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.

Spectrophotometric Estimation of Calcium

ESTIMATION OF CALCIUM

INTRODUCTION:

Although direct colorimetry can be performed in several ways, the available methods are very unselective. . To estimate small amounts of Ca (II), it is thus often preferable to use micro volumetric estimations, spectrography, or flame photometry.

With ammonium purpurate. (Muroxide)

Under suitable pH conditions, muroxide gives a yellow –red coloration with Ca (II). The absorption maximum is located at 506 nm the pure reagent shows an absorption maximum at 537 nm

The coloration disappears slowly.

Sensitivity: Molar Extinction co-efficient e=10,000 at 506 nm

Interfering ions:

Mg (II) gives a yellow coloration, but the Mg (II) muroxide compound has, at 500 nm, approximately the same extinction coefficient as the pure reagent. Consequently, Mg (II) does not interfere, unless present in an amount exceeding 10ppm when precipitation takes place..

The majority of the ions interferes and gives colorations or precipitates with the reagent.

Reagents:

Saturated solution of muroxide in water (about 0.5%) to which 2 ½ times its own volume of alcohol has been added. The reagent is stable for one day.

Caustic soda. 0.1N (1/100) or 4 g/l.

Standard solution of Ca (II) at 10ppm, prepared from pure calcium carbonate.

Operating procedure:

            The solution is neutralized if necessary. For the best results, it should contain 30mg of Ca (II). 1ml of reagent, accurately measured by means of a micro burette is added and the volume is made up to about 45ml. After a further addition of 2ml of 0.1N caustic soda, the solution is measured calorimetrically at 506 nm. The calibration curve should be checked daily.

SPECTROPHOTOMETRIC ESTIMATION OF TIN

ESTIMATION OF TIN

INTRODUCTION:

In a certain number of methods, Sn (II) is used to reduce substances, which act as redox indicators. Such methods do not permit the estimation of very slight traces, are not very accurate, and necessitate preliminary separations.

With diol (Toluene- 3,4 – dithiol).

Sn (II) gives a red precipitate, which may be estimated, calorimetrically in the colloidal state. Even small traces may be detected by this method.

Limit of detection:

Under normal conditions 1 to 6 mg or 0.1ppm of of tin can be estimated.

The coloration can be extracted with ethers and alcohols.

Interfering ions. Many ions interfere with the results. Thus Bi (III) gives a brick red precipitate, Cu (II), Ni (II), and Co (II) give black precipitates, Ag (I), Hg (II), Pb (II), Cd (II) and As (III), etc. give yellow precipitates. The NO2^- ion interferes. It is however possible to operate in the presence of 20times more Fe and Pb than Sn. Mn (II) and Zn (II) give rise to low results.

The acidity should be accurately fixed.

Sn (IV) is reduced slowly by the reagent and then undergoes reaction. The reagent is usually stabilized by the addition of thio glycolic acid, which rapidly reduces Sn (IV) to Sn (II).

Reagents:

10ml of glycolic acids are added to 0.25ml of melted dithiol. The solution is diluted to 200ml with alcohol and stored in small-stoppered bottles protected from light.

Gum Arabic in 10% solution.

Procedure:

5ml of the solution, containing less than 50mg of Sn (IV), are neutralized with ammonia and then 0.5ml of concentrated hydrochloric acid are added, followed by 0.5ml of reagent.. The mixture is placed on a boiling water bath for 1 minute and, after cooling, shaken with 2ml of gum Arabic. Colour is developed and measured at 530nm. The color is stable for 1 month in a sealed tube in the dark.

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Spectrophotometric Determination Of Iron

Spectrophotometric Determination Of Iron

INTRODUCTION

The direct-reading, single-beam spectrophotometer. Is most suitable for fast operation, and because of their single beam design they are relatively inexpensive and require a minimum of maintenance. They are particularly well suited for spectrophotometric determinations of a single component at a single wavelength where only moderate accuracy ( ±1 to 3%T) is required.

The limitations of the single-beam instrument become apparent when an absorption spectrum over a wavelength range is required. The response of the phototube, the emissivity of the light source, and the intensity of the light diffracted by the grating are all a function of wavelength.

Accordingly, in order to obtain the absorption spectrum of a compound, the instrument must be recalibrated each time the wavelength setting is changed.

HANDLING OF CUVETTES

The handling of the cuvettes is extremely important. Often two cuvettes are used simultaneously,

one for the "blank" solution and one for the samples to be measured. Yet any variation in the cuvette (such as achange in the cuvette width or curvature of the glass, stains, smudges, or scratches) will cause varying results. Thus, it is essential to follow several rules in dealing with cuvettes:

1. Do not handle the lower portion of a cuvette through which the light beam will pass.

2. Always rinse the cuvette with several portions of the solution before taking a measurement.

3. Wipe off any liquid drops or smudges on the lower half of the cuvette with a clean Kimwipe

(or other lens paper) before placing the the cuvette in the instrument. Never wipe the cuvette

with paper towels or handkerchiefs. Inspect the cuvette to ensure that nor bubbles are

clinging to the inside walls.

4. When inserting a cuvette into the sample holder:

a. To avoid any posible scratching of the cuvette in the optical path, insert the cuvette

withthe index line facing toward the front of the instrument.

b. After the cuvette is seated, line up the index lines exactly.

5. When using two cuvettes simultaneously, use one of the cuvettes always for the blank

solution and the other for the various samples being measured. Mark the cuvette ( on the

upper portion) accordingly and do not interchange the cuvettes during the remainder of the experiment.

6. Do not use a test tube brush to clean the cuvettes after the experiment is complete.

REAGENTS

1,10-phenanthroline (0.1 g of 1,10-phenanthroline monohydrate in 100 mL of distilled

water, warming to effect solution if necessary).

Hydroxylamine hydrochloride (10 g in 100 mL of distilled water).

Sodium acetate (10 g in 100 mL of distilled water).

Ferrous ammonium sulfate hexahydrate.

PART A -- Determination of Spectral Response of a Single-Beam Spectrophotometer

The purpose of this section is to familiarize you with the single beam spectro photometer operation and to provide a justification for why you must reset your instrument response each time a newwavelength has been chosen

PROCEDURE

warm-up the instrument before analysis ,read instrument manual for warm up time, latest instruments required less time for warm up before use. First, adjust the

instrument, photocell dark, with the zero control. Next, insert a cuvette filled with water into the sample holder. Take blank reading absorbance or .transmittance. Without changing either the dark current control or the 100%T control, vary the

wavelength from 340 to 900 nm in 4 nm intervals and record the instrument response. Plot the instrument response vs. wavelength.

PART B -- Determination of Iron with 1,10-phenanthroline

INTRODUCTION

The reaction between ferrous ion and 1,10-phenanthroline to form a red complex serves as a sensitive method for determining iron.

Fe2+ + 3 1,10-phenanthroline ® Fe(phen)3 2+

1,10 – phenanthrolene (Phen)

The molar absorptivity of the complex, [(C12H8N2)3Fe]2+, is 11,100 at 508 nm. The intensity of the color is independent of pH in the range 2 to 9. The complex is very stable and the color intensity does not change appreciably over long periods of time. Beer's law is obeyed.

The iron must be in the ferrous state, and hence a reducing agent is added before the color is developed. Hydroxylamine, as its hydrochloride, can be used to reduce any ferric ion that is present:

2 Fe3+ + 2 NH2OH + 2 OH- ® 2 Fe2+ + N2 + 4 H2O

The pH is adjusted to a value between 6 and 9 by addition of ammonia or sodium acetate. PROCEDURE

1. Weigh accurately about 0.07 g of pure ferrous ammonium sulfate hexahydrate, dissolve it in water, and transfer the solution to a 1-liter volumetric flask. Add 2.5 mL of concentrated sulfuric acid and dilute the solution to the mark. Calculate the concentration of the solution in mg of iron per mL. (Remember, your solution was prepared using Fe(NH4)2(SO4)2·6H2O).

2. Prepare the unknown sample as follows. Add about 0.12 g of the solid unknown and

approximately 0.25 mL concentrated sulfuric acid into a 100 mL volumetric flask and dilute to the mark. Now transfer a 1 mL aliquot of this solution to another 100 mL volumetric flask do not dilute yet. This will be referred to as the "prepared unknown".

3. Into five 100 mL volumetric flasks, pipet (volumetrically) 1, 5, 10, 25, and 50 mL portions of the standard iron solution. Put 50 mL of distilled water into another flask to serve as the blank. To each flask, including the "prepared unknown", add 1 mL of the hydroxylamine solution, 10 mL of the 1,10 phenanthroline solution and 8 mL of the sodium acetate solution. Then dilute all the solutions to the 100 mL marks and allow them to stand for 10 minutes with occasional shaking of the flasks.

4. Using the blank as a reference and any one of the iron solutions prepared above, measure the

absorbance at different wavelengths in the interval from 340 to 900 nm . (Note that it is

necessary to re-adjust the 100%T setting whenever the wavelength is changed). Take reading  about 10 nm apart except in the region of maximum absorbance where intervals of 5 nm should be used. Plot the absorbance vs. wavelength and connect the points to from a smooth curve. Select the proper wavelength to use for the determination of iron with 1,10- phenanthroline.

5. Also, calculate the molar absorption coefficient, e, at the maximum absorption on the

absorption curve (assume b = 1 cm).

6. Measure the absorbance of each of the standard solutions and the unknown at the selected

wavelength. Plot the absorbance vs. the concentration of the standards. Note whether Beer's law is obeyed. Using the absorbance of the unknown solution calculate the % (w/w) iron in your original solid sample, remember to correct for dilutions.

NORMALITY

"Normality" refers to the activity of a reagent: how many moles of active species are there per liter? For Brønsted acids and bases, normality refers to how many moles of H⁺ or OH^- there are per liter.

Thus, for hydrochloric acid (HCl) and sodium hydroxide (NaOH) the normality is equal to the molarity. But for dibasic substances like sulfuric acid (H₂SO₄) or barium hydroxide Ba(OH)₂, the normality is twice the molarity. For a tribasic substance, normality would be three times the molarity and so forth.

Preparation of 0.005M sulfuric Acid :

Normality =molarity x Basicity

for sulphuric acid 1 molar = 2 Normal

18 Molar = 36N

N        = 18 x 2 =36

 = 36N

18 Molar = 36N

 1000 x 0.1 N  =     100      = 2.777 ml = 2.8ml 

        36N                 36 

For 0.1N--------------2.8ml of sulfuric Acid is required(in 1000ml) =0.05molar

0.01N =0.005molar

        2.8 ml  of sulphuric acid / lit---------------    = 0.05m

        0.28 ml of sulphuric acid / lit--------------- = 0.005m

      0.028ml(0.03ml) of sulfuric acid for 100ml =0.005m

For standard 1000ppm K₂Cr₂O_7:

Take 1 gm of K₂Cr₂O₇ in 1000ml of standard vol flask and make up with only distill water

Note: For dilutions only, we are adding 0.005M Sulphuric acid, for standard 1000ppm we are not adding 0.005M Sulphuric acid

Preparation of 60ppm of K₂Cr₂O₇ :

0.06 gm in 1000ml is 60ppm

0.06gm---------------------1000ml (60ppm)

?-------------------------250ml

250 ´0.0 6  = 0.015gm (60ppm)

   1000

0.015gm in 250 ml std volumetric flask (60ppm) and fill with 0.005M sulfuric Acid

Preparation of 40ppm of K₂Cr₂O₇ :

0.04 gm in 1000ml is 40ppm

0.04gm---------------------1000ml (40ppm)

?-------------------------250ml

250 ´0.0 4  = 0.01gm (40ppm)

   1000

0.01gm in 250 ml std volumetric flask (40ppm) and fill with 0.005M sulfuric Acid

Preparation of 20ppm of K₂Cr₂O₇ :

0.02 gm in 1000ml is 20ppm

0.02gm---------------------1000ml (20ppm)

?-------------------------250ml

250 ´ 0.02  = 0.005 (20ppm)

   1000

0.005gm in 250 ml std volumetric flask (20ppm) and fill with 0.005M sulfuric Acid

Dilution preperation:

Available ppm(1000ppm)   X        x(vol to be transferred) = (60ppm) req ppm X(100ml)req vol

                                                              x= 60X 100     =  6000     = 6ml

1000                           1000

Take 6ml of 1000ppm in 100ml of std vol flask = 60 ppm and add 0.03ml H2S04

Preparation of 6.9 buffer:

Preparation of 6.9 buffer:

Weigh 0.85 gm of KH₂PO₄  and  0.88725 gm of Na₂HPO₄  and dissolve it in 250 ml of std volumetric flask and make up to the mark with distill water.

(or)

Weigh 3.4 gm of KH₂PO₄  and  3.549 gm of Na₂HPO₄  and dissolve it in 1000 ml of std volumetric flask and make up to the mark with distill water.

(or)

Weigh 1.7 gm of KH₂PO₄  and  1.7745 gm of Na₂HPO₄  and dissolve it in 500 ml of std volumetric flask and make up to the mark with distill water.

Procedure for making the Copper Sulfate Solution

Procedure for making the Copper Sulfate Solution

1. Add 250 g of Copper Sulfate (CuSO₄ · 5H₂O) to about 700 ml of H₂O (Ideally deionized).
2. While stirring, slowly add 10.0 ml of concentrated sulfuric acid (H₂SO₄)
3. Add H₂O (Ideally deionized) to bring the volume to 1.0 liter.

This will produce a solution of approximately 1.0 Molar copper sulfate with a pH of about 1.0.Help

Determination of pH in precipitation

4.7  Determination of pH in precipitation

4.7.1  Potentiometric method

4.7.1.1  Principle

The method is based on the determination of the potential difference between an electrode pair consisting of a glass electrode sensitive to the difference in the hydrogen ion activity in the sample solution and the internal filling solution, and a reference electrode, which is supposed to have a constant potential independent of the immersing solution. The measured potential difference is compared with the potential obtained when both electrodes are immersed in a solution or buffer with known pH or hydrogen ion concentration. The pH is defined by the formula:
          pH_(sample) = pH_(reference) + (E_(sample) – E_(reference) F/RT1n10
where E are the electrode potentials, R is the universal gas constant, T the absolute temperature and F is the Faraday constant.
This is an operationally defined pH. Buffers of known pH are specified by National Bureau of Standards, now the National Institute of Standardized Technology (NIST). The primary standard and the most widely used buffer for pH-meter calibration is 0.05 M potassium hydrogen phthalate, which has a pH of 4.00 at 20° C, and a hydrogen ion activity of 10^-4 M. This latter hydrogen ion activity is based on theoretical calculations (the Bates-Guggenheim convention).
In precipitation samples, the ionic strength will typically be in the region 10^-3 to 10^-5. The activity coefficient for monovalent cations such as the hydrogen ion will therefore be in the range 0.95-0.99. This corresponds to <0.02 pH-units difference between pH and -log(H⁺). Much more critical is the assumption of a constant reference electrode potential when going from a relatively concentrated potassium hydrogen phthalate solution to extremely dilute precipitations samples. The problem arises because of the inherent possibility of building up a liquid junction potential between the internal solution of the reference electrode, and the sample solution. This liquid junction potential may be larger if the ionic strength difference between the two solutions is large. It is reduced by making the boundary between the concentrated filling solution and the sample as sharp as possible. Various designs of pH cells meeting this criterion have been proposed. Tests of commercial electrodes against dilute acid solutions and low ionic strength buffers with known pH or hydrogen ion concentrations have shown, however, that this problem has largely been overcome with modern pH instrumentation and electrode systems.
However, it is strongly recommended to check the electrode system at regular intervals, by measuring the “apparent pH” of a solution with low ionic strength with known pH or hydrogen ion concentration. The pH readings should be within 0.02 or 0.05 pH-units of the “theoretical” result. If this is not the case, or if the reading is unstable during stirring of the solution, the reference electrode should be replaced. New glass electrodes should be tested against at least two buffers to see that the response is Nernstian.
The reference electrode should preferably be stored in dilute potassium chloride solution (0.1M).

4.7.1.2  Instrumentation

pH-meter with the possibility of reading to the nearest 0.02 pH-units or preferably to the nearest 0.01 pH-unit.
A glass electrode and a reference electrode must be used with the pH-meter. The reference electrode should be suitable for measurement in low-ionic strength solutions and preferably be of the calomel type filled with saturated potassium chloride. Other reference electrodes or combination electrodes may be used, but all electrodes should be checked for acceptable performance.
Magnetic stirrer, with teflon coated stirring bar.
Beakers used for the test solution should be made of borosilicate glass or poly­ethylene.

4.7.1.3  Chemicals

Buffer solutions for the calibration of the pH-meter. Preferably the two buffer solutions given in Section 4.7.1.4, which are recommended as standards by the U.S. National Institute of Standards and Technology (NIST).

4.7.1.4  Reagents

National Bureau of Standards solutions with known pH.

1. 0.05 M potassium hydrogen phthalate (C₆H₄ (COOH) (COOK)
   pH = 4.00 at 20 °C
   pH = 4.01 at 25 °C

Dissolve 10.12 g potassium hydrogen phthalate, C₆H₄ (COOH) (COOK), dried at 120 °C, in 1000 ml distilled water.

2. 0.025 M potassium dihydrogen phosphate (KH₂PO₄) and 0.025 M disodium hydrogen phosphate (Na₂HPO₄)
   pH = 6.88 at 20 °C
   pH = 6.86 at 25 °C

Dissolve 3.39 g potassium dihydrogen phosphate, KH₄PO₄, and 3.53 g disodium hydrogen phosphate, Na₂HPO₄, dried at 120° C, in 1000 ml distil­led water.
Instead of the anhydrous disodium hydrogen phosphate, 4.43 g of undried dihydrate, Na₂HPO₄ · 2 H₂O, may be used.

Commercial available buffer solutions may also be used, but should be checked against the primary standard buffers described above. The buffers should be kept in the dark in well closed bottles of borosilicate or polyethylene.

4.7.1.5  Calibration

Calibrate the pH-meter according to the instruction manual for the instrument using one, or preferably two, buffer solutions. The temperature of the buffer solutions must be known. The calibration should be checked after each set of samples.

4.7.1.6  Analytical procedure

Measure the pH-value of the sample according to the instruction manual for the instrument. The solution may be stirred, but not vigorously. The temperature of the sample solution must be the same as the temperature of the buffer solution used for calibration.
Rinse the electrodes thoroughly with distilled water between each measurement, and wipe off the excess water with a soft paper.
Store the electrodes in 0.1 M KCl-solution or according to the manufacturers recommendations. The reference electrode should not be stored in distilled water!

4.7.1.7  Performance test of the electrode pair

As mentioned in Section 4.7.1.1 the behaviour of the reference electrode is the main source of errors in pH-measurements, especially in low ionic strength solutions. In order to check the performance of the reference electrode, control measurements should be made on solutions of dilute acids or dilute buffers to verify that correct values are obtained for solutions of lower ionic strengths. A solution which should give a pH ~4.00 could be used for the test. A 10^-4M HC1-solution should give a pH of 3.99 ± 0.05.
Electrode pairs should also show minimal differences between measurements made in stirred and unstirred low ionic strength solutions.
Usually the liquid junction between the solution and the saturated KCl-solution in the reference electrode is formed in a porous plut of ceramic fibre. Slow stirring removes the concentrated KCl-solution which slowly runs out through this capillary.
If the stirring is too vigorous, the ionic medium in the plug itself may be diluted. This will increase the liquid junction potential, and should be avoided. The liquid junction potential may also increase if the porous plug is clogged up by impurities.

ANALYTICAL INSTRUMENTATION

Spectrophotometric Determination

                      

Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if the DNA sample is pure without significant contamination from proteins or organic solvents. The ratio of OD₂₆₀/OD₂₈₀ should be determined to assess the purity of the sample. This method is however limited by the quantity of DNA and the purity of the preparation. Accurate analysis of the DNA preparation may be impeded by the presence of impurities in the sample or if the amount of DNA is too little. In the estimation of total genomic DNA, for example, the presence of RNA, sheared DNA etc. could interfere with the accurate estimation of total high molecular weight genomic DNA.

Procedure

1.      Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260nm as well as 280nm.

2.      Add 10 ml of each DNA sample to 900ml TE (Tris-EDTA buffer) and mix well.

3.      Use TE buffer as a blank in the other cuvette of the spectrophotometer.

4.      Note the OD₂₆₀ and OD₂₈₀ values on spectrophotometer.

5.      Calculate the OD₂₆₀/OD₂₈₀ ratio.

Comments:

Ø      A ratio between 1.8-2.0 denotes that the absorption in the UV range is due to nucleic acids.

Ø      A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers.

Ø      A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or phenol. In either case (<1.8 or >2.0) it is advisable to re-precipitate the DNA.

6.      The amount of DNA can be quantified using the formula:

       DNA concentration (mg/ml) =  OD₂₆₀ x 100 (dilution factor) x 50 mg/ml

1000

Spectrophotomteric Conversions for Nucleic Acids:

1 A 260 of ds DNA                   = 50 mg/ml

1 A 260 of ss oligonucleotides = 33 mg/ml

1 A 260 of ss RNA                    = 40 mg/ml

Reference

Hoisington, D. Khairallah, M. and Gonzalez-de-Leon, D. (1994). Laboratory Protocols: CIMMYT Applied Biotechnology Center. Second Edition, Mexico, D.F.: CIMMYT.