refractive index, pH, specific gravity, viscosity, alcohol content, fineness of the particles, saponification value, Acid value, iodine value, Reducing sugars, quantitative inorganic analysis, loss on drying, determination of ash value.

1. Seminar on
“Analytical methods in Ayurvedic
formulations of BhaishajyA Kalpana ”

2. Introduction:
 Any product requires certain analytical standards or
parameters. These are more essential, particularly in
case of drugs and formulations.
 The formulations may be single drug preparation or a
compound preparation the analytical parameters are
must. However, parameters required for different
preparation may be different. Analytical standards are
the dimensions to evaluate a product.
 It is compulsory to describe a product by analytical
standards since these standards speak of quality,
authenticity and purity of the product.

3. 1.Refractive Index:
 Principle- Light passing through a substance will be slower
than the one passing through vacuum or air medium.
 Definition- A numerical expression of refractive power of a
medium , as compared with that of air, which has refractive
index of one.
 Uses- It may be used as a measure of total solids in a solution.
It is carried out in Ghrita paka, taila paka etc
 It can be used for identification of material concentration and
its purity.

4. 2.pH (Potential Hydrogen)
 In the symbol pH , p stands for ‘potenz’ which means
strength. pH indicates the strength of hydrogen i.e., ion
concentration expressed in gms per litre.
 pH is a scale representing the relative acidity or the alkalinity
of a solution , where in value 7 is neutral. A value below 7
indicates acidic nature of the liquid and the above alkaline
nature.
 pH value indicates the relative concentration of hydrogen
atoms in the solution compared with that of standard solution.
 Uses-This is useful in case of Asava-aristas , arka, dravaka,
Avaleha, Kshara, Lavana etc.

5. pH values of some materials ;
 Intestinal juice 7.5-8
 Pancreatic juice 7.5-8
 Saliva 6.35-6.85
 Urine 4.8-7.5
 Sea water 8
 Egg white 8
 Milk 6.6-6.9
 Blood 7.35-7.45
 Tears 7.4
 Beer 4.2-4.9
 Orange juice 2.6-4.3
 Vinegar 3.0
 Lemon juice 2.0

6. 3. Specific gravity:
 Definition: it is the ratio of the density of a substance
to the density of the water. The values of both are
determined at the same temperature and pressure.
 Here the density of any substance is the ratio of mass
to volume (m/v) at fixed temperature and pressure.
 Specific Gravity = Density of the substance
Density of water
 Or it can also be expressed as:
Specific Gravity = Mass of the substance
Mass of equal volume of water

7.  It is always expressed in gm/ml. it is measured at a specified
temperature , usually at 250C which indicates the temperature
of air at which substance is weighed and also temperature of
water used.
 Specific Gravity may be determined by using pycnometer,
hydrometer or Mohr-westphal balance.
 This is carried out especially in asavarishta, kwatha , Ghrita,
taila and other liquid preparations.

8. 4. Viscosity:
 Principle: If equal volumes of water and castor oil are
allowed to flow out through 10ml glass pipette under
identical conditions we observe a time difference
between the two liquids in the emptying process.
 This indicates that flow of water is faster than the flow
of castor oil. The difference in the rate of flow is
attributed to the phenomenon called viscosity.
 Fluidity is the term used to define reciprocal of
viscosity.

9.  Viscosity of a few of the liquids at 200C is as below:
 Viscosity may be carried out in any of the liquid preparations.
In asarishtas, kwatha, dravaka, Ghrita , taila and other liquid
preparations it is performed.
Liquid viscosity Centipoises (cp)
Castor oil
Chloroform
Ethyl alcohol
Glycerin (93%)
Olive oil
Water
1000
0.563
1.19
400
100
1.0019

10. 5. Alcohol content :
 There are different methods for estimating the alcohol content in any
drug preparations. One of the methods is based on the specific
gravity.
 In one of such methods 100ml of drug formulation is transferred to a
flask. A little amount of fumice powder is added to the content of the
flask. If necessary the formulation is neutralized with N/10 sodium
hydroxide using phenolphthalein indicator.
 The flask is arranged for distillation process using water condenser.
At least 90ml of the distillate is collected and is made up to 100ml
with water. The specific Gravity of this distillate is determined.
 The quantity of alcohol corresponding to this specific gravity is the
percentage of alcoholic content in the drug formulation.

11.  Percentage by volume of ethyl alcohol corresponding to the
specific gravity at 20/200 C as given below:
Specific Gravity Percentage of alcohol(v/v)
1.0000
0.9997
0.9991
0.9985
0.9976
0.9964
0.9920
0.9900
0.9848
0.00
0.20
0.60
1.0
1.61
2.43
5.58
7.12
11.36

12. 6. Fineness of the particles:
 In case of bhasma, sindura,lepa,pisti,churna etc the degree of
fineness or coarseness of a powder is measured , differentiated
and expressed by the size of the mesh of the sieve through
which the particle is able to pass.
 Sieves : the numbered sieves indicate the number of
meshes(openings) in a length of 1 inch (2.54cm) in each
transverse direction parallel to the wires. So a sieve
numbered 10 will have 10 meshes in the specified
length.

13. All particles pass through sieve no Not more than 40% pass through
 Coarse powder 10 44
 Moderately coarse powder 22 60
 Moderately fine powder 44 85
 Fine powder All pass through 85 numbered sieve
 Very fine powder All pass through 120 numbered silk sieve
 A suitable quantity of sample is weighed and transferred to the
set of sieves from number 10-85. The sieves are shaken in
sieve shakers for about 30 minutes and the residue on each
sieve is weighed separately.

14. 7. Saponification value:
 The term ‘sapo’ means pertaining to soap. Saponification
means chemically hydrolysed into soap or acid salts and
glycerol by heating with an alkali.
 Saponification value is the quantity of potassium hydroxide
required in mgs to neutralize the acid formed by hydrolysis of
fat present in the drug. This also indicates the fat content.
 Experimentally the saponification value of a given sample
may be determined by taking a weighed quantity of fat,
refluxed with excess of standard ethanoic potassium hydroxide
(KOH) solution and the unused alkali against a standard acid
solution. This indicates the saponification.

15. 8. Acid value:
 Acid value of a fat or oil indicates the amount of free fatty
acids present in it. The acid number is expressed as the number
of milligrams of potassium hydroxide required to neutralize
one gram of fat.
 It is determined by dissolving a weighed quantity of fat in
ethanol, titrating the solution against standard alkali. The acid
value of fat can give the extent of rancidity in a stored sample.
 Rancid, the literal meaning of the term is stinking or having a
disagreeable odour and taste, usually characterizing fat
undergoing oxidation or bacterial decomposition to more
volatile odoriferous substances.

16.  Many of the drug preparations may be slightly acidic in nature.
Such substances may be suitably diluted to a known volume
by water. An adequate portion may be titrated against standard
alkali and extract acidity may be calculated to find out the acid
value.
 Acid value may be measured in any of the fatty preparations
mainly Ghrita and taila.

17. 9. Iodine value :
 This indicates the unsaturation in organic compounds present
in the drug formulations. The method is based on consumption
of iodine to add across the double bonds. It is the quantity of
iodine required in mgs, to add to double bonds present in 1gm
of oil or fat.
 The extent of unsaturation in a fat or oil is expressed in terms
of its iodine value. It is identified as the number of grams of
iodine which will add to 100gms of fat or oil.
 Iodine value obviously depends on the number of double
bonds present in the acid component of glycerides. A high
iodine value indicates that the glycerides contain a large
number of double bonds, while a low iodine number implies
the presence of a few double bonds.

18. 10. Reducing sugars:
 Estimating reducing sugars is one among the significant
parameters in evaluating the standard of the pharmaceutical
products.
 Glucose for example is a reducing sugar. Such sugars may be
estimated by titration against Fehling’s solution or by
colorimeter.

19. 11. Quantitative inorganic analysis:
 Normally inorganic compounds which are present in the form
of oxides, salts or any other form may be converted into water
insoluble compounds and then estimated gravimetrically or
they may be converted into a soluble form and estimated
gravimetrically. They also may be estimated by instrumental
methods such as flame photometry.
 For example free sulphur may be converted into an insoluble
sulphate like barium sulphate and then estimated
gravimetrically.

20. 12. Qualitative identification of inorganic
elements:
 Inorganic elements are classified into different
groups. Systemic charts are available for
qualitative analysis of these elements using
different tests.

21. 13. Loss on Drying (moisture content):
 Loss on drying procedure determines the amount of
evaporative material in the drug sample.
 General method: Place about 10g of drug (without
preliminary drying and after accurate weighing) in a
special evaporating dish. Dry the material at 1050C for
5 hours. Later the sample has to be weighed again.
 This process of drying and weighing is continued until
the difference between two successive weighings
corresponds to not more than 0.25%.

22. 14. Determination of Ash value:
This includes :
 Determination of total ash
 Determination of acid insoluble ash
 Determination of water soluble ash

23. Cont….
 General method:
Incinerate about 2 to 3g accurate weighed drug.
Grid the drug and take in a dish at a temperature not
exceeding 4500C until free from carbon. Later cool it
and weigh. If the carbon free ash cannot be obtained
in this way, exhaust the charred mass with hot water,
collect the residue on an ash less filter paper,
incinerate the residue and filter paper, add the filtrate,
evaporate to dryness, and ignite at temperature not
exceeding 4500C.

24.  In acid insoluble ash, the ash obtained is boiled for 5 minutes
in gooch crucible. Whereas in water insoluble ash, the ash
obtained is boiled for 5 minutes with water for 25 minutes in
gooch crucible.
 In all the above cases the percentage of ash is calculated with
reference to the air dried drug.
 This procedure is one among the analytical parameters for all
solid dosage forms.

25. 15. Extractives:
 Alcohol soluble extractives
 Water soluble extractives
 Either soluble extractives
 General method :
Macerate 5 g of air dried drug which is coarsely powdered
with 100ml of alcohol of specified strength in a closed flask for
24hours, shaking frequently for 6 hours and allowing it to stand
for 18hours.
Filter it rapidly taking precautions against loss of solvent,
evaporate 25ml of the filtrate to dryness in a tarred flat bottomed
shallow dish and dry at 1050C to constant weight and weigh.

26.  Calculate the percentage of alcohol soluble extractive with
reference to air dried drug.
 The procedures of water soluble and either soluble extractive
are carried out with chloroform water and solvent either
respectively.

27. 16. Volatile oils:
 The determination of volatile oils in a drug is made by
distilling the drug with mixture of water and glycerine,
collecting the distillate in a graduated tube in which the
aqueous portion of the distillate is automatically separated and
turned to the distilling flask and measuring the volume of the
oil.
 A special apparatus with distilling flask, still head and spiral
condenser is used here to know the percentage of volatile oils.
 The content of volatile oil is expressed as percentage v/w.

28. 17. T L C (Thin Layer
Chromatography):
 The term chromatography has got origin from latin where
chromato + graph means to write.
 Definitions :
1. Chromatography is any one of the several processes for separating
and analysing various gaseous or dissolved chemical materials
according to difference in their absorbency with respect to a
specific substance and according to their different pigments.
2. The separation of chemical substances and particles (originally
plant pigments and other highly coloured compounds) by
differential movement through a two- phase system is called as
chromatography.

29.  The mixture of materials to be separated is percolated through
a column or sheet of a some suitable chosen absorbent (eg: an
ion-exchange material); the substances least absorbed are least
retarded and emerge the earliest; those more strongly absorbed
emerge later. Absorption, absorption or affinity
chromatography are the synonymous word for
chromatography.
 The results of chromatographic separation are expressed in
terms of Rf value. It is defined as – the distance travelled by
the sample substance / distance travelled by the solvent.

30. Types of chromatography:
 Column chromatography
 Gas chromatography
 Gas-liquid chromatography
 Gel filtration chromatography
 High performance liquid chromatography
 High pressure liquid chromatography
 Ion exchange chromatography
 Liquid-liquid chromatography
 Paper chromatography
 Partition chromatography
 Reversed phase chromatography
 Thin – layer chromatography
 Two-dimensional chromatography
 For all the solid and liquid dosage forms and raw drugs these tests of
chromatography may be performed as a standardization parameter.

31. 18.Markers:
 Markers are chemically defined constituents or group of
constituents of herbal substance, an herbal preparation or an herbal
medical product which are of interest for control purposes
independent of whether they have any therapeutic activity. Markers
serve to calculate the quantity of herbal substance or herbal
preparation in the herbal medicinal product if the marker has been
quantitatively determined in the herbal substance or herbal
preparation.
 There are two categories of markers:
 Analytical markers: Solely for analytical purposes.
 Active markers: Generally accepted to contribute to the therapeutic
effect.

32. AAS: Atomic Absorption Spectrometer
 In this technique a light source emitting a narrow
spectral line of characteristic energy is used to
analysis of trace metal impurities from inorganic,
organic, polymer, geological and biological
samples up to ppb and in some cases in ppt level.

33. ICPAES: Inductively coupled plasma atomic
emission spectroscopy:
 It is also referred to as inductively coupled plasma
optical emission spectrometry (ICPOES), is an
analytical technique used for the detection of trace
metals. It is a type of emission spectroscopy that
uses the inductively coupled plasma to produce
excited atoms and ions that emit electromagnetic
radiation at wavelengths characteristic of a
particular element. The intensity of this emission is
indicative of the concentration of the element
within the sample.

34. XRD: X-ray diffraction:
 X-ray diffraction is a versatile, non-destructive
technique that reveals detailed information about
the chemical composition and crystallographic
structure of natural and manufactured drug
material.

35. XRF: X-ray fluorescence:
 The physics behind XRF is, when materials are
exposed to short wavelength x-rays or to gamma
rays, ionization of their component atoms may
take place. Ionization consists of the ejection of
one or more electrons from the atom, and may take
place if the atom is exposed to radiation with
energy greater than its ionization potential.

36. Sl. No Aushadha kalpana Analytical parameters
01 Panchavidha kashaya kalpana 1.Organoleptic characters
2.Viscosity
3.pH value
4.Specific gravity
5.Loss on drying
02 All upakalpanas 1.Organoleptic characters
2.Viscosity
3.pH value
4.Specific gravity
5.Loss on drying
03 Churna kalpana 1.Organoleptic characters
2.Viscosity
3.Total ash/Acid insoluble ash
4.pH
5.Water soluble/alcohol soluble extractives
6.Loss on drying at 1050C
7.Test for heavy metals
8.Microbial contamination

37. 04 Granules 1.Organoleptic characters
2.Bulk density
3.Tap density
4.Compressibility
5.Flow property
6.Total ash/acid insoluble ash
7.Water soluble ash /alcohol soluble extractives
8.Total sugar
9.Test for heavy metals
10.TLC/HPTLC
11.Microbial contamination
12.Pesticide residue
05 Kshara and sattva kalpana 1.Organoleptic characters
2.Loss on drying at 1050C
3.Ash value
4.Total/Acid insoluble ash
5.pH
6.Solubility
06 Masi & Lavana kalpana 1.Organoleptic characters
2.Solubility
3.pH value

38. 07 Arka kalpana 1.Organoleptic characters
2.pH value
3.Volatile matter
4.Specific gravity at 250C
5.Clarity test
6.Sterility test
7.Assay
8.TLC/HPTLC/GLC
08 Avaleha kalpana 1.Organoleptic characters
2. Loss on drying at 1050C
3. Total ash/acid insoluble ash
4. pH value
5. Specific gravity at 250C
6.Total solid content
7.Fat content
8.Total,reducing & non-reducing
sugars
9. TLC/HPTLC
10.Test for heavy toxic metals
11.Microbial contamination

39. 09 Khanda kalpana 1.Organoleptic characters
2. Loss on drying at 1050C
3. Total ash/acid insoluble ash
4. pH value
5. Reducing & non-reducing
sugars
6. Total solid content
7. Specific gravity at 250C
8. Fat content
10 Rasakriya / Ghanasattva
kalpana
1.Organoleptic characters
2. Loss on drying at 1050C
3. Total ash/acid insoluble ash
4. pH value
5. Particle size
6.Water/Alcohol soluble
extractives
7.Test for heavy/toxic metals
8.Microbial contamination
9.Test for specific pathogens
10.Pesticide residue
11.Test for
Aflatoxins(B1B2G1G2)
12.TLC/HPTLC

40. 11 Sharkara kalpana 1.Organoleptic characters
2.Loss on drying at 1050C
3.Total ash/acid insoluble ash
4. pH value
5.Total solids
6.Total/reducing sugars
7.Weight/ml
8.TLC/HPTLC
9.Test for heavy metals
10.Microbial contamination
11.Test for specific pathogens
12.Pesticide residue
12 Vati/Varti/Guggulu kalpana 1.Organoleptic characters
2.Identification
3.Hardness
4.Disintegration time
5.Melting temperature
6.Uniformity of weight
7. Loss on drying at 1050C
8. Total ash/acid insoluble ash
9.Resin content(in case of guggulu
kalpana)
10. Water/Alcohol soluble
extractives
11.Test for heavy toxic metals
12.Microbial contamination

41. 13 Ghrita/ taila kalpana 1.Organoleptic characters
2.Rancidity
3.Weight/ml (in case of taila)
4.Refractive Index at 250C
5.Viscosity
6.Iodine value
7.Saponification value
8.Acid value
9.Peroxide value
10.Free fatty acid
11.Total fatty matter
12.GLC/TLC/HPTLC
14 Sandhana kalpana
(Asava and arista)
1.Organoleptic characters
2.pH value
3.Specific gravity
4.Total solid content
5.Alcohol percentage
6.Reducing & non reducing
sugars
7. TLC/HPTLC
8.Test for Methanol
9.Total acidity

42. 15 Lepa, Malahara and upanaha
kalpana
1.Organoleptic characters
2.Uniformity of content
3.pH
4.Particle size
5.Thermal stability
6.Total fatty matter
7. Loss on drying at 1050C
8.Spreadability
9. Microbial contamination
16 Dhupa kalpana 1.Organoleptic characters
2.Loss on drying
3.Ash value
4.Acid insoluble ash
5.Solubility

43. 17 Netra kalpana 1.Organoleptic
characters
2.pH
3.Clarity test
4.Sterility test
5.Assay
6.TLC/HPTLC/GLC
18 Nasya kalpana Depends on form of
nasya dravya

44. DISCUSSION
&
CONCLUSION
 Analytical methods are the dimensions to evaluate
a product.
 Analytical standards speaks of
 Quality
 Authenticity and
 Purity.