Partitioning Of Barbiturates Overview Biology Essay

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Phenobarbitone is an antiepileptic drug which is used to treat epilepsy. Absorbance values at different concentration of phenobarbitone can be used to plot a calibration graph which in turns can be used to find the concentration of phenobarbitone in organic phase at different pH by using the gradient of the graph. From there, the concentration of phenobarbitone in aqueous phase can be determined and hence can find the partition coefficient (P). Ptrue of phenobarbitone can be determined with the presence of the value of Papp and fu of phenobarbitone (acid). % ionization of phenobarbitone at different pH can also be calculated by knowing its pKa and this in turns can be used to determine the lipophilicity of phenobarbitone. % extraction of phenobarbitone will also determine how well does phenobarbitone passes through the organic phase (biological membrane in the body) at different pH.

'Phenobarbitone is a barbiturate used in the treatment of epilepsy. It helps to reduce seizure frequency and severity and may even stop seizures altogether.' [1] 'Phenobarbital works by acting on GABA receptors that in turn increase synaptic inhibition. This then increases the seizure threshold, hence reducing the number of seizures occurring.' [2)]

Phenobarbitone is the longest acting barbiturate. It is an acidic crystalline structure which has pka value 7.4, hence it is significantly ionised at ph values over 6. 'Lowering the pH of the solution reduces ionisation.' [4] According to Phenobarbitone structure, it has 2 hydrogen bond donors and 3 hydrogen bond receptors and the solubility of Phenobarbitone in water is 1g in 1000mL (0.1% w/v). It has low lipid solubility compared with other barbiturate. Thus it has slow onset action and longer half life.

This experiment was carried out to investigate the effect of ionisation of phenobarbitone by measuring the amount that was extracted into n-octanol from the aqueous phases of different pH values. The results collected were then used to find the concentration of the drug at equilibrium and since the pKa of the drug and the pH of the solution are known, the true partition co-efficient for phenobarbtione can be calculated. [3] 'Partition coefficient (P) is the ratio of concentration of drug in n-octanol phase divided by concentration of drug in aqueous phase. A high partition co-efficient implies that the drug is highly lipophilic.' [5]

The method used in this experiment is the shake-flask method (the most accurate measurement method). The drug, in this case phenobarbitone, is allowed to equilibrate between the NaOH solution and the n-octanol and then the concentration in both layers are been determined. [5] Since the total amount of barbiturate present in the 0.5M NaOH (which is extracted from the octanol) is known, and the UV analysis gives the amount of phenobarbitone in the octanol layer at equilibrium, we can calculate the weight left in the aqueous phase and hence the concentration of drug in each phase at equilibrium. pKa is important to determine the true partition coefficient for the barbiturate. The disadvantage for shake-flask is that it is only apply to the unionised drug which most drugs are acids or bases and are ionised in biological systems.

Experimental

0.02% w/v phenobarbitone in water was been provided as the stock solution. Using the stock solution, 50µg mL-1 solution in 0.5M NaOH [Solution A] and a 50µg mL-1 solution in water [Solution B] were been prepared.

(a) Calibration Standards

A range of calibration standards each containing 5, 10, 15, 20, 25, 30 µg mL-1 of the phenobarbitone in 0.5M NaOH were prepared using Solution A. The wavelength of maximum absorbance (λmax) at about 254nm was then determined using the 30 µg mL-1 standard. The absorbance of each standard at the λmax was read using 0.5M NaOH as the blank. A calibration curve of absorbance versus concentration for the phenobarbiturate was then constructed using the absorbance readings obtained.

(b) Partitioning Samples

Six partitioning funnels were filled as the following:

i) 10mL Solution B, 10mL 0.1M HCl, and 20mL n-octanol

ii) 10mL Solution B, 10mL pH 6.6 buffer, and 20mL n-octanol

iii) 10mL Solution B, 10mL pH 7.0 buffer, and 20mL n-octanol

iv) 10mL Solution B, 10mL pH 7.4 buffer, and 20mL n-octanol

v) 10mL Solution B, 10mL pH 8.0 buffer, and 20mL n-octanol

vi) 10mL Solution B, 10mL pH 9.0 buffer, and 20mL n-octanol

The funnels were shaken at frequent intervals for 30 minutes to allow the layers to separate fully. The organic layer was then been carefully ran off into a second separating funnel. 20mL of 0.5M NaOH was then added to the octanol and shaken for 5 minutes, allowing the layers to separate. The absorbance of the aqueous (bottom) layer was then measured by using the λmax determined previously in (a). The concentration of the phenobarbitone in the 0.5M NaOH was calculated using the calibration curve.

Results

Stock solution = 0.02% w/v

= 0.02g in 100mL

= 200 µg in 1mL

Amount of phenobarbitone required in Solution A and B

= 50 µg in 1mL

For solution A,

using C1V1=C2V2,

(200)(V1) = (500)(200)

V1 = 50mL

Amount of stock solution required to make 200mL Solution A = 50mL

For solution B,

using C1V1=C2V2,

(200)(V1) = (500)(100)

V1 = 25mL

Amount of stock solution required to make 100mL Solution B = 25mL

(a) Calibration Standards

Using C1V1=C2V2,

C1 =Concentration of Solution A = 50µg mL-1

V2 = Total volume of standard required = 50mL

(50)(V1) = (C2)(50)

Concentration of standard/ µg mL-1(C2)

Volume of Solution A / mL (V1)

5

5

10

10

15

15

20

20

25

25

30

30

Table 1.1: Preparation of Calibration Standards

Using 0.5M NaOH as the blank,

Wavelength / nm

Absorbance

251

0.777

252

0.787

253

0.793

254

0.793

255

0.789

256

0.780

257

0.766

Table 1.2: Determination of λmax using 30 µg mL-1 standard

λmax = 254nm

Using λmax = 254nm,

Concentration of standard / µg mL-1

Absorbance Reading

First

Second

Average

5

0.141

0.138

0.140

10

0.264

0.262

0.263

15

0.402

0.402

0.402

20

0.528

0.526

0.527

25

0.665

0.663

0.664

30

0.795

0.794

0.795

Table 1.3: Absorbance of Each Calibration Standards at 254nm

Graph 1: Graph of Absorbance against Concentration of Standard

(b) Partitioning Samples

Using λmax = 254nm,

Solution

Absorbance

First

Second

Average

0.1M HCl

0.578

0.580

0.579

pH 6.6 Buffer

0.546

0.544

0.545

pH 7.0 Buffer

0.531

0.528

0.530

pH 7.4 Buffer

0.464

0.468

0.466

pH 8.0 Buffer

0.422

0.424

0.423

pH 9.0 Buffer

0.368

0.370

0.369

Table 1.4: Absorbance of Each Funnels at different pH

To calculate the concentration of the barbiturate in 0.5M NaOH, Beer-Lambert equation is used.

A=A(1%,1cm).c.l

Where

A= Absorbance

A(1%,1cm) = Specific absorbance of a 1 cm layer of a 1% solution = Gradient of the

graph of absorbance against concentration of standard

c = Concentration (%)

l = path length (cm)

As the concentration used in the graph is in µg mL-1, therefore we need to convert its unit to % as required in Beer-Lambert equation to find the concentration of the standard.

Gradient of the graph =

= 263.64

Using A=A(1%,1cm).c.l ,

A(1%,1cm) = 263.64

l = 1

A=(263.64).c. (1)

Solution

Average Absorbance

Concentration of Phenobarbitone / %

0.1M HCl

0.579

2.196 X 10-3

pH 6.6 Buffer

0.545

2.067 X 10-3

pH 7.0 Buffer

0.530

2.010 X 10-3

pH 7.4 Buffer

0.466

1.768 X 10-3

pH 8.0 Buffer

0.423

1.604 X 10-3

pH 9.0 Buffer

0.369

1.400 X 10-3

Table 1.5: Concentration of Phenobarbitone in 0.5M NaOH at different pH

Weight of phenobarbitone in organic phase = Concentration of phenobarbitone X

Weight of phenobarbitone in 10mL Solution B = 50 µg X 10 = 500 µg (5 X 10-4 g)

Weight of phenobarbitone in aqueous phase

= Weight of phenobarbitone in 10mL Solution B - Weight of phenobarbitone in organic phase

Concentration of phenobarbitone in aqueous phase

= Weight of phenobarbitone in aqueous phase X

pH of 0.1M HCl = 1.1

pH

Weight of Phenobarbitone in organic phase /g

Concentration of Phenobarbitone in organic phase/%

Weight of Phenobarbitone in aqueous phase /g

Concentration of Phenobarbitone in aqueous phase / %

1.1

4.392 X 10-4

2.196 X 10-3

0.608 X 10-4

0.304 X 10-3

6.6

4.134 X 10-4

2.067 X 10-3

0.866 X 10-4

0.433 X 10-3

7.0

4.020 X 10-4

2.010 X 10-3

0.980 X 10-4

0.490 X 10-3

7.4

3.536 X 10-4

1.768 X 10-3

1.464 X 10-4

0.732 X 10-3

8.0

3.208 X 10-4

1.604 X 10-3

1.792 X 10-4

0.896 X 10-3

9.0

2.800 X 10-4

1.400 X 10-3

2.200 X 10-4

1.100 X 10-3

Table 1.6: Weight and concentration of phenobarbitone in aqueous and organic phase at different pH

fi acid =

fu acid = 1- [fi acid]

pKa of phenobarbitone = 7.4 [6]

pH

Fraction ionised

Fraction Unionised

% ionization / %

1.1

0.0000

1.0000

0.00

6.6

0.1368

0.8632

13.68

7.0

0.2847

0.7153

28.47

7.4

0.5000

0.5000

50.00

8.0

0.7992

0.2008

79.92

9.0

0.9755

0.0245

97.55

Table 1.7: % ionization of phenobarbitone at different pH

Graph 2: Graph of % Ionisation of Phenobarbitone Against pH

% of phenobarbitone extracted

= X 100%

Weight of phenobarbitone in 10mL Solution B = 50 µg X 10 = 500 µg (5 X 10-4 g)

pH

Weight of phenobarbitone in organic phase/g

% of phenobarbitone extracted / %

1.1

4.392 X 10-4

87.84

6.6

4.134 X 10-4

82.68

7.0

4.020 X 10-4

80.40

7.4

3.536 X 10-4

70.72

8.0

3.208 X 10-4

64.16

9.0

2.800 X 10-4

56.00

Table 1.8: % extracted of Phenobarbitone at different pH

Graph 3: Graph of % Extracted of Phenobarbitone Against pH

Partition Coefficient (P) =

pH

Partition Coefficient

1.1

7.224

6.6

4.774

7.0

4.102

7.4

2.415

8.0

1.790

9.0

1.273

Table 1.9: Partition Coefficient of Phenobarbitone at different pH

Ptrue =

Ptrue = 7.224/ 1

= 7.224

Discussion

In partition chromatography, molecules move from one phase to another via passive diffusion, i.e. the movement of molecules from an area of high to low concentration area without any facilitating factor. However, charged molecules are unable to move down the concentration gradient via this route. Partition chromatography of phenobarbitone mimics the movement of phenobarbitone across the biological membrane, i.e. the movement of phenobarbitone from aqueous phase, 50µg mL-1 phenobarbitone in water, to organic phase, octanol. Its chemical structure shows that there is a long hydrocarbon chain attached to the ring structure which contributes to its lipophilicity. The more lipophilic the drug, the more efficiently it is absorbed into the organic phase.

Glass separating funnels were used to avoid absorption of lipophilic drug into the container. 30 minutes after the layers are left to separate fully, the aqueous layer is carefully ran off, leaving the organic layer in the separating funnel. 20mL of 0.5M sodium hydroxide, NaOH is then added to the organic layer to separate the mixture into two immiscible layers again. This is possible because phenobarbitone is a weak acid, it reacts with the newly added base, NaOH to form aqueous phase. The concentration of phenobarbitone in the aqueous phase is then determined using a UV spectrophotometer.

From Table 1.6, at pH 1.1, the weight of phenobarbitone in organic phase is highest,

4.392 X 10-4 g; whereas at pH 9.0, the weight of phenobarbitone in organic phase marked its lowest at 2.800 X 10-4 g. This proves that the drug is most lipophilic at low pH as it is a weak acid and is unionised at low pH, therefore more able to cross into the octanol layer. Meanwhile, at pH 9.0, most of the drug is retained in the aqueous phase in its ionised form and hence unable to cross into the octanol phase as it cannot be passively diffused.

As shown in Table 1.7 and Graph 2.0, the higher the pH, more of the drug present is ionised and vice versa. Starting from pH 1.1, the percentage of phenobarbitone ionised in the aqueous phase increased slowly up to pH 6.6, followed by a steep increase from pH 6.6 to pH 8.0, and further increase less steeply from pH 8.0 to pH 9.0. As for Graph 2.0, a slow decrease is observed from pH 1.1 to pH 7.0, a steep decrease from pH 7.0 to pH 7.4, followed by a gradual decrease from pH 7.4 to pH 9.0.

Partition coefficient, P is the ratio of a drug's concentration in the octanol phase to its concentration in the aqueous phase at equilibrium with each other. A high P value hence denotes a high drug concentration in organic phase. From the results, a high P obtained at low pH proves that phenobarbitone is a highly lipophilic drug, capable of crossing lipophilic membranes in the body.

From the results section, Ptrue value at pH 1.1 is 7.74, whereas the literature value is 1.4. The comparison is made at pH 1.1 because phenobarbitone is present in its unionised form at low pH. However, the Ptrue value is much higher than the literature value. This could be due to errors that occurred during the experiment, e.g. parallax error while pipetting the stock solution, and accidentally ran off some of the organic phase while running off the aqueous phase, causing undetermined potential weight loss of phenobarbitone in the organic phase. It could also be due to insufficient time allowed for phenobarbitone to diffuse from the aqueous phase into the organic phase.

Studies revealed that the peak plasma concentration is reached 0.5 to 4 hours following an oral administration; partition chromatography mimics the diffusion of drug across biological membrane, therefore requires at least 30 minutes to reach peak plasma concentration of phenobarbitone in the organic phase. However, in the experiment, the partition was stopped at 30 minutes, not allowing more time for the mixture to separate fully. As octanol was added into the separating funnels at different times, they could not be stopped at the same time. The aqueous (bottom) layers were then run off starting from the funnel where octanol was added the earliest to the latest. The time taken to run off the aqueous phase varies as the volume of aqueous phase varies from funnel to funnel, which may have resulted in different times for each funnel to separate and hence more complete separation in the later mixtures.

The lipophilicity of phenobarbitone contributes to its absorption into the octanol phase. Likewise, it is readily absorbed across biological membranes in the body, e.g. stomach wall, cell membrane and blood-brain barrier. Phenobarbitone is administered orally; it is rapidly and fully bioavailable after oral administration as phenobarbitone is unionised in acidic environment. At pH 1.1, 87.84% of phenobarbitone was extracted into the organic phase. This signifies that 87.84% of phenobarbitone is able to cross the stomach wall into the systemic circulation. The absorption is expected to decrease with the increase of pH value down the gastrointestinal tract; the unionised fraction is smaller in the small intestine but has longer intraluminal dwell time and hence increasing absorption.

Being lipophilic, phenobarbitone crosses biological membranes readily, more preferably at low pH environment, into the bloodstream and around the body via systemic circulation and then distributed throughout the interstitial fluid. However, about 50% of the drug is bound to plasma protein, therefore neither able to travel across the blood-brain barrier into the cerebrospinal fluid nor is it able to be metabolised by the liver. From Table 1.8, at pH 7.4, 70.72% of phenobarbitone was extracted into the octanol layer. Assuming 50% is protein-bound; it can then be assumed that only about 35.36% of phenobarbitone would reach the brain at physiological pH 7.4.

Elimination is a mechanism the body utilises to rid drug, xenobiotics and waste products from the body and plasma, mainly by the kidney and the liver. The normal pH of urine ranges from pH 4.5 to pH 7.5. Phenobarbitone being lipophilic and protein-binding has a slow elimination; its lipophilicity prevents it from being filtered by the glomerulus, unless metabolised in the liver into a less lipophilic metabolite. However, 25% of phenobarbitone is still excreted in its unionised form via passive tubular secretion. This route of excretion is exaggerated when urine is alkaline or when the urine volume is increased, i.e. via forced diuresis.

Conclusion

As phenobarbitone is used in treatment of epilepsy, it needs to possess the characteristics and ability to cross the blood-brain barrier into the motor cortex to exert its depressant effect. From the partition chromatography carried out, it is evident that phenobarbitone is readily absorbed into the body across biological membranes due to phenobarbitone's lipophilic nature. It is also widely distributed in the body fluid, including the cerebrospinal fluid where it can act on the motor cortex. As for elimination, approximately 75% of the drug is metabolised by the liver before being excreted, but 25% is excreted as unionised molecules by passive tubular secretion in the kidneys. In conclusion, phenobarbitone has a relatively fast onset of action, hence it is deemed suitable to be administered via the oral route for the treatment of epilepsy.

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