Assay Methods Based on Liquid-Liquid Extraction

ASSAY METHODS BASED ON LIQUID-LIQUID EXTRACTION

A number of specific elements may be determined quantitatively based on liquid-liquid extraction method or ‘solvent-extraction’ technique, namely :

(a) Determination of copper (I) as the neo-cuproin complex,

(b) Determination of Iron (III) as the 8-hydroxyquinoline complex or Iron (III) oxinate,

(c) Determination of lead (I) by the dithizone method,

(d) Determination of molybdenum (VI) by the thiocyanate method, (e) Determination of Ni (II) :

(i) as dimethylglyoxime complex, and

(ii) by synergistic extraction.

All these assay methods shall be discussed in the following sections :

1. DETERMINATION OF COPPER (I) AS THE NEO-CUPROIN COMPLEX

Theory

‘Neo-cuproin’ (i.e., 2, 9-dimethyl-1 : 10-phenathroline) under specific experimental parameters almost behaves as a critical reagent for copper (I). The resulting complex is freely soluble in chloroform and absorbs at 457 nm.

Materials Required : hydroxyammonium chloride solution (10% w/v) : 25 ml ; sodium citrate solution (30% w/v) : 50 ml ; ammonia solution ; ‘neo-cuproin’ solution (0.1% w/v in absolute ethanol) : 50 ml ; chloroform ;

Procedure : The following steps may be adopted :

·              Transfer 10.0 ml of the sample solution (containing upto 200 mcg of copper) in a separatory funnel, add 5 ml of hydroxyammonium chloride solution to affect the reduction of Cu (II) to Cu (I),

·              To the resulting solution add 10 ml of solution citrate solution to enable complexation of any other metals that may be present,

·              Add ammonia solution gradually until the pH is about 4.0 (use Congo Red) followed by 10 ml ‘neo-cuproin’ solution,

·              Shake for about 30 seconds with 10 ml of chloroform and allow the layers to separate,

·              Repeat the extraction with a further 5 ml of chloroform, and

·              Finally, measure the absorbance at 457 nm against a blank on the reagents that have been used identically to the sample.

2. DETERMINATION OF IRON (III) AS THE 8-HYDROXY QUINOLATE COMPLEX [IRON (III) OXINATE]

Theory : Iron (III) upto an extent of 50-200 mcg can be extracted effectively from an aqueous solution with a 1% solution of 8-hydroxyquinoline (symbolized as HQ) in chloroform by carrying out a double extraction when the pH of the resulting aqueous solution ranges between 2 and 10. Evidently, between pH 2.0 to 2.5 metals like Ni, Co, Ce (III) and Al do not interfere at all. However, iron (III) oxinate is dark-coloured in chloroform and absorbs at 470 nm.

The reaction may be expressed as follows :

Materials Required : Hydrated ammonium iron (III) sulphate : 0.0266 g ; oxine solution (‘AnalaR’-Grade, 1% w/v in chloroform) : 50 ml ; chloroform ; 100 ml ;

Procedure : The following steps may be followed :

·              Weigh accurately 0.0226 g of hydrated ammonium iron (III) sulphate and dissolve it in 1 litre of DW in a volumetric flask ; 50 ml of this solution contains 100 mcg of iron,

·              Place 50 ml of the solution (≡ 100 mcg of Fe) in a 100-ml separatory funnel, and add to it 10 ml of 1% oxine solution, and shake for 1 minute,

·              Separate the chloroform layer,

·              Transfer a portion of the chloroform layer to a 1 cm absorption cell and determine the absorbance at 470 nm in a UV-spectrophotometer, employing the solvent (chloroform) as a blank or reference, and

·              Repeat the extraction with a further 10 ml quantity of 1% oxine solution, and measure the absorb-ance again so as to confirm whether all the iron was extracted or not. Usually three extractions suffice the complete extraction of Fe (III).

Note : From a glimpse of typical analytical results it may be seen that absorbance after first extraction 0.0592 ; after second extraction 0.0050 ; after third extraction 0.0010 ;

3. DETERMINATION OF LEAD (I) BY THE DITHIZONE METHOD

Theory : In solution, dithizone (diphenylthiocarbazone) exhibits tautomerism as shown below :

The enol-form of Dithizone (I) behaves as monoprotic acid having a dissociation constant pKa = 4.7 upto a pH range of about 12 : obviously, the acid proton is inherited due to the thiol moiety in (I). In reality, two kinds of ‘metal dithizonates’ are invariably formed, namely :

(a)       ‘Primary’ metal dithizonates : These are produced as per the following reaction :

They are of greater analytical value because of their high stability and greater solubility in organic solvents.

(b) ‘Secondary’ metal dithizonates : These are specifically formed by some metals, such as : Cu, Ag, Au, Hg, Bi and Pd. The second complex are produced under the following two conditions, namely :

(i) deficiency of the reagent, and

(ii) higher pH range,

and may be expressed as follows :

It is, however, pertinent to mention here that dithizone* is an extremely sensitive reagent and, there-fore, helps in the determination of lead either from a neutral or faintly alkaline medium to the extent of a few micrograms.

Materials Required : Pure lead nitrate : 0.0079 g ; ammonia-cyanide-sulphite mixture (dilute 35 ml of conc. ammonia solution having sp. gr. 0.88 and 3 ml of 10% w/v solution potassium cyanide (Caution : deadly poisonous, use protective gloves while handling) to 100 ml, and then dissolving 0.15 g of sodium sulphite in this solution) : 75 ml ; dithizone (pure) solution (0.005% w/v in chloroform)** : 7.5 ml ; chloroform : 17.5 ml ;

Procedure : Dissolve 0.0079 g of pure lead nitrate in 1 litre of DW in a volumetric flask. To 10 ml of this solution (equivalent to about 50 mcg of Pb) contained in a 250-ml separatory funnel, add 775 ml of ammonia-cyanide-mixture, and adjust the pH of the resulting solution to pH 9.5 by the careful addition of HCl. Now, add 7.5 ml of dithizone solution and 17.5 ml of chloroform rapidly. Shake the contents of the separatory funnel thoroughly for 1 minute, and allow the phases to separate. Determine the absorbance at 510 nm vis-a-vis a blank solution in a 1.0 cm absorption cell. However, a further extraction of the same solution yields zero absorption thereby indicating that complete extraction of lead has taken place.

4. DETERMINATION OF MOLYBDENUM (VI) BY THE THIOCYANATE METHOD

Theory : Molybdenum (VI) is mostly converted to molybdenum (V) when an acidic solution of the former is treated with tin (II) chloride preferably in the presence a little Fe²⁺ ion. The resulting molybdenum (V) form a red complex with thiocyanate ion as follows :

Consequently, the red-complex is extracted with either solvents possessing donor oxygen atoms, such as : 3-methyl butanol. However, Mo (VI) may also be extracted with diethyl ether-an oxygenated solvent, because it yields the maximum percentage extractive with 7.0 M NH₄ SCN as could be seen from the follow-ing Table 27.2.

a-3 : 2 volume ratio of organic to aqueous phase

b-1 : 1 volume ratio of organic to aqueous phase

c-4 : 1 volume ratio of organic to aqueous phase

The molybdenum complex exhibits maximum absorption at 465 nm.

Materials Required :

(i) Standard Molybdenum Solution : Dissolve 0.184 g of ammonium molybdate (NH₄)₆ [Mo₇O₂₄] 4H₂O in 1 litre of distilled water in a volumetric flask : this yields a 0.01% solution which can be diluted to 0.001% with 0.1 M HCl, thereby giving a Mo solution containing 100 mcg ml^–1,

(ii) Ammonium Iron (II) Sulphate Solution : Dissolve 10 g of the salt in 100 ml of very dilute sulphuric acid,

(iii) Tin (II) Chloride Solution : Dissolve 10 g of Tin (II) chloride dihydrate in 100 ml of 1 M HCl, and

(iv) Potassium Thiocyanate Solution : Prepare a 10% w/v aqueous solution from the pure salt (‘AnalaR’-Grade).

Procedure : The various steps involved are as follows :

1)          First of all construct a calibration curve by transferring accurately 1.0, 2.0, 3.0, 4.0 and 5.0 ml of the 0.001% Mo solution (i.e., containing 10, 20, 30, 40, and 50 mcg Mo respectively) in individual 50-ml separatory funnels and diluting each of them with an equal volume of water.

2)          Add to each funnel 2 ml of conc. HCl, 1 ml of ammonia iron (II) sulphate solution, and 3 ml of the potassium thiocyanate solution,

3)          Shake gently and then induce 3ml of the tin (II) chloride solution,

4)          Add water to bring the total volume in each separatory funnel to 25 ml and mix thoroughly,

5)          Introduce exactly 10 ml of redistilled 3-methyl butanol into each funnel and shake them separately for 30 seconds,

6)          Allow the two phases to separate completely and carefully drain out the lower aqueous layer,

7)          Remove the glass-stopper and pour the alcoholic extract through a small plug of purified glass wool in a small funnel and transfer the organic extract to a 1 cm absorption cell,

8)          Measure the absorbance at 465 nm in a UV spectrophotometer against a 3-methyl butanol blank,

9)          Plot the graph by taking absorbance against concentration of Mo in Mcg, thereby obtaining a straight line spreading over a range 0-50 mcg of Mo (obeying Beer’s Law), and

10)     Finally, determine the concentration of Mo in unknown samples provided and containing less than 50 mcg Mo per 10 ml ; make use of the calibration curve, and subject the unknown samples to the same treatment as the standard solutions.

5. DETERMINATIONS OF NICKEL (II)

A. As Dimethylglyoxime Complex

Theory : In ammoniacal solution, Ni (II) forms an insoluble red coordination compound with dimethylglyoxime (C₄H₈O₂N₃). Nickel dimethylglyoximate is only sparingly soluble in chloroform (35-50 mcg Ni ml^–1). It is, however, necessary to know the approximate amount of Ni present in the sample, so as to avoid adding a large excess of dimethylglyoxime, which is not very soluble in water and may precipitate easily along with the nickel-complex. The optimum pH range at which the extraction of this complex should be carried out ranges between 7-12 in the presence of citrate. It has been observed that the nickel-complex is quite bulky in nature when first precipitated and hence, shows a tendency to move up along the walls of the container. Therefore, care should be taken that the sample must not contain more than 50 mg of Ni. Lastly, the nickel complex absorbs at 366 nm and also at 465-470 nm.

The formation of nickel dimethylglyoximate complex may be expressed as follows :

Materials Required : Ammonium nickel sulphate (pure) : 0.135 g ; citric acid : 5.0 g ; dilute ammo-nia solution ; dimethylglyoxime solution (dissolve 0.50 g of dimethyl-glyoxime in 250 ml of ammonia solu-tion and diluting to 500 ml with water) : 20 ml ; chloroform : 50 ml ; Procedure

·              Weigh accurately 0.135 g of pure ammonium nickel sulphate (NiSO₄, (NH₄)₂ SO₄, 6H₂O) and dissolve in 1 litre of distilled water in a volumetric flask,

·              Transfer 10 ml of the resulting solution (Ni ~– 100 mcg) into a breaker containing 90 ml of water,

·              Add to it 5 g of citric acid, and then dilute ammonia solution carefully until the pH is 7.5,

·              Cool and transfer to a separatory funnel, add 20 ml of dimethylglyoxime solution and, after stand-ing for a minute 12 ml of chloroform,

·              Shake the contents of the funnel for 1 minute, permit the two phases to separate out completely,

·              Collect the lower red chloroform layer and determine the absorbance at 366 nm in a 1 cm absorp-tion cell against a blank, and

·              Extract once again with a 12 ml portion of chloroform and measure its absorbance at 366 nm ; usually very negligible Ni (II) may be found.

B. Synergistic Extraction

Theory : Dithizone and 1, 10-phenanthroline help in the synergistic extraction of Ni (II) both quantitatively and rapidly over a wide range of pH between 5.5 to 11.0 to give rise to a dark coloured mixed-ligand complex that absorbs at 520 nm. The resulting complex bears the following vital characteristic features, namely :

(i) It is fairly stable to allow the complete removal of excess dithizone by back-titration with 0.1 M NaOH, so as to make a ‘monocolour’ method feasible,

(ii) The molar absorptivity of the complex stands at 4.91 × 10 ⁴ mol^–1 L cm^–1, and

(iii) The synergistic method is predominantly much more sensitive as compared to any other method for the determination of Ni (II).

Materials Required : Ammonium nickel sulphate* (pure) : 0.0135 g ; phthalate or acetate (ethanoate)

buffer (pH 6.0) : 5 ml ; dilute ammonia solution ; chloroform : 15 ml ; sodium hydroxide (0.1 M) : 10.0 ml ;

Procedure

·              To 5 ml of a solution containing from 1 to 10 mcg of Nickel (II) 5 ml of a phthalate or acetate buffer,

·              In case, the sample is acidic, adjust the pH to 6.0 with dilute ammonia solution carefully,

·              To the resulting solution add 15 ml of chloroform solution of dithizone and 1, 10-phenanthroline,

·              Moderately shake the two phases for 5 minutes in a separatory funnel, allow them to separate distinctly into aqueous and chloroform (lower) layers,

·              Excess dithizone may be removed from the chloroform layer by back-extraction with 10 ml of 0.1 M NaOH, (a through shaking for 60 seconds will suffice this extraction),

·              Once again separate the chloroform layer (lower) and measure its absorbance in a 1 cm absorption cell at 520 nm Vs an identically treated blank, and

·              Finally, draw a calibration curve using standard Ni (II) solution containing 2, 4, 6, 8, and 10 mcg in 10 ml (obeying Beer’s Law).

Caution : All glassware must be rinsed with dilute acid and then thoroughly with distilled water.