Partitioning Of Barbiturates Overview

Partitioning Of Barbiturates Overview purple heart is an antiepileptic medicine which is used to administer epilepsy. Absorbance appraises at distinguish able concent proportionalityn of sodium thiopental good deal be used to plot a standardization graph which in turns drop be used to distinguish the intentness of phenobarbit matchless in ingrained bod at different pH by apply the side of the graph. From there, the concentration of phenobarbital in aqueous configuration jackpot be happend and thusly 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 cipher by knowing its pKa and this in turns can be used to determine the oleophilicity of phenobarbitone. % extraction of phenobarbitone will also determine how well does phenobarbitone passes through the thoroughgoing phase (biological membrane in the phy sical structure) at different pH.Phenobarbitone is a barbiturate used in the intervention of epilepsy. It helps to reduce raptus frequency and severity and may even stop seizures altogether. 1 Phenobarbital works by acting on GABA receptors that in turn increase synaptic inhibition. This past increases the seizure threshold, because 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 ionize at ph values over 6. clayey the pH of the dissolvent reduces ionisation. 4 According to Phenobarbitone structure, it has 2 hydrogen adhere donors and 3 hydrogen bond receptors and the solubility of Phenobarbitone in water is 1g in 1000mL (0.1% w/v). It has embarrassed lipid solubility compargond with other barbiturate. and then it has slow onset action and prolonged half life.This experiment was carried place to investigate the effect of ionisation of phenobarbitone by measuring the heart 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 dose at equilibrium and since the pKa of the drug and the pH of the resolvent are known, the true partition co-efficient for phenobarbtione can be cipher. 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 lipotropic. 5The method used in this experiment is the shake-flask method (the most(prenominal) 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 meat of barbiturate fork over in the 0.5M NaOH (which is extracted from the octanol) is known, and the UV analysis gives the amoun t 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 for each one phase at equilibrium. pKa is important to determine the true partition coefficient for the barbiturate. The loss 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.Experimental0.02% w/v phenobarbitone in water was been provided as the inception solution. Using the stock solution, 50g mL-1 solution in 0.5M NaOH Solution A and a 50g mL-1 solution in water Solution B were been prepared.(a) Calibration StandardsA range of calibration standards each containing 5, 10, 15, 20, 25, 30 g mL-1 of the phenobarbitone in 0.5M NaOH were prepared use Solution A. The wavelength of soapimum absorbance (max) at about 254nm was then determined using the 30 g mL-1 standard. The absorbance of each standard at the max was subscribe using 0.5M NaOH as th e blank. A calibration curve of absorbance versus concentration for the phenobarbiturate was then constructed using the absorbance readings obtained.(b) Partitioning SamplesSix partitioning funnel shapes were filled as the chasei) 10mL Solution B, 10mL 0.1M HCl, and 20mL n-octanolii) 10mL Solution B, 10mL pH 6.6 buffer, and 20mL n-octanoliii) 10mL Solution B, 10mL pH 7.0 buffer, and 20mL n-octanoliv) 10mL Solution B, 10mL pH 7.4 buffer, and 20mL n-octanolv) 10mL Solution B, 10mL pH 8.0 buffer, and 20mL n-octanolvi) 10mL Solution B, 10mL pH 9.0 buffer, and 20mL n-octanolThe funnels were shaken at stag intervals for 30 minutes to allow the layers to go bad fully. The constitutional layer was then been carefully ran mutilate 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 max determined previously in (a). The conc entration of the phenobarbitone in the 0.5M NaOH was calculated using the calibration curve.DiscussionIn partition chromatography, molecules collide with 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 crosswise the biological membrane, i.e. the movement of phenobarbitone from aqueous phase, 50g mL-1 phenobarbitone in water, to perfect phase, octanol. Its chemical structure shows that there is a long hydrocarbon chain attached to the ring structure which contributes to its lipophilicity. The much lipophilic the drug, the more efficiently it is absorbed into the organic phase.Glass separating funnels were used to subdue absorption of lipophilic drug into the container. 30 minutes after the layers a re left to separate fully, the aqueous layer is carefully ran finish, 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 clear acid, it reacts with the newly added base, NaOH to ashes aqueous phase. The concentration of phenobarbitone in the aqueous phase is then determined using a UV spectrophotometer.From control board 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 well-kept in the aqueous phase in its ionised form and hence unable to cross into the octanol phase as it cannot be passively dispersed.As shown in Table 1.7 and represent 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 come on increase less steeply from pH 8.0 to pH 9.0. As for Graph 2.0, a slow settle is observed from pH 1.1 to pH 7.0, a steep decrease from pH 7.0 to pH 7.4, followed by a in small stages decrease from pH 7.4 to pH 9.0.Partition coefficient, P is the ratio of a drugs 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 lit erature value is 1.4. The comparison is make 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 virtually 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 succession allowed for phenobarbitone to diffuse from the aqueous phase into the organic phase.Studies revealed that the peak plasma concentration is tump overed 0.5 to 4 hours following an oral administration partition chromatography mimics the diffusion of drug crosswise biological membrane, therefore requires at least 30 minutes to flip over 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 subsequent 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 p hase. 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 approximately 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 pass across the blood-brain barrier into the cerebrospinal fluid nor is it able to be metabolised by the colorful. 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 p hysiological pH 7.4.Elimination is a appliance the body utilises to rid drug, xenobiotics and waste products from the body and plasma, mainly by the kidney and the liver. The sane pH of urine ranges from pH 4.5 to pH 7.5. Phenobarbitone organism 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 vasiform secretion. This route of excretion is mislead when urine is alkaline or when the urine volume is increased, i.e. via forced diuresis. oddmentAs phenobarbitone is used in treatment of epilepsy, it needs to throw the characteristics and ability to cross the blood-brain barrier into the motor cortex to exert its somnific effect. From the partition chromatography carried out, it is evident that phenobarbitone is readily absorbed into the body across biological me mbranes due to phenobarbitones 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 riotous onset of action, hence it is deemed suitable to be administered via the oral route for the treatment of epilepsy.