Application of Analytical techniques in characterization ofCalx, a dosage form of traditional systems of medicine

Introduction

Traditional systems of medicine (TSM) use drugs of minerals and biological origin. Most metals/ minerals and some biological origin drugs intended for internal use are taken in calcined form called calx¹ . Calx is a traditional method of preparation where metals are converted into digestible oxide or carbonate. Metals in calx form have enhanced bioavailability and also act as catalyst for physiological processes^2,3. The quality of raw materials and good manufacturing practice determine the purity and quality of calx. Use of inferior grade raw material, adulteration and deviations in standard manufacturing practices either intentionally or accidentally, leads to the production of inferior quality products, which can be hazardous to use. Analytical techniques help in ensuring the purity, efficacy and safety of the end product⁴ . Although, the concept of testing purity and perfectness of calx is there in ancient literature, the classical methods are not effective for detecting the traces of impurity and toxicity in the preparation. Analytical techniques are of utmost importance like X-ray Diffraction (XRD), X-ray Fluorescence (XRF), Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Atomic Absorption Spectroscopy (AAS), Flame Atomic Absorption Spectroscopy (FAAS), Graphite Furnace Atomic Absorption Spectroscopy (GFAAS), Scanning Electron Microscope (SEM), Transmission Electron microscopy (TEM), Electro Probe Micro analyzer/ European Powder Metallurgy Association (EPMA), Electron Spectroscopy for Chemical Analysis (ESCA), Thermo gravimetric analysis (TGA), Energy Dispersive X-Ray (EDX/EDAX), Energy Dispersive Spectroscopy (EDS), Differential Scanning Calorimetry (DSC), Microwave PlasmaAtomic Emission Spectrometer (MP-AES), Dynamic Light scattering method (DLS), Fourier Transform Infrared Spectroscopy (FTIR), Raman spectroscopy, Mossbauer spectroscopy, U-V light, Ion Chromatography (IC) etc.

Modern analytical techniques which takes into account the qualitative and quantitative difference in inorganic as well as organic chemical constituents in calx, particle size, elemental phase detection, functional group, estimation of heavy metals (qualitative as well as quantitative), elemental composition, surface morphology and surface topography etc. is more effective than the traditional ones⁵ . This review describes the details and application of these analytical techniques in characterizing calx. In this paper, authors have tried to explain the basic principles, advantages, disadvantages and application of various analytical techniques in the process of characterization of calx.

2. Material and methods

A review of literature on calx and application of modern analytical techniques was undertaken using the bibliographic database viz. Pub Med, Google Scholar, Science Direct, Scopus, Relevant articles, periodicals, peer reviewed indexed journals and other published works available on the journals which follow COPE (Committee on publication Ethics: www. publication ethics.org) guidelines and other standard internet sources were used to regain online literature. The search was conducted using the terms ‘Kushta, Calx, Bhasma, Parpam, XRD, XRF, ICP-MS, ICP-AES, ICP-OES, AAS, FAAS, GFAAS, SEM, TEM, EPMA, ESCA, TGA, EDAX, DSC, MP-AES, FTIR etc. Books published in Urdu and English were used to compile classical information.

3. Observation and Results

3.1.1 Analysis of the perfectly prepared calx in TSM: Perfectly prepared calx in TSM is ensured by the following tests: 1) there should be no metallic lustre when calx is kept on sunlight. 2) In finger test, when small pinch of calx taken between index finger and thumb and spread, it should be as fine as to get easily into finger lines. 3) In floating test, when a small quantity of calx is spread on cold and still water, it should float on the surface. 4) The calx should not revert to the original state. 5) In wall stick test, when a pinch of calx thrown on the wall, it should sticks on the wall indicate perfect and standard calx. 6) In curd test, when small quantity of calx is put in a petri dish to see a color change. 7) In lemon test, small amount of calx is put in lemon juice in a test tube to see color change. In both curd test and lemon test if the color is not changed means calx is perfectly prepared and genuine. Calx, unless otherwise specified in individual formulations, are generally whitish, yellowish, black and red colored, depending upon the predominant drugs as well as the other drugs used in the process of preparation. The calx should be always odorless and tasteless, which are the important parameters for characterization of calx in TSM⁶ .

3.1.2 Purity assessment of various Calx in TSM⁷ .

3.2 X-RD: X-RD is a technique through which the special arrangement of structural units of substance in crystalline state is known. This technique is used for the determination of crystal structure, chemical analysis, stress measurement, study of phase equilibria, and measurement of particle size determination of the orientation of one crystal or orientations in a polycrystalline aggregate. It can be applied for differentiation among various Oxides/ Sulphide of metals, sample can be easily prepared, large library of known structure is available for comparison, it is not a destructive method. It works on the Principle of the Bragg law. The particle size (D) of the phase in sample can be calculated using the Scherrer’s formula: D = 0.9 λ / βcosθ, where λ is the wavelength of the X-ray use (1.5406 Å), β is the full width at half maximum of the Bragg peak, and θ is the Bragg angle of the Bragg peak (peak position). Basic relationship for determination of particle size using XRD pattern is 0.9λ/βcosθ. There are some limitations of XRD, it does not help in case of amorphous solid and trace element detection is often difficult^8,9.

Pareek et al., (2018) reported a study in which XRD of raw metal showed crystallite peaks of Zinc metal whereas that of Yashada bhasma sample showed hexagonal crystallite ZnO peaks in its very effective Zincite form. The average crystallite size of ZnO nano particle from Scherer's formula was found to be 32.79 nm. The absence of any crystallite zinc metal peaks and presence of nano size particles is confirmed. It is observed that zinc which was present in raw metal had converted into ZnO nanoparticles after proper incineration process and this gets easily absorbed at targeted site as it has particle size of 32 nm as particles less than 100 nm can be easily absorbed by intestine. Particle size analysis of both samples shows reduction in size, as the mean particle diameter of raw metal was found to be 2063 nm and that of Yashada bhasma was found to be 340 nm¹⁰.

3.3 XRF: XRF is an analytical technique used to determine the chemical composition of all kinds of materials. The materials can be in solid, liquid, powder, filtered or other form. XRF can be also sometimes used to determine the thickness and composition of layers and coatings. The method is fast, accurate and non-destructive, and usually requires only a minimum of sample preparation. In XRF, X-rays produced by a source irradiates the sample. In most cases, the source is an X-ray tube but alternatively it could be a radioactive material. The elements present in the sample will emit fluorescent X-ray radiation with discrete energies that are characteristic for these elements. By measuring the energies of the radiation emitted by the sample it is possible to determine which of the elements is present. This step is called qualitative analysis. By measuring the intensities of the emitted energies (colors) it is possible to determine how much of each element is present in the sample. This step is called quantitative analysis¹¹.

Gupta et al., (2014) reported XRF analysis Yasad sample have 98.20% zinc metal along with other trace elements like Pb-0.63%, Sn-0.11%, Fe-0.56%, Ca-0.07%, Al-0.09%, Cr- 0.06% and in Yasad bhasma 98.20% zinc oxide was present along with trace element like Fe2O3-2.6%, K2O-0.8%, Al2O3-0.32%, PbO-0.2% etc. XRF showed that during process of Yasad bhasma formation zinc element converted to zinc oxide, Jarita Yasad showed higher percentage of zinc oxide than Yasad bhasma this indicated that maximum oxidation of Yasad took place in jarana process. Yasad bhasma have higher percentage of Iron oxide than Jarita Yasad this showed that oxidation of iron took place in marana process. So, oxidation of zinc takes place at lower temperature i.e. during Jaran process of Yasad and oxidation of iron take place at higher temperature i.e. during Maran process of Yasad. Beside this percentage of lead, tin, silica and potassium oxide decreased in Yasad bhasma than Jarita Yasad which might be due to chemical reaction taking place during Jaran and Maran process¹².

3.4 AAS: AAS measures the concentrations of metals in the samples. AAS is so sensitive that it can measure down to ppb of a gram in a sample. The technique makes use of the wavelengths of light specifically absorbed by an element. In their elemental form, metals will absorb UV when they are excited by heat. Each metal has a characteristic wavelength that will be absorbed. The AAS looks for a particular metal by focusing a beam of UV light at a specific wavelength through a flame and into a detector. The sample of interest is aspirated into the flame. If that metal is present in the sample, it will absorb some of the light, thus reducing its intensity. These instruments measure the change in intensity. A computer data system converts the change in intensity into an absorbance¹³.

Saha D et al., (2017) reported a study in which AAS was helpful in the quantitative determination of metal which is present in Kasisa Bhasma. The analysis was done by using the different lamp for different heavy metals to detect their presence in the sample and to determine the iron percentage of each sample. The percentage of iron determined by this method was found to be 27.6% in raw, 43.1% in Shodhita and 63.3% in Bhasma of Kasisa whereas zinc, cadmium, and lead was found to be very less amount in all samples¹⁴.

3.5 ICP-MS: ICP-MS is a multi elemental analytical method. Advantages of ICP-MS is very low limits of detection in the range of ppm or below for solid sample in the range of ppb or below for solution sample¹⁵. In ICP-MS, the aim is to maximize singly charged ions and minimize multiply charged ions. Most ICP-MS detection systems use electron multipliers, which convert ion currents into electrical signals. The magnitude of the electrical signal is proportional to the number of analytes ions present in the sample¹⁶. Using ICP-MS, all kinds of materials can be measured; the ICP source converts the atoms of the elements into the sample to ions. These ions are then separated and detected by the mass spectrometer. Solutions are vaporized using a nebulizer, while solids can be sampled using laser ablation. Gases can be sampled directly. To cover the whole mass range for all elements, a simple quadruple mass analyzer provides ample resolution to detect all the isotopes of an element differing by 1 atomic mass unit¹⁷. Disadvantages of ICP-MS of the lower-cost ICP-MS systems utilize single quadruple mass analyzer systems, which are inherently sequential systems, and have relatively low mass resolution. The presence of oxides and doubly charged ions in the plasma deteriorates the quantitative capability of ICP- MS in ultra trace analysis. ICP-MS instruments are more susceptible to instability than ICP-AES instruments when running samples with higher levels of total dissolved solids¹⁸.

Ali, et al., (2018) conducted a study in which results of ICP-OES and ICP-MS analysis by both the processes showed the absence of mercury (Hg < 0.1 mg/kg) but showed the presence of Fe and Al in mg/kg. Aluminum was present in better quantity than iron, whereas presence of arsenic (As) was also noted in ppm levels. In ICP-OES analysis, the results were on borderline of permissible limit of As, that is, 3 ppm. The percentage of sulfur in KSCM and KSMFM was found to be 24.05% and 22.08%, respectively. Sulfur percentage is more in KSCM as compared to KSMFM, indicating probably less evaporation in classical method because of less heat exposure in comparison to muffle furnace method¹⁹.

3.6 ICP-AES: ICP-AES is a technique used for the detection of chemical elements. 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. It is a flame technique with a flame temperature in a range from 6000 to 10,000 K. The intensity of this emission is indicative of the concentration of the element within the sample.

Chandran S et al., (2017) reported a study on Particle size estimation and elemental analysis of Yashada Bhasma. Elements such as Cu, Fe, Mg, P, Al, Pb, and Zn were present in ≤1% and elements such as Ti, Co, Mn, Ni, Cr, and Cd were detected in ppm level in both JMY and PMY samples, but 0.309% of arsenic and 31.1 ppm mercury were detected in PMY sample only²⁰.

Bhowmick et al. (2009) studied physicochemical analysis of Jasada bhasma using ICP-AES and EDAX analysis techniques. They have reported 88.32 wt% soluble matters in the Jasada bhasma and remaining 11.70% oxygen analyzed by EDAXSEM. X-ray diffraction result showed the absence of crystalline zinc metal in Jasada bhasma. There was shortage of oxygen at the surface of the bhasma which was supported by X-ray photon spectroscopy analysis²¹.

3.7 ICP-OES: ICP-OES is a powerful and widely used multi element analysis technique used for the determination of traces elements using inductively coupled plasma source to dissociate the sample into its constituent atoms or ions, exciting them to a level where they emit light at a characteristic wavelength²². Detection limits are in single and sub-parts ppb range. Limitations are moderate to low detection limits (but often much better than FAAS), Spectral interferences possible, some element limitations²³. Kantham, L et al., (2017) conducted a study on Acute, Sub-acute, and Subchronic oral toxicity studies of Nandhi Mezhugu. ICP-OES detection of heavy metal traces in animal tissue samples in sub-chronic toxicity study (high dose group) was analyzed. Acute toxicity study showed no mortality and no treatment-related toxicity signs. Similarly, sub-acute and sub-chronic toxicity studies showed no treatment-related abnormalities at the high dose levels such as 90 and 110 mg/kg body weight in rats respectively²⁴.

3.8 FAAS: The sample atomization occurs when a liquid sample is drawn into a flame. The flame serves as a “sample holder”, as the light passes through the atoms and flame simultaneously, and the absorbance of light calculated. We let through the fire a light beam with such a wavelength that can be absorbed by ground state atoms and thus we measure the decrease of light intensity. FAAS instrument has high sensitivity and the capability to analyze many elements in complex samples. These include the following elements: As Ca Cd Co Cu Fe Mg K Ag Na Zn. For most of these elements, a Beer’s law relationship will hold between approximately 0.5 and 15-20 ppm. This means that FAAS will not be able to determine the concentration of an analyte that is below or above this range. For this reason, the dilution of an unknown sample is frequently required. The analysis of this diluted solution, the original (non-diluted) concentration may be calculated. Although AFS is rather sensitive, the potential of the method compared to FAAS only for a few elements is comparable, thus it is relatively less popular. Recently flameless atomization (graphite furnace atomic spectroscopy, GFAAS) keeps getting more and more significant since it has small sample volume demand, high sensitivity and good detection limits²⁵.

Uzdavinien D et al., (2007) reported a study on determination of calcium in mineral waters by FAAS. The determination of calcium by FAAS method was investigated and optimized. It can be seen that the concentration of Ca in the tested water samples ranges from 4.5 to 220 mg/L. It is quite a wide range of Ca concentration; thus, the accuracy and reliability of the results were assessed by analyzing the model types of mineral water. The findings of the analysis suggested Ca determination method provides quite reliable results. The Ca ranges from 2.4 to 205 mg/L, and the standard deviation ranges from 1.5 to 4.0%²⁶.

3.9 GFAAS: GFAAS is an analytical technique designed to perform the quantitative analysis of metals in a wide variety of samples. Solid sampling GFAAS has been used for the determination of traces of heavy metals (Cd, Pb, Cu, Cr, Ni, V and As) in barites over a wide concentration range, e.g. Cd from 0.023 to 27.0 μg/g and Pb from 1.54 to 3509 μg/g²⁷. Metals in solution may be readily determined by GFAA. The method is simple, quick, and applicable to a large number of metals in environmental samples. With the exception of the analyses for dissolved constituents, all samples require digestion prior to analysis. Analysis for dissolved elements does not require digestion if the sample has been filtered and then acidified. It exhibits high sensitivity primarily because there are no flame gases to dilute the free, gaseous atoms that are analyzed. Principle: The GFAAS experiment follows the Beer’s laws of Spectrophotometry²⁸.

Khalid RS et al., (2016) reported a study on reliability of GFAAS as alternative method for trace analysis of arsenic in natural medicinal products. GFAAS technique had a substantial improvement in analytical sensitivity after the introduction of matrix modifiers Pd-Mg salts. GFAAS has good detection limits for a majority of elements, with a small sample size for analysis 20 μl and minimum requirements for sample preparation²⁹.

3.10 SEM: High-energy electron beam is focused to the sample which is kept inside the microscopes vacuum column evaporator. Because of the interactions of electrons and the surface of the sample, emission of electrons takes place which is collected by an appropriate detector. SEM is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that can be detected and that contain information about the sample's surface morphology, topography and composition. SEM can achieve resolution better than 1 nanometer. Specimens can be observed in high vacuum, in low vacuum, in wet conditions (in environmental SEM), and at a wide range of cryogenic or elevated temperatures. Due to the very narrow electron beam, SEM micrographs have a large depth of field yielding a characteristic three dimensional appearance useful for understanding the surface structure of a sample. Characteristic X-rays are emitted when the electron beam removes an inner shell electron from the sample, causing a higher energy electron to fill the shell and release energy. These characteristic X-rays are used to identify the composition and measure the abundance of elements in the sample³⁰.

Kale B et al., (2017) conducted a study on Synthesis and characterization of Vanga bhasma. The study reveals that the synthesized Bhasma was converted into its nontoxic oxide form and had a highly reduced particle size observed from SEM images. These studies reveal that Vanga Bhasma prepared by traditional method of heating has 50% nanoparticles (NPs) (150-300 nm range) that prepared by using electric muffle furnace has 100% nanoparticles (50-100 nm range) while commercial  samples has 50% NPs (100-300 nm range). Study also confirmed the formation of organometallic compound (SnO2) at the end of the manufacturing process. The percentage bio accessibility for gastrointestinal digestion is more than the gastric digestion³¹.

3.11 TEM: TEM uses high energy electrons to penetrate through a thin (≤100 nm) sample. This offers increased spatial resolution in imaging (down to individual atoms) as well as the possibility of carrying out diffraction from nano sized volumes³². The TEM operates on the same basic principles as the light microscope but uses electrons instead of light. TEMs are capable of imaging at a significantly higher resolution than light microscopes, owing to the small de Broglie wavelength of electrons. TEM forms a major analysis method in a range of scientific fields, in both physical and biological sciences. TEMs find application in cancer research, virology and nanotechnology research³³.

Lassoued, A et al (2017) conducted a study on control of the shape and size of iron oxide (α-Fe2O3) NPs synthesized through the chemical precipitation method. Size and morphology of the NPs are determined by analyzing the recorded TEM images. The TEM images of α -Fe2O3 NPs obtained from precipitation method and calcined at 700 oC for 4 h. After the heat treatment at 700 oC for 4 h, the α-Fe2O3 particles were found in the range of 21-82 nm. It is clear that hematite NPs are mainly present as granules with small and big spherical shaped particles and are well crystalline in nature. The NPs size depends largely on the concentration of precursor used in the synthesis of hematite, which proved that the particle size increased with the rise of the concentration of precursor (FeCl3, 6H2O) because the reactant with the higher concentration enhanced the merge of crystal nucleus and agglomeration of particles³⁴.

3.12 EPMA: EPMA is also informally called an electron microprobe, or just probe. An EPMA is a micro beam instrument used primarily for the in situ nondestructive chemical analysis of minute solid samples. It is fundamentally the same as an SEM, with the added capability of chemical analysis. The primary importance of an EPMA is the ability to acquire precise, quantitative elemental analyses at very small "spot" sizes (as little as 1-2 microns). The spatial scale of analysis, combined with the ability to create detailed images of the sample, makes it possible to analyze geological materials in situ and to resolve complex chemical variation within single phases. The technique is capable of high spatial resolution (~1um) and relatively high analytical sensitivity (<0.5% for major elements) and detection limits (~100 ppm for trace elements). It is normally equipped with up to 5 wavelength dispersive spectrometers. They also contain: an electron gun, a high vacuum (10-6 torr), scanning coils, secondary electron detectors, backscattered electron detectors, cathode-luminescence (CL) detectors, EDS detectors³⁵.

3.13 ESCA: X-ray Photoelectron Spectroscopy (XPS) is also known as ESCA. ESCA is based on the photoelectron effect. A high energy X-ray photon can ionize an atom, producing an ejected free electron with kinetic energy. ESCA uses a probe beam of X-rays of a single energy. Since it is difficult to focus X-rays, the beam diameter is typically 5-10 mm for a traditional X-ray source and 0.5-3 mm for a monochromatic X-ray source. The X-rays penetrate several micrometers into the sample at typical ESCA source energies, liberating electrons from the atoms of the sample. Measuring the kinetic energy of the electron as it is collected therefore allows the binding energy to be computed. The binding energy tells us not only what element the electron came from, but also was the chemical state. ESCA provides unique information about chemical composition and chemical state of a surface. Advantages are surface sensitive, wide range of solids and relatively non-destructive. Disadvantages are expensive, slow, poor spatial resolution, and requires high vacuum³⁶.

Salama W, et al (2018) conducted a study in which XPS survey scans indicate the presence of Fe, O, C, N, Na, Cl, Ca and Si in all type of laminae, while S, Zn, Ti and P are only restricted to the green laminae. The high resolution of the Fe 2p3/2 indicates that Fe is linked to OH ligand in the yellowish-brown laminae that correspond to goethite, while Fe is linked to SO4 -2 ligand in the green laminae. The XPS survey scans of types 2 & 3 indicate that Fe is linked to O2 ligand that corresponds to hematite³⁷.

3.14 EDAX: EDAX analysis helps to carry out compositional analysis of sample. The data produced by the EDAX analysis consists of the spectra containing the elements present in the given sample which was being analyzed ^{[38, 39]}.

Kannan N, et al (2017) conducted a study on structural and elemental characterization of traditional Indian Siddha formulation: Thalagak karuppu. The selected portion of the EDX spectrum reveals the presence of As (46.65%), S (29.02%), O (23.41%) and Ca (0.92%), the result suggest that the formulation may contain As, S and O. The proportion of sulphur is more than oxygen to bind with As⁴⁰.

3.15 DSC: DSC is a fundamental tool in thermal analysis, it is a technique for measuring the energy necessary to establish nearly zero temperature deference between a substance and an inert reference material, as the two specimens are subjected to identical temperature regimes in an environment heated or cooled at a controlled rate. DSC is also monitors heat effects associated with phase transitions and chemical reactions as a function of temperature. In a DSC the difference in heat flow to the sample and a reference at the same temperature, is recorded as a function of temperature. It can be used pharmaceuticals, to nano materials and food products. Use to understand amorphous and crystalline behaviour, polymorph and eutectic transitions⁴¹. In heat flux DSC, the sample and reference are connected by a low resistance heat flow path. The assembly is enclosed in a single furnace. Enthalpy or heat capacity changes in the sample cause deference in its temperature relative to the reference; the resulting heat flow is small compared with that in deferential thermal analysis because the sample and reference are in good thermal contact. The temperature deference is recorded and related to enthalpy change in the sample using calibration experiments⁴². Singh et al. reported a study on Naga Bhasma in which TGA measurements accompanied with the DSC scan shows weight loss from beginning. The weight loss increases with temperature and nearly 12% weight loss is observed up to 850°. This weight loss is high enough and cannot be explained only on the basis of the evolution of hydrogen which suggests desorption of hydrocarbon gases as well. It is notable that desorption of hydrocarbon gases are not due to oxidation of carbon compounds but this may be due to thermal cracking phenomenon⁴³.

3.16 FTIR: FTIR provides specific information about the vibration and rotation of the chemical bonding and molecular structures, making it useful for analyzing organic materials and certain inorganic materials. An infrared spectrum represents a fingerprint of a sample with absorption peaks which correspond to the frequencies of vibrations between the bonds of the atoms making up the material. Because each different material is a unique combination of atoms, no two compounds produce the exact same infrared spectrum. Therefore, infrared spectroscopy can result in a positive identification (qualitative analysis) of every different kind of material. In addition, the size of the peaks in the spectrum is a direct indication of the amount of material present. With modern software algorithms, infrared is an excellent tool for quantitative analysis. The IR region is commonly divided into three smaller areas: near-IR (400-10 cm-1), mid-IR (4000-400 cm-1), and far-IR (14000-4000 cm-1). Chemical bonds vibrate at characteristic frequencies, and when exposed to infrared radiation, they absorb the radiation at frequencies that match their vibration modes. Measuring the radiation absorption frequency produces a spectrum that can be used to identify functional groups and compounds. Some impurities produce their own characteristic bands in infrared region. The FTIR spectrometer has several major advantages over the dispersive instrument. Its sensitivity is better because it measures all frequencies simultaneously rather than scanning through the individual frequencies. Less energy is needed from the source and less time (typically 1 to 2 seconds) is needed for a scan. Several scans can be completed in a few seconds and averaged to improve the signal. Few limitations of FTIR spectrometer are listed as, minimal elemental information is given for most samples, background solvent or solid matrix must be relatively transparent in the spectral region of interest and molecule must be active in the IR region^44,45.

Singh RK, et al (2017) conducted a study on physical properties of Ayurvedic nano crystalline Tamra Bhasma by employing modern scientific tools. X-RD analysis and SEM results revealed that the crystalline size of Bhasma powder was less than 100 nm and nano crystallites of agglomerated size in micrometer. The single carbon and chlorine (C-Cl) bond with stretching vibration were in the range of 80-700 cm-1. The carbon oxygen stretching bond (C-O-C) vibration were found at 1200 cm-1 and a number of inorganic groups such as sulfate, phosphate and carbonate were also observed at wavelength below 1200 cm1⁴⁶.

Discussion

Calx is an established dosage form of Ayurveda, Unani, Siddha and some other traditional system of medicine and is claimed to be active with minimum dose and maximum efficacy, but most of the calx have not been standardized. Since, most of the calx is made up of metals and minerals which are more toxic than plant origin drug; therefore there is need of standardization of calx^1,2. Classical methods of evaluation of calx such as color, odor, taste, luster, fineness, etc. are not sufficient enough to set parameters for standardization. Classical tests are not sufficient because these tests are nonspecific as color of a particular calx can be compared with that mentioned in the classical book. Similarly, fineness, luster, stickiness etc is common to many calx, by using these tests significant conclusion cannot be drawn. Organoleptic, crude and subjective parameters do not give satisfactory results² . Presently certain analytical techniques developed for otherwise purposes have fortunately been applied in case of calx also. Such techniques give accurate results and set objective parameters. These techniques also analyze particle size; elements phase detection, functional group, heavy metals, elemental composition, surface morphology and surface topography, etc¹⁹.

Since, a single technique cannot be applicable for many of these characteristics therefore more than a dozen techniques can be used for a single calx for different characteristics such as X-RD gives better result for particle size estimation, phase element detection and crystallanity but not that of amorphous compound (non crystalline) and trace elements. Similarly ICP-MS detects heavy metals, trace elements but not that of functional groups and oxidation state, and so on. By applying two or more than two such techniques most of the calx can be standardized. In certain studies researchers have tried to evaluate calx by various analytical techniques mentioned in this paper. But, such studies are limited. Analysis of these studies revealed that the researchers have tried to set parameters for standardization of some calx^1,2,19.

These studies give an idea about analysis of particle size, elemental phase detection, functional group, estimation of heavy metals, elemental composition, surface morphology and surface topography, oxidation number, etc. These studies also addressed and correlate toxicity study related issues and a step ahead of establishing the rationale behind the safety and efficacy of this traditional dosage form.

The rationale of studying calx with these analytical methods may better be understood by citing some reported studies. Study conducted on ‘Trace Metals Analysis in Selected Pharmaceutical Multi mineral Formulations’, twelve pharmaceutical multi mineral formulations were analyzed for calcium, copper, iron, magnesium, manganese, potassium and zinc contents using AAS technique. Cu, Mn and Zn contents were low in most of the formulations⁴⁷. In a study conducted on ‘an Approach towards Standardization of Swarna Makshik Bhasma’, tried to assure the quality of bhasma, rasa shastra quality control tests like nischandratva, varitara, amla pariksha, etc., followed by using XRD and TGA which revealed presence of Fe2 O3 , FeS2 , CuS and SiO2 ⁴⁸. Study reported on ‘Synthesis, Characterization and Histopathological Study of a Lead-Based Indian Traditional Drug: Naga Bhasma’, the crude drug was purified as per classical method and analyzed by XRD, FTIR, EDAX and XPS. The result showed that it contained lead in nano-crystalline (~60 nm) lead sulfide form (Pb2+), found safe in histopathology of skin, small intestine, pancreas, testis, brain, lung, kidney and liver at 6 mg/100 g/day for 40 days study⁴⁹. A study conducted on ‘Analytical study of raw Swarna Makshika (Chalcopyrite) and its Bhasma through TEM and EDAX’. TEM and EDAX analysis revealed the presence of iron, copper, and sulphur in the Bhasma. In addition, Potassium, Magnesium, Aluminum, and Silicon in trace amount were also found⁵⁰. In a study conducted on ‘Characterization of Tarakeshwara Rasa: An Ayurvedic herbomineral formulation’, the XRD studies indicated Fe2 O3 in major phase and SnO2 , HgS, SiO2 , HgO as minor phases. SEM revealed size of the majority of particles between 0.5 and 2 μm⁵¹. Study reported on ‘Preliminary physicochemical evaluation of Kushta tutia; a Unani Formulation’, finished product was evaluated for physicochemical characteristics including preliminary tests mentioned in classical literature and conventional methods. Findings may be considered as standard reference for quality⁵². A study conducted on ‘Physico-Chemical Characterization of Lead Based Indian Traditional Medicine-Naga Bhasma’, samples from three different batches from the same manufacturer were procured and analyzed. TGA showed that Naga bhasma sample was thermally stable up to 900o C, indicating the absence of free organic molecules. The FTIR spectra revealed that all the samples contained organic moieties probably in the form of complexes. Particle size and surface area analysis indicated presence of micron-sized particles. Elemental analysis indicated the presence of arsenic impurity in the samples. Electron microscopy studies revealed that bhasma contained particles in micron and sub-micron ranges. EDX analysis too showed the presence of arsenic along with Lead. XRD showed the lead oxide phase in all three samples⁵³. A study reported on ‘Analytical study of Yashada Bhasma with Ayurvedic and Modern Parameters’, XRD identified the final product as Zinc oxide (ZnO). SEM revealed the amorphous nature of the bhasma with particle size range 5-20 micrometer. ICPAES showed the presence of Zinc in major portion (95.08ppm) and other elements like Sn (0.27), Pb (0.14), Fe (1.69), Ca (1.82), Mg (1.00), Cu, Co and Mn < 0.5 ppm in the final product⁵⁴. A study conducted on ‘Poora Parpam’. The aim of the study was to determine the compound by qualitative and quantitative analysis using ICP-MS, FTIR, MS, SEM and TEM. The study revealed some specific compounds were responsible for different pharmacological activity⁵⁵. In a study conducted on ‘A Green approach for the synthesis of nano-sized iron oxide, by Indian Ayurvedic modified bhasmikaran method’, XRD was used to monitor phase transformation from α- to γ- phase and characterizations were done using TGA, TEM and FTIR⁵⁶. In a study conducted on ‘Green Synthesis of Gold nanoparticles using Cinnamomum verum, Syzygium aromaticum and Piper nigrum extract’, the UV-visible spectrum of Au NPs showed a broad band in the region 543- 546 nm corresponding to the surface plasmon resonance band (SPR) of Au NPs. Furthermore, the rate of formation of nanoparticles using different spice extracts as reducing agent give an indication towards the concentration of active components present in them . FESEM confirmed the size range, poly dispersity and existence of NPs as separated particles. DLS confirmed their poly dispersity and size in the range between 2.9-3.6 nm⁵⁷. The present study reviewed various useful techniques for setting in the objective parameters for calx standardization which have been used for human use for the cure of various diseases in traditional complimentary medicine. It is hoped that a rational choice of more than one technique will help in better standardization of the calx.