Question:

Discuss Capillary Electrophoresis Is a Separation Technique.

Answer to Question: Capillary Electrophoresis Technique

Pharmacy

1 a. The separation technique of Capillary Electrophoresis involves the use differential migration to separate substances in an applied field (1).

The capillary tube has a narrow diameter and the separations take place.

The electrosmotic flow (2) describes the flow of solution through a capillary tube with a narrow diameter.

The flow allows all species to move through a capillary tube. This allows for the injection of analytes at one end of a capillary tube to be eluted on the other.

The capillary tubes are filled with an aqueous buffer that carries the analytes between the anode and the cathode.

The analytes are moved to the cathode via the buffer solution’s electronosmotic circulation.

Electrophoretic flow is a process of differential migration that results in the separation of components. It depends on the species’ charge (3).

Electroosmotic, and electrophoretic movements are the main contributors to the net movement.

Cations are first eliminated because their electrophoretic flow is in the exact same direction that electroosmotic movement.

The buffer solution is eluted second, while neutral charges move in the opposite direction.

Because anions’ electroosmotic flow is opposite to that of the buffer solution, they are eluted at the end.

Capillary electrophoresis separation of a mixture that contains paracetamol (caffeine) and salicylic would yield a mixture containing caffeine, paracetamol, as well as caffeine. Paracetamol is then followed by salicylic acid.

The peak 1 value is caffeine. Peak 2 and 3 are for paracetamol. Peak 3 are for salicylic acids.

Pka of coffee is 0.52 (5).

Paracetamol’s Pka is 9.78 and salicylic acid’s Pka is 2.98 (6).

Caffeine is neutral at PH 9 because it is a weakly simple solution. Therefore it is eluted last.

Paracetamol can be 50% ionized at PH9 to form anionic materials. Salicylic acid is fully ionized, producing anionic species.

You can reverse the migration sequences of paracetamol (caffeine), salicylic acid and caffeine by decreasing buffer pH.

A low buffer PH will cause caffeine’s protonation to form conjugate acid ((4)).

Protonation is caused by the presence of a nitrogen-atom in the molecule, which acts as a proton absorbor.

Paracetamol is also protonated by acidic conditions due to the presence of a nitrogen-atom in its structure.

A very small proportion of caffeine’s Pka value means that only a fraction of it is protonated in order to form its conjugate acids (5).

Because it has a Pka Value of 2.98, salicylic Acid will be neutral under acidic conditions.

Because salicylic acids are neutral at low Ph levels, they will be eluted first.

Paracetamol at PH 2 will also be neutralized and eluted.

A small amount will also be protonated, creating anionic species.

Two distinct advantages can be found in capillary electrophoresis compared to RP-HPLC.

Capillary electrophoresis offers a higher resolution than the RP-HPLC ((8)).

The nature of flow and velocity in the mobile phase determine the resolution.

The smaller diameter of capillary tubes reduces the effects of temperature differences and lateral diffuse.

In this way, buffer solution velocity is constant.

Capillary electrophoresis is also significantly less effective at reducing band widening, compared to RPHPLC (9).

In RPHPLC, the mobile phases are pumped under pressure resulting is laminarflow.

Contrary to popular belief, electroosmotic flows are fluid and independent of pressure. Flat flow results.

RP-HPLC therefore has a lower velocity for the mobile phase at the interface between walls of the tube, and mobile phase. This results in a velocity profile which bulges in the middle, which causes band widening.

A capillary electrophoresis is superior to RP-HPLC because it has a higher selectivity (10).

Capillary electrophoresis is able to adjust the PH as well as the nature of capillary to allow for a good separation.

The disadvantage of capillary electronesis over RP HPLC is its inability to be robust (11).

The types of analytes that capillary electronesis can analyze are limited because they separate species based upon their charge.

RPHPLC can, however, be used to analyze different types of analytes by changing their type of detector.

This level of robustness is not possible with capillary electronesis.

PD MiniTrap G-10 uses a gel filtration chromatographic technique (12) to separate molecules based in part on their molecular sizes.

Sephadex G-10 is used in the column of PD MiniTrap G-10. The Sephadex G-10 quickly separates molecules with higher molecular masses from those with smaller molecular masses.

Any molecules larger than the pores on the Sephadex mat are expelled first from the mixtures. They are then eluted first using the chromatographic column.

Molecules smaller than the pores of Sephadex matrix penetrate into the pores to different levels.

Based on the size of the molecules, elutes take place at different times. Larger molecules (13) are first.

PD MiniTrap G-10 allows for buffer exchange and cleaning-up of biological specimens (13).

The cleaning of biological samples such as peptides, small proteins and oligosaccharides is done to remove radioactive dyes and labels.

Because they are larger than the pores in the Sephadex, contaminants can be excluded during separation (14).

On the contrary, biological samples penetrate through the pores and are separated in order of their size.

Because they are larger, buffer molecules penetrate into the Sephadex matrix.

The column is thus free from contaminants and impurities, so the buffer can be eluted.

PD MiniTrap G-10 provides significant advantages when compared to ion exchanging resins (13).

It is quick to clean up carbohydrates, proteins, peptides, and other contaminants.

Because it uses gel filtration, the device is also more effective in removing contaminants.

PD MiniTrap G-10’s desalting capabilities are higher than ion-exchange.

Additionally, the device can work with small volumes of samples, usually between 100 microliters and 1 mil.

4 a. Benzylpenicillin remains stable at PH 6.8 to 6.8 and temperatures below 4 (15).

Hydrolysis in the lactam ring is the primary cause of penicillin instability (16).

Temperature and pH can influence penicillin’s hydrolysis and subsequent instabilities.

Above PH 6.8, benzyl penicillin’s carbonyl groups are subject to necleophilic attacks by the hydroxyl anion. The result is penicilloic, which is stable.

Hydrolysis takes place when the PH is below 3.

First, the protonation process of the nitrogen atom takes place. Then the nucleophilic attack by the acryl on the carbonyl carbon follows.

Subsequently the lactam-ring opens, destabilizing the thiazole.

The acid catalyzed acid reaction causes the destabilized thiazole to ring to open, which in turn forms penicillanic acid.

Penicillanic Acid is formed under acidic conditions.

However, the temperature has an effect on the rate that benzylpenicillin hydrolyzes.

Low temperatures below 4 degrees Celsius will result in very low hydrolysis rates.

The rate of hydrolysis increases with an increase in temperature above 4.

The oxidation of penicillin begins at higher temperatures.

Oxidation means that benzylpenicillin is added to oxygen at the nitrogen.

To improve the stability, benzyl penicillin must be modified to have a polar amide sidechain. In this way, benzyl penicillin can resist acid-catalyzed hydrlysis.

One way to improve the stability of benzylpenicillin is to substitute an electro withdrawing group at its alpha position (18).

Amino, phenoxy, as well as halo groups are all possible electron withdrawing compounds that can be used in benzylpenicillin.

Substitutions of electron-drawing groups in the structure of benzylpenicillin can stabilize the molecule, reducing acid catalyzed hydrillysis.

The decreased nucleophilicity (19) of the amide-carbonyl oxygen group leads to an increase in stability.

Because the amide is less sensitive to nucleophilic damage, protonation doesn’t occur.

Phenoxybenzyl penicillin, for example, is more stable that benzylpenicillin.

Another structural modification that can improve the chemical stability of benzylpenicillin is the incorporation an acidic substituent or a pole group at the alpha of the sidechain benzyl carbon penicillin (20).

The possibility of the benzyl ring opening would be reduced if the highlighted groups are included in the sidechain. This would increase chemical stability.

By introducing potassium or salt into the structure, benzylpenicillin’s chemical stability can be increased.

Hydrolysis is more common in potassium and sodium benzyl benzyl penicillins.

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