Scholarly document

1. Department of Pharmacy
School of Medicine
University of Zambia
Medicinal Chemistry (PMY 440)
Laboratory Manual
By: Lungwani T.M. Muungo
(2003)
[This manual Has been prepared to cover Molecular Modeling, Synthesis and Pharmacopoeial
Assays]

2. Serial No. Content Page
1 Tutorial for Chemsketch 3
2 AutoDock 4 and ADT: Hands on Docking 12
3
Tutorial For Chimera
20
Preparation of Molecules for DOCKing
4 Tutorial For Pymol 39
5 Synthesis of Benzimidazole 45
6 Synthesis of 2-phenyl Indole 48
7 Synthesis of Benztriazole 51
8 Synthesis of 7-hydroxy-4methyl coumarin 54
9 Synthesis of Paracetamol 57
10 Synthesis of Phenytoin 61
11 Assay of Aspirin Tablets 63
12 Assay of Ascorbic Acid Tablets 66
13 Assay of Paracetamol tablets 68
14 Assay Of Promethazine Hydrochloride tablets 70
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3. Tutorial for Chemsketch.
Chemical drawing is very basic step for chemistry students as well as for drug designing
subjects. In this chapter a very basic art of chemical drawing has been explained, for adwanced
drawing students are adwised to go to the link
https://tigadalamsatu.files.wordpress.com/2012/04/chemsk_t.pdf
In chem. sketch there are 2 tabs named drawing tab and structure tab for chemical
drawing students should mainly work in structure tab.
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12. AutoDock Tutorial
AutoDock 4 and ADT: Hands on Docking
Overview
Working with AutoDock4 includes 3 steps:
1. Preparation of receptor & ligand files.
2. Calculation of affinity maps by using a 3D grid around the receptor & ligand.
3. Defining the docking parameters and running the docking simulation. The preparation step
starts with pdb files of receptor (R.pdb) and ligand (L.pdb), which are added hydrogens and then
saved as RH.pdb & LH.pdb. The calculation of affinity maps in the "Grid" section requires the
above pdb files to be assigned charges & atom types, and also that the nonpolar hydrogens are
merged. This is done automatically by ADT, and the resulting files need to be saved as RH.pdbqt
& LH.pdbqt, which is the only format AutoGrid & AutoDock can work with. Calculation of
affinity maps is done by AutoGrid, and then docking can be done by AutoDock. The newest
docking algorithm is LGA (Lamarckian Genetic Algorithm).
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13. Preparing and Running a Docking job
A. Preparing the protein
1. Opening file: [Right-click "PMV molecules"] → [choose file].
2. Color by atom: [Click ◊ under "Atom"].
3. Eliminate water: Select → Select from string → [write HOH* in "Residue" line
and * in the "Atom" line] → Add → Dismiss → Edit → Delete → Delete AtomSet.
4. Find missing atom and repairing them: File → Load module → [Pmv;
repairCommands] → Edit → Misc. → Check for missing atoms → Edit → Misc. →
Repair missing atoms.
5. Add hydrogens: Edit → Hydrogens → Add → [choose "All hydrogens", "no bond
order", and "Yes" to renumbering]. under "showMolecules"].
6. Hide protein: [Click on the gray (Note: if you are planning rigid docking (i.e. no
flexible parts in the protein), save the protein as RH.pdb for now)
B. Preparing the ligand
1. Make sure the ligand has all hydrogens added before working with ADT.
2. [Toggle the "AutoDock Tools" button].
3. Opening file: Ligand → Input → Open → All Files → [choose file] → Open.
(ADT now automatically computes Gasteiger charges, merges nonpolar hydrogens, and
assigns Autodock Type to each atom.)
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14. (Note: if there are problems with the automatic charge assignment on any residue, this could be
addressed by: Edit → Charges → Check Totals on residues. The molecule is now shown with all
the assigned charges, and they could be changed manually. Alternatively, the deficit charge can
be spread over the entire residue.)
4. Define torsions:
* Ligand → Torsion Tree → Detect Root (this is the rigid part of the ligand)
* Ligand → Torsion Tree → Choose Torsions → [either choose from the viewer
specific bonds, or use the widget to make certain bond types active (rotatable) or inactive (non-
rotatable). Amide bonds should NOT be active (colored pink)] → Done.
* Ligand → Torsion Tree → Set Number of Torsions → [choose the number of
rotatable bonds that move the 'fewest' or 'most' atoms].
5. Save ligand file:
* Ligand → Output → Save as PDBQT → [save with L.pdbqt].
6. Hide the ligand, as explained in (A5) for the protein.
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15. C. Preparing the flexible residue file
(Note: if you are planning rigid docking, ignore this section and do the following:
Grid → Macromolecule → Open → [choose RH.pdb].
(AutoDock will automatically add charges and merge hydrogens. Save the object as RH.pdbqt
and move to section D.)
1. Flexible residues → Input → Choose molecule → [choose the original protein
R.pdb] → Yes to merge nonpolar hydrogens (AutoDock assigns charges + atom
types to R.pdb, and merges nonpolar hydrogens).
2. Select the residues to be flexible: Select → Select from string → ARG8 → Add →
Dismiss.
3. Define the rotatable bonds: Flexible residues → Choose torsions in currently
selectedresidues → [click on rotatable bonds to inactivate them or vice versa].
4. Save the flexible residues: Flexible residues → Output → Save flexible PDBQT →
[save as R_flex.pdbqt].
5. Save the rigid residues: Flexible residues → Output → Save rigid PDBQT → [save
as R_rigid.pdbqt].
6. Delete this version of protein: Edit → Delete → Delete Molecule → [choose protein
(R)] → Delete → Dismiss.
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16. D. Running AutoGrid calculation
The purpose of this section is to define the search grid and produce grid maps used later by
Autodock.
1. Open the rigid protein: Grid → Macromolecule → Open → [choose the rigid protein]
→ Yes to preserving the existing charges.
(Note: if you are doing rigid docking, choose RH.pdbqt)
2. Prepare grid parameter file: Grid → Set Map Types → Choose Ligand → [choose
the ligand already opened] → Accept.
3. Set grid properties: Grid → Grid Box → [Set the grid dimensions, spacing, and
center] → File → Close Saving Current.;
4. Save the grid settings as GPf file: Grid → Output → Save GPF → [save as R.gpf].
5. [Make sure the AutoGrid executable is in the same directory as the input files].
6. Running: Run → Run AutoGrid → [make sure the program name has the right
path, and that it is where the input files are] → Launch → [in the command prompt
prompt, type "tail –f hsg1.glg" to follow the process]
(Note: the AutoGrid calculation can be started directly from the command prompt by typing
"autogrid4 –p hsg1.gpf –l hsg1.glg &")
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17. E. Preparing the docking parameter file (.dpf)
1. Specifying the rigid molecule: Docking → Macromolecule → Set Rigid Filename →
[choose R_rigid.pdbqt]. (or RH.pdbqt for rigid docking)
2. Specifying the ligand: Docking → Ligand → Choose → [choose L.pdbqt] → [here
you can set the initial location of the ligand] → Accept.
3. Specifying the flexible residues: Docking → Macromolecule → Set flexible Residues
Filename → [choose R_flex.pdbqt].
4. Setting the parameters for the chosen docking method: Docking → Search Parameters
→ Genetic Algorithm → [for 1st time, use the short number of evaluations (250,000),
and for other runs choose the medium or long] → Accept.
5. Setting docking parameters: Docking → Docking Parameters → [choose the defaults].
6. Specifying the name of the ligand dpf file to be formed, containing the
docking instructions: Docking → Output → Lamarckian GA → [type L.dpf].
7. Confirming the details of docking: Docking → Edit DPF → [make sure the right
ligand pdbqt file name appears after the word "move", and that the right number
of active torsions is specified].
Note: "outlev" specifies the level of output detail in the docking result file, in the section
where the conformations are analyzed for similarity. For LGA docking, outlev=0 is sufficient.
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18. F. Running AutoDock4
1. [Make sure the AutoDock executable is in the same directory as the
macromolecule, ligand, GPF, DPF and flex files (in case of flexible docking)].
2. Running: Run → Run AutoDock... → Launch.
When RH and LH already exist
1. Protein:
Grid → Macromolecule → choose RH.pdb → (charges & atom types assigned,
nonpolar hydrogen merged) → File → save → write PDBQT → save as RH.pdbqt
2. Ligand:
Ligand → Input → Open → All Files → choose LH.pdb → (charges & atom types
assigned, nonpolar hydrogen merged) → save as LH.pdbqt
3. Set the rest of the grid parameters & calculate map
4. Setting Docking parameters:
Docking → Macromolecule → Set Rigid Filename → choose either RH.pdbqt or
RH_rigid.pdbqt → Docking → Ligand → Choose → choose LH.pdbqt → set the rest
of the docking parameters.
5. Running docking simulation.
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19. Viewing Docking Results
A. Reading the docking log file (.dlg)
1. [Toggle the "AutoDock Tools" button].
2. Analyze → Dockings → Open → [choose L.dlg].
3. Analyze → Conformations → Load → [double-click on each conformation
to view it on screen].
B. Visualizing docked conformations
1. Analyze → Conformations → Play... (Note: & allows changing the ligand's
color)
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20. Tutorial For Chimera
Preparation of Molecules for DOCKing
This tutorial describes the steps required to prepare receptor and ligand molecules as inputs for
DOCK calculations that predict orientations of a ligand in a receptor active site. It studies the
complex L-Arabinose-Binding Protein bound to L-Arabinose (PDB code 1ABE ) as an example
system. However, these techniques should be transferable to any protein-ligand system.
This tutorial uses the program Chimera (Snapshot Release 1.2309), which can be downloaded
from the UCSF Computer Graphics Lab athttp://www.cgl.ucsf.edu/chimera. Chimera is freely
available to academics and relatively simple for novice users to learn. However, a variety of
other packaged programs and script libraries are available to perform these types of
modifications to the structure files (see DOCK Related Links for more information).
For a receptor, an overview of the general procedure is to visualize the source file of the target,
to remove extraneous atoms, such as alternate conformations, ligands, ions, solvent molecules,
cofactors, etc., to add missing atoms, such as, hydrogens, incomplete side chains, etc., to assign
atom types and partial charges, to create a final mol2 file, and to create a final pdb file without
hydrogens. During all these steps, one should keep in mind one's scientific model. For assigning
atom types and partial charges, Dock uses Sybyl atom type labels but Amber force field
parameters. For a ligand, the general procedure is simpler but similar.
STEP 0: Examine the pdb file.
In this case, the file we shall be using is 1ABE.pdb. A visualization of this file can be seen
below:
Image generated using Chimera (http://www.cgl.ucsf.edu/chimera)
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21. This file contains Cartesian coordinates for the L-arabinose-binding protein (red ribbon),
crystallographic waters (purple), and two conformations of the ligand L-arabinose (green and
orange). Each of these components must be dealt with during preparation for DOCKing.
STEP 1: Prepare the receptor file.
1a) Open the 1ABE.pdb file in Chimera
1b) Select and delete the ligands (L-arabinose) from the complex
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23. 1c) Use the Dock Prep tool to complete the receptor preparation. For more information on the
Dock Prep module, see the Chimera documentation. Note that recent versions of Chimera
provide additional features compared with those in the screen shot below. In particular,
'Mutate residues with incomplete side chains to ALA (if CB present) or GLY' is a popular
feature. The screen shots will be updated soon.
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24. 1d) Select the method for adding hydrogens; in this case we will allow the hydrogen to be
optimized by the hydrogen bonding network, and we will allow the method to determine the
protonation state
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25. 1e) Examine warnings from the Dock Prep procedure
1f) Resolve issues that are causing warnings in the Dock Prep procedure
As you can see, there are a few warnings about non-standard atoms for this receptor. You
can use Chimera to take a closer look at the problem residues using the Command Line.
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26. This action will open the Command Line interface. Type the following commands into the
command line to isolate the first residue in the warning--LYS 306.
~display
display :306
focus
color byelement
linewidth 3
rlabel
This series of commands will 1) undisplay the entire receptor, 2) display residue 306 only, 3)
refocus the screen on residue 306, 4) color the atoms based on element, 5) increase the size of the
bonds for easier viewing, and 6) label the residue. For more information on other Command Line
options, see the Chimera documentation for the Basic Function: Commands.
It should now be obvious that there is a problem with this lysine residue as compared to a
normal lysine--LYS 300.
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27. Most likely, the crystallographer knew the terminal residue was a LYS, but did not see any
electron density for the LYS 306 side chain. As a result, only the backbone was built into the
structure. Because Chimera is being told this residue is a LYS, the charges for the LYS
template are being loaded resulting in non-integral charges for the residue and causing the
warning message.
The best way to fix this situation is to mutate the incomplete LYS residue to a GLY residue.
GLY residues have the appropriate number of atoms, which will result in an integral set of
charges for the residue, and the structure will still comply with the experimental data. To
mutate the LYS residue, type the following command into the Command Line:
swapaa gly :306
This command will change the LYS 306 to a GLY in the same orientation.
You need to repeat this procedure for the ASN 2 residue, which is the source of the
remaining warnings.
swapaa gly :2
1g) Save the receptor in mol2 format
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28. Once all the warnings have been resolved, the receptor can be saved in mol2 format. The Dock
Prep procedure should be run again to incorporate the mutated residues (see STEP 1c). Make
sure the Write Mol2 box is checked at this point. The final molecule can then be written to
file, in this case as rec_charged.mol2.
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29. NOTE: When saving, make sure all the boxes in the Save As dialog box are checked!
1h) Strip hydrogens from the mutated receptor and save in pdb format (this step is necessary
for molecular surface generation in the Sphere Generation and Selection Tutorial)
To perform this step, first select all the hydrogens from the molecule and then delete them.
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31. The receptor should now be saved in pdb format: rec_noH.pdb.
NOTE: When saving, make sure the "Use untransformed coordinates " box is checked in the
Save As dialog box!
STEP 2: Prepare the ligand file.
We will only prepare the L-arabinose A conformation in the pdb file for simplicity. Selection
of the conformation A over the conformation B was arbitrary.
2a) Open the 1ABE.pdb file in Chimera
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32. 2b) Select and delete everything BUT the ligand from the complex
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34. 2c) Remove the B conformation of the ligand
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35. 2d) Make the molecule easier to see; use these Command Line (see STEP 1f) commands or
hunt and peck to perform the equivalent with the mouse
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36. focus
color byelement
linewidth 3
2e) Add hydrogens
2f) At this point, we recommend one of two options for completing preparing the ligand
depending on your needs and application:
OPTION 1: Calculate charges using the Chimera Add Charge tool.
The Add Charge tool is a call to the antechamber program. Antechamber is a set of auxiliary
programs for molecular mechanic (MM) studies. This software package addresses the
following issues in MM calculations:
(1) recognizing the atom type
(2) recognizing the bond type
(3) judging the atomic equivalence
(4) generating the residue topology file
(5) finding missing force field parameters and supplying reasonable and similar substitutes
Antechamber can generate input automatically for most organic molecules in a database.
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37. (A) To complete this option, first activate the Add Charge tool
(B) To seed the charge calculation, Chimera needs the formal charge of the molecule.
Chimera will estimate the value based on the atom types and bonding. Here, Chimera has
estimated accurately, so we will calculate AM1BCC charges.
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38. (C) Save the molecule in mol2 format
OPTION 2: Prepare your ligand(s) using the ZINC database.
ZINC is a free database of commercially-available compounds for virtual screening. It contains
over 35 million compounds in ready-to-dock, 3D formats (Irwin, Sterling, Mysinger, Bolstad and
Coleman, J. Chem. Inf. Model. 2012. DOI: 10.1021/ci3001277). We recommend this procedure
if you have a large library of ligands to prepare or if you would like a protonation state or
conformation expansion. If you utilize this option, be sure to save your ligand preparation work
from the previous parts of STEP 2: save the ligand in Tripos mol2 format (see part C of OPTION
1 of STEP 2f above). The ZINC web site is zinc.docking.org.
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39. Tutorial For Pymol
Download the Protein from www.pdb.org ( e.g. 21ty, 1DB1 etc)
Open it in pymol
Then Remove water as following : Protein : Action : Remove waters
SCREEN SHOT AFTER WATER REMOVAL
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40. Add Hydrogen by following step: Protein : Action : Hydrogen : Add
SCREEN SHOT AFTER HYDROGEN ADDITION:
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41. Steps for Ligand sites: 1db1: action : preset: ligand sites: cartoon :
SCREEN SHOT AFTER ligand sites:
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42. Select the ligand by clicking on the ligand:
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43. Extract the ligand by following: sele : action : extract object
General Notes:
 For zooming right click and drag

SCREEN SHOT AFTER EXTRACTION
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44. Save the extracted ligand and protein one by one: File save molecule:
DATE :
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45. EXERCISE NO. :
OBJECT : To synthesize and submit Benzimidazole and determine its
% yield and melting point.
REQUIREMENTS : o-Phenylenediamine, Formic acid, Sodium hydroxide,
Beaker, Conical flask, Pipette, Measuring cylinder etc.
PRINCIPLE :
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46. FORMULA(E) :
Theoretical Yield
Percentage Yield = -------------------------- x 100
Practical yield
CALCULATION :
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47. Theoretical Yield :
Practical Yield :
Percentage Yield :
PROCEDUR
E
: Place 27 g ( 0.25 mol) of o-phenylenediamine in a 250 ml RBF and add
17.5 g (16 ml, 0.34 mol) of 90 % formic acid. Heat the mixture at water bath
at 100 oC for 2 hours. Cool, add 10% sodium hydroxide solution slowly,
with constant stirring of the flask until the reaction is just alkaline to litmus.
Filter off the crude Benzimidazole at the pump. Wash with ice-cold water,
drain well and wash again with 25 ml of cold water. Dissolve the crude
product in 400 ml of boiling water, add 2g of decolorizing carbon and digest
for 15 min. Filter rapidly at the pump through a preheated Buchner funnel
and flask. Cool the filtrate to about 10 oC, filter off the Benzimidazole, wash
with 25 ml of cold water and dry at 100 oC .
RESULT : % YIELD______________
M.P.__________________
USES :
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48. DATE :
EXERCISE NO. :
OBJECT : To synthesize and submit 2-Phenyl Indole and determine its
% yield and melting point.
REQUIREMENTS : Acetophenone, Phenyl hydrazine, Ethanol, Glacial Acetic acid,
Polyphosphoric acid, Beaker, Conical flask, Pipette, Measuring cylinder
etc.
PRINCIPLE :
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49. FORMULA(E) :
Theoretical Yield
Percentage Yield = -------------------------- x 100
Practical yield
CALCULATION :
Theoretical Yield :
Practical Yield :
Percentage Yield :
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50. PROCEDURE : (A) Acetophenone Phenyl Hydrazone: Warm a mixture of 20 g (0.167
mol ) Acetophenone and 18 gm of Phenyl Hydrazine with 60 ml of ethanol
and a few drops of glacial acetic acid. Filter the cold reaction mixture, wash
the solid with hydrochloric acid followed by about 12 ml of cold rectified
spirit. Recrystallize a small portion with ethanol and thus obtain a sample of
pure acetophenone phenyl hydrazone as a white solid.
(B) 2-Phenyl Indole : Place 28 g of the crude phenyl hydrazone in a 250 ml
beaker containing 180 g of Polyphosphoric acid. Heat on a boiling water
bath, stir with a thermometer and maintain at 100-120
0
C for 10 min. (the
reaction is exothermic). Add 450 ml of cold water and stirr well to complete
the solution of Polyphosphoric acid. Filter at pump and wash well with
water. Heat the crude solid under reflux with 300 ml of rectified spirit. Add
little decolorizing charcoal and filter through a preheated Buchner Funnel,
Wash the residue with 40 ml of hot rectified spirit. Cool the combined
filtrates to room temperature; filter off the 2-Phenyl Indole and wash it
three-times with 10 ml portion of cold alcohol. Dry in a vacuum desiccator
over anhydrous Calcium chloride.
RESULT : % YIELD ______________
M.P. ______________
USES/CATEGORY :
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51. DATE :
EXERCISE NO. :
OBJECT : To synthesize and submit Benzotriazole and determine its
% yield and melting point.
REQUIREMENTS : o-Phenylenediamine, Glacial acetic acid, Sodium nitrite, Benzene,
Beaker, Conical flask, Pipette, Measuring cylinder etc.
PRINCIPLE :
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52. FORMULA(E) :
Theoretical Yield
Percentage Yield = -------------------------- x 100
Practical yield
CALCULATION :
Theoretical Yield :
Practical Yield :
Percentage Yield :
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53. PROCEDUR
E
: Dissolve 10.8 g ( 0.1 mol) of o-phenylenediamine in a mixture of 12 g
(11.5 ml, 0.2 mol) of glacial acetic acid and 30 ml of water contained in a
250 ml Beaker, slight warming may be necessary. Cool the clear solution to
10 oC. Stirr magnetically and then add a solution of 7.5 g (0.11 mol) of
sodium nitrite in 15 ml of water in one portion. The reaction mixture
becomes warm and within 2-3 min. reaches a temp. of about 85 0C and then
begins to cool while the color changes from deep red to pale brown.
Continue stirring for 15 min by which time the temperature will have
dropped to 35-400Cand then thoroughly chill in an ice water bath for 30
min. collect by vacuum filtration the pale brown solid which separates and
wash with three 30 ml. Portions of ice cold water. Dissolved the solid in
about 130 ml of boiling water, add decolorizing charcoal, filter and allow
the filtrate to cool to about 50 0C before adding a few crystals of the crude
Benzotriazole which have been retained for seeding. Allow the mixture to
attain room temp. slowly (to avoide the separation of the material as oil) and
then thoroughly. Chill in ice and collect the Benzotriazole, which separates
as pale straw colored needles. Second crop may be obtained by
concentrating the filtrate. The Benzotriazole may be recrystallized from
benzene.
RESULT : % YIELD______________
M.P. ______________
USES :
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54. DATE :
EXERCISE NO. :
OBJECT : To synthesize and submit 7-hydroxy-4methyl coumarin from
Resorcinol & Ethyl Acetoacetate, determine its % yield &
melting point.
REQUIREMENTS : Ethyl acetoacetate, Resorcinol, Pipette, Measuring
cylinder, RBF, etc.
PRINCIPLE :
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55. FORMULA(E) :
Theoretical Yield
Percentage Yield = -------------------------- x 100
Practical yield
CALCULATION :
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56. Theoretical Yield :
Practical Yield :
Percentage Yield :
PROCEDURE : Stirr 15 ml of Conc. H2SO4 mechanically in a flask with external cooling by
ice up to 4 -5
o
C, meanwhile add 3.7 g powdered resorcinol to 4.4 ml of
ethyl acetoacetate, stir the solution to obtain a complete solution. Add this
solution slowly to H2SO4 so that the temperature of the flask does not rise
above 10
o
C, then continue stirring for 30 min. Pour the mixture on crushed
ice (100g) in a thin stream with strirring, the solid 7-hydroxy-4methyl
coumarin separates. Filter off the product on a buchner funnel under
suction. Wash the product with a spray of cold water.
RESULT : % YIELD______________
M.P.__________________
USES :
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57. DATE :
EXERCISE NO. :
OBJECT : To synthesize and submit p-acetylaminophenol (Paracetamol)
from p-aminophenol, determine its % yield & melting point.
REQUIREMENTS : p-aminophenol, Acetic anhydride, Pipette, Measuring
cylinder, conical flask, etc.
PRINCIPLE :
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58. FORMULA(E) :
Theoretical Yield
Percentage Yield = -------------------------- x 100
Practical yield
CALCULATION :
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59. Theoretical Yield :
Practical Yield :
Percentage Yield :
PROCEDURE : Suspend 5.5 gm of p-aminophenol in 15 ml of water contained in a conical
flask and 6 ml of acetic anhydride. Shake the mixture vigorously and warm on
water bath. The solid dissolves after 10 min., cool the mixture. The crystals are
formed filter it and wash with little cold water. Recrystallise from hot water and
dry in air.
RESULT : % YIELD______________
M.P.__________________
USES :
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60. DATE :
EXERCISE NO. :
OBJECT : To synthesize and submit 5,5 Diphenyl hydantoin or Phenytoin
and determine its % yield and melting point.
REQUIREMENTS : Benzoin, Conc. HNo3, Urea, 30% NaOH, Absolute Ethanol,
Conc. HCl, Methylated spirit, etc.
PRINCIPLE :
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61. FORMULA(E) :
Theoretical Yield
Percentage Yield = -------------------------- x 100
Practical yield
CALCULATION :
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62. Theoretical Yield :
Practical Yield :
Percentage Yield :
PROCEDURE : (A) Preperation of Benzil from benzoin: Take 10 g of Benzoin & add in
25 ml of conc. Hno3, Reflux it for 1 hr, whenever it converts into Oil layer,
stop the reaction at this point. Pour it into cold water, crystal of benzil
separate out with having yellow colour, filter & air-dry. Recrystallized in
ethanol, these are the crystal of Benzil.
(B) Preperation of Phenytoin from Benzil : Took 5.3 g of benzil , 3g of
urea, 15 ml of 30% NaOH & 75 ml of absolute ethanol in RBF (Round
Bottom Flask). Preferably reflux it over hot plate for about 2 hrs. Poured
this reaction mixture into 125 ml of cold water. Keep out it for 15 min,
insoluble byproduct get settled and filter it. Acidified with conc. HCl &
Phenytoin get precipitated. Filter at pump and wash well with water.
Recrystallized the product in ethanol.
RESULT : % YIELD
M.P.
______________
______________
USES/CATEGORY :
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63. Name: Date:
THE ASSAY OF ASPIRIN
(WORKSHEET)
OBJECTIVE:
PROCEDURE: Quickly titrate a sample of aspirin with a portion of NaOH using
phenolphthalein as the indicator. Add an equivalent amount of NaOH plus excess. Titrate the
excess with standardized HCl. Use these values to calculate the percentage of aspirin in the
sample.
DATA:
Molarity of NaOH = Molarity of HCl =
Mass of Aspirin Trial #1 Volume of NaOH Volume of NaOH Volume of HCl
Used (I Titration) (II Titration)
Start
Finish
Total
Mass of Aspirin Trial #1 Volume of NaOH Volume of NaOH Volume of HCl
Used (I Titration) (II Titration)
Start
Finish
Total
Mass of Aspirin Trial #1 Volume of NaOH Volume of NaOH Volume of HCl
Used (I Titration) (II Titration)
Start
Finish
Total
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64. Aspirin Assay: Sample Calculations
1)Sample mass sample wt. g (A)
2)First base titration initial reading .
final reading .
first volume ml
3)Second base increment final reading .
total added base ml ( = B - convert to liters)
4)Back titration initial reading .
final reading .
Vol. of HCl ml ( = C - convert to liters)
5)Molarity of NaOH ( = D)
6)Molarity of HCl (from bottles) ( = E)
7)Total moles of base consumed by aspirin (B x D) - (C x E ) = ( = F)
8) % Aspirin = [½ x 180.15 x 100] x F / A =
9) Second sample: Repeat calculations up to step 8 ±
10) Third sample: Repeat calculations up to step 8 ±
11) Average % aspirin = (8 + 9 + 10) / 3 =
RESULTS:
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65. Detail Procedure:
1. Get a sample of powdered aspirin from the instructional assistant or the instructor and
accurately weigh three samples of 0.5 gm each into 150 mL Erlenmeyer flasks. Do not
dry the samples, Dissolve each sample in turn in 15 mL of absolute alcohol, add 4 drops
phenolphthalein indicator, and titrate each sample quickly to the first persistent faint pink
color with standard 0.1 M NaOH.
2. Record this volume and then add, from your buret, that same volume again + 5 mL
excess. Place the flasks on the steam bath for 15 minutes to allow reaction (2) to proceed
to completion. Then back-titrate the excess base with your standard 0.1 M HCl.
3. From the total titration volumes you may calculate the percentage aspirin in your sample.
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66. THE ASSAY OF ASCORBIC ACID
OBJECTIVE:
Procedure: Titrate the solution of ascorbic acid with Iodine solution of
known concentration using 0.5 % starch solution as indicator.
Solutions Needed:

Iodine solution: (0.01 M). Weigh 2 g of potassium iodide into a 100 mL beaker. Weigh
1.3 g of iodine and add it into the same beaker. Add a few mL of distilled water and swirl
for a few minutes until iodine is dissolved. Transfer iodine solution to a 500 ml
volumetric flask, making sure to rinse all traces of solution into the volumetric flask
using distilled water. Make the solution up to the 500 mark with distilled water.


Starch indicator solution: (0.5%). Weigh 0.25 g of soluble starch and add it to 50 mL of near
boiling water in a 100 mL conical flask. Stir to dissolve and cool before using.


For sample preperation: Weigh the tablet equivalent to 0.5 gm of Vitamin C and dissolve in
500 mL of distilled water (in a volumetric flask).

Titration
1. Pipette a 10 mL aliquot of the sample solution into a 250 mL conical flask and add about 150
mL of distilled water and 1 mL of starch indicator solution.
2. Titrate the sample with 0.01 M iodine solution. The endpoint of the titration is identified as
the first permanent trace of a dark blue-black colour due to the starch-iodine complex.
3. Repeat the titration with further aliquots of sample solution until you obtain concordant
results (titres agreeing within 0.1 mL
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67. Calculation:
Theoretically
1 mole of iodine solution ≡ 1 mole of C6H8O6
1000 ml 0f 1M iodine solution ≡ 176 g of vit c
1 ml 0f 1 M iodine solution ≡ 0.176 g of vit c
1 ml 0f 0.01 M iodine solution ≡ 0.00176 g of vit c
2 ml 0f 0.01M iodine solution ≡ 2 x 0.00176 g of vit c
V(ml) of 0.01M iodine solution ≡ V x 0.00176 g of vit c = X g of vit C
% Purity = × 100ℎ
Result:
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68. THE ASSAY OF PARACETAMOL By UV SPECTROSCOPY
OBJECTIVE:
Procedure:
Diluent preparation
Methanol and water (15:85, v/v) used as a diluent.
Standard preparation
10 mg drug was dissolved in 15 ml methanol and was shaken well. Then 85 ml water was added
to it to adjust the volume up to 100 ml (100 ppm). From that 5 ml was taken and volume was
adjusted up to 50 ml with diluents.
Test preparation
20 tablets were weighed and powdered. Powdered tablet equivalent to 100 mg of paracetamol
was weighed and taken into 100 ml volumetric flask then 15 ml of methanol was added and
shaken well to dissolve it after that 85 ml of water was added to adjust the volume up to 100 ml.
From that 1 ml of solution was withdrawn and taken in 100 ml volumetric flask. The volume was
adjusted with diluent up to 100 ml.
Formula:
The single point standardization procedure involves the measurement of the absorbance of a
sample solution and of a standard solution of the reference substance. The concentration of the
substances in the sample is calculated from the proportional relationship that exists between
absorbance and concentration.
Ctest= (Atest×Cstd)/Astd
Where Ctest and Cstd are the concentrations in the sample and standard solutions respectively
and Atest and Astd are the absorbances of the sample and standard solutions respectively.
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69. Calculation
%=
Mean Test concentration
× 100Mean Std concentration
Results:
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70. The Assay Of Promethazine Hydrochloride tablets
OBJECTIVE:
Procedure:
 Carry out the following procedure protected from Light.

 Triturate a quantity of powder containing 50 mg of Promethazine Hydrochloride with 10
ml of 2M HCl and add 200 ml water.

 Shake for 15 minutes, add sufficient water to produce 500 ml and centrifuge about 50 ml
of the mixture, to 5 ml of clear supernatant liquid add 10 ml 0f 0.1 M HCl and sufficient
water to produce 500 ml.

 Measure the absorbance of the resulting solutionat maximum at 249 nm, Calculate the
content of Promethazine Hydrochloride taking 910 as the value of A (1%, 1 cm) at
maximum at 249.
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71. Calculation
%=
Mean Test concentration
× 100Mean Std concentration
Results:
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