Dr. Puspendra Kumar

Lab Manual Pharm. Analysis-II

By Dr. Puspendra Kumar M. Pharm., Ph.D.

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Dr. Puspendra Kumar

List of Experiment 1. To determine the Rf value of given sample of paracetamol and compare with standard paracetamol by TLC. 2. To develop and optimize the TLC solvent system for given sample of Caffeine sample. 3. Determination of hardness of water by complexometric titration. 4. To demonstrate the methodology of column chromatography. 5. Preparation and standardization of EDTA solution. 6. To isolate the compounds from a mixture by column chromatography. 7. Preparation and standardization of perchloric acid by non-aqueous titration. 8. Preparation and standardization of sodium/potassium methoxide/Lithium methoxide solutions by non-aqueous titration. 9. Assay of magnesium hydroxide/ magnesium sulfate by complexometric titrations. 10. Determination of Alum by Complexometric titration. 11. To separate the amino acid mixture by paper chromatography. 12. To separate the amino acid mixture by radial paper chromatography. 13. Demonstration of HPLC. 14. Prepare and standardization of NaNO2 by diazotization titration. 15. Assay of sulpha drugs by diazotization. 16. Determination of end point of redox titration by potentiometry. 17. Assay of moisture content in a drug by Karl Fischer Titration method.

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Dr. Puspendra Kumar EXPERIMENT NO. 01 Aim: To determine the Rf value of given sample of paracetamol and compare with standard paracetamol by TLC. Requirement: TLC plate, TLC chamber, TLC development chamber, Solvents for mobile phase, Silica gel G, Forceps, Detecting reagents/UV chamber, Paracetamol. References: Sethi PD and Charegaonkar D. “Identification of Drugs in Pharmaceutical Formulations by Thin Layer Chromatography”, Second Edition, 2005, CBS Publishers and Distributors, New Delhi, page: 1-27, 83. Theory Principle: Thin layer chromatography is a method of analysis in which the stationary phase (a finely divided solid) is spread as a thin layer on a solid rigid supporting plate; and the mobile phase: a liquid is allowed to migrate across the surface of the plate by the capillary action and separation takes places due to adsorption phenomenon and gives different R f values for each sample compound. Rf= Distance travel by solute/Distance travel by solvent Stationary Phase Silica gel, the most commonly used stationary phase, has the empirical formula SiO2. However, at the surface of the silica gel particles, the dangling oxygen atoms are bound to protons. The presence of these hydroxyl groups renders the surface of silica gel highly polar. Thus, polar functionality in the organic analyte interacts strongly with the surface of the gel particle and non polar functionality interacts only weakly. Mobile Phase For silica gel chromatography, the mobile phase is an organic solvent or mixture of organic solvents. As the mobile phase moves past the surface of the silica gel it transports the analyte past the particles of the stationary phase. However, the analyte molecules are only free to move with the solvent if they are not bound to the surface of the silica gel. Thus, the fraction of the time that the analyte is bound to the surface of the silica gel relative to the time it spends in solution determines the retention factor of the analyte. The ability of an analyte to bind to the surface of the silica gel in the presence of a particular solvent or mixture of solvents can be viewed as a the sum of two competitive interactions. First, polar groups in the solvent can compete with the analyte for binding sites on the surface of the silica gel. Therefore, if a highly polar solvent is used, it will interact strongly with the surface of the silica gel and will leave few sites on the stationary phase free to bind Dr. Puspendra Classes: https://www.youtube.com/user/puspendra007

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Dr. Puspendra Kumar with the analyte. The analyte will, therefore, move quickly past the stationary phase. Similarly, polar groups in the solvent can interact strongly with polar functionality in the analyte and prevent interaction of the analyte with the surface of the silica gel. This effect also leads to rapid movement of the analyte past the stationary phase. Method: Preparation of TLC plate Suspend 100 g of silica gel G in 200-250 ml of water, mix with a stirrer to get homogeneous slurry. Take the air dried TLC glass plates or dried in oven at 1100C and pour the silica gel G slurry into the glass plate. (Thickness should be around 250 μm) Slurry should be used within 2 minute of preparation otherwise slurry will dry and needs more water to maintain the fluidity. Dry the plate in a TLC chamber until complete drying occurs. Dried TLC plates are activated in oven at 1100C for 30 minutes and immediately used for development after cooling. Pre-coated plates: With the availability of pre-coated plates commercially, the use of laboratory hand-made plates is on decline. The pre-coated plates with different support material (glass, aluminium, plastic) and with different sorbent layers are available in different format and thickness by various manufacturers. Usually plates with sorbent thickness of 100-250μm are used for qualitative and quantitative analysis. Pre-coated plates also require activation. Sample Preparation For TLC on silica gel, the use of least polar solvent which allows quantitative dissolving and spotting of sample and there is no preliminary development and separation within the initial spot at the origin, is recommended. Application of sample Sample application is most critical step for obtaining good resolution. The sample should be completely transferred to the layer, however, under no circumstances; the application process should damage the layer, as damaged layer results in unevenly shaped spots. Wherever possible, use of automatic application device is recommended. The sample should be applied through clean smaller diameter capillary.

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Dr. Puspendra Kumar Selection of mobile phase First level: A neat solvent from different selectivity areas is tested. Within a selectivity area, solvents may give similar separation. Usually diethyl ether, ethanol, methanol, tetrahydrofuran, dimethyl formamide, dichloromethane, ethylacetate, acetonitrile, methyl-ethyl ketone, toluene and chloroform are used as neat solvents. If acceptable resolution and medium Rf value range is achieved, the analyst can directly third level, if first level does not yield satisfactory result, then proceed with second level. Second level: From first level, solvents which leave the main fraction/component of the analyte near the starting point or close to the solvent front, are required to adjusted. If R f values are too high, solvent strength should be decreased by adding non polar solvents and polar solvents for too low Rf values. Third level: Mixtures of solvents from different selectivity group are investigated; the strength is adjusted, if required. These mixtures can be binary, tertiary or even quaternary, but binary mixtures are preferred one. At this level, addition of small amount of acidic (acetic acid) or basis (triethyl amine) modifiers significantly enhance the separation efficiency of mobile phase. Fourth level: At this level, final optimization of mobile phase to be used for a particular separation is made. To get the best separation, small variations in the proportions of different solvents may have to be made. Preconditioning Chamber saturation has pronounced influence on the separation profile. When the plate is introduced into an unsaturated chamber, during the course of development, the solvent evaporates from the plate mainly at the solvent front. Therefore larger quantity of the solvent shall be required for a given distance; hence resulting is increase in Rf values. If the tank is saturated (by lining with filter paper) prior to development, solvent vapours soon get uniformly distributed throughout the chamber. As soon as the plate is placed in such a saturated chamber, it soon gets preloaded with solvent vapours, hence less solvent shall be required to travel a particular distance, resulting in lower Rf values. Development and drying Develop the chromatogram in twin trough chamber or other TLC development chamber until and unless solvent reaches the three fourth distance of the plate. Dry the developed plate in chromatographic drying chamber. Evaluation of thin layer chromatogram First of all spots of TLC are detected by using suitable detecting reagent or physical methods. Dr. Puspendra Classes: https://www.youtube.com/user/puspendra007

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Dr. Puspendra Kumar The evaluation depends on the purpose of a chromatographic analysis. For quantitative determination often localization of substances is sufficient. This can be easily achieved by parallel runs with reference substances. Rf values: A parameter often used for qualitative evaluation is the Rf value (retention factor). The Rf value is defined as follows: Rf =

Distance travel by solute (spot) Distance travel by solvent front

i.e. Rf values are between 0 and 1, best between 0.1 and 0.8. Chromatographic condition for Paracetamol Standard solution: Dissolve appropriate quantity of paracetamol (i.e. approximate 10μg/ml) in methanol for spotting. Sample solution: Suspend powdered sample in methanol, sonicate for 5 minute, filter through whatman filter paper and use the filtrate for spotting. Mobile phase: Chloroform: Acetone: Toluene (6.5: 2.5: 1.0) Chamber saturation time: 30 minutes. Migration distance: 70 mm (If 10x10 cm plate is used) Detection: UV light (254nm) or iodine vapours. Method: 1. Take the given mobile phase ration in a TLC chamber and saturate the chamber for 30 min. 2. Take a dried and activated TLC plate. 3. Take the prepared standard solution and apply the spot in TLC plate around 1 cm above from the bottom. 4. Take the selected test solution and apply the spot in same TLC plate parallel to the standard spot. 5. Wait for drying of spot and kept the TLC plate tilted in the saturated TLC chamber. 6. Develop the TLC plate up to 80-90% of the stationary phase. 7. Remove the TLC plate from the chamber and kept for air drying of mobile phase. 8. View the spot(s) under UV chamber or Iodine chamber. 9. Calculate the Rf values of the selected paracetamol standard and sample. Results: Rf value of the standard and sample paracetamol was found to be ........ and ......... respectively.

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Dr. Puspendra Kumar EXPERIMENT NO. 02 Aim: To develop and optimize the TLC solvent system for given sample of Caffeine sample. Requirement: TLC plate, TLC chamber, TLC development chamber, Solvents for mobile phase, Silica gel G, Forceps, Detecting reagents/UV chamber, Caffeine. References: Sethi PD and Charegaonkar D. “Identification of Drugs in Pharmaceutical Formulations by Thin Layer Chromatography”, Second Edition, 2005, CBS Publishers and Distributors, New Delhi, page: 1-27, 83. Theory Principle: Thin layer chromatography is a method of analysis in which the stationary phase (a finely divided solid) is spread as a thin layer on a solid rigid supporting plate; and the mobile phase: a liquid is allowed to migrate across the surface of the plate by the capillary action and separation takes places due to adsorption phenomenon and gives different R f values for each sample compound. Rf= Distance travel by solute/Distance travel by solvent Stationary Phase Silica gel, the most commonly used stationary phase, has the empirical formula SiO2. However, at the surface of the silica gel particles, the dangling oxygen atoms are bound to protons. The presence of these hydroxyl groups renders the surface of silica gel highly polar. Thus, polar functionality in the organic analyte interacts strongly with the surface of the gel particle and non polar functionality interacts only weakly. Mobile Phase For silica gel chromatography, the mobile phase is an organic solvent or mixture of organic solvents. As the mobile phase moves past the surface of the silica gel it transports the analyte past the particles of the stationary phase. However, the analyte molecules are only free to move with the solvent if they are not bound to the surface of the silica gel. Thus, the fraction of the time that the analyte is bound to the surface of the silica gel relative to the time it spends in solution determines the retention factor of the analyte. The ability of an analyte to bind to the surface of the silica gel in the presence of a particular solvent or mixture of solvents can be viewed as a the sum of two competitive interactions. First, polar groups in the solvent can compete with the analyte for binding sites on the surface of the silica gel. Therefore, if a highly polar solvent is used, it will interact strongly with the surface of the silica gel and will leave few sites on the stationary phase free to bind with the analyte. The analyte will, therefore, move quickly past the stationary phase. Dr. Puspendra Classes: https://www.youtube.com/user/puspendra007

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Dr. Puspendra Kumar Similarly, polar groups in the solvent can interact strongly with polar functionality in the analyte and prevent interaction of the analyte with the surface of the silica gel. This effect also leads to rapid movement of the analyte past the stationary phase. Method: Preparation of TLC plate Suspend 100 g of silica gel G in 200-250 ml of water, mix with a stirrer to get homogeneous slurry. Take the air dried TLC glass plates or dried in oven at 1100C and pour the silica gel G slurry into the glass plate. (Thickness should be around 250 μm) Slurry should be used within 2 minute of preparation otherwise slurry will dry and needs more water to maintain the fluidity. Dry the plate in a TLC chamber until complete drying occurs. Dried TLC plates are activated in oven at 1100C for 30 minutes and immediately used for development after cooling. Pre-coated plates: With the availability of pre-coated plates commercially, the use of laboratory hand-made plates is on decline. The pre-coated plates with different support material (glass, aluminium, plastic) and with different sorbent layers are available in different format and thickness by various manufacturers. Usually plates with sorbent thickness of 100-250μm are used for qualitative and quantitative analysis. Pre-coated plates also require activation. Sample Preparation For TLC on silica gel, the use of least polar solvent which allows quantitative dissolving and spotting of sample and there is no preliminary development and separation within the initial spot at the origin, is recommended. Application of sample Sample application is most critical step for obtaining good resolution. The sample should be completely transferred to the layer, however, under no circumstances; the application process should damage the layer, as damaged layer results in unevenly shaped spots. Wherever possible, use of automatic application device is recommended. The sample should be applied through clean smaller diameter capillary.

Dr. Puspendra Classes: https://www.youtube.com/user/puspendra007

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Dr. Puspendra Kumar Selection of mobile phase First level: A neat solvent from different selectivity areas is tested. Within a selectivity area, solvents may give similar separation. Usually diethyl ether, ethanol, methanol, tetrahydrofuran, dimethyl formamide, dichloromethane, ethylacetate, acetonitrile, methyl-ethyl ketone, toluene and chloroform are used as neat solvents. If acceptable resolution and medium Rf value range is achieved, the analyst can directly third level, if first level does not yield satisfactory result, then proceed with second level. Second level: From first level, solvents which leave the main fraction/component of the analyte near the starting point or close to the solvent front, are required to adjusted. If R f values are too high, solvent strength should be decreased by adding non polar solvents and polar solvents for too low Rf values. Third level: Mixtures of solvents from different selectivity group are investigated; the strength is adjusted, if required. These mixtures can be binary, tertiary or even quaternary, but binary mixtures are preferred one. At this level, addition of small amount of acidic (acetic acid) or basis (triethyl amine) modifiers significantly enhance the separation efficiency of mobile phase. Fourth level: At this level, final optimization of mobile phase to be used for a particular separation is made. To get the best separation, small variations in the proportions of different solvents may have to be made. Preconditioning Chamber saturation has pronounced influence on the separation profile. When the plate is introduced into an unsaturated chamber, during the course of development, the solvent evaporates from the plate mainly at the solvent front. Therefore larger quantity of the solvent shall be required for a given distance; hence resulting is increase in Rf values. If the tank is saturated (by lining with filter paper) prior to development, solvent vapours soon get uniformly distributed throughout the chamber. As soon as the plate is placed in such a saturated chamber, it soon gets preloaded with solvent vapours, hence less solvent shall be required to travel a particular distance, resulting in lower Rf values. Development and drying Develop the chromatogram in twin trough chamber or other TLC development chamber until and unless solvent reaches the three fourth distance of the plate. Dry the developed plate in chromatographic drying chamber. Evaluation of thin layer chromatogram First of all spots of TLC are detected by using suitable detecting reagent or physical methods. Dr. Puspendra Classes: https://www.youtube.com/user/puspendra007

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Dr. Puspendra Kumar The evaluation depends on the purpose of a chromatographic analysis. For quantitative determination often localization of substances is sufficient. This can be easily achieved by parallel runs with reference substances. Rf values: A parameter often used for qualitative evaluation is the Rf value (retention factor). The Rf value is defined as follows: Rf =

Distance travel by solute (spot) Distance travel by solvent front

i.e. Rf values are between 0 and 1, best between 0.1 and 0.8. Method: 1. Take the given mobile phase ration in a TLC chamber and saturate the chamber for 30 min. 2. Take a dried and activated TLC plate. 3. Take the prepared standard solution and apply the spot in TLC plate around 1 cm above from the bottom. 4. Wait for drying of spot and kept the TLC plate tilted in the saturated TLC chamber. 5. Develop the TLC plate up to 80-90% of the stationary phase. 6. Remove the TLC plate from the chamber and kept for air drying of mobile phase. 7. View the spot(s) under UV chamber or Iodine chamber. 8. Calculate the Rf values of the selected Caffeine standard. Results: Rf value of the standard and sample paracetamol was found to be ........ and ......... respectively.

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Dr. Puspendra Kumar EXPERIMENT NO. 03 Aim: Determination of hardness of water by complexometric titration. Requirement: Water for testing, Distilled Water, Eriochrome Black Bl ck T Indicator, EDTA, Volumetric flask, Conical flask, Burette, Pipette etc. References: Kar A. “Pharmaceutical Pharmaceutical Drug Analysis”, Analysis Third Edition, 2015, 5, New Age International (P) Ltd Publishers,, New Delhi, page: 171-172. 1  Theory: Complexometric titr titration (sometimes chelatometry) is a form of volumetric analysis in which the formation ation of a colored complex is used sed to indicate the end point of a titration.  Complexometric titrations ns are pparticularly useful for the determination ination of a mixture of different metal tal ions in solution.  An indicator tor capable of producing an una unambiguous color change is usually used to detect the end-point of the titration. Principle: In theory, any complexation plexation reaction can be used as a volu volumetric technique echnique provided that:  The reaction reaches equilibrium rapidly after each portion of titrant is added..  Interfering situations do not arise. For instance, the stepwise formation ation of several different complexes of the metal m ion with the titrant, resulting in the presence esence of more than one complex in solution dduring the titration process.  A complexometric etric indicator capable capa of locating equivalence point with fair accuracy is available.  In practice, the use of EDTA as a titrant is well established esta Ca+2/Mg+2 + Eriochrome Black T/Mordant Black T (Indicator) (Wine Red Color)

(Titrant: Ethylene diamine tetra acetic acid/ EDTA)

Ca-EDTA Complex / Mg-EDTA EDTA Complex + Free Eriochrome Black T Indicator (Colorless)

+

(Blue Color)

Dr. Puspendra Kumar (Final Color at the end point Blue) Method: Weigh accurately 18.6 g of disodium ethylene diamine tetra acetae, dissolve in sufficient DW in a 1 litre volumetric flask and make up the volume upto the mark. Calculations:

Standardization of 0.05 M Disodium Edetate Solution 

Weigh accurately about 0.8 g of granulated zinc dissolve by gentle warming in 12 ml of dilute hydrochloric acid and 5 drops of bromine water.



Boil to remove excess bromine, cool and add sufficient DW to produce 200 ml in a volumetric flask.



Pipette 20 ml of the resulting solution into a flask and neutralize carefully with 2N sodium hydroxide.



Dilute to about 150 ml with DW, add to it sufficient ammonia buffer (pH 10.0) to dissolve the precipitate and add a further 5 ml quantity in excess.



Finally add 50 mg of Mordant Black II mixture (mixture of 0.2 part Mordant Black II with 100 parts of NaCl)



Titrate with the disodium edetate solution until the solution turns green.



Each 0.003269 g of granulated zinc is equivalent to 1 ml of 0.05 M disodium ethylene diamine tetra acetate.

Calculations:

Determination of Calcium and Magnesium ion to determine the hardness of water 

Water hardness due to Ca and Mg is expressed as the amount of Ca and Mg ions in ppm.



Actually, the hardness is due to both Ca and Mg salts but these two are determined together in the titration.

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Dr. Puspendra Kumar 

The total Ca and Mg is titrated with standard EDTA solution using eriochrome black T as indicator.



Titration



Take 20 ml of normal water for test in a conical flask and add 5 ml of ammonia buffer solution (pH 10)



Add 3-4 drops of Eriochrome black T indicator solution and titrate against the appearance of light blue colour from starting light wine red or light pink colour.



Calculate the amount of Ca and Mg present in the water sample.

Calculation: 0.08g of Zn is present in the 20ml solution. So, 0.08/0.003269 = 24.47ml 0.05 M EDTA. Actual Molarity of EDTA = 0.05 x 24.47/ Vol. of EDTA consumed. Calculation for Hardness of water: 0 to 0.60 mmol/L:

Soft water

0.61 to 1.20 mmol/L: Moderately hard water 12.1 to 1.80 mmol/L: Hard water >1.80 mmol/L: 2+

Very hard water 2+

Conc. of M (Ca + Mg2+) = Molarity of EDTA x Vol. consumed (Burette reading)/ 20ml Results: Molarity of the water is found to be .......... and the water sample was ..................

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Dr. Puspendra Kumar EXPERIMENT NO. 04 Aim: To demonstrate the methodology of column chromatography. Requirement: Column, solvents for mobile phase, Silica gel for column. References: Skoog, Holler and Crouch, “Instrumental analysis” Tenth Edition, page: 419420. Theory Types of columns 

Gravity Columns: The mobile phase move through the stationary phase by gravity force.



Flash Columns (Air or nitrogen pressure): The mobile phase is pushed by stream of air or nitrogen using special special values (Adaptors).



Low and Medium Pressure Columns (pumped): The movement of mobile phase is accelerated by using pumps that generate low or medium pressure. The increase in the flow rate shorten the time of separation.



Vacuum Columns [Vacuum liquid chromatography (VLC)]: The adsorbent is applied dry into a sintered glass funnel. The sample is applied by dry method or as solution. Then the mobile phase is added portion by portion and vacuum is applied after each portion to collect each fraction.



High pressure Columns (HPLC): In this columns we use very fine silica gel so great increase in separation

power.

However, the flow rate of the mobile phase is

severely decreased. High pressure pumps are used to push the solvent through the column which in this case must be made of stainless steel. Adsorbent  The most common solid adsorbents are alumina (aluminum oxide) and silica gel (silicon dioxide).  A variety of grades and activities is available depending upon the specific application.  Silica gel is slightly acidic while alumina may be acidic, neutral or basic.  For greatest effectiveness the particles of solid adsorbent should be of uniform size and large surface area (for instance, 150 mesh alumina has a surface area of 155 m2/g).  The weight of adsorbent used is usually 20 - 50 times the sample weight, the greater ratio used for more difficult separations.  The strength of adsorption depends upon the compounds involved.

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Dr. Puspendra Kumar  Since the adsorbents are polar, the more polar compounds are adsorbed more strongly. Thus, non-polar compounds are eluted first.  The order of elution from a column usually follows the series:  Alkyl halides < saturated hydrocarbons < unsaturated hydrocarbons < ethers < esters < ketones < amines < alcohols < phenols < acids.  Polymeric compounds and salts will often not elute. Characteristics of Adsorbent  Adsorbent must not undergo any reaction with the substance(s) under separation.  Adsorbent must not catalyze in any way.  Adsorbent should have uniform particle size, shape and be colorless.  Adsorbent should not be much expensive.  Adsorbent should have high degree of mechanical stability. Activity of Adsorbent  Strong Adsorbent: Eg. Alumina, Fuller’s earth, Activated charcoal.  Intermediate Adsorbent: Eg. Calcium carbonate, Silica gel, Calcium phosphate, Magnesia  Weak Adsorbent: Eg. Cellulose, Starch, Sucrose powder, Talc. Packing of Columns  Slurry packing (Wet method): The adsorbent is suspended in the mobile phase and stirred very well to drive off all air bubbles. The resulted slurry is then poured into the column. In gel chromatography the adsorbent must be soaked in the mobile phase overnight to absorb the mobile phase and swell.  Dry Packing: In this method the dry adsorbent is poured to the column directly. Vibration is the applied to get rid of air bubbles then the mobile phase as passed through the adsorbent. This method cannot be applied in gel Chromatography Mobile phase: It is a mixture of organic solvents (unusually one solvent only) the choice of the column mobile phase is achieved after TLC study in different solvent systems. Isocratic system: Using the same mobile phase from the beginning to the end of the separation. Gradient: The polarity of the system increased gradually during separation by increasing the proportion of the more polar solvent. A typical gradient may be start with CHCl 3, followed by CHCl3/MeOH mixtures with gradual increase in % of MeOH till all spots are eluted from the system.

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Dr. Puspendra Kumar Sample Application  Wet application: Dissolve the sample in the initial mobile phase and apply by pipette to the top of the column. This is very good method but in most of cases the samples are not soluble in the initial mobile phase.  Dry loading:

Dissolve sample in any volatile solvent. The sample solution is then

adsorbed on small weight of adsorbent and the solvent is allowed to evaporate. The dry adsorbent loaded with the sample is then applied to the column. Column chromatography is one of the most common and useful ways to separate mixtures of materials and isolate large amounts of the individual components in pure form. In this experiment liquid chromatography will be employed to separate the components of a mixture. Much like TLC, the stationary phase will be silica gel and the mobile phase will be a solvent, or mixture of solvents. In contrast to TLC, where the components of a mixture were separated from top to bottom using capillary forces to draw solvent onto the bottom of a TLC plate, column chromatography is performed using gravity to draw solvent downward through a glass tube containing the silica gel. Column chromatography allows separation of relatively large amounts of a product mixture into pure individual compounds, each of which can be isolated, characterized, and perhaps used for other experiments or syntheses. This type of purification and isolation is crucial to all synthetic organic chemistry and the modern pharmaceutical industry could simply not exist without it. Various types of liquid chromatography are also widely used in separations and preparations of biological macromolecules such as proteins, carbohydrates, and DNA. Method: 1.

Clamp your column securely but gently to a ring stand, leaving enough space at the bottom to allow for easy switching of the flasks or beakers you will use to collect the eluted solvent. Try to clamp the column such that it is as straight as possible so the compounds will run evenly down the column. Place a funnel on the top of the column and place a beaker underneath. Also have ready several clean, labeled sample vials for collection of the compound containing fractions.

2.

Use a wet–pack method to fill the column. Weigh the required amount of silica gel into a small beaker; suspend into initial solvent which will be used for elution and pour into the column slowly. Care should be taken to avoid the entrapment of air bubble.

3.

Very carefully open the stopcock and let some of the solvent drip through.

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Dr. Puspendra Kumar 4.

Important that the column does not ever run dry during this process. Little cracks and channels are formed when air is allowed to enter the column, and separation is greatly compromised as a result.

5.

Close the stopcock when the solvent level is just covering the top of the column. If the top surface of the packed silica appears crooked, tap gently on the side of the column until it flattens out.

6.

Run the column and identify the fractions

Result: Methodology of column packing and separation were done.

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Dr. Puspendra Kumar EXPERIMENT NO. 05 Aim: Preparation and standardization of EDTA solution. Requirement: Distilled Water, Eriochrome Black Bl T Indicator, EDTA, Volumetric flask, Conical flask, Burette, Pipette ipette etc. References: Kar A. “Pharmaceutical Pharmaceutical Drug Analysis”, Analysis Third Edition, 2015, 5, New Age International (P) Ltd Publishers,, New Delhi, page: 171-172. 1 Theory:  Complexometric titration (so (sometimes chelatometry) is a form of volumetric etric analysis in which the formation of a colored ccomplex is used sed to indicate the end point of a titration.  Complexometric titrations ns are pparticularly useful for the determination ination of a mixture of different metal tal ions in solution.  An indicator tor capable of producing an una unambiguous color change is usually used to detect the end-point of the titration. Principle: In theory, any complexation plexation reaction can be used as a volu volumetric technique echnique provided that:  The reaction reaches equilibrium rapidly after each portion of titrant is added..  Interfering situations do not arise. For instance, the stepwise formation ation of several different complexes of the metal m ion with the titrant, resulting in the presence esence of more than one complex in solution dduring the titration process.  A complexometric etric indicator capable capa of locating equivalence point with fair accuracy is available.  In practice, the use of EDTA as a titrant is well established esta Ca+2/Mg+2 + Eriochrome Black T/Mordant Black T (Indicator) (Wine Red Color)

(Titrant: Ethylene diamine tetra acetic acid/ EDTA)

Ca-EDTA Complex / Mg-EDTA EDTA Complex + Free Eriochrome Black T Indicator (Colorless)

+

(Blue Color)

Dr. Puspendra Kumar (Final Color at the end point Blue) Method: Weigh accurately 18.6 g of disodium ethylene diamine tetra acetae, dissolve in sufficient DW in a 1 litre volumetric flask and make up the volume upto the mark. Calculations:

Standardization of 0.05 M Disodium Edetate Solution 

Weigh accurately about 0.8 g of granulated zinc dissolve by gentle warming in 12 ml of dilute hydrochloric acid and 5 drops of bromine water.



Boil to remove excess bromine, cool and add sufficient DW to produce 200 ml in a volumetric flask.



Pipette 20 ml of the resulting solution into a flask and neutralize carefully with 2N sodium hydroxide.



Dilute to about 150 ml with DW, add to it sufficient ammonia buffer (pH 10.0) to dissolve the precipitate and add a further 5 ml quantity in excess.



Finally add 50 mg of Mordant Black II mixture (mixture of 0.2 part Mordant Black II with 100 parts of NaCl)



Titrate with the disodium edetate solution until the solution turns green.



Each 0.003269 g of granulated zinc is equivalent to 1 ml of 0.05 M disodium ethylene diamine tetra acetate.

Calculations:

Calculation: 0.08g of Zn is present in the 20ml solution. So, 0.08/0.003269 = 24.47ml 0.05 M EDTA. Actual Molarity of EDTA = 0.05 x 24.47/ Vol. of EDTA consumed. Results: Molarity of the prepared EDTA was found to be ......... M.

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