 80.0 90.0 80.0
90.0 82.0 92.0 81.0 92.0 82.0 87.0 82.0 88.0 81.0 91.5 80.5
absorbance of the excess of iron(II1) and of the iron(IIIt glycine complex. This shorter wavelength was found to lie in the range 250 + 2 nm. It should therefore be separately determined for each experimental arrangement. Table 1 shows the effect of primary amino-acids. e.g., glycine and alanine. The amino-acid:IDA mole ratio was varied from 1:4 to 1:l. It is evident from the results that the method is very promising in these cases too. Probably the metal complexes of the primary amino-acids are not stable at pH 3-3.5 or else AA then is negligible. It is sometimes necessary to determine IDA in the presence of metal ions, so the effect of some chosen metal ions was also studied. Metal ion complexes of IDA which are not stable at the pH range used or from which the metal ion can be easily displaced by ferric ion, do not
Summary-Iminodiacetic acid down to lo-‘M of primary amino-acids by complex-formation fairly accurate, rapid and free from interference
Error, %
Error, %
rf:0.0 +25 +2.2 +1.3 + 2.2 + 2.5 -3.6 + 2.5 -2.2 +1.3 +1.7 + 0.6
interfere. Table 2 shows some typical results; it is evident that the metal ions under study do not interfere seriously. It can be claimed that the method is very simple, reproducible and is easily adaptable. Acknowledgement-The authors are grateful A. K. Saha for his kind encouragement.
to Professor
REFERENCES
1. N. Furman, H. Morrison and F. Wagner, Anal. Chem., 1950, 22, 1561. 2. S. Moore and W. H. Stein, J. Biol. Chem., 1948, 176, 367. 3. E. Critchfield and B. Johnson, Ad. Gem., 1956, 28, 436.
is determined spectrophotometrically in the presence with iron(II1) at pH 3.s3.5. The method is simple, from most common metal ions.