Introduction to Capillary Electrophoresis

Discuss about the Capillary Electrophoresis Is A Separation Technique.

1 a. Capillary Electrophoresis is a separation technique in which substances are separated by differential migration in an applied field (1). The separations occur in capillary tube of narrow diameter. During separation, the flow of the solution occurs from the anodic end to the cathodic end of the capillary tube know as electrosmotic flow (2). The flow results in the movement of all species through the capillary tube thus allowing analytes injected at one end of the capillary tube to be eluted at the other end. The capillary tube is filled with aqueous buffer solution which carries the analytes from the anode to the cathode. Electroosmotic flow of the buffer solution moves the entire components of the analyte towards the cathode. However, separation of the components occurs as a result of differential migration, known as electrophoretic flow, which depends on the charge of the species (3).  The net movement of the charged species is therefore contributed by electroosmotic and electrophoretic movement. Cations are eluted first since their electrophoretic movement is in the same direction as electroosmotic flow. On the other hand, neutral charges move with the same speed as the buffer solution thus eluted next. Anions are eluted last since their electrophorectic flow is opposite electroosmotic flow (4).

In capillary electrophoresis separation of a mixture containing paracetamol, caffeine and salicylic acid, caffeine would be eluted first followed by paracetamol and lastly salicylic acid. Therefore peak 1 is that of caffeine, peak 2 is for paracetamol, and peak 3 is for salicylic acid. The Pka of caffeine is 0.52 (5). On the other hand, Pka of paracetamol is 9.78 while that of salicylic acid is 2.98 (6). At PH 9, caffeine is neutral since it is a weakly basic solution therefore is eluted first. Paracetamol is 50% ionized at PH 9 to form anionic species thus eluted next while salicylic acid is fully ionized produced anionic species thus eluted last.

1. The separation parameters that can be employed to reverse the migration sequence of paracetamol, caffeine, and salicylic acid are decreasing the buffer PH. At low PH, caffeine will be protonated to produce it conjugate acid (4). Protonation of caffeine is due to the presence of nitrogen atom in the molecule that acts as a proton acceptor. Similarly, the presence of nitrogen atom in the structure of paracetamol makes it a proton aceptor and is therefore protonated under acidic conditions to form anionic species. Due to the very small Pka value of caffeine very little of the molecule is protonated to form its conjugate acid (5). On the other hand, salicylic acid will remain neutral under acidic conditions since it has a Pka value of 2.98. Being neutral at low Ph values salicylic acid will be eluted first. Similarly, paracetamol will neutral at PH 2 thus eluted next. On the other hand, a small portion of caffeine will be protonated forming anionic species thus will be eluted last (6).
2. Capillary electrophoresis offers two distinct advantages over RP-HPLC. Firstly, capillary electrophoresis gives a better resolution than RP-HPLC (8). Resolution is dependent on the nature of flow and velocity of the mobile phase. For capillary electrophoresis, the smaller diameter of the capillary tubes minimizes the effects of temperature difference and lateral diffusion. As a consequence the velocity of buffer solution is constant. In addition, band broadening is significantly reduced in capillary electrophoresis compared to RP-HPLC (9). In RP-HPLC, the mobile phase is pumped under pressure resulting in laminar flow. On the contrary, the velocity of electroosmotic flow is independent of pressure and the capillary diameter leading to flat flow. Therefore, in RP-HPLC the velocity of the mobile phase is lower at the interface between the walls of the tube and the mobile phase leading to a velocity profile that bulges at the center causing band broadening. Another advantage of capillary electrophoresis over RP-HPLC is higher selectivity (10). In capillary electrophoresis, the PH and the nature of the capillary can be adjusted to provide a good separation of components. On the other hand, the disadvantage of capillary electrophoresis over RP-HPLC is its lack of robustness (11). Since capillary electrophoresis separates species based on their charge, the types of analytes can be analyzed by this method is limited. On the other hand, RP-HPLC can be used to analyze several types of analytes by changing the type of detector. This kind of robustness cannot be achieved with capillary electrophoresis.
3. PD MiniTrap G-10 uses gel filtration chromatographic technique to separate molecules based on the differences in molecular size (12). The column of PD MiniTrap G-10 is composed of Sephadex G-10 which rapidly separates molecules with higher molecular sizes from those with smaller molecular sizes. Molecules whose sizes are larger than the pores of Sephadex matrix are excluded first from the mixtures and eluted first from the chromatographic column. Molecules whose sizes are smaller than the pores of the Sephadex matrix penetrate into the pores to different degrees. The molecules are eluted at different times depending on their sizes starting which bigger molecules (13).

PD MiniTrap G-10 is used in buffer exchange and clean-up of biological samples (13). Biological samples such as peptides, oligosaccharides, and small proteins are cleaned to remove contaminants such as radioactive labels and dyes. During separation, the contaminants are excluded from the Sephadex matrix given that they have larger sizes than the pores of the matrix (14). On the other hand, the biological samples penetrate into the pores and are eluted in order of size. As regards buffer exchange, the buffer molecules penetrate into the Sephadex matrix while the impurities are excluded and eluted from the buffer owing to their larger sizes. The buffer is therefore eluted from the column free from impurities and contaminants.

Compared to ion exchange resins, PD MiniTrap G-10 offers significant advantages (13). To begin with, the device offers rapid clean-up of carbohydrates, proteins, and peptides. In addition, the device is more efficient in removal of contaminants since it is based on gel filtration. Further, PD MiniTrap G-10 has a higher desalting capacity compared to ion exchange. Finally, the device can be used with relatively smaller volumes of samples usually between 100microliters to 1 milliliter.

4 a. Benzyl penicillin is stable at PH between 6 and 6.8 and temperature between below 4  (15). The principal cause of instability of penicillin is hydrolysis of the lactam ring (16). Hydrolysis and subsequent instability of penicillin is influenced by PH and temperature. Above PH 6.8, the carbonyl group of benzyl penicillin undergoes necleophilic attack by hydroxyl ion resulting I formation of penicilloic acid which is stable. For PH below 3, benzyl penicillin undergoes hydrolysis. First, the nitrogen atom undergoes protonation which is followed by nucleophilic attack of the acryl carbon on the carbonyl carbon. Subsequently, the lactam ring opens causing destabilization of the thiazole ring. The destabilized thiazole ring as well undergoes ring opening which is acid catalyzed to form penicillanic acid which unstable. The formation of penicillanic acid under acidic conditions accounts for the instability of benzyl penicillin (17). On the other hand, temperature affects the rate of hydrolysis of benzyl penicillin. At low temperatures, below 4  , the rate of hydrolysis is very low. On the other hand, increase in temperature above 4 increases the rate of hydrolysis. Also, higher temperatures initiate the oxidation of benzyl penicillin. Oxidation is the addition of oxygen to benzyl penicillin at the nitrogen.

1. Improvement of stability of benzyl penicillin involves manipulation of the polar amide side chain so that benzyl penicillin is resistant to acid-catalyzed hydrolysis. Firstly, the stability of benzyl penicillin can be improved by substituting an electro withdrawing group at the alpha position of benzyl penicillin (18). Electron withdrawing groups that can be substituted in benzyl penicillin include amino, phenoxy, and halo groups. Substitution of an electron withdrawing group in the structure of benzyl penicillin stabilizes the molecule by reducing the chances of acid catalyzed hydrolysis. The increased stability that is imparted by substitution of electron withdrawing groups results from decreased nucleophilicity of the amide carbonyl oxygen atom (19). When the amide group is less susceptible to nucleophilic attack, protonation does not occur and therefore the formation of penicillanic acid that is responsible for instability of benzyl penicillin is hindered. For instance, amino benzyl penicillin and phenoxybenzyl penicillin are more stable than benzyl penicillin.

The other structural modification that can be done on benzyl penicillin to improve its chemical stability is incorporation of acidic substituent, or a polar group at the alpha position of the side chain benzyl carbon atom of benzyl penicillin (20). The incorporation of the highlighted groups in the side chain of benzyl penicillin would reduce the chances of benzyl ring opening up hence imparting on chemical stability. Finally, the chemical stability of benzyl penicillin can be improved by introducing potassium or sodium into the structure of benzyl penicillin. Sodium and potassium benzyl penicillin are more resistant to hydrolysis.

References

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