Capillary Electrophoresis Technique

Question:

Talk about Capillary Electrophoresis: A Separation Technique.

Answer:

Pharmacy

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1 a. Capillary Electrophoresis, a separation method in which substances are separated using differential migration in an application field (1).

A capillary tube with a narrow diameter is used for separation.

Electrosmotic flow (2) is the process by which the solution flows from the anodic and the cathodic ends.

The flow causes the movement of all species within the capillary tubes, which allows analytes to be eluted from one end of the tube.

The capillary tube contains an aqueous buffer which transports analytes to the cathode.

The entire analyte is moved towards the cathode by the buffer solution’s electronosmotic movement.

But, the component’s are separated by differential migration (also known as electrophoretic flows), which is dependent on the species’ charged (3).

Therefore, electroosmotic or electrophoretic movement contributes to the net movement.

Since their electrophoretic movement follows the same direction as that of electroosmotic flows, cations will be eluted first.

Neutral charges, on the other hand move with the same speed of the buffer solution so they are eluted last.

Because anions’ electroosmotic flow opposes that of the buffer solution, anions must be eluted first.

A mixture consisting of caffeine, paracetamol and salicylic Acid would be separated by capillary electrophoresis. First, the caffeine would be removed, followed by paracetamol. Finally, the salicylic Acid would be extracted.

Thus, peak 1 is for caffeine, peak 2 for paracetamol, peak 3 for salicylic acid.

Pka of caffeine equals 0.52 (5).

However, the Pka of paracetamol (a weakly acid solution) is 9.78 (5) and that of salicylic acids (2.98 (6).

Since caffeine is a weakly base solution at PH 9, it is neutral and therefore is eluted as the first.

Paracetamol, which is 50% ionized at PH 9, forms anionic compounds. Salicylic Acid is fully ionized produces anionic species and is eluted last.

To reverse the migration of paracetamol and caffeine, you can use buffer PH to reduce the separation parameters.

The pH of the buffer will decrease and caffeine will be protonated to make it conjugate acid.

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

Paracetamol, which also has a nitrogen atom in its structure, is protonated under acidic conditions to make anionic species.

The Pka value for caffeine is very low so only a small amount of the molecule can be protonated to create its conjugate acid (5).

However, salicylic acids will remain neutral in acidic conditions due to its Pka value 2.98.

Low Ph values will cause salicylic acid to be neutralized first.

Paracetamol will also neutralize at PH 2. It will therefore be eluted second.

However, small amounts of caffeine may be protonated to form anionic substances.

Capillary electrophoresis has two distinct advantages over RPHPLC.

First, capillary electronesis has a better resolution that RP-HPLC (8).

Resolved depends on the flow velocity and nature of the mobile phase.

The capillary electrophoresis tubes with a smaller diameter reduce the temperature difference and lateral diffusive effects.

Therefore, the buffer solution velocity remains constant.

In capillary electrophoresis, band widening is much lower than in RP-HPLC (9).

Laminar flow occurs when the mobile phase of RP-HPLC is pumped under pressure.

However, electroosmotic flow’s velocity is independent of pressure or the diameter of the capillary, resulting in flat flow.

RPHPLC’s velocity is lower between the walls of a tube and the mobile Phase, resulting in a velocity profile that bulges around the center. This causes band broadening.

Higher selectivity (10 is another advantage of capillary electrodephoresis over RPHPLC).

Capillary electrophoresis allows for the adjustment of the PH and nature of the capillary to achieve a good separation.

Capillary electrophoresis has a disadvantage over RPHPLC in that it lacks robustness (11).

Because capillary electrophoresis distinguishes species according to their charge, it is not possible to analyze all types of analytes by this method.

RP-HPLC is able to analyze many types of analytes, depending on the type and detector.

This level of robustness is not possible with capillary electronesis.

PD MiniTrap G-10 uses a gel filtration chromatographic procedure to separate molecules based purely on differences in molecular size (12).

Sephadex G-10 composes the column of PD MiniTrap G-10. It quickly separates molecules of larger molecular size from those of smaller molecular.

All molecules with sizes greater than the pores in the Sephadex matrix are removed first from the mixtures, and then eluted from the chromatographic columns.

Molecules smaller than the pores in the Sephadex matrix penetrate into the pores to different degrees.

Depending on the size of the molecules, they are eluted at different times. Larger molecules (13) are first.

The PD MiniTrap G-10 buffer exchanger is used to clean up biological samples (13).

For biological samples like peptides and oligosaccharides as well as small proteins, radioactive dyes and radioactive labels are removed.

The contaminants are removed from the Sephadex matrix because they have larger pores (14).

However, biological samples penetrate the pores and are separated in order of their size.

Buffer exchange is where the buffer molecules penetrate into Sephadex matrix and the impurities are excluded from buffer.

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

PD MiniTrap G-10 is a better option than ion-exchange resins (13).

First, it provides fast clean-up of carbohydrates and proteins.

The gel filtration technology makes it more efficient to remove contaminants.

PD MiniTrap G-10 is more salt-tolerant than ion exchanging.

Additionally, the device can handle smaller samples volumes, typically between 100microliters and 1 milliliter.

4 a. Benzylpenicillin is stable at PHs between 6 and 6.8 (and temperatures below 4 (15).

Hydrolysis of lactam rings (16) is the principal reason for penicillin’s instability.

The temperature and pH are important factors in the hydrolysis and subsequent instability penicillin.

Above PH 6.8, carbonyl groups of benzylpenicillin are subject to necleophilic attacks by the hydroxyl ion. This results in the formation of stable penicilloic acids.

Hydrolysis occurs when benzylpenicillin is below 3.

First, the nitrogenatom undergoes protonation. This is followed up by nucleophilic attacks of the acrylcarbon on the carbonylcarbon.

The lactam rings then open, which causes destabilization of thiazole.

As well, the thiazole ring that has been destabilized undergoes a ring opening. This is acid catalyzed to make penicillanic Acid which is unstable.

Penicillanic acids are formed in acidic conditions. This is what causes the instability of benzylpenicillin (17).

The temperature, however, has an impact on the rate of hydrolysis.

Hydrolysis rates are very low at temperatures below 4 °C.

However, hydrolysis is more efficient when the temperature rises above 4.

Additionally, higher temperatures can cause benzylpenicillin to oxidize.

Oxidation is when oxygen is added to benzyl penicillin at nitrogen.

The modification of the polaramide side chains can improve the stability of benzylpenicillin. This will allow it to resist acid-catalyzed hemolysis.

First, an electro withdrawing agent can be substituted at the alpha of benzylpenicillin (18).

You can substitute benzylpenicillin with amino, penoxy, or halo electron withdrawing group.

Substitutions of electron withdrawing groups in benzylpenicillin’s structure stabilize the molecule and reduce the likelihood of acid-catalyzed hydrolysis.

Increased stability is achieved by substituting electron withdrawing elements. This is due to a decreased nucleophilicity for the amidecarbonyl oxygen atom (19).

Protonation occurs less frequently when the amide group resists nucleophilic attack. This prevents the formation of penicillanic acids that are responsible for the instability of benzyl penicillin.

The stability of benzylpenicillin and aminobenzylpenicillin are higher than that of phenoxybenzyl.

Incorporation of an acidic substitute, or a polar atom at the alpha position on the side chain benzylcarbon atom of penicillin (20) is another structural modification that can be made to benzylpenicillin in order to improve its chemical stability.

The inclusion of the highlighted elements in the sidechain of benzyl penicillin will reduce the likelihood of the benzylring opening up, thereby increasing chemical stability.

In addition, potassium and sodium can be added to the structure of benzylpenicillin to improve its chemical stability.

Hydrolysis is more severe for sodium and potassium benzylpenicillin.

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