PRACTICAL 8 MINERALS - CALCIUM,

PHOSPHOROUS AND IRON

Structure
8.1 Introduction
8.2 Calcium: The Macronutrient
8.3 Phosphorous: The Macromineral
8.4 Iron: The Micronutrient

Experiment 1 :Estimation of Calcium in a Given Sample Using EDTATitrimetric Method

Experiment 2 :Determination of Inorganic Phosphorus in given solution, Serum and
Food sample
Experiment 3 :Estimation of the Iron Content in the Given Solution
Experiment 4 : Estimation o f the Haemoglobin Content in Blood by Cyan-
methemoglobin Method

8.1 INTRODUCTION
Minerals are inorganic substances. This means they are not formed by living matter and
contain no carbon. Minerals used in the body, we have already studied, are classified as:
major minerals, also called "macrominerals," and
trace minerals, also referred to as micronutrients.
Major minerals, as we already know, include calcium, phosphorus, magnesium, potassium,
sulfur, chloride and sodium. The trace minerals are iron, zinc, copper, iodine, fluoride,
chromium, selenium, manganese and molybdenum. All tissues and internal fluids of our
body contain varying quantities of minerals. Minerals are constituents of the bones,
teeth, soft tissue, muscle, blood and nerve cells. They are vital to overall mental and
physical well-being.
Although minerals are of vital importance, they make up only about 4% of the body's
weight, all of the trace elements account for only about 0.01 % of total body weight. In
this practical, we shall learn about the methods used to estimate mineral content of food
or any given sample. We shall limit our learning to the study of two macrominerals,
namely calcium and phosphorous and to one vital micronutrient i.e. iron.

After going through this practical and the experiments given herewith, you should be

describe the methods used to estimate the calcium, phosphorous and iron content
of any given sample,
estimate the calcium, phosphorous and iron content of any given sample in the
laboratory, and
estimate the haemoglobin content in blood using the cyanmethaemoglobin method.

8.2 CALCIUM - THE MACRONUTRIENT

Calcium is an important mineral found mainly in bone and teeth. It is widely distributed
in food substances such as milk, cheese, egg yolk, beans, lentils, nuts, figs, cabbage etc.
The total calcium in the body is 25-35 mols (100-170g). About 99% of it is found in
bones. It exists as carbonate or phosphate of calcium. About 0.5% is in soft tissues and
0.1% in ECF. The normal levels of plasma calcium are 9-1 1mgldl.
The calcium in the plasma is of three types:
a ) Ionized calcium
b) Protein bound calcium
c) Complexed calcium 193
Nutritional About 40% oftotal calcium is in ionized form. Albumin is the major protein with which
Biochemistry calcium is bound. All the three forms of calcium in plasma remain in equilibrium with
each other.

Let us next try to understand the principle involved in the estimation of calcium.

EDTA Titrimetric Method for Estimation of Calcium

The amount of calcium in any sample can be estimated by EDTA titrimetric method.
The principle behind this method is described next.

Principle
Calcium at pH I0 in presence of dye eriochrome black T (indicator) is wine red. The
affinity of ethylene diamine tetraecetate (EDTA) for calcium is greater than the affinity
of the indicator for calcium and so the indicator releases its metal to EDTA. When
completely complexed (chelated) with EDTA, the indicator is released from its
combination with calcium. This free indicator gives a blue colour to the solution indicating
that the end point is reached. Magnesium must be present for satisfactory end point and
is added as Mg EDTA. End point sharpness increases with pH but high pH may cause
precipitation of Ca(OH), and cause colour changes of dye. A pH of 10.0 +_ 0.1 is a
satisfactory compromise: A NH,OH - NH,CI buffer is used to maintain this pH. Limit
of 5 minutes for titration minimizes precipitation.

The reaction involved in this estimation is presented herewith:

CaIn + EDTA CaEDTA + In

(Calcium indicator) (colourless)
(red)
So in the estimation ofcalcium, the solution containing calcium is titrated against EDTA
solution. EDTA can form a chelate complex with calcium. It is a six-coordinate Mg-
EDTA complex. A small amount of indicator (In) is added to the Ca2+to form a red
complex as indicated in the reaction above. As EDTA is added, it reacts first with free
colourless Ca" and then with the red CaIn complex. The EDTA binds to Ca2+better
than the indicator binds to Ca2'. The indicator thus releases its metal to EDTA. The
change from the red CaIn to the blue of unbound indicator (In) signals the end point of
titration. It is important to note that the colour of the free indicator is pH dependent.
Hence most indicators can be used only in certain pH range. In order to maintain pH we
therefore use buffers.
A metal ion indicator is a compound which changes its colour on binding t6 metal ion.
The detailed procedure involved in the estimation of calcium is described in Experiment
1 later in this practical. Read the procedure carefully before you start the experiment.
Next, we shall study about phosphorus and the methods employed for its estimation.

8.3 PHOSPHORUS: THE MACROMINERAL

The body contains about 620 g (20 moles) of phosphorus. Of this 87% is present in
bones. It is thus more widely distributed in the body than calcium as part of it is also found
i n soft tissues. Phosphorus is present in nucleic acids and is also present in some proteins,
lipids and enzymes. It is essential in storage and liberation of energy and in the intermediate

1 . Inorganic phosphorus in the f o p of HPO, or H,PO,

2. Organic or ester phosphorus such as glycerophosphate, nucleotide
phosphate etc.
3. Lipid phosphorus such as lecithin, cephalin etc.
194 4. A small amount of residual phosphorus
Phosphate is absorbed from the jejunum with calcium and is deposited in the Minerals -Calcium,
skeleton or mobilized from it in a fixed proportion to calcium. It is excreted by Phosphorus and Iron
kidneys following glomerular filtration and active tubular reabsorption. The latter
process is inhibited by PTH which thereby increases phosphate excretion. Serum
inorganic phosphorus levels are measured in terms of phosphate ions, for ionised
free phosphate does not circulate.
Let us study a little more about the estimation of phosphorus next.

Estimation of Inorganr.. Phosphorus

Most methods estimating inorganic phosphorus use the reaction between the
phosphate and an acid molybdate. The hexavalent molybdenum of the phospho-
molybdic acid formed absorbs light at 340nm and can be reduced to give molybdenum
blue whereas the reducing agent has negligible effect on the uncomplexed molybdic
acid. A variety of reducing agents have.been used in the past and recent years by
different methods and some of them are 1,2,4 amino napthol sulphonic acid (ANSA)
in a mixture of sodium bisulphate and sodium sulphite (Fiske and Subbarow Method)
or p methyl amino sulphate (Gomorri Method), etc.
These mkthods can also be used to assess phosphorus status in the body by
measuring phosphorus excretion or fecal phosphorus. The phosphorus excretion
method uses a suitable dilution, usually 1 to 10 and any of the abave methods for
estimation. Fecal phosphorus includes both organic and inorganic phosphorus and
thus usually estimated as total phosphorus in metabolic studies.
Let us look at the method of estimation of phosphorus. The method used is the
colorimetric method which is described next.

Colorimetric Method for the Estimation of Phosphorous

Estimation of phosphorous is based upon the principle of colorimetry. Many methods
for quantitative analysis are based upom the production of coloured solutions in
such a way that the intensity or depth of colour so obtained may be used as a
measure of concentration of substance being determined. Such use of colour as an
iridex of concentration is the science of colorimetry as you have already studied
earlier in Practical 2 and the instrument used for colour evaluation is the colorimeter.
Let us understand the principle behind colorimetric estimation of phosphorous next.

The proteins in the blood are precipitated with the help of trichloroacetic acid. The
protein free filtrate containing inorganic phosphorus is then treated with molybdic
acid reagent and phosphomolybdic acid is formed. The phosphomolybdic acid is
reduced to blue coloured molybdenum blue or hetropolymolybdenum blue
(NH4)3{Po4(Mo03),,)) by 1 amino 2 napthol4 sulfonic acid (ANSA). The intensity
of the colour formed is directly proportional to the amount of inorganic phosphorus
present. A mild reducing agent like ANSA is employed in order to avoid reduction
of the excess of molybdate present.

8.4 IRON: THE MICRONUTRIENT

Iron, as you may be aware, is a trace element present in the body. The body contains
approximately 3 - 3.5 g of iron of which about two thirds is physiologically active and
remainder is storage iron. Out of the physiological iron, virtually 90% is present as
haemoglobin and about 5% as myoglobin and I % as haem enzymes such as cytochromes.
Iron is a maior costituent of a red-coloured compound haemoglobin present in the blood.
Haemoglobin, we already know, is the complex protein-iron compound in the blood that
carries oxygen to the cells from the lungs and carbon dioxide away from the cells to the
195
lungs. Blood haemoglobin concentration serves as the indicator for anaemia. Nutritional
Nutritional anaemia, as we already know, is defined as a condition in which the haemoglobin
Biochemistry . concentration is below the level that is normal, for a given individual, due to
deficiency ofone or more o f t k nutrients required for haemopoiesis and conversely,
as a condition in which the haemoglobin concentration can be raised by increasing the
amount of nutrient(s) absorbed. Therefore estimation of haemoglobin is an important
tool for assessment of anaemia. In this practical, activity 4 we shall learn about the
process of haemoglobin estimation.
It is estimated that upto half of all anaemia found in population group is caused due to
dietary deficit of iron. Just a little amount required, but iron perform some very signifi-
cant functions in our body. The study of iron, its functions, sources and estimation in
food items or any sample is therefore very fascinating. In this practical, 3 we shall get to
learn about the procedure we can use in the laboratory for estimation of iron. Let us
then get started and learn about the methods and the principle involved in estimation
of iron and haemoglobin.

Methods for Estimation of Iron and Haemoglobin

In this section we shall study the procedure involved in estimation of iron in any sample
first followed by a review on the cyanmethaemoglobin method used for estimation of
haemoglobin.

A. Estimation of Iron in any Solution

Iron can be determined calorimetrically. The principle is described next.
Principle
The iron is determined in the solution or digested sample by converting iron to ferric
form using an oxidizing agent like potassium persulphate or hydrogen peroxide and treating
it with potassium thiocyanate to form red ferric thiocyanate which is measured
colorimetrically at 490 nm.

B. Estimation of haemoglobin in blood

Several methods are used to determine haemoglobin. In these methods hemoglobin is
measured as oxyhaemoglobin, carboxyhaemoglobin,cyanmethaemoglobinand acid and
alkaline haemin by its oxygen carrying capacity and by its iron content. Out of all these
techniques the cyanmethaemoglobin method is now accepted as the standard method
for haemoglobin estimation. It is routinely used for all haemoglobin estimations and gives
reproducible results. The principle behind the cyanmethemoglobin method is described
next.
Principle
The haemoglobin is treated with a reagent containing potassium cyanide and sodium
carbonate. With haemoglobin ferricyanide forms methaemoglobin which is converted to
cyanmethaemoglobin or haemiglobincyanide (HiCN) with cyanide. The absorbance is
measured at 540 nm. The reaction is presented herewith:
Haemoglobin + potassium ferricyanide ------+Methaemoglobin
Mefhaemoglobin + potassium cyanide ------+
Cyanmethaemoglobin (HiCN)
in solution the ferrous ions of the haemoglobin are oxidized to the ferric state by potassium
fericyanide to form methaemoglobin. In turn methaemoglobin reacts with cyanide ions
(CN-) provided by potassl'um cyanide to form HiCN. The time required for full colour
development is shortened by if dihydrogen potassium phosphate is substituted for sodium
bicarbonate in the classic Drabkin's reagent. The colour is stable for more than 24 hours
in well stoppered tubes kept in dark.
An HiCN standard can be used to calibrate the spectrophotometer, The concentration
of HiCN in the sample is calculated from the absorbance in a calibrated spectrophotometer
196 or alternatively with the help of a standard curve.
Minerals -Calcium,
Phosphorus and Iron
 ESTIMATION OF CALCIUM IN A GIVEN SAMPLE

Principle and Reaction

Write the principle and reaction on your own in the space provided herewith.

Apparatus
Burette - 50 ml
Pipettes 5 ml, 10 ml
Conical flasks 100 ml
Volumetric flasks 100 ml
Measuring cylinders
Beakers
Glass marker

Reagents
1. Buffer solution: Dissolve 16.9 g NH4CI in 143 ml NH40H. To this add 50 ml of
solution containing 1.17g Na,EDTA. 2H,O and 0.644 g MgCI,. 6 H,O and dilute to
250 ml with distilled water.
2. Indicator: This contains a mixture of 0.5 g Eriochrome black T and 100 g NaCI. It
is used in a dry form.
3. EDTA solution (0.01 M): Weigh 3.723 g Na, EDTA. 2 H,O and dilute to one liter
with distilled water.
4. Standard calcium solution: Calcium carbonate solution containing 1mg/ml of calcium
carbonate.
5. Distilled water

Procedure
Carry out the experiment following the steps enumerated herewith:
1. ,Tt'tan:lm-d ttirntionPipette 10 ml standard calcium solution into a 250 ml conical
--

flask. To this add 50 ml of distilled water. Add 2 ml buffer solution and 150-200 mg
indicator powder. Titrate with given EDTA solution stirring continuously until the
reddish tinge disappears and the colour becomes blue which is the end point. The
198
 Buret reading (ml)
S.No.
Initial Final Difference
Pilot
1
+ 2
3

r
Buret reading (ml)
S.No.
Initial Final Difference
Pilot
I
2
3
Now submit this experiment for evaluation.

...................................
Counsellor signature
DETERMINATION OF INORGANIC PHOSPHORUS IN
GIVEN SOLUTION, SERUM AND FOOD SAMPLE
Date: ... ......................
Aim: To plot a standard curve for phosphorous and estimate the amount ofphosphorous
in the given solution.

Apparatus and Equipment

15 ml Borosil glass tubes & test tube stand Stickers
Test tube stand
Tissue paper roll
Spectrophotometer/colorimeter
Volumetric flasks 100 ml Cuvettes
Wash bottle with double distilled water Single Pan Balance
Glass marker Butter paper for weighing

1. Trichloroacetic acid (10%) - Dissolve 100 g of high grade crystalline acid in

distilled water and dilute to 1 Litre.
2. 10 NSulphuric acid - Slowly add 450 ml of concentrated sulphuric acid to 1300 ml
distilled water in a pyrex container. Allow to cool. Store in a glass stoppered pyrex

3. Ammonium molybdate solution 2.5% in 3 N sulphuric acid. Dissolve 12.5 g of

ammonium molybdate in I00 ml distilled water in a 500 ml volumetric flask. Add
160 ml of 10 N sulphuric acid solution and dilute to 500 ml with distilled water.
4. 15%sodium bisulphite solution - Add 30 g of reagent grade sodium bisulphate in
distilled water and dilute to 200 ml. Use only clear supernatant.
5. 20% sodium sulphite solution - Dissolve 20 g of reagent grade anhydrous sodium
sulphite in distilled water and dilute to 100 ml. Filter if necessary and keep in a well
stoppered flask.
6 . Amino napthol sulphonic acid solution (ANSA) - Place 195 ml of the sodium
bisulphate solution in a suitable glass stoppered container and add 0.5 g of 1 , 2 , 4
amino napthol sulphonic acid and 5 ml of sodium sulphite solution. Stopper and
shake until dissolved. If it is not completely dissolved add more of sulphite, 1 ml at
a time till completely dissolved. Pour in a dark glass bottle and store in a refrigerator.

Write the principle behind the colorimetricestimation of phosphorous in the space provided.

Follow the procedure enumerated herewith for estimation of phosphorus in serum:

1. Label a series of test tubes as : blank (B), standard (Sl, S2, S3, S4, and S5), Sample
 Add 7.6 ml of distilled water to each tube. Shake well.
Keep the tubes at room temperature for 10 minutes for colour development.
2. Preparation of standard solution of phosphorous: Weigh 20 mg of potassium
dihydrogen phosphate (KH,PO,). Transfer the weighed amount into a 100 ml
volumetric flask and make the volume to the mark with distilled water. Mix the
solution well before use. This is the standard solution of phosphorous. Next:
Pipette different volumes of standard phosphorous solution (0.2,0.4,0.6,0.8,
1.0 ml) in the standard tubes (S 1- S5), respectively.
Now add 0.8,0.6,0.4,0.2 ml distilled water in tubes Sl - S4, respectively. No
distilled water will be added in tube S5.
Add Iml of molybdic acid solution and 0.4 ml ANSA reagent to each tube.
Now add 0.8,0.6,0.4,0.2 ml distilled water in tubes S 1 - S4, respectively. No
distilled water will be added in tube S5.
Add 1 ml of molybdate solution and 0.4 ml ANSA reagent to each tube.
Add 7.6 ml of distilled water to each tube. Shake well.
Keep the tubes at room temperature for 10 minutes for colour development.
3. Preparation of Blank: Prepare the blank tube (B) with 1 ml distilled water, 1 ml
molybdic acid solution, 0.4 ml ANSA reagent and 7.6 ml distilled water. Mix well
after the addition of each solution.
4 . Preparation of Unknown solution: Dilute the given unknown solution to 100 ml
mark with distilled water. Take the 2 tube (S6, S7) for unknown solution. Take 1 ml
of unknown solution in each tube and add all reagents as you did in the standard
tubes. Keep the tubes at room temperature for 10 minutes for colour development.
5. Measurement of Optical Density: Read in a spectrophotometer or colorirneter at
540 nm. First measure the optical density after setting the colorirneter at 100%
transmittance using the blank solution. Take the reading of each solution measuring
the 0 . D from the lower concentration to the higher concentration.
6 . Standard Curve: Plot a graph between concentration of phosphorous and the optical

7. Calculate the concentration of the unknown solution.

Vol of Conc. of Distilled Molybdate ANSA Distilled OD for 0.0092

Std Sol. Std (mg) Water (ml) Solution (ml) OD H,O mg phos.

Precautions
1. All reagents should be added in the order mentioned.
2. Allow 10 minutes for the colour to develop in each tube.
3. Take the reading in increasing order of concentration.
4. Before placing the cuvette in the cuvette holder of the colorirneter, wipe the
outside of the cuvette with a piece of tissue paper to remove traces of solution,
dust and finger prints.

Calculations and Standard Curve Plotting

Record your observations and do the calculations as suggested herewith:
i

I . Preparation of Standard Phosphorous Solution

i) Molecular weight of KH,P04 = 136 g
136 mg of KH,PO, contains 3 1 mg phosphorous (P)

:. 20 mg of KH2P04contains =
136
= 4.56 mg P

b $ 20 mg of KH,P04 was dissolved in distilled water and the volume was made upto
I00 ml.
:. 100 ml of KH,P04 contains = 4.56 mg of phosphorous
:. 1 ml of KH2P0, contains = 0.0456 mg P
/ hi Now I .O ml of standard &,Po, solution contains 0.046 mg of phosphorous

11. Colorimetric Reading for Standard Phosphorous Solution

iv) Record the values as indicated in the format given herewith.
--
I 11 111 IV v vI VII VIlI I
Vol o f Conc. o f Distilled Molybdate ANSA Distilled Optical O D for
KH,PO, P (mg) Water(m1) Sol. (ml) (ml) H,O Density at 0.0092 mg
590 nm phospho-
rous.
0.2

0.4

0.6

0.8

1.o

Mean O D for 0.009 mg P = ... ... +. .. . ...+......t. ......+ . . . .. = .4 = ... . ing

5
Now prepare the standard curve for phosphorous (on a graph paper) :s :rh ,:~nre!:tra+:i?n

11I . Colorimetric Reading for Unknown Solution

v) O D of the Unknown solution, Sample 1 =
Sample 2 =
Mean OD of the unknown sample = Z =
I
IV. Determination of Phosphorous Content from Standfrrd C'urt~t.
:
1
On the standard curve prepared earlier, plot the OD of the unknown soiurlon dn the Y
axis. Now check the corresponding concentration of phosphorous for thls 017 o.i the ,'
....................................
Counsellor signature
Date: ................... ......
Aim: To plot a standard curve for iron and estimate the amount of iron present in the
given sample.

Principle
(Look up the principle given earlier in section 8.4 and write the principle here in the
space provided)

Apparatus and Equipment

Test tubes Borosil glass (6x314 inch) Spectrophoto meter
Test tube stand Single pan balance
Conical flasks Cuvettes
Pipettes 1 ml, 5 ml, 10 ml Butter paper for weighing
Beakers Glass markerlpermanent marker
Measuring cylinder Labels
Funnel

Note: All apparatus should be acid washed. Immerse all glassware in dilute hydrochloric
acid ( 1 x2) or chromosulphuric acid for 1-2 hours. Wash with water and rinse with distilled
water 4-5 times. Put upside down in a plastic basket and not in metal containers and
heat dry in a windowless room.

Reagents
1 . Conc. sulphuric acid (iron free)
2. Conc. potassium permanganate solution
3. Saturated potassium persulphate (K,S20,) solution:
Shake 7-8 g of reagent grade iron free potassium persulphate with 100 ml of water
in a glass stoppered bottle. Compensate for loss by decomposition. Shake briefly
before use. Keep the reagent in the refrigerator.
4. 3N potassium thiocyanate solution:
Dissolve 146 g of potassium thiocyanate in 480 ml ofdistilled water. Filter ifturbid.
Add 20 ml of acetone to improve keeping quality.

Procedure
Carry out the experiment following the steps enumerated herewith:
1. Weigh 702 mg of reagent grade ferrous ammonium sulphate (NH,),Fe (SO,),.
Dissolve it in 1000 ml distilled water. Add 5 ml ofconc. sulphuric acid (or 3ml of 18
M sulphuric acid). 1 ml contains 0.100 mg of ferrous iron. Warm slightly and add 5
&
r!@
: , +$' 7
ml concentrated potassium permanganate (KMnO,) drop by drop until it produces a
206 #
@
,?
,- permanent colour (light pink). KMnO, is added to oxidize the ferrous to ferric
 Transfer this solution to a 1 litre volumetric flask and make the volume to 1 litre
mark with distilled water. This solution contains 0.1 rng ironlml.
2. Label a series of Borosil test tubes with blank (B). standard (Sl-S6) and sample
(SA 1-4) and place them in a rack.
3. Take different volumes (0.2,0.4,0.6,0.8, 1.0 and 1.2 ml) of standard solution in the
labeled tubes and make the volume to 2rnl with distilled water. Add 0.5 ml conc.
sulphuric acid, 1ml potassium persulphate and 2 ml of potassium thiocyanate to all
tubes. Mix. Keep for 5 minutes for colour development. Add 9.5 ml distilled water
to all tubes, mix and measure the optical density at 490nm.
4. For unknown sample make up the volume upto I OOml of the given solution. Take
0.6 ml and 0.8 ml of the solution in duplicate and proceed just as in case of standard.
5. Draw the standard curve and calculate the amount of unknown solution present.

Precautions
1. Washing of glassware for iron estimation is important. Insufficient washing for iron
removal may cause fluctuation in the result. Immerse all glassware in acid as
mentioned above in advance.
2. Use only distilled water.

Observations, Calculations and Plotting of Standard Cu w e

Record your observations and do the calculations as suggested herewith:

1. Preparation of Standard Ferrous Ammonium Sulphate Solution

i) 700 mg of ferrous ammonium sulphate dissolved in distilled water and the volume
made upto 1000 ml. After adding 5 ml conc. sulphuric acid and 5 ml KMNO,
this solution is transfer to a 1 litre volumetric flask and the volume made to 1 litre
mark with distilled water.
This solution contains = 0.1 rng ironlml. The calculations are:
a) Molecular weight of ferrous ammonium sulphate NH, Fe (SO,),= 392.14 mg
392.14 mg of (NH, Fe (SO,), contains 56 mg Iron (Fe)

:. 700 mg of NH, Fe(SO,), contains = 700 56 = 99.96 = approximately 100 mg Fe

392.14
b) 700 mg of NH, Fe(SO,), was dissolved in distilled water and the volume
was made upto 1000 ml.
:. 1000 ml of NH, Fe(SO,), contains = 100 mg Fe
:. 1 ml of NH, Fe(SO,), contains = 0.1 mg Fe
i Now 1.0 ml of standard NH, Fe (SO,), solution contaict, 0.1 mg of Iron
.'. 0.2 ml standard solution contains = Oe21.o = 0.02 mg = 20 pg
0.4 ml of standard solution contains =
0.6 ml of standard solution contains =
0.8 ml of standard solution contains =
I .O ml of standard solution contains =
1.2 ml of standard solution contains =

1 . Colorimetric Reading for Standard Solution

iii) Record the values as indicated in the i.,):~z!argiven here i i t h -
 I II m IV v VI W wr
Vol. of Conc of Distilled Conc. K2S,0, Potassium 0 p t i c a 1 Optical
( N H ,F e Fe (pg) Water H2S04 solution thiocynate Density Density for
(SO,),, (ml) (ml) (m~) at490nrn 40pg
solution
0.2

0.4
/

0.6

0.8

1.o

1.2

Mean OD for 40 pg solution = ...... +.......+........+ ........+.......+....... = A =

.......mg
6
Now prepare the standard curve for solution (on a graph paper) with concentration of
iron (figure included in item I1 above) on X-axis and the optical density (figures in item
V11) on Y-axis. Stick the graph on page 209.

III. Colorimetric Reading for Unknown Solution

iv) OD of the Unknown solution, Sample 1 =
Sample 2 =
Mean OD ofthe unknown sample = Zl =
Sample 3, =
Sample 4 =
Mean OD of the unknown sample = Z 2=

IK Determination of Iron Content from Slandard Curve

On the standard curve prepared earlier, plot the OD of the unknown solutions on the y-
axis. Now check the corresponding concentration of iron for this OD on the x-axis. Say
the value obtained is E and E l .

So 0.6 ml of unknown solution contains E ml of Iron

Similarly 0.8 ml of unknown solution contains El ml of Iron

:. 100 ml of unknown solution will contain = -
0.8
= F2= .....mg.

Using this calculation now you calculate the Iron content in the unknown samples given
to you. Write your calculations here in the space provided.

Observed Value = F = .............

208 Expected value = D =..,.........(Take it From the counselor)
Now submit this experiment for evaluation.

...............................
Counsellor signature
ESTIMATION OF THE HAEMOGLOBIN CONTENT

Date: ..........................
Aim: To estimate the haemoglobin content of blood by cyanmethaemoglobin method.

(Look up section 8.4 and wrjte the principle for the cyanmethaemoglobin method here
in the space provided)

Apparatus & instruments

15 ml Test tubes (Borosil) Spectrophotometer/colorimeter
Pipettes 20 ml, 1 ml, 5 ml Cuvettes
Wash bottle Tmue roll
Single pan balance for weighting
Test tube stand
Butter paper for weighing
Lancet

1. Ferricyanide-cyanide reagent or Drabkin's solution:

'Dissolve 0.2 g potassium ferricyanide, 0.05 g potassium cyanide and 1.0 g sodium
bicarbonate in about 100-150 ml distilled water. Add 1 ml of nonionic detergent like
Triton X-100. Make this to 1000 ml with distilled water. This solution should be made
fresh once a month and stored in a brown bottle as it is unstable in light. Reagent should
be pipetted carefully and care should be taken not to discard cyanide solution into sinks.
Commercial Drabkin's solution is also available. We can use this commercial solution

2. Cyanmet&aemoglobin s$andard:
Certified standard haemoglobin solution may be obtained from certified or reputed
laboratory supply finns. Sterile standard solutions are small quantity packs.1t should be
stored in the dark between 4- 1O°C. A typical value of 60 mgldl is generally used. This is
equivalent to haemoglobin concentration of 1 5gIdl.

Procedwre
Carry out the experiment following the steps enumerated herewith:

1. Preparation of the standard curve for haemoglobin

o. Label 6 clean dry rest tubes and label them as S 1-S6
Add 5 ml of Drabkin's solution to test tube numbers S2 - S6
Add 5 mC of standard solution to test tube no 1 (S 1). Note, this tube does not
contain Drabkin's solution.
Add 5 ml of standard solution to test tube no S2 containingthe Drabkin's solution.
Mix well. 21 1
 Proportionately reduce the standard concentration from tubes ~ 3 - tos plot ~ a
standard curve. This can be done by transfering 5 ml of the content in S2 tube to
S3 tube in which Drabkin's solution is already present. Again transfer 5 ml of
this mixture to test tube 4 and mix well. Similarly proceed from test tube No 4 to
5. Mix the contents of the tubes well.
Test tube no. S6 serves as blank. This tube contains only 5 ml of the Drabkin's

Mix contents of all the tubes.

Let the tubes stand for 4 minutes for colour development.

2) Sample preparation
Wipe the index finger or middle finger with a cotton swab soaked in spirit.
Prick the finger with a lancet opersting device after a gentle massage of the
finger.
Discard the first drop and with the help of a haemoglobin pipette take 20pl blood
for analysis.
Add the 20 pl of blood to 5.0 ml of the Drabkin's reagent in the test tube marked
Sa. Mix. Let it stand for at least 4 minutes and read against a reagent blank at
540 nm.

Calculation
Haemoglobin in the blood can be calculated by the following two methods
i) Using mean OD

Blood hemoglobin (ddl) = Readin of unknown

Readingeof
standard
4
dilution factor conc. of standard

where , dilution factor = 25 1

ii) Using standard curve.

A standard curve can be prepared by suitably diluting the standard with the reagent (as
mentioned in the procedure) A linear graph may be obtained by plotting the known
hemoglobin concentration against the colorimetric optical density reading. Hemoglobin
concentration of the unknown sample can then be directly read from the calibration curve
by knowing the optical density of the unknown blood sample.

Precautions
1. Potassium cyanide is highly toxic and hence drabkin's solution should not be
pippeted by mouth.
2. Drawing of blood should be perfected before determination in an actual sample.

Observations, Calculations and Plotting of Standard Curve

Record your observations and do the calculations as suggested herewith:

i) Preparation of Standard Cyanmethaemoglobin

Strength of standard cyanmethaemoglobin solution =...........mg/dl
.............g/dl Hb
-
-

Tubes
1 2 3 4 5 6

Drabkin Solution (ml)

Standard Solution (ml)

212
ii) Colorimetric Reading for Standard Solution
Record the values under the different heads indicated in the format given herewith. To
give you a clue we have written some of the vlaues. Complete the table and calculate
the Optical density in column V for 15.0 mg/dl HiCN as indicated:

Test-lh be Dilution of HiCN optical Optical Density

Density at for 15.0 mg/dl

- 60

Now calculate theMean OD from the above values obtained in column V

Mean OD =

= A = ............mg/dl.

Using the values obtained above prepare the standard curve (on a graph paper) by
plotting the known haemoglobin concentration (figure included in item 111above) on X-
axis against the colorimetric optical density (figures in item IV) on Y-axis. Stick the
graph on page later.

i) Colorimetric Reading for Test Sample

Kecord the 01) reading of your sample and of any other four samples (of the fellow
,tuctents) in tlic format given herewith:
-
S. No
w
Sample 1 Sample 2 Sample 3 Sarnde 4 Sample 5
OD

iv) Calculation of Haemoglobin Content Using the OD

Wrrte tllc equation given above for calculation of Hb from OD in the space provided

Hb content =

Now using the formulae you have written above calculate the Hb content of the test
sample in the space provided herewith:

Sample 1 ) Hb content =
Sample 2) Hb content = Y

Sample 3) Hb c o m f =

Sample 4) Hb content =

Sample 5) Hb content =

v) Calculation of Huemoglobin Content From Standard C u m

Paste the calibration curve prepared for the standard solution (under point iii)here in the
space provided. Now from this curve, haemoglobin concentration of the unknown sample
can be directly read by plotting the optical density of the unknown blood sample on the
y-axis and then checking the corresponding concentration of Hb for this OD on the x-
axis.

Observe the Hb concentration for each sample taken and record the value in the
caliberated graph and write the value here in the space provided:

Standard curve between concentration of HiCN and OD

 Strength o f standard cyanmethaemoglobin (HiCN) solution = 60 mgldl
Concentration of Hb. = 6014 = 15 g/dl Hb
:. Imgldl HiCN = 0.25 gl dl Hb
Similarly calculate for the sample 1-5:
Concentration of Haemoglobin in Sample 1 =
Concentration of HiCN of O D .............. = ......mg/dl
:. Concentration of haemoglobin = ........... d d 1

Concentration of Haemoglobin in Sample 2 =

Concentration o f HiCN of O D .............. = ......mg/dl
:. Concentration o f haemoglobin = ........... g/dl
Concentration of Haemoglobin in Sample 3 =
Concentration of HiCN of O D .............. = ......mg/dl
:. Concentration of haemoglobin = ........... g/dl
con cent ratio^ of Haemoglobin in Sample 4 =
Concentration of HiCN of O D .............. = ......mgldl
:. Concentration of haemoglobin = ........... d d 1
Concentration of Haemoglobin in Sample 5 =
Concentration of HiCN of O D .............. = ......mg/dl
:. Concentration o f haemoglobin = ........... d d 1

The haemoglobin content of blood as estimated by cyanmethaemoglobin method of 5

subjects is found to be in the range of ........................................................
From standard OD and ..........................................................from
standard curve.

Now submit this experiment for evaluation.

.................................
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