METHODS USED

FOR OFFICIAL ASSAY

PART1
PharChem 3
■ Objectives
■ To demonstrate the different methods used in official assay
Standardization

■ The process of determining the exact concentration

(molarity) of a solution.
■ Titration is one type of analytical procedure often
used in standardization.
■ In a titration, an exact volume of one substance is
reacted with a known amount of another substance.
Assay
■ is an analysis done to determine the presence
of a substance and the amount of that
substance.
■ a test or appraisal to determine the
components of a substance
Classification of Methods Used for
Official Assay
1. Titrimetric methods (volumetric analysis)
2. Gravimetric methods
3. Spectrometric methods
4. Electrometric methods
5. Chromatographic methods
6. Miscellaneous methods
 Types of Titration: Classified based on the type
of reaction involved
1. Acid-base titrations, in which an acidic or basic titrant reacts with
an analyte that is a base or an acid. Ex. HCl + NaOH → H2O + NaCl
2. Precipitation titrations, in which the analyte and titrant react to
form a precipitate. Ex. Ag+ + X- → AgX(s)
3. Redox titrations, where the titrant is an oxidizing or reducing agent.
Ex. 5NaC2O4 + 2 KMnO4 + 6H+ → 10CO2 + 2Mn2+ +8H2O
4. Complexometric titrations, involving a metal-ligand complexation
reaction. Chelating agent EDTA is commonly used to titrate metal
ions in solution.Not anatically useful.
 1. Titrimetric Method of Analysis
■ Titration (also called titrimetry and volumetric analysis) is a
common laboratory method of quantitative chemical analysis to
determine the concentration of an identified analyte (a substance
to be analyzed, or active constituent, also called the titrate or
titrand).
■ A reagent, called the titrant or titrator, is prepared as a standard
solution of known concentration and volume.
■ The titrant reacts with a solution of analyte to determine the
analyte's concentration.
■ The volume of titrant that reacted with the analyte is called
the titration volume or also called the titer
■ Endpoint- the point at which the reaction is complete in a titration
■ Indicator – is a chemical used which changes color to indicate
(signal) the endpoint
■ Titrant/titrator : solution of known concentration.
■ Titrand/titrate/analyte: solution being titrated in order to determine
its concentration.
■ Titre/titration volume : total volume of solution delivered by the
burette (buret) to reach the end point of the titration.
■ Ex. HCl + NaOH  NaCl + H2O
Analyte Titrant

Phenolphthalein - indicator
■ In the example given, the indicator changes color when equivalent quantities of
NaOH and HCl have reacted.
■ Since the quantity of NaOH can be easily calculated, the amount of HCl in the
sample can be readily determined.
■ Components:
– Titration
– Indicator
– Stoichiometric or equivalence point
– End point
■ Types of Titration according to the number of titrants:
– Direct titration – one titrant
– Indirect or back titration- uses two titrants
■ Back-Titration = a known amount of excess standard reagent
is added to the sample. Then a second standard reagent is
used to titrate the excess of the first reagent.
■ involves two separate reactions.
1. The concentration of a substance to be analyzed is
determined by reacting it with a known amount of excess
reagent (A).
2. The remaining excess reagent is then titrated with another
second reagent (B) .
■ Indicators
 to determine points in neutralization process
 to determine hydrogen ion concentration
 to indicate a desired change in pH has been
effected thru the change in color
Theories of acid-base indicators

■ 2 theories proposed to explain the change of color of

acid-base indicators with change in pH:
■ 1. Ostwald’s theory
■ 2. Quinonoid theory
The color change is due to ionization of the acid-base
indicator.the unionized form has different color than
the ionized form.
■ Standardization
– A. Primary standard
– B. Secondary standard
■ Titer
■ Standard acid solutions: HCl and H2SO4
■ Standard alkali solutions: NaOH, KOH, and Ba(OH)2

■ BLANK DETERMINATION
Primary standard is a reagent that is extremely pure, stable, has no
waters of hydration, and has a high molecular weight.
Some features of a primary standard are;
■ High purity
■ Stability (low reactivity)
■ Low hygroscopicity and efflorescence
■ High solubility (if used in titration)
■ High equivalent weight
Ex, Sodium Carbonate (Na2CO3), Potassium dichromate (K2Cr2O7)
Potassium hydrogen phthalate (KHP) (KHC8H4O4)
■ A secondary standard solution is a chemical term
that refers to a solution that has its concentration
measured by titration with a primary standard
solution.
■ The amount of chemical reactants in the primary
standard solution is known beforehand.
■ A blank titration is a titration without an analyte
being present, only the solvent used in the analyte
solution.
■ It is carried out like a regular titration but with no
analyte present
■ Buret used in titration: Mohr and Geissler
■ Cleaning solutions of buret and glasswares:
1. Sodium dichromate in sulfuric acid
2. Ammonium peroxydisulfate in sulfuric acid
3. Trisodium phosphate solution
4. Synthetic detergents
: M1V1 = M2V2, where M1 is the concentration in molarity
(moles/Liters) of the concentrated solution, V1 is the volume of
the concentrated solution, M2 is the concentration in molarity of
the dilute solution (after more solvent has been added), and V2
is the volume of the dilute solution.
Titration Problem Solving

1. Write the balanced chemical equation

2. Identify number of mols of known or standard solution
M=mol/L , mol=MxL
3. Use mole ratio to solve for moles of unknown
4. Solve for unknown M or L , M=mol/L, L=mol/M also
expressed as C=n/v
Sample problem 1
■ How many mL of 0.45M HCl must be added to 25mL of 1.00M KOH
to make a neutral solution?
1. Write the balanced equation
HCl + KOH  KCl + H2O
0.45M 1.00M
?ml 25ml
2. Find mols of known solution.  KOH M=mol/L,
mol=MxL, 1.00M x 0.025L = 0.025mol KOH
3. Use mols ratio to find mols of unknown
0.025mol KOH x 1mol HCL/1mol KOH = 0.025mol HCL
4. Solve for the unknown vol (HCl) M=mol/L
L=mol/M = 0.025 mol HCl/0.45M HCl = 0.056L = 56ml
 Titrimetric (Volumetric) Method of Analysis
■ PROBLEMS: Standardization:
1. A 0.128 g sample of KHP (HKC8H4O4 mw=204.23) required
28.54 mL of NaOH solution to reach a phenolphthalein
endpoint. Calculate the molarity of the NaOH.

• HKC8H4O4 + NaOH -----> NaKC8H4O4 + H2O

• (0.128 g KHP)(1 mol / 204.23 g KHP ) = 6.267 x 10-4 mol KHP
• (6.267 x 10-4 mol KHP)(1 mol NaOH / 1 mol KHP) = 6.267 x 10-4 mol NaOH
• 6.267 x 10-4 mol NaOH / 0.02854 L NaOH = 0.0220 M NaOH
■ PROBLEMS: Standardization:

2. A 20.00 mL sample of HCl was titrated with the NaOH

solution from Example1. To reach the endpoint required
23.72 mL of the NaOH. Calculate the molarity of the HCl.
■ PROBLEMS: Standardization:
1. A solution of HCl is standardized and found to be 1.183 M.
Calculate the volume of this solution that diluted to 1.000 L
in a volumetric flask, produces a 0.1000 M solution of HCl.

2. Exactly 46.32 mL of NaOH is used to titrate a 1.200 mg

sample of primary standard potassium acid phthalate, or
KHP (MW=204.2). Calculate the molarity of sodium
hydroxide.
■ In a titration, 27.4 ml of a 0.0154M solution of Ba(OH)2 is
needed to neutralize 20.0mL of HCl. What was the concentration
of the acid solution?
1. 1 Ba(OH)2 + 2HCl  BaCl2 + 2H2O
2. M=m/L; m=ML ; m=0.0154(.0274L)
= 0.000422mol Ba(OH)2
3. moles HCl= moles Ba(OH)2 x 2HCl / 1 Ba(OH)2
=0.000422 x 2/1
= 0.000844mol HCl
4. MHCl =m/L= 0.000844/0.020L = 0.0422M HCl
***MVb=MVa  shortcut way but is not always the best way
figured out by looking at the balanced equation
Practice Problems

■ Study this
■ If 25.21 mL of NaOH solution is required to react
completely with 0.550 g KHP, what is the molarity
of the NaOH solution?
Study this
■ A 2.55g AgNO3 was dissolved in 300ml water and
gives 0.05 M silver nitrate solution and was used to
titrate 30ml of HCl w/ phenolphthalein as indicator
where 15ml of AgNO3 were used to obtain the
endpoint.
a) Calculate how the 0.05M silver nitrate was attained.
b) What is the molar concentration of the unknown
chloride solution?
■ Study this
■ Calculate the molarity of each of these solutions.
a. A 5.623-g sample of NaHCO3 is dissolved in enough
water to make 250.0 mL of solution.
b. A 184.6-g sample of K2Cr2O7 is dissolved in enough
water to make 500.0 mL of solution.
Study this
1. A solution was prepared adding 23.512g of NaCl to
enough water to obtain 250.00mL of solution. A
10.0mL aliquot of the solution was taken. Calculate the
moles of NaCl in the aliquot.
2. A 25.0 mL sample of 0.105 M HCl was titrated with
315 mL of NaOH. What is the concentration of the
NaOH?
 % PURITY CALCULATION
■ To 'assay' means to analyze a sample for its purity
■ Purity is very important where impurities could have
dangerous side effects in a drug or medicine
• However in any chemical process it is almost
impossible to get 100.00% purity and so samples are
always analyzed in industry to monitor the quality of the
product.
• The more a product is processed e.g. by distillation or
crystallization, the more costly the process, but the
purer the product gets.
 % PURITY CALCULATION
■ % purity is the percentage of the material which is the
actually desired chemical in a sample of it.
MASS of USEFUL PRODUCT
■ PERCENT PURITY = 100 x ------------------------------------------------
in TOTAL MASS of SAMPLE

■ %Purity= mass of pure subs/mass of impure subs x 100

 % PURITY CALCULATION
■ The titration of an impure sample of KHP found that 36.0
mL 0.100 M NaOH was required to react completely with
0.765 g of sample. What is the percentage of KHP in this
sample?
Explanation:
The equation for the reaction with NaOH is
KHC8H4O4+NaOH→KNaC8H4O4+H2O
mllKHPmll+NaOH→mllKNaPml+H2O
■ Let's start by calculating the moles of NaOH (known)
■ Moles of NaOH=0.0360L NaOH×0.100 mol NaOH/1L
NaOH=0.003 60 mol NaOH
Now we can calculate the moles of KHP neutralized by
the NaOH.
Moles of KHP=0.00360mol NaOH×1 mol KHP/1mol
NaOH=0.003 60 mol KHP
And now we can calculate the mass of the KHP.
Mass=0.003 60mol KHP×204.22 g KHP1mol KHP=0.7352 g
KHP
Finally, we calculate the purity of the KHP.
Purity=mass of pure KHP /mass of impure
KHP×100%=0.7352g0.765g×100%=96.1%
The KHP is 96.1 % pure.
%Purity Calculation
■ A sample of impure KHP weighing 2.1283 g required
42.58 mL of a 0.1084 M NaOH solution for titration
to the phenolphthalein end point. Calculate the
%purity of the sample.
Prob.
1. Suppose a 300mg ASA tablet was dissolved in 30ml
ethanol (its not very soluble). If it took 16.45ml of
0.1M NaOH to neutralize it, calculate the % purity of
ASA.
■ A 1.80 g of impure sample of Na2CO3 was dissolved in 250
mL of water. A 25 ml aliquot of this solution required
24.3mL of a 0.110 mol/L solution of HCl for complete
neutralization. The equation for neutralization reaction is
Na2CO3 + 2HCl  2 NaCl + H2O + CO2
Calculate the % purity of the original sample of Na2CO3.