– Sumit Goel.

distillate at 25 oC = B



“It should be a matter of conscience with him to be thoroughly convinced in every case that the patient always takes the right medicine and therefore he must give the patient the correctly chosen medicine prepared, moreover, by himself”.

APHORISM 265, 6TH EDITION, ORGANON OF MEDICINE The problems of quality control with homoeopathic potencies serve one of the areas of greatest challenge. The extremely high dilutions of homoeopathic potencies make it almost impossible to apply analytical tests by conventional methods in the laboratory. No existing tests have yet been able to standardize the high potencies of homoeopathic medicines. If homoeopathic preparations could be reproducibly measured by scientific instrumentation, this would be an important gain in quality assurance of the final product An analysis of homoeopathic potencies cannot reveal either the identity of the medication, neither the potency. Only mother tinctures can be subjected to a more comprehensive analysis, both qualitative and quantitative. The “in-process” quality control, embracing every step of the preparation, from raw material to finished products, is critical in ensuring the purity and safety of homoeopathic medicines.

Thus a system of quality control is even more vital in their manufacture than with their allopathic counterparts. As the homoeopathic medicinal agents consist of highly diluted and potentized medicines, care at each step of manufacture adds on to the quality and reliability of the final product.

Finished products in homoeopathic medicinal preparations include the mother tincture, liquid homoeopathic potencies and powder potencies and triturates.


Mother tincture is pharmaceutically prepared from a drug substance of plant or animal kingdom by the process of extraction (maceration or percolation) using a suitable menstruum, in a definite proportion as per pharmacopoeia. The mother tincture represents drug strength of 1/10, when prepared according to the modified method of preparation of mother tinctures.

Mother tinctures can be subjected to a qualitative and quantitative analysis that evaluates the identity, potency, purity and stability of the preparation. The following tests are conducted –

A. Alcohol content determination

B. Weight per ml

C. pH value

D. Total solids

E. UV absorbance

F. Identification and Assay

A. DETERMINATION OF ALCOHOL CONTENT Estimation of the alcohol content of the mother tincture is a test for the potency of the mother tincture and an evaluation of the extraction process. Alcohol content within the normal range indicates that proportion of drug and vehicle in the tincture is as per pharmacopoeial guidelines and that the tincture has uniform drug strength. Specific gravity of the distillate is determined pycnometrically and the corresponding alcohol contents in percent by volume read off in the alcoholometric tables.


* Sample taken at 25 oC = 25 ml

* Wt of empty specific gravity bottle = A

* Wt of specific gravity bottle + distillate at 25 oC = B

* Wt of specific gravity bottle + water at 25 oC = C

Specific gravity = B – A / C – A

Alcohol Content, expressed as Pecent v/v is obtained from reference table in pharmacopoeia.


WEIGHT PER MILLILITER Weight per milliliter of a liquid is determined by dividing the weight in air, expressed in grams, of the quantity of the liquid which fills a pycnometer at 20o or 25o by the capacity of the pycnometer at 20o or 25o respectively, expressed in milliliters.

The capacity of the pycnometer at these temperatures is ascertained from the weight in grams of the quantity of water required to fill the pycnometer.

The following data are assumed: – Wt. of 1 ml of water at

20o – – – – – – – – – – – – – – 0.99719 g

25o – – – – – – – – – – – – – – 0.99602 g

Ordinary deviations in the density of air do not affect the result of a determination significantly for pharmacopoeial purposes.


Weight per ml – at 25 oC

Wt of empty specific gravity bottle = A

Wt of specific gravity bottle + sample at 25 oC = B

Volume of specific gravity bottle at 25 oC = C

Wt per ml = ((B – A) / C) at 25 oC


The specific gravity of a substance is the weight of a given volume of that substance at a stated temperature as compared with the weight of an equal volume of water at the same temperature, all weighings being taken in air. A suitable pycnometer may be used fore the determination.


The pH value of an aqueous liquid may be defined as the common logarithm of the reciprocal of the hydrogen ion concentration expressed in gram per liter. This definition provides a fairly useful and practical means for quantitative indication of the acidity or alkalinity of a solution.

* The acid base balance in a liquid is important from the point of view of stability of a liquid in terms of its properties.

* Storage, exposure to temperature and pressure tend to alter the pH, thus making the liquid unstable.

* It also helps to detect adulteration.

* The pH must be controlled within pharmacopoeial limits, to ensure optimum stability and activity of the medicament. During the course of manufacture, the pH of the product can be influenced in various ways and estimation of pH is useful in determination of purity. Glass is a very useful material for storage, but it may impart alkalinity to products stored in glass containers. Precipitation can sometimes occur if the pH of the solution is changed significantly. The two most important methods of measuring pH are by the use of colored indicators or of special electrodes. According to HPI, the pH value of a liquid is determined potentiometrically by means of the glass electrode and a suitable pH meter. For the potentiometric measurements, the apparatus consists of a potentiometer – a circuit designed to give meter-needle deflection as a function of pH. Numerous indicator electrodes are available for the determination of pH, but the glass electrode is most widely used.

The glass electrode consists of a thin glass bulb of special glass blown at the end of a glass tube and the bulb is filled with dilute acid. A silver – silver chloride makes the necessary electrical connection with the acid whose pH remains constant. When the glass bulb is immersed in a solution of unknown pH, a potential is set up across the glass. To measure the pH of a solution, the bulb of the glass electrode and a suitable reference electrode is immersed in a sample of the solution and the two electrodes connected to a pH meter. The reference electrode is essential for the second electrical contact with the solution and must have a constant potential irrespective of the pH of the solution.

The electrical zero of the pH meter is adjusted, if necessary, and, with the electrodes immersed in a buffer solution of standard pH, the asymmetry potential control is altered until the meter reads the known pH of the buffer solution.

These standardized electrodes, after rinsing with distilled water, are then immersed in the test solution and the pH of the solution read directly in case of deflection meters.


The term ‘total solids’ is applied to the residue obtained when the prescribed amount of the mother tincture is dried to constant weight under specified conditions.


* Shallow, flat bottomed flanged dishes about 75 mm in diameter and about 25 mm deep made of Nickel or other suitable metal of high heat conductivity and which is not affected by boiling water.

* Water bath; hot air oven


* Weigh accurately or measure an accurate quantity of the preparation.

* Place it in a tarred dish, evaporate at as low a temperature as possible until the alcohol is removed.

* Heat on a water-bath until the residue is apparently dry.

* Transfer to an oven and dry to constant weight at 105 o. Owing to the hygroscopic nature of certain residues, it may be necessary to use dishes provided with well-fitting covers.

* Cool in an efficient desiccator.


* Weight of empty glass dish = A

* Sample taken = 10 ml

* Weight of dish + dry residue = B

Total solids = (B – A) X 100 / 10, expressed as percentage

E. (>) MAX

Molecules have translational, rotational, or vibrational modes. Molecules as result of these movements have energy. The energy from ground level gets excited if light or electromagnetic waves are passed through it. It is possible to effect a change in a particular type of molecular energy using appropriate frequency and wavelength of the incident radiation and measure them. This is the basis of spectroscopy.

Colored substances owe their colours due to presence of one or more unsaturated linkage (C=C, C=O, N=N, etc.) which are called chromophores. A substitution on chromophore may lead to increase in colour (C-Br, C-OH, C-NH2, etc.). They are called auxochrome. Normally such substitution leads to red-shift called bathochromic effect. Result of these leads to change in absorption. Intensity of emitted light will decrease as the thickness or concentration of absorbing media (drug solution) increase. The wavelength corresponding to maximum absorptivity is denoted by ?max.

The measurement of light absorption is made with spectrophotometers. The wavelength at which measurement is to be made may be in the visible or in the ultra-violent region as specified in the main text of the monograph. An instrument should be used which is suitable for the desired wavelength. Care should be taken to see that the solvent used for making solutions is free from fluorescence at the desired wavelength or wavelengths. The solvent used in the solvent cell must be from the same batch as the one used for preparing the solution for test.

Determination of Light absorption

When radiation is passed through a homogeneous solution containing an absorbing substance, part of the radiation is absorbed and the intensity of the radiation emerging from the solution is less than the intensity of the radiation entering it. The extent to which radiation absorbed in passing through a layer of an absorbing substance is expressed in terms of the extinction, E, defined by the E = log10 (Io)/I; expression where Io is the intensity of the radiation entering the absorbing layer; I is the intensity of the radiation emerging from the absorbing layer.

The extent of absorption in case of each absorbing substance depends on its concentration in the solution and the thickness of the absorbing layer taken for measurement. For convenience of reference and for each in calculations, the Extinction of a 1 cm layer of a 1 percent w/v solution of the substance has been given in this pharmacopoeia in the case of a few substances. For each absorbing substance there is one wavelength, or there are a few wavelengths, at which maximum absorptions take place and the values differ from one wavelength to another. It is therefore necessary to specify the wavelength at which the measurement is made. This property of light absorption is at times utilized for identifying substances, and assays where solutions can be obtained free from interfering materials and simpler methods were not found satisfactory.


Many substances – for example, quinine in solution in dilute sulphuric acid – when suitably illuminated, emit light of a different wavelength or colour from that which falls on them. This emitted light (fluorescence) ceases when the exciting light is removed.

Analytical tests based on fluorescence in daylight are not much used, as they are usually unreliable. Fluorescence lamps are usually fitted with a suitable filter that eliminates visible radiation from the lamp and transmits ultraviolet radiation of the desired wavelength.

* Aconitine – light blue

* Berberine – yellow

* Emetine – orange

* Cinchona bark – luminous yellow patches with a few light blue ones

* Inner surface of cinchona bark + dilute sulphuric acid – blue

* Wood of hydrastis rhizome – golden yellow

* Areca nuts, cut – light blue endosperm

* Most oils, fats and waxes show some fluorescence when examined in filtered UV light.

* The location of separated compounds on paper and TLC by use of UV light is extensively employed.


Chromatography is a separation process based upon the differential distribution of a mixture between two phases, one of which is percolated through other. It is a process for separating the components of a mixture by producing different rates of flow or movement for each component in a counter current system. In this, a liquid flows over a stationary solid phase, carrying with it solutes that have varying degrees of affinity for the stationary phase. Different rates of flow are thus produced for each solute and physical separation is achieved.

There are various methods of chromatography study – paper, thin layer, columnar, gas, etc. Each method has its special advantage. The fixed phase is called the stationary phase and the other is termed as the mobile phase. Specific requirements for chromatographic tests of drugs, including adsorbent and developing solvents, are given in individual monographs.

Chromatographic methods can be classified according to the nature of the stationary and mobile phases. The difference between adsorption and partition chromatography can be ascribed to the nature of the forces that influence the distribution of the solutes between the two phases.

* If the stationary phase is a solid, the process is called adsorption chromatography.

* If the stationary phase is a liquid, the process is called partition chromatography.

The types of chromatography useful in qualitative and quantitative analysis that are employed in the H.P.I. assays and tests are Paper Chromatography and Thin Layer Chromatography.

Use of reference substances in Identity Tests Rf value In paper and thin layer chromatography, the ratio of the distance traveled on the medium by a given compound to the distance traveled by the front of the mobile phase, from the point of the application of the test substance, is designated as the Rf value of the compound.

Rr value

The ratio between the distances traveled by a given compound and a reference substance is the Rr value.

Rf values vary with the experimental conditions, and thus identification is best accomplished where an authentic specimen of the compound in question is used as a reference substance. For this purpose, chromatograms are prepared by spotting on the thin layer adsorbant or on the paper in a straight line, parallel to the edge of the chromatographic plate or paper, solutions of the substance to be identified, the authentic specimen, and a mixture of nearly equal amounts of the substance to be identified and authentic specimen. Each sample application contains approximately the same quantity by weight of material to be chromatographed. If the substance to be identified and authentic specimen are identical, all chromatograms agree in color and Rf value and the mixed chromatogram yields a single spot, i.e. Rr is 1.0.

Location of the Spots

The spots produced by the chromatographed materials may be located by:

(1) Direct inspection if the compounds are visible under white or ultraviolet light.

(2) Inspection in white or UV light after treatment with reagents that will make the spots visible in paper and thin layer chromatography – for example, Dragendorff’s agent, iodine vapour, antimony trichloride in chloroform, etc.


In paper chromatography the adsorbant is a sheet of paper of suitable texture and thickness. The paper chromatography is of following types:

Descending Chromatography

Separation of chemical compounds by descending chromatography is accomplished by a procedure of allowing the mobile phase to flow downward on the chromatographic sheet.

* The substances to be analyzed are dissolved in a suitable solvent.

* Convenient volumes, delivered from suitable micro pipettes, of the resulting solution, normally containing 1 to 20 microgram of the compound, are placed in 6 to 10 mm spots along the pencil line not less than 13 cm apart. If the total volume to be applied would produce spots of a diameter greater than 6 to 10 mm, it is applied in separate portions to the same spot, each portion being allowed to dry before the next is added.

* The spotted chromatographic sheet is suspended in the chamber by use of the antisiphoning rod, which holds the upper end of the sheet in the solvent trough.

* The bottom of the chamber is covered with the prescribed solvent system.

* It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber without touching the rack or the chamber walls or the fluid on the bottom of the chamber.

* The chamber is sealed to allow saturation of the chamber and the paper with the solvent vapour. Any excess pressure is released as necessary. For large chambers, saturation over night may be necessary.

* After saturation of the chamber the prepared solvent is introduced into the trough. The solvent is allowed to travel down the paper to the desired distance. Precautions must be taken against allowing the solvent to run down the sheet when opening the chamber and removing the chromatogram.

* The location of the solvent front is quickly marked, and the sheets are dried, the spots visualized and Rf values calculated.

* If the compounds being separated are colourless, their positions on the paper may be determined by spraying the paper with a suitable reagent that produces a colour.

Ascending Chromatography In ascending chromatography the lower edge of the sheet is dipped into the mobile phase, to permit the mobile phase to rise on the chromatographic sheet.

* The test materials are applied to the chromatographic sheets as directed under Descending Chromatography, and above the level to which the paper is dipped into the developing solvent.

* Empty solvent troughs are placed on the bottom of the chamber, and the chromatographic sheets are suspended so that the end on which the spots have been added hangs free inside the empty trough.

* The chamber is sealed, and saturation is allowed to proceed as directed under Descending Chromatography.

* Then the solvent is added through the inlet to the trough in excess of the solvent required for complete moistening of the chromatographic sheet.

* The chamber is resealed when the solvent front has reached the desired height, the chamber is opened and the sheet is removed and dried.

* Further procedure maybe conducted as described under Descending chromatography.


In thin layer chromatography, the adsorbant is a powdered material applied usually to a glass plate. Silica gel is slightly acidic and therefore is best applied to the separation of neutral and acidic substances. Alumina on the other hand is basic and should be used for the separation of basic compounds. The separations achieved may be based upon adsorption, partition or a combination of both effects, depending on its use with different solvents. Quantitative measurements are possible by removing the spots from the plate with a suitable solvent. For two dimensional thin layer chromatography, the chromatographed plate is turned at a right angle and again chromatographed, usually in another chamber saturated with a different solvent system.

In short, the method consists of preparing, on a suitable glass plate, a thin layer of material, which may be either an adsorbent as used in column adsorption chromatography or an inert support that holds an aqueous phase as in column partition chromatography. The mother tincture / mixtures to be resolved are dissolved in a suitable solvent and placed as a series of spots on the film towards one end of the plate; this end is dipped in a suitable solvent mixture and the whole enclosed in an airtight container. The solvent front travels up the film and after a suitable time the plate is removed, the solvent front marked, the solvent allowed to evaporate and the positions of the separated compounds determined by suitable means.

* Arrange the neat and clean plates on the aligning tray, and secure them so that they will not slip during the application of the adsorbant.

* Mix appropriate quantities of adsorbant and liquid, usually water, which when shaken for 30 seconds give a smooth slurry that will spread evenly with the aid of spreader. Generally an analytical plate has an adsorbant thickness of 250 micrometer to 500 micrometer, while a preparative plate has a thickness of 500 micrometer to 2000 micrometer. Allow the plates to remain undisturbed for 15 minutes and then dry at 105o for 30 minutes. Store the prepared plates in a desiccator.

* Apply the sample solution and the standard solution by means of suitable micro pipettes at points about 1.5 cm apart and about 2 cm from the lower edge of the plate, and allow to dry.

* Place the plate in the developing chamber. The solvent in the chamber must be deep enough to reach the lower edge of the adsorbant, but must not touch the spot points.

* Seal the cover in place and maintain the system until the solvent front ascends; this commonly requires from 15 minutes to 1 hour.

* Remove the plates, dry them in air and observe first under ultraviolet light.

* Measure and record the distance of each spot from the point of origin.

* If further directed, spray the spots with the reagent specified, observe, and compare the sample with the standard chromatogram.

Till such time the Rf of individual drug is prescribed, all mother tinctures should pass Co-TLC with the reported main constituents subject to the condition that they are not less than 1 per cent w/w in raw material. Co-TLC with tinctures made from authenticated raw material is also permitted.


Recent advances in instrumental methods of analysis have helped to establish these techniques for analysis of homoeopathic medicines.


The electromagnetic vibrations utilized in spectroscopic analysis can be roughly divided, according to wavelength, into the ultraviolet (185 – 380 nm), the visible (380 – 780 nm), the near infrared (780 – 3000 nm) and the infrared (3 – 40 ?m) regions. Spectroscopic analysis is concerned with the capacity of certain molecules to absorb vibrations at specific wavelengths. In the ultraviolet and visible regions, the characteristic absorption spectrum of a molecule is produced by changes in the electronic levels associated with various chromophoric groups within the molecule. These changes involve the absorption of relatively high amounts of energy and are also accompanied by changes in vibrational and rotational energy changes within the molecule.


* Principal applications

Structure determination and identity of organic and inorganic compounds – quantitative analysis.

* Molecular Phenomenon

Excitation of molecular vibrations by light absorption.


* Principal applications

Quantitative analysis, especially as end methods in chemical analysis schemes.

* Molecular Phenomenon

Excitation of loosely bonded electrons


* Principal applications

Structure determination and identity of organic compounds; symmetry of molecular groups in solid state.

* Molecular Phenomenon

Excitation of molecular vibrations by light scattering.


* Principal applications

Structure determination and identity of organic compounds; molecular conformation

* Molecular Phenomenon

Reorientation of magnetic nuclei in a magnetic field


* Principal applications

General multicomponent quantitative analysis of volatile organics. Highly efficient separation technique.

* Molecular Phenomenon – partitioning between vapour phase and substrate


* Principal applications

Identification of crystalline substances, especially inorganic. Determination of crystallinity, especially polymers.

* Molecular Phenomenon

Diffraction of X-rays from crystal planes.


* Principal applications

Precision quantitative analysis for a given metal. Trace analysis for a given metal.

* Molecular Phenomenon

Absorption of atomic resonance line.


* HPLC is a liquid column chromatography system that employs relatively narrow columns operating at ambient temperature or upto about 200oC at pressures upto 200 atm (20000 kPa).

* HPLC can give much more improved and more rapid separations.


* This consists of a liquid stationary phase and a mobile gaseous phase. Some pharmacognostical examples of its application include examination of many volatile oils, camphor, etc.














4. Chromatography

(TLC on Silica Gel G)


Ref Std:

Solvent System:

Spray Reagent:


ASSAY FOR PURITY Alcohol Content:

(v/v at 20oC)


Wt per ml at 25oC:

Total Solids:

(>)(>) max:





Alcohol content : 38.0 to 42.0 Pecent v/v

pH : Between 5.1 and 6.1

Specific gravity : From 0.933 to 0.970

Total solids : Not less than 1.8 Pecent w/w

Lambda max : 256 and 290 nm

Identification : Colour Test –

To 1 ml of chloroform layer of mother tincture, add one drop of sulphuric acid, the chloroform layer turns green.

Carry out TLC of mother tincture using Chloroform methanol (8:2 v/v) as mobile phase. Three spots appear at Rf 0.03, 0.11 and 0.98

* CANTHARIS : Mother tincture

Alcohol content : 87.0 to 91.0 Pecent v/v

pH : Between 9.5 and 10.2

Specific gravity : From 0.810 to 0.840

Total solids : Not less than 1.2 Pecent w/w

Lambda max : 265 and 223 nm

Identification : Carry out TLC of mother tincture using cyclohexane – acetone

(1:1 v/v) as mobile phase and spray with 2:4 dinitrophenyl hydrazine solution. Four spots appear at Rf 0.52, 0.68, 0.86 and 0.94, all are red. 0.94 corresponds with Cantharidine.

OR Carry out Co-TLC of mother tincture on Silica Gel (G) with cantharidine using cyclohexane acetone (9:1 v/v) as mobile phase and 2:4 dinitrophenyl hydrazine solution for spray. Red spots corresponding to Cantharidine apear.


* What is standardization? Discuss the parameters for standardization of homoeopathic mother tinctures.

* Discuss Identification tests for standardization of mother tinctures.


1. Identification of the mother tincture is confirmed by

(a) Alcohol content determination

(b) pH value

(c) Total solids

(d) Chromatography

2. Determination of alcohol content is a test for

(a) Identity of mother tincture

(b) Potency of mother tincture

(c) Purity of mother tincture

(d) Stability of mother tincture

3. The residue obtained when the prescribed amount of the mother tincture is dried to constant weight under specified conditions is termed as

(a) Total solids

(b) Foreign organic matter

(c) Acid insoluble ash

(d) Adulterant

4. Aconitine exhibits fluorescence of

(a) Orange color

(b) Yellow color

(c) Light blue color

(d) Golden yellow color

5. The ratio of the distance travelled on the medium by a given compound to the distance travelled by the front of the mobile phase, from the point of the application of the test substance is called as

(a) Rr value

(b) Rf value

(c) Specific gravity

(d) pH value

ANSWERS: 1 (d); 2 (b); 3 (a); 4 (c); 5 (b) .

Dr.Devendra Kumar MD(Homeo)
International Homeopathic Consultant at Ushahomeopathy
I am a Homeopathic Physician. I am practicing Homeopathy since 20 years. I treat all kinds of Chronic and Acute complaints with Homeopathic Medicines. Even Emergency conditions can be treated with Homeopathy if case is properly managed. know more about me and my research on my blog https://www.homeoresearch.com/about-me/
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