Download EFFECT OF pH ON THE PARTITION CO

EFFECT OF pH ON THE PARTITION CO-EFFICIENT
AIM:
To determine the partition co-efficient of aspirin and the effect of different pH on the
partition co-efficient of aspirin
MATERIALS REQUIRED:
Chemicals: ASPIRIN-50mg, 25 mg, 10mg, n-hexane, phosphate buffer, hydrochloric acid
and sodium hydroxide
Instruments and apparatus: UV-spectrometer, pipette, pH meter, separating funnel.
PRINCIPLE:
Partition co-efficient and log P are used in QSAR studies and rational drug design as a
measure of molecular hydrophobicity. Partition co-efficient provides a thermodynamic measure
of its hydrophilicity-lipophilicity balance. Hydrophobicity affects drug absorption,
bioavailability, hydrophobic drug-receptor interactions, metabolism of molecules as well as their
toxicity.
Partition co-efficient is the ratio of the concentration of a chemical species dissolved by
one phase to the concentration of the species in the other phase. It can calculate by dividing the
amount of compound present in the aqueous phase and applying logarithm.
Concentration of solute in oil phase
K = ------------------------------------------------Concentration of solute in water phase
Liphophilicity is a key factor determining in vivo behavior of drugs; determining
pharmacokinetics factors (permeation of physiological membrane, plasma protein binding,
volume of distribution) and pharmacodynamic factors (target recognition, target affinity, target
specificity)
PROCEDURE:
In three separating funnels 10 ml of Phosphate buffer of different pH (4.0, 7.4 and 9.2)
were taken. To this 50 mg, 25 mg, 10 mg aspirin were weighed and dissolved. To these solutions
each 10 ml of n-hexane was added. These flasks were then shaken for 15 mins. The aqueous and
organic layers were separated. The concentration of solute present in each layer was measured
using UV spectroscopy at 277nm wavelength. These concentrations were used to calculate the
ratio i.e., partition co-efficient of aspirin. Partition coefficient values of aspirin at different pH
were plotted in graph.
SOLUBILITY OF DIFFERENT DRUGS
AIM:
To determine the volume of solvents required to dissolve glucose and caffeine according to
the standards of Indian pharmacopoeia
PRINCIPLE:
The absolute and relative solubility of drugs in aqueous and lipid phases of the body are
physical properties of primary importance in providing effective concentration of drugs at their
sites of action. Solubility involves distribution between solid or liquid and saturated solution.
The increase in biological activity roughly parallels to the decrease in water-solubility and
increase in lipid solubility (partition coefficient), which may be associated with the availability
of the compound for the cell where the action occurs. According to Indian pharmacopoeia
solubilities are indicated by a descriptive phase and are intended to apply at 20 to 30 degrees.
The following table indicates the meanings of the terms used in statements of approximate
solubilities.
DESCRIPTIVE TERMS
Very soluble
APPOXIMATE VOLUME OF SOLVENTS
IN MILLILITRES PER GRAM OF
SOLUTE
Less than 1
Freely soluble
From 1 to 10
Soluble
From 10 to 30
Sparingly soluble
From 30 to 100
Slightly soluble
From 100 to 1000
Very slightly soluble
From 1000 to 10000
Insoluble or practically
insoluble
More than 10000
The term ‘partly soluble’ is used to describe a mixture of which only some of the
components dissolve
PROCEDURE
Take 1 gram of the solute and add the solvent with which the solute solubility has to be
determined. Keep on adding the solvent till the solute get completely solubilized in the solvent.
Then calculate how much amount of solvent was required to solubilize the solute and compare it
with the IP standard to determine the solubility. Repeat this procedure to find the solubility of
glucose and caffeine with different solvent like distilled water, HCl, NaOH, and ethanol.
ESTIMATION OF PROTEIN IN DIFFERENT TISSUES
AIM:
To estimate the amount of protein in different tissue samples by using Lowry’s method
PRINCIPLE:
The phenolic group of tyrosine and trytophan residues (amino acids) in a protein will
produce a blue purple color complex with Folin- Ciocalteau reagent which consists of sodium
tungstate molybdate and phosphate.
It has maximum absorption in the region of 660 nm wavelength, thus the intensity of
color depends on the amount of these aromatic amino acids present and will thus vary for
different proteins.
Most proteins estimation techniques use Bovin Serum Albumin (BSA) as a standard
protein, because of its high purity, ready availability. The method is sensitive down to about 10
μg/ml .The reaction is also dependent on pH and a working range of pH 9 to10.5 is essential.
REAGENTS:
1. Stock standard solution (1 mg//ml)
1 mg of Bovine serum albumin dissolved in one ml of distilled water.
2. Solution A
0.1 g of sodium carbonate and 0.2g of potassium hydroxide dissolved in 50 ml distilled
water.
3. Solution B
5% copper sulphate in 100ml of distilled water
4. Solution C
10% sodium potassium tartarate in 10ml of distilled water 0.5 ml of copper sulphate (B)
and 0.5 ml of 10% sodium potassium tartarate (C) was mixed with 4 ml of distilled water
1 ml of this B +C mixture was mixed with 50 ml of solution A.
5. Folin’s reagent
SAMPLE PREPARATION:
1 g of the given sample was weighed accurately and homogenized with 10% ice cold Potassium
chloride and then centrifuged at 5000 rpm for 5 minutes and the supernatant was taken for
estimation. In case of plasma the blood sample was collected with anticoagulants (10% sodium
citrate) then centrifuged at 2000 rpm and plasma separated.
PROCEDURE:
Concentration of 10g, 20g 40g, 80g and 160g of standard protein solution was
pipette out into a series of test tubes and the volume of each test tube was made upto 1 ml with
distilled water. 1 ml of distilled water served as blank. 1 l of the sample was taken and made
upto 1 ml with distilled water. 5 ml of alkaline solution (solution C) was added to all the test
tubes, mixed well and allowed to stand for 10 minutes. Then a 0.5 ml of Folin’s reagent was
added to all the test tubes mixed well and incubated for 30 minutes. The developed blue color
was measured at 660 nm in the spectrophotometer against a reagent blank. A standard graph was
plotted by taking concentration of protein on the X-axis and the optical density on the Y-axis.
From the standard graph, the amounts of protein in the given samples were calculated by
extrapolation.
REPORT:
OBSERVATION:
S.No
CONCENTRATION
(µg/ml)
1
2
3
4
5
6
Samples
a)
b)
c)
ABSORBANCE
DETERMINATION OF pH, log p, log D and pKa VALUE OF KNOWN DRUGS
AIM:
To determine the pH, log p, log D and pKa value of known drugs
PRINCIPLE
Physio-chemical properties of a drug is highly related with its biological action .Various physiochemical properties like hydrogen bonding, ionization, oxidation, reduction potential, surface
action and iso-sterism can modify the drug action .
pH:
pH is defined as minus the decimal logarithm of the hydrogen ion activity in an aqueous
solution. A figure of 7 is regarded as neutral; figures below this indicate the decree of acidity and
above alkalinity.
pH = -log[H+]
Majority of all the drugs are weak electrolyte so their ionization depends on the pH
ie weakly acidic drugs gets ionized in basic pH of intestine and they remains unionized in acidic
pH of stomach. Hence, their absorption takes place from acidic stomach pH
In case of weakly basic drugs, it stays unionized in basic pH of intestine. Hence their absorption
takes place from basic intestine pH.
Ka value and pKa valueThe Ka value is a value used to describe the tendency of compounds or ions to dissociate. The
Ka value is also called the dissociation constant, the ionization constant and the acid constant.
The pKa value is related to the Ka value in a logic way. PKa values are easier to remember than
Ka values and pKa values are in many cases easier to use than Ka values for fast approximations
of concentrations of compounds and ions in equilibriums.
The pKa value is defined from Ka and can be calculated from the Ka value from the equation
pKa = -Log10 (Ka)
Log D
Log D is the ratio of the equilibrium concentrations of all species (unionized and ionized) of a
molecule in octanol to same species in the water phase at a given temperature, normally 25° C. It
differs from LogP in that ionized species are considered as well as the neutral form of the
molecule.
Distribution Coefficient, D = [Unionized] (o) / [Unionized] (aq) + [Ionised (aq)
Log D = log10 (Distribution Coefficient)
LogD is related to LogP and the pKa by the following equations:
For acids
For bases
Partition Coefficient and log P
The Partition Coefficient is defined as the ratio of concentration of compound in aqueous phase
to the concentration in an immiscible solvent, as a neutral molecule…
Partition Coefficient, P = [Organic] / [Aqueous]
Log P= log10 (Partition Coefficient)
PROCEDURE:
pH DETERMINATION
-Prepare the drug solution of 1 M concentration with distilled water.
- Measure the Ph of the solution with the help of Ph meter.
-From the ph of the solution (H+) was determined and further Ka and Pka values were
calculated from the bellow formulas
(H+)2
Ka =
Ca
And pka =-logka
-Then 1% v/v solution of of sample drug was prepared to find out the λ max
PARTITION COEFFICIENT
- 20 ml of n-hexane and 20 ml of water along with the sample drug were transferred into a
separating flask and were shaked for 15 mints prior to the separation of organic and aqueous
layer.
-From both the layers the absorbance values were determined to get the drug concentration
present in both the layers
-Further from the concentration of drug present in both the layers, partition coefficient of drug
was determined from the below formula..
-Partition Coefficient, P = [Organic] / [Aqueous
DISSOCIATION CONSTANT
- From log P i.e partition coefficient log D value was calculated from the below formula
OBSERVATION
S.No
PHYSIOCHEMICAL
PARAMETERS
pH
pKa
Log p
Log D
REPORT:
ASPIRIN
NEOSTIGMINE
ESTIMATION OF G SCORE FOR LIGAND AND RECEPTOR
OBJECTIVES:
To estimate the G value for known ligant and receptors
PRINCIPLE:
The ligand and receptor interactions depend on their physicochemical properties as well the selectivity
of the compounds to the targets. On every ligand and target (protein) interactions energy will be
released. Further, the pose of the molecule can differ and the depending on the pose the energy release
can also get altered. This Glide program will indicate the energy level after ligand target interactions.
The best fit or selective molecule will have better energy and pose.
METHODS:
PROTEIN PREPARATION:
 Protein cocrystallized with ligand (1X7j) structure was imported
in to maestro in PDB format.
 Multimeric complexes were simplified.
 Water molecule in the active site was kept and other molecules were deleted.
 Protein structure was corrected and minimization was carried out using protein preparation
wizard.

LIGAND PREPARATION



Structure was drawn and it was converted in to 3D structure using LigPrep.
Bond lengths and bond angles has been corrected
Then the stereoisomers and tautomers were generated using LigPrep.
GRID GENERATION:

Receptor grid generation from the glide submenu of the application menu was opened and
grid was generated by defining the active site of the receptor by specifying the ligand in the cocrystallised structure.
GLIDE DOCKING:




The receptor grid file has been specified the prepared ligands.file name also specified in the
docking panel. the docking was carried out Standard Precision mode
The results were viewed.
The H- bonding between the ligand and receptor also observed
G score was noted.
H- BONDING
Glu 305
His 475
Arg 346
Score
-10.194
RECEPTOR:
Estrogen receptor –beta
LIGAND:
Estrogen
PREPARATION OF STANDARD CALIBRATION CURVE F0R PROTEIN
OBJECTIVE:
To prepare the standard calibration curve by using bovine serum albumin
PRINCIPLE:
It is based on the biuret reaction and the poorly understood folin-ciocalteau reaction. Cu2+
ions are reduced to Cu+ by the reaction with peptide bond under alkaline conditions. In turn Cu2+
reacts with the Folin’s reagent catalyzing oxidation of aromatic amino acids with concomitant
reduction of phosphomolyb tunstate to heteropolybdenum blue. It intensity of the blue colour
developed can be measured by absorbance at either 750 nm (low expected protein concentration
below 100g/ml) or 550 nm (high protein concentration 100-200 g/ml).
REAGENTS:
1. Stock standard solution (1 mg//ml)
1 mg of Bovine serum albumin per ml of distilled water.
2. Solution A
0.1 g of sodium carbonate and 0.2g of potassium hydroxide dissolved in 50 ml distilled
water.
3. Solution B
5% copper sulphate in 100ml of distilled water
4. Solution C
10% sodium potassium tartarate in 10ml of distilled water 0.5 ml of copper sulphate (B)
and 0.5 ml of 10% sodium potassium tartarate (C) was mixed with 4 ml of distilled water
1 ml of this B +C mixture was mixed with 50 ml of solution A.
5. Folin’s reagent
PROCEDURE:
Concentration of 50 g, 100 g, 200 g, 400 g and 800g, of standard protein
solution was pipetted out into a series of test tubes and the volume of each test tubes was made
upto 5 ml with distilled water.
A volume of 2 ml of alkaline solution (solution C) was added to all the test tubes, mixed
well and allowed to stand for 10 minutes.
Then a volume of 0.2 ml of Folin’s reagent was added to all the test tubes mixed well and
incubated for 30 minutes.
The blue color development measured at 660 nm in the spectrophotometer against a
reagent blank. A standard graph was plotted by taking concentration of protein on the X-axis and
the optical density on the Y-axis..
CONCLUSTION:
at 50% of absorbance 114 µg of bovine serum albumin is present.
TABLE:1
BSA
WATER
(ml)
(ml)
SABPLE
CONC (µg)
SAMPLE
VOL
(ml)
ALK.CuSO4
(ml)
LOWRY
REAGENT
W.L
660nm
(ml)
0.05
4.95
10
5
2
0.2
“
0.1
4.9
20
5
2
0.2
“
0.2
4.8
40
5
2
0.2
“
0.4
4.6
80
5
2
0.2
“
0.8
4.2
160
5
2
0.2
“
TABLE:2
Conc
ABSORBANCE
(µg)
1
2
3
4
5
6
7
8
0.044
0.042
0.043
0.039
0.052
0.044
0.042
0.043
20
0.085
0.091
0.071
0.089
0.101
0.0103
0.098
0.089
40
0.163
0.081
0.221
0.161
0.225
0.232
0.162
0.185
80
0.329
0.351
0.437
0.329
0.411
0.421
0.323
0.354
160
0.541
0.664
0.717
0.542
0.656
0.482
0.462
0.562
10
TABLE:3
CONC
―
(µg)
X
10
0.043
0.00373
0.0215
20
0.090
0.00945
0.0343
40
0.191
0.0302
0.0302
80
0.365
0.0462
0.0462
160
0.602
0.0813
0.1008
S.D
SEM
CHARACTERIZATION OF PHYSIOCHEMICAL PARAMETER OF UNKNOWN
DRUG
AIM:
To determine the physicochmeical parameter of the given chemical molecule using pH, log p,
log D and pKa values
PRINCIPLE:
Physicochemical properties of a drug is highly related with its biological action .Various
physicochemical properties like hydrogen bonding, ionization, oxidation, reduction potential,
surface action and iso-sterism can modify the drug action .
pH
pH is defined as negative logarithm of hydrogen ion concentration in an aqueous solution. A
figure of 7 is regarded as neutral; figures below this indicate the degree of acidity and above
alkalinity.
pH = -log[H+]
Majority of the drugs are weak electrolyte so their ionization depends on the pH
ie weakly acidic drugs gets ionized in basic pH of intestine and they remains unionized in acidic
pH of stomach. Hence, their absorption takes place from acidic stomach pH. In case of weakly
basic drugs, it stays unionized in basic pH of intestine. Hence their absorption takes place from
basic intestine pH.
Ka value and pKa valueThe Ka value is a value used to describe the tendency of compounds or ions to dissociate. The
Ka value is also called the dissociation constant, the ionization constant and the acid constant.
The pKa value is related to the Ka value in a logic way. pKa values are easier to remember than
Ka values and pKa values are in many cases easier to use than Ka values for fast approximations
of concentrations of compounds and ions in equilibriums.
The pKa value is defined from Ka and can be calculated from the Ka value from the equation
pKa = -Log10 (Ka)
Log D
Log D is the ratio of the equilibrium concentrations of all species (unionized and ionized) of a
molecule in octanol to same species in the water phase at a given temperature, normally 25° C. It
differs from LogP in that ionized species are considered as well as the neutral form of the
molecule.
Distribution Coefficient, D = [Unionized] (o) / [Unionized] (aq) + [Ionised (aq)
Log D = log10 (Distribution Coefficient)
LogD is related to LogP and the pKa by the following equations:
For acids
For bases
Partition Coefficient log P
The Partition Coefficient is defined as the ratio of concentration of compound in aqueous phase
to the concentration in an immiscible solvent, as a neutral molecule…
Partition Coefficient, P = [Organic] / [Aqueous]
Log P= log10 (Partition Coefficient)
PROCEDURE:
SOLUBILITY:
Weigh 10 mg of unknown sample and determined its solubility in distilled water as per the IP
standard procedure.
pH DETERMINATION
-Prepare the drug solution of 1 M concentration with distilled water.
- Measure the pH of the solution with the help of pH meter.
-From the pH of the solution (H+) ion concentration was determined to calculate Ka and Pka
values. The Ka and PKa values are calculated using the formula
Ka =
[H+]2
Ca
And Pka =-logka
-Then 1% v/v solution of sample drug was prepared to find out the λ max
PARTITION COEFFICIENT
20 ml of n-hexane and 20 ml of water along with the sample drug were transferred into a
separating flask and were shaked for 15 mints prior to the separation of organic and aqueous
layer. The UV absorbance were measured based on the λ max of the drug solution to determine
the drug concentration present in organic and aqueous layers
From the drug concentration the partition coefficient of drug was determined from the below
formula..
Partition Coefficient, P = [Organic] / [Aqueous
DISSOCIATION CONSTANT
From log P, log D value was calculated from the below formula
OBSERVATION