HAEMOGLOBIN DETERMINATION

Procedure for the determination of Haemoglobin

Hemoglobin concentration can be measured in venous or capillary blood by colorimetric determination of derivatives of hemoglobin such as cyanmethemoglobin, oxyhemoglobin, or acid hematin. Various automated methods exist that are based on some of these principles. The preferred method, as recommended by the International Committee of Standardization in Hematology (14), involves the conversion of ail hemoglobin derivatives except sulfhemoglobin to cyanmethemoglobin by dilution of blood in a solution containing potassium cyanide and potassium ferricyanide. Absorbance is then measured in a photoelectric colorimeter or spectrophotometer at a wavelength of 540 nm. Reference standards of cyanmethemoglobin that conform to the specifications of the ICSH are commercially available.

Comparable information to that obtained from hemoglobin determinations can be obtained by the measurement of the packed red-cell volume, a simple technique that only requires a micro centrifuge and capillary tubes. However, due to changes in the mean corpuscular hemoglobin concentration that occur in iron deficiency anemia, changes in hemoglobin concentration are more marked than those in the packed cell volume.

Spectrophotometric Estimation of Aluminium at 389nm

Estimation of Aluminium

Introduction:

Provided a suitable pH is employed, a large number of colorimetric agents, among which aluminon may be particularly distinguished, combine with Al (III) to give coloration which are due not only to definite compounds but often also to adsorption compounds (lakes). The corresponding colorimetric determinations are not very precise since the results vary with the time, the grain size, etc. The pH is a very important factor. Many other substances, in particular Fe (III), give analogous coloration and in view of this, they should be separated out or complexed.

Oxinate:

Aluminium oxinate is yellow when dissolved in chloroform, carbon tetrachloride or benzene. The coloration is not very appreciable to the eye, but the adsorption is high in violet and ultraviolet light.

Sensitivity:

Ε=6,700 at 390nm and 80,000 at 260nm

Interfering ions:

Separation by extraction of the oxinate:

Aluminium oxinate can be extracted quantitatively at pH 4.5-5.0 (acetic buffer). In this way, aluminium may be separated from a number of elements, including Be(II), Th(IV) etc.

More specific separation may be obtained by operating in an ammoniacal medium at pH

9 in the presence of tartrate , cyanide and hydrogen peroxide. Separation from the following may thus be achieved : Cu(II), Co(II), Ni(II), Zn(II), Cd(II), Fe(III), Ti(IV), V(V), V(IV), U(VI), Mn(II), Cr(III), Mo(VI), Sn(IV), and Ag(I). Less than 10mg of Zr(IV) or  Nb (V) does not interfere.

Reagents:

Oxine, 2g in100ml of chloroform

Potassium cyanide, 13% in water

Sodium sulphite, 20% in water

Tartaric acid, 10% inn water

Hydrogen peroxide, 10volume

Standard solution of Al(III): 1g of 99% aluminium dissolved in hydrochloric acid.

Operating procedure:

2 ml of tartaric acid and 1ml of hydrogen peroxide are added to 25ml of a weekly acidic solution 10-150µg of Al(III). The solution is allowed to stand for 5min, and 5ml of sulphite are then added. After standing for a further 3 minutes, 10ml of cyanide are added and the solution is heated about 70°C-80°C and then cooled to 25-30°C. Finally, 2g of ammonium nitrate are added, and pH is adjusted to 8.9 ±0.3 with ammonia or Hcl. The solution is then transferred to a separating funnel, 5ml of oxine are added  ,and the funnel is shaken for 2min. The solution is allowed to settle, and the organic phase is collected in a measuring flask. The extraction is repeated three times, the solution being made upto 50ml with chloroform.

Finally , a colorimetric estimation is performed at 389nm in comparison with a blank.

Spectrophotometric Estimation of Bismuth 465nm or at UV region 337nm

Estimation of Bismuth

Introduction:

Iodide Complxes:

The iodine Complxes   of Bismuth (III) are orange colored, and Beers law is obeyed in the presence of an excess of I- ions (concentration of potassium iodide greater than   1%)

The complex is soluble in alcohols, esters, and ketones.

Sensitivity:

The molar coefficient ε ~ 34,000 at 337 nm in water

The coloration is stable for three to four hrs. The acidity should be fixed between the limit 1-2 N H2SO4

Interfering ions:

Oxidizing agents liberate iodine and should be reduced with e.g.: Sulpurous acid. CuI and AgI can be separated by precipitation without Loss of Bi (III), but PbI2 retains Bi (3) and interferences. Large amounts of   Cd (II) consume (2) I- by the formation of complexes .Hg (II) does so to an even greater extent .1000ppm of iron, 100 ppm of pb (II), 20 ppm of Cu (II) and 400 ppm of As, F-. and tartarate ions do not interfere

Pt (IV), Pd (II), Sn (IV) and Sb (III) produce interfering colors, but Sb (III) only interferes above 200 ppm, and the same doubtless applies to Sn (IV) .Sb (III) and Bi (III) can be estimated simultaneously, Cl- and F- weaken the coloration

REAGENTS:

Potassium iodide, 10% in water;

Sulphurous Acid solution, 5% freshly prepared;

Hypo phosphorous acid, 30% in water

Operating Procedure:

The initial solution consists of 10-20ml, containing from 5-50μg of bismuth (III). The acidity should be adjusted to 1-2N H2SO4. Then .0.1ml

Of sulphurous acid, 1ml of hypo phosphorous acid, and 3ml of iodide are added and the volume made up to 25ml. Colorimetry is performed at 465nm, or alternatively in the UV at 337nm

The blank test or the calibration curve should be determined under identical conditions with respect to acidity and the concentration of salts and iodine 

Spectrophotometric Estimation of Molybdenum at 475nm

ESTIMATION OF MOLYBDENUM

Complex thiocyanate  of Mo(V)

The controlled reduction of Mo(VI) in the presence of thiocyanate ions leads to the formation of a complex orange –red Mo(V) thiocyanate. The reduction should  not be too vigorous and the acidity should be fixed at a definite level, since several alternative reductions may occur. Thus for example , Mo(III) may be formed or, in a weakly acidic solution ‘molybdenum blue’ may appear.

Sensitivity:

Interfering Ions:

Colored ions may be interfere if the molybdenum is not first isolated by extraction . large amounts of Cr(III) should be separated in the form of CrO₂Cl₂

Certain other ions give rise to extractable colored thiocyanate complexes; moderate quantities of Fe(III) are reduced and to do interfere. Pt(IV) interferes. Co(II) interfere if its content exceeds half that of the Mo(IV). Cu(II precipitates in the form of cuprous thiocyanate and may be separated in this way. W(IV) should be complexed by citrate or tartrate ions, and Ti(IV) by F ^–

Large amounts of Bi(III), V(V) and P(V) interfere. Re(VII) gives the same reaction. U(VI) interferes

Reagents:

Potassium thiocyanate, 10%

Stannous chloride: 10g of SnCL₂. 2 H₂O are dissolved in 10ml of concentrated HCL and the solution is made up to 100ml with water.

Solutions of ferrous ion:  1 g of Mohr’s salt is dissolved in 100ml of 0.2N( I/80) sulphuric acid.

Isoamyl alcohol.

Standard solution of molybdenum: 750 mg of guaranteed purity MoO₃ are dissolved in a few ml of dilute caustic soda. The solution is made slightly acid with HCl and the volume adjusted to 500ml. The solution id diluted to the point where 1 ml contains 1 µg of Mo(VI).

Operating Procedure:

2.0 ml of concentrated HCL . 1ml of ferrous solution, 3.ml of thiocyanate, and 3ml of stannous chloride are added to 15ml of solution containing from 1 to50µg of molybdenum. The solution is diluted to 25ml and an accurately measured 10ml portion of iso-amyl alcohol is added. After shaking vigorously for 1 minute and allowing to settle , the colorimetry is performed at 475nm

Spectrophotometric Estimation of Gold at 565nm

ESTIMATION OF GOLD

Using Rhodamine B

In the form of AuCl₄^-, Au(III) gives rise to a violet complex  with the cation of rhodamine can be extracted with  benzene and iso- propyl ether . The extracted is governed by the concentration of hydrochloric acid and other chlorides.

Interfering ions:

Sb(V), Tl(III), W (VI), Hg(II) (> 250), Fe(III) (>100 µg),  and  Sn (IV) (10µg) also  give the extractable  coloration. When these metals are present , the gold may be separated by precipitation with hydroxylamine hydrochloride , using  tellurium as an entraining agent .

Reagents:

1.      Hydrochloride acid, 6M 250ml of water are added to 250ml of concentrated hydrochloric acid.

2.      Ammonium chloride , saturated: 150g of ammonium chloride are dissolved in 500ml of water .

3.      Rhodamine B, 0.04% : 200mg of rhodamine B are dissolved in 500ml of water.

4.      Iso propyl ether.

Operating procedure:

2.5ml of 6M hydrochloric acid and 5.0ml of ammonium chloride are added to 5ml of solution containing 10 to 20µg of gold: the volume is made up to 15ml.  5ml of rhodamine B are added, followed by 10ml (accurately measured) of iso propyl ether. The mixture is vigorously shaken 100times, allowed to settle and the colorimetric determination is then performed at 565nm.

Spectrophotometric Estimation of Antimony

ESTIMATION OF ANTIMONY

Using Rhodamine B

In fairly concentrated hydrochloric acid in the presence of Rhodamine B, Sb (V) gives a violet-red compound RH SbCl₆ which can be extracted, in particular, by isopropyl ether and benzene.

 Sensitivity:

Co-efficient of molar extinction co-efficient e»40,000at at 545nm in isopropyl ether.

Interfering ions:

Oxidizing agents such as the nitrate, ion, can destroy the dye.

Au (III), Ti (III), Fe(III), Ga(III) give analogous reactions . W (IV) is precipitated.

Alternatively, Sb (V) first separated with isopropyl ether from 1-2 N HCL. Colorimetrically is then carried out directly in the solvent after the addition of reagent., excess of which remains in aqueous solution. The operating procedure for this method will be described below.

Fe (III), which interferes, may be reduced by hydroxylamine.

In this way it is possible to estimate 2 mg of antimony in the presence of 30mg of iron. Ti (III), As (III), and Au (III) interfere when present in the amounts exceeding 250mg.

Oxidation of Sb (III) to Sb (V)

In general, Sb is initially present in its trivalent form, which may be oxidized by ceric salts.

Oxidation can only take place in sufficiently concentrated hydrochloric acid 6M HCL. The solutions often contain Sb (IV) and since this is only oxidized very slowly under these conditions. The excess of oxidizing agent is subsequently removed by reaction with additions of hydroxylamine.

The compositions of Sb (V) solutions change very rapidly on standing, doubtless to condensation.

Reagents

Sodium Sulphite 1%

Hydrochloric acid:

Ceric Sulphate: 3.3g of anhydrous ceric sulphate are dissolved in 100ml of 0.5M(3/100) sulphuric acid

Hydroxylamine hydrochloride, 1 %;

Washing solution: I g of hydroxylamine hydrochloride in 100ml of M hydrochloric acid;

Rhodamine B:

200mg in 100ml of M hydrochloric acid

Isopropyl ether saturated with acid by shaking with M Hcl

Standard Solution of antimony: A quantity of Sb₂ O₃ of guaranteed purity is weighed out and dissolved in 100ml of 6N (1/2) hydrochloric acid. The solution is made up to 100ml of 6 N (1/2) HCL. The solution is made up to 1000ml. Under these conditions, 10197g of Sb₂O₃ corresponds to 1000mg of antimony per ml. This is diluted with N HCL to obtain 2 mg /ml

Operating procedure:

Oxidation of Sb (III).

10ml of concentrated hydrochloric acid and 2ml of sulphite are added t 10ml of a solution containing 5-50mg of antimony, and the mixture is shaken with 3ml of ceric salt. 10 drops of hydroxylamine solution are then added; the solution is further agitated, and followed to stand for one minute.

Separation of Sb (v)

The solution as prepared above is transferred to a separating funnel with 60ml of water and 5ml of isopropyl ether, and is shaken for 30 seconds. The aqueous phase is separated, 2ml of the washing solution are added to the organic phase, and the funnel shaken for 1-2 seconds the aqueous phase is then drawn off . 2ml of  hydrochloric acid are added to the solvent the funnel is shaken for a further few seconds and the aqueous phase separated

Colorimetry/Spectrophotometry.

2ml of Rhodamine are added to the solvent and shaken for 10 sec. The solvent phase is then transferred into a 25 ml measuring flask, made up to volume with the solvent, and estimated colorimetrically at 550 nm

Spectrophotometric Estimation of Magnesium

ESTIMATION OF MAGNESIUM

INTRODUCTION:

I) Thiazole Yellow:

Thiazole yellow and titan yellow dissolve in water giving a yellow coloration. In the presence of colloidal magnesium hydroxide they are adsorbed, with a pink colouration.

The method possesses the disadvantages associated with all adsorption techniques: coloration is affected by many variables such a time, temperature, concentration, operating procedure, pH etc. The results may also vary with the origin of the dye. The colloid may be stabilized by the addition of starch, hydroxylamine hydrochloride, polyvinyl alcohol, or sodium phosphate. And is then stable for 2 days in the absence of light.

 Sensitivity:

(Molar extinction coefficient) e =1500 at 535nm

The method is not, however, accurate (±2 to 5 %)

Interfering ions:

Interference is obtained from many ions and preliminary separations are often essential. P(V)  interferes above 100ppm, Ca(II) below 500ppm enhances the colour , but can be complexed with mannitol. Many ions (Cu (II), Ni (II) and Al (III), since Mg (OH) 2 adsorbs AlO₂^- , Also Sn(IV), Ag(I), Hg(I), (II), Cd(II), Co(II), Pb(II), Si(IV), Li(I), Fe(III), Zn(II).And La(III) precipitates, sometimes giving colorations by adsorbing the dye. Ti (IV) can be complexed with H₂O₂ to give a colorless solution at pH ³12. Mn (II) oxidizes in air but this may be prevented by adding hydroxylamine hydrochloride which in addition stabilizes the coloration due tp Mg (II). Sb (III), As (III), As (V) prevent the appearance of the coloration to some extent. NH₄⁺ present in large amounts (> 500ppm), interfere because of its buffering effect on the solution. Proteins interfere. C₂O₄^2- has no appreciable effect.

Reagents:

Caustic soda: 10N (400g per litre)

Hydroxylamine hydroxide, 5% in water

Polyvinyl alcohol, 2% in water.

Thiazole Yellow, 0.5% in 50% alcohol, kept in a brown bottle

Thiazole Yellow, 0.01%, 2ml of the preceding solution are mixed with 5ml of 0.5%polyvinyl alcohol and made upto100ml with water.

Operating procedure:

5ml of hydroxylamine hydrochloride, 4ml of polyvinyl alcohol, 5ml of 0.05% thiazole yellow, and 3.5ml of 10N caustic soda are added to 30ml of neutral solution containing 30-200mg of magnesium. The solution is allowed to stand fro 15min at 25±0.5°, and colorimetrically is carried out at 540 nm within the next half hour.

II) By Extraction of oxinate.

Hydrated magnesium oxinate is soluble in 5% butyl cello solve, from which it may be extracted by chloroform. Mg+2 can be extacted by chloroform at pH II ± 0.5 by a chloroform in the presence of n-butyl amine.

Sensitivity:

e =5,600 at 380nm

Interfering ions:

Fluorides and EDTA prevent the extraction. Oxalates, cyanides, sulphates, tartrates and citrates do not interfere.

Concentrations of Oxine and of the n-butyl amine are suck that the alkali metals and the alkali earth metals and Cr, Mo, W, As, Sb, B, Se, Te, Be do not interfere.

Sn (IV) prevent the extraction of Mg+ when present in amounts exceeding 5mg

Moderate amounts of Ti (IV), V (V), U (VI) can be complexed with H₂O₂; Zn (II), Cd(II), Ni(II), Co(II), Fe(III) can be complexed with cyanide .

15mg of Al (III) doesn’t interfere in the presence of triethlylene.

The following interfere and should be separated with oxine before the addition of n-Butyl amine: In (III), Ga (III), Tl (III), Sn (II), Pb (II), Zr (IV), Th (IV), Bi (III), Nb (V), Ta (V), Mn (II), and the rare earth metals.

The operating procedure describes permits Mg⁺² to be determined in the presence of small residues of interfering ions.

Reagents:

Oxine solution, 0.1% in chloroform.

Potassium sodium tartrate, 20%in water

Ammonia solution, M (1/12)

n-butyl amine

Potassium cyanide

Hydrogen peroxide, 30%(110-volume)

Operating procedure:

            To 30ml of solution containing 20 to 200 mg of magnesium, are added 5ml of tartrate and {V (V), Ti (IV), or U (VI) is present}, 2ml of hydrogen peroxide. 1 M ammonia solution is added to give a pH of about 9 and the compound is extracted with 20ml of the oxine solution; the extraction is repeated until the chloroform phase becomes colorless. 0.5 to 1 g of potassium cyanide and 1 ml of n-butyl amine are added to the aqueous solution, and the pH is adjusted to 11.0 ± 0.5 with concentrated ammonia; the mixture is shaken with 50ml of oxine for 1 minute and colorimetrically is performed at 380 nm automatically, the solution may be extracted twice with 20ml of the oxine solution, shaking each time for 30seconds. The extracts are transferred to a 50ml-measuring flask, 2ml of methanol are added, and the volume is made up to 50ml with chloroform.