The Partitioning Of Quinalbarbitone Solution Biology Essay

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Partition coefficient plays an important role with regards to absorption, distribution and elimination of barbiturates. Partition coefficient is defined as the equilibrium concentration ratio of unionized drug distributed between a lipid phase and an aqueous phase. Barbiturates are weak acids with pKa values between 7 and 8. So, they are significantly ionized at pH more than 6. Measurement of partition coefficient enables us to make prediction about the behaviour of barbiturates in the body.

The concentration of the each Quinalbarbitone partitioning sample is obtained by using the absorbance value in UV spectrophotometer and determined by a calibration graph. The % ionisation of Quinalbarbitone and % of Quinalbarbitone extracted are calculated. The partition coefficient value is determined to identify the liphophilicity of the drug.

From the result obtained in this experiment, the % of unionisation of Quinalbarbitone in 0.1M Hydrochloride acid (HCl) is 93.45%w/v. This experiment shows more un-ionisation form of Quinalbarbitone in lowest pH buffer solution where most acidic condition. Quinalbarbitone in un-ionised form is diffuses from the gastro-intestinal tract through the lipoprotein membrane into the blood plasma. The absorbed drug is removed by the bloodstream to the active sites of the body to exert its effects. High partition coefficient value indicates highly lipophilic drug where the drug can cross the biological membrane for absorption, distribution and elimination purposes. The P true value of phenobarbitone is 2.28.

1. Introduction:

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Barbiturates are derivatives of Barbituric Acid, which is formed from malonic acid and urea. Synthesis of barbiturates began in 1903. In 1912 the first barbiturate that became available was Phenobarbital. They are classified as hypnotics and anxiolytics and exhibit very broad effects, ranging from mild sedation to complete anaesthesia. They are invariably used in the treatment of insomnia, convulsions and pre-operative medication.

Barbiturates are weak acids that are highly ionisable at pH values over 6. This is because pka is between7-8. In this experiment, we are using quinalbarbitone to investigate the effect of ionization on the apparent partition coefficient by measuring the amount extracted into the octanol from aqueous phases of different pH values. Quinalbarbitone's formula is

Measurement of partition coefficient enables us to make prediction about the behavior of a compound in the body. Partition coefficient only applies to the unionized form of compound. Partition coefficient is defined as the equilibrium concentration ratio of unionized drug distributed between a lipid phase and an aqueous phase:

(P) =

The ratio of ionized to unionized drug in an aqueous phase have to obey the Handerson-Hasselbalch equation. The ionized form of drug can be present in the aqueous phase, but not in the octanol as it is a non-polar solvent that cannot stabilize ionic charge. Thus, ionization in the aqueous phase will reduce the amount of unionized form to distribute into octanol. High partition coefficient implies a highly lipophillic drug. Hydrophobicity affects drug absorption, bioavailabilty, hydrophobic drug-receptor interactions, metabolism of molecules, as well as their toxicity.

The majority of drugs are weak acids or bases and depending on their pH, exist in a unionized or ionized form. The apparent partition coefficient is defined as the ratio of the drug concentration in octanol to the total concentration (ionized and unionized drug in aqueous phase). may vary from 0 to , depending on the degree of ionization at different pH values of aqueous phase. For the efficient extraction of an acid into an organic solvent from an aqueous environment, the pH of the aqueous phase should be at least one pH unit lower than the pKa of the acid. Similarly, for efficient extraction of a base, the pH of the aqueous medium should be at least one pH unit higher than the pKa of the base.

= x

Where:

= fraction of total amount of solute unionized at the pH; ≤ 1

From the Henderson-Hasselbalch equation, pH = pKa + log [base]/ [acid]

Rearranging the Henderson-Hasselbalch equation, [acid]/ [base] = antilog (pH - pKa)

This leads to [acid]/[total] = (acid) = 1/1+ antilog(pH - pKa). Hence,

For an acid: = 1/[1 + antilog (pH - pKa)]

For a base: = 1/[1 + antilog (pKa -pH)]

Extend of ionization of a drug can be calculated If the pKa of the drug and the pH of the solution are known.

Therefore, this allows the true partition coefficient for the barbitone to be calculated.

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For an acidic drug,

%ionised (acid) = 100/[1+anitlog (pKa - pH)]

For basic drug,

%ionised (base) = 100/[1+antilog (pH - pKa)]

One of the most common ways of measuring partition coefficients is to use the shake flask method. This relies on the equilibrium distribution of a drug between oil and aqueous phase should be saturated with the oil phase and vice versa. The experiment should be carried out at constant temperature. The drug should be added to the aqueous pahse and oil phase which, in the case of octanol, as it is less dense than water, will sit on top of the water. The system is mixed and then left to reach equilibrium. The two phases are separated and the concentration of the drug is measured in each phase and a partition coefficient is calculated.

As barbiturates absorb most effectively in alkaline solutions, so all the UV measurements are carried out in 0.5M NaOH solution. Since the total amount of barbiturate present are known, and the UV analysis gives the amount in the octanol layer at the equilibrium, the weight left in the aqueous phase and hence the concentration of the drug in each phase at equilibrium can be calculated.

2. Experimental:

2.1. Materials:

An ultraviolet molecular spectrophotometer was used in this experiment. 0.5M of Sodium hydroxide (NaOH) solution was used as blank. Stock solution was 0.02%w/v of Quinalbarbitone. The other solutions used in this experiment were distilled water, 0.1M Hydrochloride acid (HCL), pH 6.6 buffer solutions, pH 7.0 buffer solutions, pH 7.4 buffer solution, pH 8.0 buffer solution, pH 9.0 buffer solution, Octanol as organic phase.

The apparatus used were six separating funnels, beakers, 5mL, 10mL, 15mL, 20mL, 25mL and 30mL pipettes and cuvettes were used to fill the blank solution and the Quinalbarbitone solutions.

2.2. Methods:

Preparation of Solution A and Solution B:

Firstly, 50ml of 0.02%w/v of Quinalbarbitone was used to prepare a 50μg ml-1 Solution A and made up to 200mL using 0.5M of NaOH. 50μg ml-1 Solution B was then prepared by using 25mL of 0.02%w/v of Quinalbarbitone and made up to 100mL using distilled water.

Calibration Standards:

A range of calibration standards containing 5, 10, 15, 20, 25 and 30 μg ml-1 of the Quinalbarbitone were prepared using Solution A and made up to 50ml using distilled water. The wavelength of the maximum absorbance (λmax) at about 254nm was determined using the prepared 30μg ml-1 standard. The absorbance of each standard at the λmax using 0.5M as the blank was recorded. A calibration curve of absorbance versus concentration for the Quinalbarbitone was plotted.

Partitioning Samples:

Samples were prepared into six separating funnels according to below:

10mL Solution B, 10mL 0.1M HCL, and 20mL octanol.

10mL Solution B, 10mL pH 6.6 buffer and 20mL octanol.

10mL Solution B, 10mL pH 7.0 buffer and 20mL octanol.

10mL Solution B, 10mL pH 7.4 buffer and 20mL octanol.

10mL Solution B, 10mL pH 8.0 buffer and 20mL octanol.

10mL Solution B, 10mL pH 9.0 buffer and 20mL octanol.

All the funnels were shaken at frequent intervals for 30 minutes to allow the layers to separate fully. Funnels were shaken carefully to prevent the formation of octanol- water emulsion. Aqueous layer was then carefully run off into a second separating funnel. 0.5ml NaOH was added to the remaining octanol in each separating funnel. Separating funnels were shaken for further 5 minutes to allow the separation of layers. The absorbance of the aqueous (lower layer) was measured at the λmax determined previously. The concentration of the Quinalbarbitone in the 0.5M of NaOH was calculated using the calibration curve.

3. Results:

(1) The volume of solution A required to prepare 50ml of standard solutions in concentration of 5- 30 μgmL-1 in 0.5M Sodium hydroxide.

Concentration of Quinalbarbitone in

0.5M NaOH (μgmL-1)

Volume of solution A required to make 50ml of standard solutions

5

5

10

10

15

15

20

20

25

25

30

30

Table 1: The volume of solution A required to prepare 50ml of standard solutions according to the concentrations of Quinalbarbitone (μgmL-1) in 0.5M NaOH.

(2) Determination of the maximum absorbance (λmax) at wavelength 252nm to 256nm using 30μg ml-1 standard.

Wavelength (nm)

1streadings of Absorbance (A)

2ndreadings of Absorbance (A)

Mean Absorbance (A)

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252

0.835

0.836

0.836

253

0.841

0.840

0.840

254

0.842

0.841

0.843

255

0.836

0.840

0.838

256

0.831

0.831

0.831

Table 2: The absorbance (A) at wavelength 252nm to 256nm using 30μg ml-1 standard.

(3) Determination of the absorbance at maximum wavelength 254nm in different concentration of Quinalbarbitone (μgmL-1) in 0.5M NaOH.

Concentration of Quinalbarbitone in

0.5M NaOH (μgmL-1)

1streadings of Absorbance (A)

2ndreadings of Absorbance (A)

Mean Absorbance (A)

5

0.160

0.162

0.161

10

0.287

0.289

0.288

15

0.434

0.436

0.435

20

0.576

0.578

0.577

25

0.712

0.713

0.712

30

0.814

0.816

0.815

Table 3: The absorbance (A) at wavelength 254nm using different concentration of Quinalbarbitone in 0.5M NaOH (μgmL-1).

(4) Determination of the absorbance at maximum wavelength 254nm in different partitioning samples.

Partitioning samples

1streadings of Absorbance (A)

2ndreadings of Absorbance (A)

Mean Absorbance (A)

10ml Solution B, 10ml 0.1M HCL and 20ml octanol

0.656

0.657

0.657

10ml Solution B, 10ml pH6.6 buffer and 20ml octanol

0.659

0.657

0.658

10ml Solution B, 10ml pH7.0 and 20ml octanol

0.633

0.634

0.634

10ml Solution B, 10ml pH7.4 and 20ml octanol

0.586

0.586

0.586

10ml Solution B, 10ml pH8.0 and 20ml octanol

0.566

0.564

0.565

10ml Solution B, 10ml pH9.0 and 20ml octanol

0.565

0.565

0.565

Table 4: The absorbance (A) at wavelength 254nm using different partitioning samples.

(5) Determination of the concentration of Quinalbarbitone in different partitioning samples.

pH of the partitioning samples

Concentration of Quinalbarbitone (µgml-1)

1.0

23.3613

6.6

23.4708

7.0

22.5219

7.4

20.8066

8.0

20.0766

9.0

20.0401

Table 5: The concentration of Quinalbarbitone (µgml-1) of each partitioning sample with different pH.

(6) Determination of the amount of Quinalbarbitone extracted in different partitioning samples.

Partitioning sample with pH

Amount of Quinalbarbitone extracted (μg)

1.0

467.23

6.6

469.42

7.0

450.44

7.4

416.13

8.0

401.53

9.0

400.80

Table 6: Amount of Quinalbarbitone (μg) extracted for each solution with different pH value.

(7) Determination of the % ionisation of Quinalbarbitone and % of Quinalbarbitone extracted in different partitioning samples.

Partitioning sample with pH

Percentage ionisation of Quinalbarbitone (%)

Percentage extraction (%)

1.0

6.55

93.45

6.6

6.12

93.88

7.0

9.91

90.09

7.4

16.77

83.23

8.0

19.69

80.31

9.0

19.84

80.16

Table 7: Percentage ionisation and percentage extraction of Quinalbarbitone at different pH value of each partitioning sample.

Calculation:

(1)Preparation of Solution A and Solution B:

Calculation by using the equation of:

M1V1=M2V2

Whereby, M1 = concentration of Quinalbarbitone

M2 = concentration of Solution A/B

V1 = require volume of 0.02%w/v Quinalbarbitone

V2 = volume of Solution A/B

0.02%w/v of Quinalbarbitone = 0.02g in 100ml

= 0.0002g in 1ml

= 200μg in 1ml of Quinalbarbitone

Preparation of Solution A:

(200μg ml-1) V1= (50μg ml-1) (200ml)

V1= 50mL

Preparation of Solution B:

(200μg ml-1) V1= (50μg ml-1) (100ml)

V1= 25mL

(2) Calibration Standards:

Calculation for the standards by using the equation of:

M1V1=M2V2

Whereby, M1 = concentration of Solution A

M2= concentration of standard.

V1 = required volume of Solution A

V2 = volume of standard.

5 μg ml-1

(50μg ml-1) V1= (5μg ml-1) (50ml)

V1= 5ml

10 μg ml-1

(50μg ml-1) V1= (10μg ml-1) (50ml)

V1= 10ml

15 μg ml-1

(50μg ml-1) V1= (15μg ml-1) (50ml)

V1= 15ml

20 μg ml-1

(50μg ml-1) V1= (20μg ml-1) (50ml)

V1= 20ml

25 μg ml-1

(50μg ml-1) V1= (25μg ml-1) (50ml)

V1= 25ml

30μg ml-1

(50μg ml-1) V1= (30μg ml-1) (50ml)

V1= 30ml

(3) Determine of concentration for each partitioning sample:

From Graph 1, the equation obtained is y=0.0274x + 0.0159. By using this equation, we can calculate the concentration of Quinalbarbitone in µgml-1.

y=0.0274x + 0.0159

Whereby, y= Absorbance (A)

x= Concentration of Quinalbarbitone (µgml-1)

i) Sample at pH 1.0

0.656 = 0.0274x + 0.0159

x = 23.3613 µgml-1

ii) Sample at pH 6.6

0.659 = 0.0274x + 0.0159

x = 23.4708 µgml-1

iii) Sample at pH 7.0

0.633 = 0.0274x + 0.0159

x = 22.5219 µgml-1

iv) Sample at pH 7.4

0.586 = 0.0274x + 0.0159

x = 20.8066 µgml-1

v) Sample at pH 8.0

0.566 = 0.0274x + 0.0159

x = 20.0766 µgml-1

vi) Sample at pH 9.0

0.565 = 0.0274x + 0.0159

x = 20.0401 µgml-1

(4) Calculate the % Extraction of Quinalbarbitone and % ionisation of Quinalbarbitone:

As the concentration of Solution B is 50µgml-1, so we can calculate the total amount of Quinalbarbitone in each funnel. In each funnel, 10mL of the Solution B was added. So the total amount of Quinalbarbitone present in each funnel is 500µg.

50µgml-1 = 50 µg in 1ml

So 10ml of Solution B contain 500 µgml-1 of Quinalbarbitone.

For each funnel, aqueous layer was run off into a second separating funnel. 20mL 0.5M of NaOH was then added to the octanol layer. Therefore, the amount of Quinalbarbitone extracted can be calculated.

As the volume of octanol is 20mL so we can multiply the volume with concentration of Quinalbarbitone in octanol layer calculated previously to get the amount of Quinalbarbitone extracted.

Extracted Quinalbarbitone = Concentration of Quinalbarbitone in octanol x 20ml

As drug can only ionized in aqueous layer and so unionized form remain in the organic layer. Percentage of extraction indicated the amount of Quinalbarbitone remain in the octanol (organic layer).

To calculate the Percentage Extraction of Quinalbarbitone, the below equation can be use:

Percentage Extraction = Amount of Quinalbarbitone extracted x 100%

Total amount of Quinalbarbitone

i) Solution at pH 1.0

Percentage of extraction = 467.23x 100%

500

= 93.45 %

Percentage of ionization = 100% - 93.45%

= 6.55%

ii) Solution at pH 6.6

Percentage of extraction = 469.42 x 100%

500

= 93.88%

Percentage of ionisation = 100% - 93.88%

= 6.12%

iii) Solution at pH 7.0

Percentage of extraction = 450.44 x 100%

500

= 90.09%

Percentage of ionisation = 100% - 90.09%

= 9.91%

iv) Solution at pH 7.4

Percentage of extraction = 416.13x 100%

500

= 83.23%

Percentage of ionisation = 100% - 83.23%

= 16.77%

v) Solution at pH 8.0

Percentage of extraction = 401.53x 100%

500

= 80.31%

Percentage of ionisation = 100% - 80.31%

= 19.69%

vi) Solution at pH 9.0

Percentage of extraction = 400.80 x 100%

500

= 80.16%

Percentage of ionisation = 100% - 80.16%

= 19.84%

(5) Calculate the partition coefficient value of each partitioning sample of Quinalbarbitone:

Formulae: Partition coefficient, P = [drug] org/ [drug] aq

i) Solution at pH 1.0

Partition coefficient,P = [(1000-(20x 23.36))/10]/ 23.36

= 2.28

ii) Solution at pH 6.6

Partition coefficient,P = [(1000-(20x 23.47))/10]/ 23.47

= 2.26

iii) Solution at pH 7.0

Partition coefficient,P = [(1000-(20x 22.52))/10]/ 22.52

= 2.44

iv) Solution at pH 7.4

Partition coefficient,P = [(1000-(20x 20.81))/10]/ 20.81

= 2.81

v) Solution at pH 8.0

Partition coefficient,P = [(1000-(20x 20.08))/10]/ 20.08

= 2.98

vi) Solution at pH 9.0

Partition coefficient,P = [(1000-(20x 20.04))/10]/ 20.04

= 2.99

(5) Calculate the Partition coefficient (P true) value of Quinalbarbitone:

Quinalbarbitone with pKa value of 7.8 is extracted from 20mL of a 50μgmL-1aqueous solution at pH into 20mL of Octanol. The concentration found in the aqueous phase after extraction is 23.36μgmL-1.

The calculation of true partition co-efficient:

pKa value of Quinalbarbitone: 7.8

pH of the solution: 1.0

Volume of Octanol as organic phase: 20mL

[Drug] org = [(1000-(23.36 x 20))]/10 = 53.28μgmL-1

Papp = 53.28/23.36 = 2.28

Fu = 1/(1+ antilog (pH- pKa)) = 1/(1+ antilog (1-7.8)) = 0.9999%

Ptrue = 2.28/ 0.9999 =2.28

The P true value calculated is 2.28; it is slightly higher compared to the literature value which is 2.0 obtained in Clarke's Analysis of Drugs and Poisons.

Graph 1: Graph of absorbance against concentration of Quinalbarbitone.

Graph 1: Graph of % ionisation and % extraction of Quinalbarbitone in each pH of partitioning sample.

4. Discussion:

The major process for absorption of most drugs is passive diffusion. It is primarily applies to non- charged species. The drugs have to dissolve in biological fluid to be absorbed. Only lipid soluble drugs or lipophilicity will diffuse through the biological membrane either move both forwards and backwards until the concentrations on both extracellular and intracellular sides are balanced, this is known as dynamic equilibrium. Molecules of drug must be in un-ionised form to diffuse through biological membrane. Ionisation of drug depends on the pKa of the drug and the pH of the biological fluid. At equilibrium, both compartments contain an equal concentration of non-ionised form of drugs. The compartment in which the drug is most ionised will contain the highest drug concentration. For the weak acid drug, Quinalbarbitone will be more concentrated in the compartment with the lowest pH where acidic condition. Partition coefficient defines the equilibrium of drug concentration in organic phase against drug concentration if aqueous phase. High partition coefficient implies a highly lipophilic drug. The concentration of drug in aqueous phase can be determined through separation of two immiscible liquids. Octanol is hydrophobic agent which mimics the biological membrane and it has good lipophilicity.

From the result obtained in this experiment, the % of unionisation of Quinalbarbitone in 0.1M Hydrochloride acid (HCl) is 93.45%w/v, the % of unionisation of Quinalbarbitone in 10mL pH 6.6 buffer solution is 93.88%w/v, the % of unionisation of Quinalbarbitone in 10mL pH 7.0 buffer solution is 90.09%w/v, the % of unionisation of Quinalbarbitone in 10mL pH 7.4 buffer solution is 83.23%w/v, the % of unionisation of Quinalbarbitone in 10mL pH 8.0 buffer solution is 80.31%w/v and the % of unionisation of Quinalbarbitone in 10mL pH 9.0 buffer solution is 80.16%w/v. This experiment shows more un-ionisation form of Quinalbarbitone in lowest pH buffer solution where most acidic condition. Quinalbarbitone in un-ionised form is diffuses from the gastro-intestinal tract through the lipoprotein membrane into the blood plasma. The absorbed drug is removed by the bloodstream to the active sites of the body to exert its effects.

The literature P true value obtained is 2.0 which stated in Clarke's Analysis of Drugs and Poisons. The P true value calculated is 2.28; it is slightly higher compared to the literature value found in the book. It is difficult to measure the P true value as almost no drug would be left in the aqueous phase. The true partition coefficient value or P true value indicates for the un-ionised species of drug molecule.

Partition coefficient of a drug is the main component in absorption, metabolism and elimination process in pharmacokinetics of a drug. The drug molecule has to cross lipophilic membranes in order to reach "active site" to exert its pharmacological effect. Other than this, the drug molecule has to cross cell membrane in the kidney to be metabolised to active form if it is a pro-drug, the drug has to be metabolised to promote absorption and to be eliminated from the body. The accumulation of drug in the plasma which not be excreted from the body will cause toxicity. From the result obtained in this experiment, high partition coefficient implies a highly lipophilic drug. Quinalbarbitone is a hydrophobic or lipophilic drug which crosses the biological membranes easily for the absorption purpose. The partition coefficient value, P is used to predict the onset or the drug and the duration of action of the drug within the body. It is important in rational drug design in medicinal chemistry to determine the relationship between the biological membranes and the drug.

Shake flask method is a classical method to determine the partition coefficient value. It is consider a most reliable and accurate method which consists of two phase system, organic phase and aqueous phase for measuring the partition coefficient of a drug compound. The concentration of drug is measured by using the absorbance obtained from UV spectroscopy through a calibration graph which plotted. The disadvantages of this method are time consuming which shake for 30 minutes per sample, if large samples are used, more time required. Other than this, the octanol as organic phase is formed emulsion easily with aqueous phase when shake, this will cause problem in phase separation in the end. If some drug is extremely lipophilic or hydrophobic, the concentration in either organic phase or aqueous phase will be relatively small and thus cause problem in quantified.

5. Conclusions:

The absorption, distribution and elimination of a drug are affected by the lipophilicity of the drug molecules and the % of ionisation of drugs in the biological membrane. The drug with high un-ionised form is diffuse through lipoprotein membrane into the bloodstream if the drug is administered orally. Weak acid drug such as Quinalbarbitone is more lipophilic in basic environment which indicates highest true partition coefficient value. Equilibrium has to be achieved in partitioning process for both aqueous phase and organic phase. Shake flask method is reliable and accurate method for this experiment because the samples to be determined are small. Other than shake flask method, measurement of partition coefficient can be done by thin layer chromatography (TLC) or the use of reversed phase, high performance liquid chromatography (HPLC).