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Determining Osmotic Potential Using Density Gradient

A cell, when exposed to an environment where the external water potential is less negative than the internal water potential, will loose water by osmosis down a concentration gradient. Conversely, when exposed to an environment where the internal water potential is less negative than the external water potential the cell will take in water from the surrounding solution. In the case of the former this has the effect of the cell loosing its ability to exert pressure on the cell wall and become flaccid. If water loss is consequential the cell will eventually plasmolyse. The point at which the cell is neither turgid nor flaccid and the net movement of water has reached dynamic equilibrium is known as insipient plasmolysis. It is at this point that the osmotic potential of the cell is equal to the osmotic potential of the surrounding solute. In a more concentrated solution, plasmolysis will continue, causing the protoplasm to pull away from the cell wall leaving a space which gradually fills with the external surrounding fluid. As the osmoticum enters the gap between the protoplasm and the cell wall, the cell density increases. Because the osmoticum of sucrose is denser than water, the plasmolysed cell is therefore denser than the non plasmolysed cell and will travel further and at a quicker rate through a density gradient.
To construct and utilise a density gradient to plot a graph from which the point of insipient plasmolysis can be ascertained, and hence the osmotic potential of a plant cell found.
As script – A mean of two values was taken as time did not permit for the experiment to be run three times.
A graph was then plotted of the mean distance travelled by each stem section against the molar concentration in which it had been equilibrated. The graph was then analysed to see at which point the gradient changed significantly and the point of insipient plasmolysis was found by interpolation thus giving the osmotic potential of the cell.
Figure 1 shows the stems fell at a steady rate in a gradual decline until the 0.3m point where the graph dips sharply to the 0.2m point. Suggesting that the point of insipient plasmolysis is around 0.2m as the steep change in direction to the 03.m point implies that the cells have increased in density thus travelling further and more quickly. The readings at 0.1m do not fit the general trend of the graph suggesting that they are anomalies in the data.
Discussion and Evaluation
The change in the graph occurs because cell membranes in the tissue start to pull away from the cell walls, at the 0.2m concentration. At the 0.3m solution point, more water has left the cells by osmosis in an attempt to achieve equilibrium in the surrounding fluid, however in doing so the cells have become plasmolysed, allowing the sucrose solution to enter the space between the cell membrane and cell wall, therefore it is here the initial increase in density is seen as a sharp increase in the distance travelled by the stem sections. As the cells become further plasmolysed due to immersion in increasing extracellular concentrations, more sucrose solution enters the space in the cells causing them to become denser and hence the stem sections travel further. Insipient plasmolysis was shown to occur when the stems were equilibrated in 0.2 molar sucrose solution; hence because the solute potential of a solution is proportional to its molarity (Campbell Reece et al.) the osmotic potential of the solution was 0.2 moles. At the point of insipient plasmolysis the osmotic potential of the cell is equal to the osmotic potential of the surrounding fluid and therefore the osmotic potential of a plant cell is 0.2moles. The readings taken for the stem in the 0.1 molar solution show that the stem travelled quite some way, this should not have occurred as the cells should not have started to plasmolyse and they should in fact have been turgid at this point as the osmotic potential of the cell is 0.2m and as such has a less negative water potential than the surrounding fluid, encouraging uptake of water into the cell from the surrounding fluid. The stems were prepared in the group it may have been that the stems were not uniformly cut and possibly weighed heavier in the first instance. It would have been more prudent to run the experiment a few more times to gain a more accurate mean for the readings. However, the readings obtained are sufficient to produce a graph from which we can identify the point of insipient plasmolysis.
The Osmotic potential of plant cells is equal to that of insipient plasmolysis which is, 0.2moles
Campbell, R., Reece, J., Urry, L., Cain, M., Wasserman, A., Minorsky, P., and Jackson, R. (2008) Biology, 8th edition, Pearson International: Benjamin Cummings
Bioskills Practical book
Enzyme Hydrolysis of Glycogen by Alpha and Beta Amylase
After a meal carbohydrates are stored in the liver as Glycogen. Glycogen is a branched polymer of glucose where glucose residues are linked by alpha 1-4 glycosidic bonds in linear chains and branched points are linked by alpha 1-6 glycosidic bonds. When required this Glycogen is released back into the bloodstream but first needs to broken down into smaller ‘usable’ disaccharides. Alpha amylases catalyse the hydrolysis of glycogen at the 1-4 linkages, producing Maltose and Maltotriose. Beta amylase also acts in the same manner, but only acts at the non reducing end of the polysaccharide as it is an exo-amylase. Once a branch is reached a limit dextrin is produced as hydrolysis stops. Glycogen digestion by enzymes can be ascertained by determining the amount of product produced during hydrolysis. The resulting product being a reducing sugar, which reduces yellow DNS dye to produce an orange red colour (3-amino-5 nitrosalicylic acid). The more reducing sugar produced, the darker and denser the colour produced during the reduction reaction. A spectrophotometer is used in order to measure the density of the resulting solution as density increases so does absorbance at 540nm.
To determine which if any of two enzymes, Alpha and Beta Amylase digests glygogen most efficiently.
Method – As Script
Maltose concentrations were converted into micromoles per ml and a calibration curve was constructed. A regression line was added and an equation for the line found which was used later in order to find concentrations for each enzyme after the assay had been run and absorbance’s found. These concentrations were then plotted on a separate graph and the graph analysed to ascertain which enzyme performed most efficiently.

The results in Figure 3 show that alpha amylase yields the most product reaching over 2.5 micromoles over time but the graphs also show a similar curve suggesting that the reaction for both enzymes is progressing at a similar rate.
If a gradient is taken for the initial activity for both enzymes it is found that they both produce 0.1 micromoles of product per ml per minute and hence the rate of reaction appears to be the same for both enzymes. However alpha amylase clearly produces more reducing sugar, due to its reaction within the glycogen compound and the initial rate must therefore be faster than that of Beta amylase which only reacts at the reducing ends of the polysaccharide and is also inhibited by its own product maltose. ( 11.4.10) This suggests that t0 is not t0, as suggested.
During the experiment the alpha amylase gave absorbance readings at 540nm at over 1 as did the maltose during the making of the calibration curve, as the absorbance of radiation at a particular wavelength by a solution is ‘directly proportional to the concentration of the absorbing solute’ the readings over 1 are highly likely to be inaccurate as the linear relationship only applies up to a certain concentration, and above this concentration the relationship becomes non linear. As can be seen in figure 2 most of the absorbances for alpha amylase were over one and as such should be questioned as to their validity.
On this basis the alpha amylase should have been diluted further to give absorbances of less than 1 and then this multiplied by the dilution factor to give the absorbance of the original solution.
From the curves in Figure 3 it is very apparent that t0 is not t0 and the majority of the reactions in both cases took place almost instantaneously. To find t0 further experimentation should be carried out during the time the curve represents a zero order reaction. I.e. where the rate is constant with time. The substrate concentrations should be the variable factor with multiple readings taken, and the velocity measured for each one. This data should then be plotted and the two parameters which define enzyme kinetics, Km and V max found. This information can then be applied to the Lineweaver-Burke model and the point at which the line crosses the y axis is the point of 1/V0. This figure can then be differentiated to find t0.
It would appear the alpha amylase is the most efficient enzyme for digestion of glycogen.

Apparent Partition Coefficient of Quinalbarbitone

The effect of ionization on the apparent partition coefficient of quinalbarbitone was investigated using the shake-flask method. High partition coefficient reflected the high lipophilicity of the drug. In the shake-flask experiment, calibration curve was constructed for the determination of concentration of quinalbarbitone in solutions of different pH values. Low ionization of quinalbarbitone in acidic environment will result in high apparent partition coefficient as it is extracted more into the n-octanol layer. This is the case when quinalbarbitone was absorbed through GI tract and crossed the blood-brain-barrier easily.
Quinalbarbitone is weak acid with pKa 7.9 and hence is significantly ionized at pH values over 6. Chemically, it is known as 5-allyl-5-(1-methylbutyl) barbiturate.[1]For all the drug formulations, it is converted to sodium salt, which is more water-soluble for administration to the patients. Quinalbarbitone sodium is very soluble in water, soluble in alcohol, and practically insoluble in ether.[1]Quinalbarbitone is a short-acting barbiturate and it is used for severe intractable insomnia only in patients already taking barbiturates (hypnotic).[5]
In this experiment, we are investigating the apparent partition coefficient of quinalbarbitone. The general principle of the shake-flask method is based on the drug partitioning. Drug is allowed to equilibrate between two immiscible liquids, then the concentration in both layers are determined after they have been separated.
An understanding of partition coefficient and the effect of pH on partition coefficient is useful in relation to the extraction and chromatography of drugs. The partition coefficient for a compound (P) can be simply defined as,[4]
where Co = concentration of the substance in an organic phase
Cw = concentration of the substance in water
In other words, partition coefficient reflects the lipophilicity relative to the hydrophilicity. The greater the P, the more a substance has an affinity for the organic media. P is often quoted as a log-P value. N-octanol is more commonly used as an organic phase experimentally because, to some extent, it resembles the biological membrane in our body.
Papp is the apparent partition coefficient that varies with pH. From the Henderson-Hasselbalch equation:[4]
For acids: Papp =
Experimental (Materials and Methods): 200mL of Solution A and 100mL of Solution B with 50 µg mL-1 quinalbarbitone solution in 0.5M NaOH and in water respectively were prepared from the 0.02%w/v stock solution. For example, 50mL of stock solution was pipetted out and mixed with 150mL of 0.5M NaOH in order to make 200mL of Solution A.
A range of 50mL calibration standards containing 5, 10, 15, 20, 25 and 30 µg mL-1 of quinalbarbitone in 0.5M NaOH was prepared using the Solution A made. For example, 5mL of Solution A was pipetted out and mixed with 45mL of 0.5M NaOH in order to make 50mL of 5 µgmL-1 solution in a round-bottomed flask.
Using the 30 µg mL-1 standard, absorbance of different wavelengths near the expected λmax(254nm) were recorded and the one which gave maximum absorbance was confirmed as the λmax. The UV spectrophotometer was set to 254nm for the absorbance measurement of each standard using 0.5M NaOH as the blank. All the data gathered was used to plot a calibration curve of absorbance against concentration for the quinalbarbitone.
10mL of Solution B, 10mL of 0.1M HCl or buffer solutions with pH 6.6, 7.0, 7.4, 8.0 or 9.0 and 20mL of n-octanol were added into six separating funnels. This provided a system with the drug, aqueous phase and organic phase. The funnels were shaken at frequent intervals for 30 minutes to allow the layers to separate fully. Vigorous shaking should be avoided. After that, the aqueous layer was run off and left with the organic layer. 20mL of 0.5M NaOH was added and it was further shaken for 5 minutes. Finally, the absorbance of the aqueous (bottom) layer was measured for the six partitioning samples. The concentration of quinalbarbitone in the 0.5M NaOH (which was extracted into n-octanol) can be calculated based on the absorbance reading.
As a hypnotic, quinalbarbitone must be a weak acid in order to ionize in blood plasma and lipophilic enough to cross the blood-brain-barrier in order to exert its therapeutic effect. Quinalbarbitone is significantly ionized at pH value greater than 6. This is shown in Table 8 where the percentage of ionization increases from 0% to 92.64% as the pH values of aqueous phase increases from 1 to 9. At low pH, quinalbarbitone is less ionized and therefore, the large amount of unionised species which are much more lipid soluble, will cross the biological membranes much more rapidly than the ionised species. This suggests that the quinalbarbitone will get absorbed more efficiently across a membrane especially in the stomach with pH 1 to 2. Degree of ionization is not the only factor that influences the drug’s absorption. It basically explains the behaviour of a drug only. The other factor is the partition coefficient.[2]
Partition coefficient defines the equilibrium of the drug between the organic and the aqueous phases, depending on the relative affinity for each phase.[2] Greater lipid solubility is reflected as a larger partition coefficient. There is an optimum balance between aqueous and lipid solubility for maximum biological activity at maximum value of partition coefficient.[3] The coefficient is determined only for drugs at less than their saturation concentration in both phases.[3] However, partition coefficient applies only to unionised drugs and it assumes equilibrium state.
Hansch et al. showed that a range of non-specific hypnotic drugs with widely different types of structure were found to have a log P values around 2, provided they were not rapidly metabolized or eliminated.[2] As a rule of thumb, drugs that are targeted for central nervous system (CNS) should have a log P value of approximately 2 so that they can be transported across the CNS quicker. Besides, the therapeutic use of different barbiturates reflects the importance of partition coefficients. For example, quinalbarbitone with pKa 7.9 and Papp 2.0, is used as a short-acting hypnotic because it rapidly enters brain tissues (CNS), whereas phenobarbital with similar pKa 7.4 and Papp 1.4, is used for the chronic treatment of epilepsy, rather than treating insomnia. Furthermore, the two R groups on the barbiturates will also confer to their lipophilicity. Quinalbarbitone with long alkyl side chains is more lipid-soluble, hence it has high percentage of extraction and higher Papp than phenobarbitone.
The literature log P value for quinalbarbitone is 2.0. The experimental log P value is 1.29, which is lower than the literature value. The factor that is most likely to contribute to the difference in both values is due to the inefficiency in extraction. In this experiment, only one extraction is done which limits the efficiency in extraction of the drug into the organic phase. Other than that, we did not perform the shaking with constant magnitude and frequency which will limit the extraction of drug. We can achieve the most efficient extraction by performing large number of extractions with small portions of extracting liquid and using a machine with set intensity to perform the shaking.[7] Besides, the aqueous layer might not be completely run off which causes inaccuracies in determination of concentration of quinalbarbitone in n-octanol.
Another reason might due to the presence of impurities (other solutes, normally salts) as they might affect the results (% of extraction) by forming complex with solute or by salting out one of the phase.[7] This definitely lowers the percentage of extraction and thus, lowering the P value. Other factor influencing the P value (and log P value) is the choice of partition solvent in which the solvent (n-octanol) used in this experiment donate and accept hydrogen bonds and is known as amphiprotic.[8] This is because the H-bonding reduces P as water solubility of the drug increases. The drug activity and its partition coefficient are strong correlated and the accuracy of the correlation depends upon the solvent system used as the model.[2] In this experiment, n-octanol gives consistent results for drugs absorbed in the GI tract though n-octanol is not the same as our biological membranes.
From Table 7 and Table 8, the percentage of ionization is closely related to the percentage of extraction into the organic solvent. The lower the pH of the aqueous solvent, the lower the percentage of ionization, the larger the amount of unionized species in the mixture, the more lipid soluble the solute or the drug is. This means more of the drug will get extracted from the aqueous phase into the organic phase resulting in higher percentage of extraction as compared to those where the drugs are dissolved in solution of higher pH values. Higher pH will results in higher degree of ionization and hence, more drugs will remain in the aqueous phase and will not get extracted into the organic solvent.
The method used in this experiment is known as “shake-flask” method. There are some limitations associated with this method. First, it is time consuming (> 30 minutes per sample). Next, complete solubility must be attained, and it could be difficult to detect small amounts of undissolved material. If the compound is extremely lipophilic or hydrophilic, the concentration in one of the phases will be exceedingly small, and thus difficult to quantify. Lastly, relative to chromatographic methods, large amounts of material are required.
The pharmacokinetic phase of drug action includes the Absorption, Distribution, Metabolism and Excretion (ADME). Absorption is defined as the passage of drug from its site of administration into the bloodstream after enteral administration.[3] Distribution is the transport of the drug from the area of absorption to its site of action.[3] Metabolism is the biotransformation of the drug into a more water-soluble form to be excreted in urine.[3] Excretion is the elimination of unwanted substances from the body.[3]
In term of clinical significance of partitioning with regard to absorption of quinalbarbitone, the extremely acidic environment in the stomach (pH of 1-2) cause less quinalbarbitone to get ionized and behaves much more lipid soluble as the P value is higher as compared to environment with higher pH values. Table 9 shows the Papp values for each six mixtures. The higher the Papp (log Papp) values imply a highly lipophilic drug, where they are more likely to move through the lipid bilayer of the biological membranes. This eventually leads to more quinalbarbitone molecules absorbed from the stomach lining into the bloodstream by passive diffusion. This also suggests that the quinalbarbitone molecules easily and rapidly get across the blood-brain-barrier and reach the target site to exert their therapeutic effect.
Although the theory states that weak acids like quinalbarbitone will preferentially be absorbed from the stomach, these drugs are basically absorbed quite effectively from the small intestine even if they exist in a predominantly ionized form.[6] Gastric emptying will accelerate and hasten the passage of the drugs into the upper intestine with its higher pH environment and much larger surface area which are designed for drug absorption. Small amount of unionised drugs is absorbed and continually being carried away by the rich blood supply of the gut. This leads to equilibrium established and more unionised drugs get absorbed.[6]
Lipophilicity is the dominant factor that affects its distribution in the body.[2] The higher partition coefficient of the drug which means it is much more lipid soluble, the more rapid it passes through the membrane of the tissue and therefore it is more distributed throughout the body. The degree of protein binding is also a function of lipophilicity. Quinalbarbitone has 70% protein binding in plasma.[1] Even though it has a reasonably high P values, its distribution is limited by the plasma protein binding. Thus, it has a low volume of distribution 1.5L/kg as most of quinalbarbitone are bounded to the plasma protein and do not get distributed widely throughout the whole body system. It gets absorbed in the stomach due to extremely acidic environment and thus, it is distributed more widely in the stomach. Due to the fact that small intestine is the organ designed for drug absorption,[6]the resulting volume of distribution is fairly reasonable to say that quinalbarbitone is fairly distributed in the body. In short, quinalbarbitone has the highest lipophilicity, highest plasma and brain protein binding, and the shortest duration of action as compared to other barbiturates.
Most drugs are metabolized first in liver into a more water-soluble form before being eliminated from the body. Oxidation is the most common phase I metabolic pathway. During drug oxidation, a hydroxyl group is added to the lipophilic part of the drug or a short alkyl group is removed, usually is the methyl group.[2] This process is catalyzed by cytochrome P450.[3] The major metabolic reactions involved in quinalbarbitone are hydroxylation of both R groups with further oxidation of the ω-position on the butyl side-chain.[2]
Quinalbarbitone is excreted via renal excretion. Renal excretion involves both filtration at the glomerulus and secretion along the nephron.[3] By virtue of the relationship between pKa and pH, the slightly basic urine will increase the percentage of ionization of the quinalbarbitone, thereby reducing the amount of filtered drug reabsorbed through luminal surface of nephron and therefore increasing its renal elimination.[2] The percentage of extraction is reduced as the degree of ionization increases. The resulting P values become small indicating low lipophilicity which means quinalbarbitone hardly to get through the membrane and back into the circulation. This is the basis of the management of barbiturate overdose. As urine is acidic, quinalbarbitone will be excreted unchanged in it as the percentage ionized is small or even negligible. In fact, it is less than 5% of an oral dose is excreted unchanged in the urine as quinalbarbitone alone is lipid-soluble.[1] It is extensively metabolised to the more water-soluble form of metabolites for excretion.
Conclusions: Partition coefficient defines the equilibrium of the drug between the organic and the aqueous phases, depending on the relative affinity for each phase. Highly unionized (low % ionization) quinalbarbitone means that it is more lipid-soluble which is reflected in high partition coefficient (high % extraction) can rapidly cross the blood-brain-barrier and exert its hypnotic effect. Log P determined is 1.29 which is slightly lower than the literature log P value, 2.0.
In terms of ADME, quinalbarbitone is absorbed in GI tract, highly distributed in our body, mostly hydroxylated and excreted via urine. Since the concentration of quinalbarbitone is determined using UV-visible spectroscopy, therefore the analysis should be done at maximum wavelength so that the absorbance will be high and constant around the chosen wavelength.