The document provides instructions for performing an assay of calcium gluconate by complexometry, including preparing standard EDTA and magnesium sulfate solutions, titrating calcium gluconate against EDTA while using magnesium and an indicator to identify the endpoint, and calculating the percentage purity of calcium gluconate based on the titration results. The titration is a replacement complexometric titration that uses the stable magnesium-indicator complex to indirectly determine the endpoint of the calcium-EDTA reaction.

1. PA-I Practical
Presented By
Mrs.Smita P.Shelke
Assistant Professor, Department of Pharmaceutical Chemistry,
GES’s Sir Dr.M.S.Gosavi College of Pharmaceutical Education and
Research, Nashik-05.(Maharashtra

2. Experiment No: 10
Title: To perform assay of clacium gluconate by complexometry.

3. Experiment No: 10
This experiment is performed in two parts i.e.
A: Preparation and standardization of 0.05 M EDTA solution.
B: Assay of calcium gluconate

4. Requirements
• Glasswares: Burette, burette stand, conical flask, volumetric pipette, beaker,
volumetric flask, funnel, glass rod, and wash bottle, etc.
• Chemicals: LR grade calcium gluconate (C12H22CaO14), disodium edetate
(EDTA), ammonia (NH3), calcium chloride (CaCl2), ammonium chloride
(NH4Cl), magnesium sulphate ( MgSO4), hydrochloric acid (HCL), and
solochrome black-T indicator or mordant black II, etc.
• Apparatus: Digital/analytical balance, and Ultrasonicator.

5. Theory:
The assay of calcium gluconate is based upon a replacement complexometric titration.
Magnesium forms a complex with the mixture of indicator mordant black II which indicates
the first color.
The magnesium-indicator complex is more stable compared to the calcium-indicator complex;
hence calcium does not affect the magnesium-indicator complex. When titrated against
disodium edetate, calcium and EDTA formed.

6. Principle
When all of the calcium has been consumed, the drop of EDTA breaks the
magnesium-indicator complex, allowing the free indicator to form a complex
with the magnesium. The endpoint is determined by detecting the second color
at that time.
The following is the reaction that is involved in this titration.

7. Structure of EDTA: Ethylene Diamine Tetra Acetic Acid
The EDTA molecule is represented as “H4Y”. Each acid hydrogen
on EDTA can be removed, producing H3YI1, H2YI2, HYI3, and
YI4 ions. The disodium dihydrate of EDTA, Na2H2Y•2H2O is
commonly used to prepare standard EDTA solutions.

8. Eriochrome black T(EBT) indicator
The end point of an EDTA titration is determined with a metallochromic
indicator These indicators are complexing agents that change color when
combined with metal ions. A variety of! indicators can be used for EDTA
titrations In this experiment we will use Eriochrome black T (EBT) indicator,
having the structure shown below.

9. Mechanism of Titration
This indicator (shown as H2In- in the equations below) changes from blue to
red when combined with a metal ion, forming a complex ion:
M2+ + H2In- + 2H2O <---> MIn- + 2H3O+
Blue Red

10. Mechanism of Titration
EDTA is a stronger complexing agent than the indicator, and displaces the
indicator from the metal ion allowing the indicator to return (through shades
of
violet) to a pure blue color, indicating the end of the reaction.
MIn- + Y-
4 <----> MY2- +H2In-
Red Blue

11. Mechanism of Titration
Calcium ion (Ca+2) does not form a stable red complex with the EBT indicator;
therefore the direct titration of Ca2+ by EDTA may not cause a sharp color
change of EBT indicator at the end point. The magnesium complex with EBT
is stable and the Kf of Mg2+ with EDTA is lower than the Kf of Ca2+ with
EDTA. Thus a displacement titration of Ca2+ by the mixture of Mg2+ and
EDTA will help to determine the end point with the following mechanism:
CaIn- +MgY2- <----> CaY2- +MgIn-

12. Mechanism of Titration
To accomplish this displacement titration, a small amount of Mg2+
will be mixed with the EDTA solution.The EDTA - Mg mixture
will titrate the unknown Ca2+ solution. At the end point, Mg2+ will
be released from the EBT indicator and complexed with EDTA,
causing the color change from red to blue.

13. Solutions required for this experiments are
• 0.05M EDTA
• 12M HCl
• 8.5M Ammonia ammonium chloride buffer solution
• Eriochrome Black T

14. Preparation of 0.05 M magnesium
sulphate:
Mole wt of EDTA in 1000 ml is 1M solution
372.2 g of EDTA in 1000 ml is 1M solution
37.22 g of EDTA in 100 ml is 1M solution
1.861 g of EDTA in 100 ml is 0.05 M solution

15. Preparation of Ammonia 10 M
• Take few ml of DW in 100 ml volumetric flask
• Add 75 ml of strong ammonia solution and make up the volume up to 100
ml.

16. Preparation of strong ammonia-ammonium chloride
solution: pH 10
• Take 10 ml of water and 35 ml of 10 M ammonia
solution in a beaker
• Add 5.4g of ammonium chloride dissolve it.
• Make up the volume upto 100 ml.

17. Preparation of 2 M sodium hydroxide solution
• Take few ml of water in 100 ml VF.
• Add 0.8g of sodium hydroxide dissolve it completely.
• Make up the volume upto 100 ml.

18. Titration procedure
All glassware should be cleaned and dried according to standard laboratory
procedures.
Before filling the burette for the titration, rinse it with distilled water and then
pre-rinse it with a portion of the titrant solution. Pre-rinsing is required to make
sure that all solution in the burette is the desired solution, not a contaminated or
diluted solution.
Take the unknown stock solution of titrant in a clean and dry beaker then fill the
burette using the funnel.
Remove air bubbles from the burette and adjust the reading to zero.

19. Standardization of disodium EDTA
• The titration is based on titration of EDTA with standard zinc chloride
sloution prepared from granulated Zinc.
• Bromine water is added to ensure oxidation of trace iron impurities.(Fe++
ions are oxidized to fe+++ ions. This Fe+++ ions forms less stable complex
than Fe++ with EDTA.)
• Sodium hydroxide is added to neutralize the solution but it causes the
precipitation of Zinc hydroxide.
• To dissolve this PPTammonia buffer is added.
• Ammonia buffer is added in excess because it maintain th pH that is suitable
for formation of Zn EDTA complex

20. Standardization of disodium EDTA
Zn+2 + HCl ZnCl2 + H2
ZnCl2 + EDTA ZnH2Y + NaCl
1 mole of Zn = 1 mole of EDTA
65.38 g of Zn = 1000ml of 1 M EDTA
65.38 g of Zn = 1000ml of 1 M EDTA
0.06538 of Zn = 1 ml of 1 M EDTA
0.00327 g of Zn = 1 ml of 0.05 M EDTA

21. Standardization of disodium EDTA
• Weigh accurately 0.8g granulated Zinc.
• Dissolve in Dil HCl solution by gentle warming.Add 0.1 ml of bromine
water in to it.
• Boil the solution to remove excess of bromine. Make up the vol. 200ml by DW.
• Take 20 ml above soln netralize it 2M NaOH solution.Dil to 150 ml by DW.
• Add sufficient ammonia buffer to dissolve PPT.
• Add Mordant Black II.
• Titrate with Prepared EDTA till green colour appears.

22. • Direct or Back titration is not posssible for Ca so replacement titration is done.
• Ammonium chloride buffer is added calcium solution to this solution
standard solution of EDTA is added.
• Therfore stable Ca-EDTA complex are formed.
• Mg-EDTA complexes are less stable.
• To this Magnesium Sulphate solution is added.
• Mg ions are liberated which are titrated with EDTA.
Assay of Clacium Gluconate

23. 1 mole of Calcium gluconate = 1 mole og EDTA
448.40 g of Calcium gluconate = 1000 ml of 1 M EDTA
44.80 g of Calcium gluconate = 100 ml of 1 M EDTA
4.480 g of Calcium gluconate = 10 ml of 1 M EDTA
0.4480 g of Calcium gluconate = 1 ml of 1 M EDTA
0.02242 g of Calcium gluconate = 1 ml of 0.05 M EDTA
Assay of Calcium Gluconate

24. • Take 0.5 gm of calcium gluconate and pour it into a conical flask, and
dissolve in 50 ml of warm water.
• Add 05 ml of 0.05 M magnesium sulphate solution.
• Add 10.00 ml of strong ammonia-ammonium chloride solution.
• Titrate with EDTA.
• Near to the end point add indicator, add 2 drops of mordant black II
mixture and properly mix it.
• Titrate ill colour change is from pink to blue
• Substract the vol of magnesium sulphate from from C B R.
Assay of Clacium Gluconate

25. • Titrate the sample solution with standardized disodium edetate
until the endpoint is reached. The actual endpoint is indicated by a
change in the color of the solution.
• Properly record the readings of the burette.
• To get accurate results, repeat the titration three times.
• Take their mean and calculate the percentage purity of calcium
gluconate.

26. Observations for standardization of EDTA
Solution in Burette: EDTA
Conical flask: 20ml of ZnCl2 +130 ml H2O +NH3 Buffer
Indicator: Mordant Black II
Colour Change: Blue to green
Constant Burette readings:
Sr. No. Burette reading Volume of
titrant
1 16.1 16.1 16.1
2 17.5 17.5 17.5
3 18.0 18.0 18.0
Mean 17.2

27. Calculations:
Cross multiplication
1 x 0.05 x 0.08 = Avg C B R x X M x 0.00327
1 x 0.05 x 0.08 = 17.2 x X M x 0.00327
1 x 0.05 x 0.08 0.004
X M = ------------------------- = --------- = 0.0711 M
17.2 X 0.00327 0.056244
1 ml of 0.05 M EDTA = 0.00327 g of Zn
17.2 ml of X M EDTA = 0.08 g of Zn

28. Observations for Assay of ca gluconate
Solution in Burette: EDTA
Conical flask: 0.5 g Ca Glu+ 5 ml og MgSO4+10ml Buffer solution
Indicator: Mordant Black II
Colour Change: Pink to Blue
Constant Burette readings:
Sr. No. Burette reading Volume of
titrant
1 19.4 19.4 19.4
2 20.2 20.2 20.2
3 21.1 21.1 21.1
Mean 20.23
CBR - ml of MgSO4 is the actual vol consumed by Ca gluconate
20.3 - 5 = 15.3 ml

29. Calculations:
Cross multiplication
1 x 0.05 x W = 15.3 x 0.0711 M x 0.02242
15.3 x 0.0711 x 0.02242 0.02438
W g = ------------------------- = --------- = 0.4876 g
0.05 0.05
1 ml of 0.05 M EDTA = 0.02242 g of Calcium gluconate
15.3 ml of 0.0711 M EDTA = W g of Calcium gluconate

30. Calculations of percentage purity of calcium gluconate sample
Assay result is 0.4876 g of calcium gluconate
Actual sample weight taken was 0.5 g
i. e. 0.5 g is ---- 100 %
0.4876 g is ----- X %
0.4876 x 100
X = --------------- = 97.52 %
0.5

31. Reporting of Results
Result: Percentage purity of given calcium gluconate sample was
found to be 97.52 %
IP standard: NLT NMT
Conclusion: The given sample of calcium gluconate passes the
IP limits of standard.

32. Video link
https://youtu.be/g7tr2h0gu7o