Simultaneous Estimation of Some Drugs in Bulk and in their Formulation by Chromatographic Methods.

Master's Thesis, 2013

106 Pages



1 Introduction

2 Drug Profile

3 Aims and Objectives

4 Plan of Work

5 Results and Discussion

6 Experimental

7 References


Introduction to analytical chemistry (1-4)

Analytical chemistry may be defined as the “science and art of determining the composition of materials in terms of the elements or compounds contained”. Large number of drug/s introduced in the pharmaceutical market is increasing per year. These drugs may be either new entities or partial structural modification/s of the existing one. Very often there is a time delay from the date of introduction of a drug in the market to the date of its inclusion in pharmacopoeias. This is because of the possible uncertainties in the continuous and wider usage of these drug/s, reports of new toxicities (resulting in their withdrawal from the market), development and introduction of better drug/s by competitor/s. Under these conditions, standard/s and analytical procedure/s for these drugs may not be available in the pharmacopoeia/s. It therefore becomes necessary to develop newer analytical methods for such drugs.

Quality control is a concept, which strives to produce a perfect product by series of measures designed to prevent and eliminate errors at different stages of production. With the growth of pharmaceutical industry during last many years, there has been fast development in the field of pharmaceutical analysis involving complex instrumentation. Providing simple, rapid analytical method for complex formulation is a matter of importance.

Important reasons for the improvement of newer drug analysis methods are:

- The drug or combinations of drug may not be official in any pharmacopoeias.
- An appropriate analytical method for the drug may not be available in the literature due to patent regulations.
- Suitable analytical procedure may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
- Analytical method/s for the evaluation of the drug in biological fluids may not be available.
- Analytical method for a drug in combination with other drugs may not be available.
- The existing analytical procedures may require expensive solvents and reagents. It may also involve burdensome extraction, separation procedures and these may not be reliable.


Highly specific and sensitive analytical techniques hold the key in the designing, development, standardization and quality control of medicinal products. They are equally important in drug metabolism and pharmacokinetics studies, both of which are fundamental to the evaluation of bioavailability and duration of clinical response.

Modern physical methods of analysis are very sensitive, precise and providing thorough information from minute samples of material. They are for the most part quickly applied and in general are readily amenable to automation. For these reasons they are now widely used in product development, control of manufacture, quality control, as a check on stability during storage and monitoring the use of drugs and medicines.


1) Chemical Methods

a) Titrimetric Methods; They involves
i. acid base reactions, ii. precipitations, iii. redox reactions iv. complexomeric reactions,
v. large cation reagents
b) Gravimetric Methods are
i. weighing the active ingredients after separation, ii. weighing of the residue after ignition of the sample, iii. precipitation and weighing of the derivatives of the active ingredients

2) Instrumental methods

a) Spectroscopic techniques; There are,
i. ultraviolet and visible spectrophotometry, ii. fluorescence and phosphorescence spectrophotometry, iii. atomic spectrometry (emission and absorption), iv. infrared spectrophotometry, v. raman spectroscopy, vi. X-ray spectroscopy, vii. radiochemical techniques including activation analysis, viii. nuclear magnetic resonance spectroscopy, ix. electron spin resonance spectroscopy
b) Electrochemical techniques cover,
i. potentiometry (pH and ion selective electrodes), ii. conductance techniques, iii. voltammetric techniques, iv. stripping techniques, v. coulometry, vi. electrogravimetry.
c) Chromatographic techniques; Some commonly used chromatographic techniques are
i. gas chromatography, ii. high performance liquid chromatography (HPLC), iii. high performance thin layer chromatography (HPTLC).
d) Miscellaneous techniques
i. Thermal analysis, ii. Mass spectrometry, iii. Kinetic techniques.
e) Hyphenated techniques
i. GC - MS (gas chromatography - mass spectrophotometry), ii. ICP - MS (Inductively coupled plasma - mass spectrophotometry), iii. GC - IR (gas chromatography - infrared spectroscopy), iv. MS - MS (mass - mass spectroscopy)

UV-Visible Spectroscopy: (5-6)

In electromagnetic spectrum wavelength range from 190 - 800 nm is called as Ultraviolet- Visible (UV-VIS) region. The transitions that result in the absorption of radiation are transitions between the electronic energy levels, in the UV - Visible spectroscopy

For an atom that absorbs UV - VIS radiation, the absorption spectrum sometimes consists of very sharp lines, as it would be expected from a quantized process that is occurring between two discrete energy levels. But for molecules, the UV - VIS absorption occurs over a wide range of wavelengths that results in a broad band of absorption centered near the wavelength of maximum absorption (λmax). The fundamental law that governs the quantitative spectrophotometric analysis is Beer - Lambert’s Law.

Beer’s law

Beer observed a relationship holds between transmittance and the concentration of a solution, i.e., the intensity of a beam of monochromatic light decreases exponentially with the increase in concentration of the absorbing substance arithmetically.

Lambert’s law

When a beam of light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is proportinal to the intensity of the light.

The combination of these two laws yields the Beer - Lambert’s Law.

Mathematically it can be expressed as,

A= log (I0/I) = εcl (for a given wavelength) …eq. 1


A = absorbance, I0 = intensity of light incident upon sample cell,

I = intensity of light leaving sample cell, c = molar concentration of solute,

l = length of sample cell (cm), ε = molar absorptivity.

The assay of single sample, which contains other absorbing substance/s, can be calculated from the measured absorbance by using one of three principal procedures. They are, 1) Standard absorptivity value, 2) Calibration graph and 3) Single or double point standardization. Some of the methods used for the assay of multi component sample are, 1) Simultaneous equation method, 2) Multi component mode, 3) Derivative spectrum method, 4) Absorbance correction method.

Chromatography (7-18)

Earlier, chromatography used to be a separation technique, but now it can be used for quantitation also. It involves the separation of components of a mixture which are scattered among two phases, the mobile phase and the stationary phase. The mobile phase moves over or through the surface of the stationary phase. As different components of the mixture have different affinities for each phase they differ in their retention on the stationary phase, which leads to their separation. The separation of components is determined by the chemical and physical properties of the two phases and the experimental conditions (temperature and pressure).

Modern pharmaceutical formulations are complex mixtures containing one or more therapeutically active ingredients and a number of inert materials like disintegrant, colors, excipients, and flavors. So as to ensure quality and stability of the final product, the pharmaceutical analyst must be able to separate the mixtures into individual components prior to quantitative analysis. Chromatography is the powerful technique to separate the mixture into different components.

Chromatographic methods can be classified according to the nature of the stationary and mobile phases. Different types of chromatography are: 1) Adsorption, 2) Partition, 3) Ion exchange, 4) Size exclusion or gel permeation. The modern instrumental techniques of GLC and HPLC provide excellent separation and allow accurate assay of very low concentrations of wide variety of substances in complex mixtures.

High Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is used in almost all sectors. Most of the drugs in dosage forms can be analyzed by this technique because of several advantages like accuracy, precision, specificity, and ease of automation.

There are different modes of separation in HPLC. They are normal phase and reversed phase, reverse phase ion pair, affinity chromatography and size exclusion chromatography (gel permeation and gel filtration).

In the normal phase, the mobile phase is nonpolar and the stationary phase is polar in nature. In this method, nonpolar compound/s travel quicker and are eluted first, because of the lower attraction between the nonpolar compound/s and the stationary phase. Polar compound/s are retained for longer period of times because of their higher affinity with the stationary phase. These compounds, therefore take more time to elute. Hence, normal phase separation is not commonly used for pharmaceutical applications because most of the drug molecules are polar in nature and hence take longer time to elute. In reversed phase, the stationary phase is nonpolar and the mobile phase is polar.

An aqueous mobile phase allows the use of secondary solute chemical equilibrium (such as ionization control, ion pairing, ion suppression and complexation) to control retention and selectivity. The polar compounds get eluted first in this mode and nonpolar compounds are retained for longer time of period. As nearly all the drug/s and pharmaceuticals are polar with varying degree in nature, they are not retained for longer times and thus elute more rapidly. The different HPLC columns used are C4, C8, octa decyl silane (C18 or ODS) etc.

In ion exchange chromatography, the stationary phase contains ionic groups like NR3+ or SO3- , which interact with the oppositely charged ionic groups of the sample molecules which is suitable for the separation of charged molecules only. Altering the salt concentration and pH can change the retention.

Ion pair chromatography may be used for the separation of ionic compounds and this method can also be a substitute for ion exchange chromatography. In reversed phase ion pair chromatography or soap chromatography, strong basic and acidic compound/s may be separated by reversed phase mode by forming ion pairs (columbic association species formed between two ions of opposite electric charge) with appropriate counter ions.

Affinity chromatography uses highly specific biochemical interactions for separation process. The stationary phase contains specific groups of molecules which can adsorb the sample if certain steric and charge related conditions are satisfied. This technique can be used to isolate enzymes, proteins, antibodies from complex mixtures.

Size exclusion chromatography separates molecules according to their molecular mass. Molecules with highest molecular mass are eluted first and the smallest molecular mass in the last. This technique is usually used when a mixture contains compounds with a molecular mass difference of at least 10%. This mode can be further subdivided into gel permeation chromatography (with organic solvents) and gel filtration chromatography (with aqueous solvents).

Column packing

The packing used in modern HPLC consists of small, rigid particles having a narrow particle size distribution. The different types of column packing in HPLC are

1. Porous, polymeric beds
Porous, polymeric beds based on styrene divinyl benzene co-polymers. They are used in ion exchange and size exclusion chromatography. For analytical purpose these are replaced by silica based packing which are more efficient.
2. Porous layer beds
Consisting of a thin shell (1-3 μm) of silica or modified silica on a spherical inert core (e.g. glass). After the development of totally porous micro particulate packing, these have not been used in HPLC.
3. Totally porous silica particles (dia. < 10 μm)
These packing have been widely used for analytical HPLC in recent years. Particles of diameter > 20 μm are usually dry packed, while particles of diameter < 20 μm are slurry packed in which particles are suspended in a suitable solvent and the slurry so obtained is driven into the column under pressure.


The function of the detector in HPLC is to monitor the mobile phase as it merges from the column. Usually, detectors are of two types:

1. Bulk property detectors

It compares overall changes in a physical property of the mobile phase with or without an eluting solute. e.g. dielectric constant or density, refractive index.

2. Solute property detectors

It responds to a physical property of the solute. e.g. Fluorescence, UV absorbance or diffusion current. These detectors are about 1000 times extra sensitive than the bulk property detectors.

Quantitative analysis in HPLC

Three methods are generally used for quantitative analysis. They are as follows,

1. External standard method

The external standard method involves the use of a single standard. The peak area or the height of the sample and the standard used are compared.

2. Internal standard method

A widely used technique of quantitation involves the addition of an internal standard to compensate for various analytical errors. In this method, a known drug of a fixed concentration is added to the known amount of samples to give separate peaks in the chromatogram to compensate for the loss of the compounds of interest during pretreatment of the sample. Loss of the component of interest will be accompanied by the loss of an equivalent fraction of the internal standard. The correctness of this method clearly depends on the structural equivalence of the compounds of interest and the internal standard. The necessities for an internal standard are:

- It must give a completely resolved peak with no interferences.
- It should elute close to the compound of interest.
- It must behave equivalent to the compound of interest for analysis like pretreatments, derivative formations etc.
- It should be added at a concentration that will produce a peak area or peak height ratio of about unity with the compound.
- It should not be present in the original sample.
- It must be stable, nonreactive with column packing, the mobile phase, sample components, and
- It is desirable that this compound is commercially obtainable in high purity.

The internal standard should be added to the sample prior to sample preparation procedure and homogenized with it. To be able to recalculate the sample component concentration in the original sample, the response factor should be demonstrated first. The response factor (RF) is the ratio of peak areas of sample component (Ax) and the internal standard (AISTD) obtained by injecting the same quantity on molar basis. It can be calculated by using the formula,

RF = Ax / AISTD …eq. 2

On the basis of the response factor and strength of the internal standard (NISTD), the amount of the analyte in the original sample can be calculated using the formula,

X =Ax × N / RF × AISTD …eq. 3

The calculations described above can be used after proving the linearity of the calibration curve for the internal standard and the analytical reference standard of the compound of concern. While more than one component is to be analyzed from the sample, the response factor of each component should be determined. One can also use the slope of the calibration curve based on standard that contain known concentrations of the compound of interest. When more than one component is to be analyzed from the sample, the response factor of each component should be determined in the calculations using similar formula.

3. Standard addition method

In this technique, a known quantity of the standard compound is added to the sample solution to be determined. Standard addition technique is suitable if sufficient amount of the sample is available and is more realistic in the sense that it allows calibration in the presence of excipients or other components. As gradient elution can be a source of additional error in quantitative study. It is also essential to maintain the flow rate and the mobile phase composition steady. The sample must be dissolved in the mobile phase. If the solvent used in preparing the sample solution and the mobile phase are not the same, the study can become less precise.

System suitability Parameters:

To ensure that the data obtained from HPLC system and procedure followed is of acceptable quality, system suitability testing should be performed. System suitability testing is an integral part of chrmatographic methods. A few commonly used system suitability parameters and what they mean to analysis is given below. System suitability tests must be performed on a regular basis.

i) Resolution (RS): -

It is a measure of quality of separation of adjacent bands in a chromatogram; obviously overlapping bands have small Rs values. Resolution is calculated from the retention time and the width of two adjacent peaks.

Ideally RS should be greater than 1.5

Abbildung in dieser Leseprobe nicht enthalten

Where t1 and t2 are the retention time of the first and second eluting adjacent bands, where W1 and W2 are their respective baseline widths. Reliability of calculation is poor if Rs is < 2.0.

ii) Capacity factor (k): -

It is the measure of the position of a sample peak in the chromatogram, being specific for a given compound. This is a parameter that specifies the extent of the retention of substances to be separated. It depends on the mobile phase, quality of column packing, stationary phase and temperature.

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iii) Selectivity factor:

The selectivity of the chromatographic system is a measure of the difference in retention time between two given peak. The selectivity factor (α) of a column for the two species A and B is defined as;

α = KB / KA …eq. 7

Where KB is distribution constant for more strongly retained species B, and KA is distribution constant for less strongly or more rapidly eluted species A. Alpha is always greater than unity.

iv) Number of theoretical plates (N): -

The column efficiency can be expressed as the plate number (N) and Height equivalent to theoretical plate [HETP]

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Design of Separation Method

To develop a method for analyzing single or multiple components of a formulation one should know the nature of sample present like, molecular weight, polarity, ionic character and solubility. Development of a method involves considerable trial and error procedures. Generally, one starts with reversed phase chromatography which involves non polar stationary phase, when the compounds are hydrophilic in nature and are water soluble.

The organic solvent concentration required for the mobile phase can be estimated by gradient elution technique. In case of aqueous sample mixtures, the best way to start is with gradient reversed phase chromatography. Gradient can be started with 5 - 10 % organic solvent in the mobile phase and the organic solvent concentration can be increased up to 100 % within 30 - 45 min. Separation can then be optimized by changing the initial mobile phase composition and the slope of the gradient according to the chromatogram obtained from the preliminary chromatographic run. Initial mobile phase composition can be estimated on the basis of where the compounds of interest eluted, namely at what mobile phase composition. Changing the polarity of mobile phase can alter elution of drug/s. Elution strength of a mobile phase depends upon its polarity. Ionic samples (acidic/ basic) can be separated, if they are present in unionized form. Dissociation of ionic samples may be suppressed by the proper selection of pH. The pH of the mobile phase has to be selected in such a way that the compounds do not ionize. If the retention times are too short, the organic phase concentration needs to be decreased. If the retention time is very high, organic phase concentration needs to be increased.

Selection of detection wavelength is an important activity to get good analytical results. To select a wavelength of detection one should have the knowledge of UV spectrum of each component in the sample. UV spectra can be measured for standards prior to method development. It is not always necessary to use λmax for the detection. For simultaneous method development, a wavelength where all the components of the sample have considerable absorption should be selected considering the ratio of components in the formulation. The addition of peak modifiers to the mobile phase can affect the separation of ionic samples. For examples, the retention of the basic and acidic compounds can be influenced by the addition of small amounts of triethylamine and acetic acid (a peak modifier), respectively. This can lead to valuable changes in selectivity. Tailing or fronting indicates that the mobile phase is not totally compatible with the solutes. Ion - pair chromatography can be used, when the peak shape does not improve by lower (2 - 3) or higher (8 - 9) pH. For basic compounds, anionic ion - pair molecules at lower pH and acidic compounds, cationic ion pair molecules at higher pH can be used. In case of amphoteric solutes or a mixture of acidic and basic compounds, ion-pair chromatography is the technique of choice. The low solubility of the sample in the mobile phase can also results in to bad peak shapes. It is always suitable to use the same solvents for the preparation of sample solution as the mobile phase to avoid precipitation of the compounds in the system. Optimization can be started only after a reasonable chromatogram (chromatogram with more or less symmetrical peaks) has been obtained. By small alter of the mobile phase composition, the peak position can be predicted within the range of investigated alterations. An optimized chromatogram is the one in which all the peaks are symmetrical and are well resolved in less run time. The peak resolution can be increased by using a more efficient column (column with higher theoretical plate number, N) which can be obtained by means of a column of smaller particle size, or a longer length. However, these factors, will enhance the analysis time.

High Performance Thin Layer Chromatography (HPTLC)

HPTLC is a simple separation technique in which different samples are applied to the stationary phase before it comes in contact with the mobile phase resulting in sample migration. After development the mobile phase is removed by evaporation and detection is performed on the stationary phase. The record of the detector response is plotted against the separation distance is called a densitogram. Availability of different stationary phases is an important difference between TLC and column chromatography. Each run needs use of new stationary phase which eliminates the cross contamination from previous samples. Only the sample components that are eluted out of the column can be detected in the column chromatography and the components that remain on the column may be easily overlooked but in TLC components cannot be usually overlooked. As the mobile phase is evaporated before the detection process, it does not interfere with the measurement of components of the mixture. In classical TLC, mobile phase moves through the stationary phase by capillary forces. There are many modifications to the classical TLC approach in which flow of mobile phase is forced through the layer which are collectively called as forced flow methods. Some of the forced flow methods are electro-planar chromatography (EPC), over pressure layer chromatography or optimum performance laminar chromatography (OPLC), rotation planar chromatography (RPC) etc. Capillary forces are stronger in the narrow inter-particle channels, leading to more rapid advancement of the mobile phase. During the chromatographic process a solvent gradient in the mobile phase is produced as the solvent front migrates through the adsorbent layer. This is particularly true for mixed mobile phases where more polar component is more selectively adsorbed. If vapor and mobile phase are not in equilibrium, evaporation causes loss of mobile phase from the plate surface.

TLC provides for separations in the milligram to picogram range. Separated substances that are alternately identified by TLC can be isolated for further characterization by other techniques, such as gas chromatography (GC), high performance liquid chromatography (HPLC), visible, ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), mass spectrometry (MS), and electrophoresis. Eluted substances can also be quantified by procedure such as these, but in situ densitometry is the most convenient, accurate, and precise approach for quantitative TLC. The fundamental parameter used to characterize the position of a spot in a TLC chromatogram is the Retardation factor or Rf value.

The Retardation Factor (Rf)

It is the quotient of the distance of the substance zone from the sample origin to the front of the mobile phase (Zf).

Abbildung in dieser Leseprobe nicht enthalten

Fig. No.1 Calculation of Rf Value

By definition, the Rf value cannot exceed 1.0. To avoid the decimal point, the Rf value is sometimes multiplied by 100 and then described as the hRf value. Systematic error in the measurement of the Rf value arises from the difficulty in locating the precise position of the solvent front. If saturation of the development of the chamber is not done, adsorbent layer, mobile phase and vapor phase will be in equilibrium, then the condensation of vapor phase or the evaporation of mobile phase in the region of the solvent front will give erroneous Rf value.

The ultimate chromatographic performance of a TLC plate, and therefore resolution, is dependent on certain parameters like the velocity constant of mobile phase, diffusion coefficient of the substance in mobile phase, mean particle size and particle size distribution of the stationary phase. The mobile phase velocity is determined by particle size while chromatographic efficiency is dependent upon the coarser particles and the performance is improved by using particles of narrow size distribution. However, spot broadening in HPTLC is controlled by molecular diffusion. Performance of TLC can be evaluated in terms of the number of theoretical plates (N), height equivalent to theoretical plate (HETP), and separation number (SN).

In column chromatographic techniques, all substances travel same migration distance (the length of column) but have different diffusion time (retention time on the column). This is opposite to TLC where all substances have same diffusion time (the plate is developed for the fixed time) but migration distance varies. The chromatographic measures of performance in TLC (N, HETP, and SN) are all correlated to the migration distance of substance.

Advantages of HPTLC

They are; 1) short development time, 2) wide choice of stationary phases, 3) early recovery of separated components, 4) superior separation effects, 5) easy visualization of separated compounds.

Steps involved in HPTLC analysis

i) Sample preparation: -

For normal phase chromatography using silica gel / alumina precoated plates, solvent generally should be nonpolar and of volatile type. For reverse phase chromatography usually polar solvents are used.

ii) Selection of chromatographic layer: -

There are at least 25 types of sorbents available for TLC. Silica gel or aluminium oxide is useful in many applications. They also can be split into different types depending on the pore size, particle size and pH. Selection of layer depends on the nature of material to be separated like polarity, solubility, ionizability, molecular weight, shape and size. These properties are also important for selecting the solvents for preparation of sample and development. Types of sorbents are given below,

Abbildung in dieser Leseprobe nicht enthalten

(Peter E Wall et al., Thin Layer Chromatography – A modern practical approach, © The Royal Society of Chemistry 2005.pp. 7)

iii) Plates: -

Standard size plates for HPTLC are manufactured by various companies which are most satisfactory. Generally plates of 20 X 20 cm, 10 X 10 cm or 5 X 7.5 cm size having 100 - 250 µ adsorbent thickness are used for quantitative analysis. Silica gel 60F254 having a pore size 6 µ with fluorescent indicator as a coat material is widely used. The basic difference in TLC and HPTLC plate is particle size of coated material which is 5 - 20 µm for TLC and 4 - 8 µm for HPTLC.

iv) Pre-washing: -

Plates need to be prewashed to remove water vapors or other volatile impurities, which might get trapped in the plates. These give dirty zones and spots on the plates. To avoid this, plates are cleaned by using methanol as solvent by ascending or descending or by dipping mode.

v) Conditioning: -

The prewashed plates exposed to humidity and surroundings are needed to be activated by placing them in an oven at 110° C for 15 to 20 minutes. This process is known as conditioning. This allows the active centers of coating materials attenuated for better separation of sample material.

vi) Sample application: -

It is most important step for obtaining good resolution and results. Application of 1.0 - 5 µL is most satisfactory, for HPTLC, application of the sample and standard as a band gives better separation, equal Rf values and less spot broadening. This sample application is carried out by Linomat type applicator on the plates which gives uniform, accurate results. In Linomat applicator nitrogen gas is used for the sample application. Flow of nitrogen is adjusted according to vehicle used for sample preparation.

vii) Pre conditioning (Chamber saturation): -

This has profound influence on the effective separation of sample. For low polarity mobile phase there is no need of saturation, however, saturation is desirable in case of highly polar mobile phases. Partial saturation is recommended for mobile phase composition leading to phase separation. For reverse phase chromatography it is essential to saturate the chamber with methanol or polar solvent.

viii) Mobile phase: -

The selection of appropriate mobile phase is based on chemical properties of solute and solvent, solubility of analyte, absorbent layer etc.

ix) Chromatographic development: -

Various forms of chromatographic development like ascending, descending, horizontal, continuous, and gradient can be tried. For HPTLC plates, migration distance of 5- 6 cm is sufficient. After development, plates are removed from the chamber and dried to remove traces of mobile phase.

There are some special development techniques in TLC, they include

- Continuous development
- Multiple development
- Stepwise development

1) Two dimensional separation
2) Three dimensional separation
3) Wedged tip technique

Common problems encountered during chromatographic development are as follows;


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Simultaneous Estimation of Some Drugs in Bulk and in their Formulation by Chromatographic Methods.
MASTER OF PHARMACY (Quality Assurance Techniques)
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simultaneous, estimation, some, drugs, bulk, formulation, chromatographic, methods
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Dr. Satish Gabhe (Author)Dr. Kakasaheb Mahadik (Author)Sachin Potawale (Author)Anirudh Thati (Author), 2013, Simultaneous Estimation of Some Drugs in Bulk and in their Formulation by Chromatographic Methods., Munich, GRIN Verlag,


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