Capillary Electrophoresis Technique

Table of Contents

Question:

Discuss the Capillary Electrophoresis is a Separation Technique.

Answer:

Pharmacy

1 a. Capillary Electrophoresis (or Capillary Electrophoresis) is a separation technique where substances are separated through differential migration in an applied field (1).

Capillary tubes with narrow diameter are used to perform the separations.

The flow of the solution from the anodic to the cathodic ends of the capillary tube is known as electrosmotic flows (2).

This causes all species to move through the capillary tube, allowing for analytes that were injected at one end to be eluted at another end.

The capillary tube is filled by aqueous buffer solution, which transports the analytes from one end to the other.

The buffer solution’s electroosmotic flow moves all components of the analyte towards its cathode.

The differential migration of the buffer solution, also known as electrophoretic flux, causes the components to separate. This depends on the species’ charge (3).

Electroosmotic movement and electrophoretic motion are responsible for the net movement of charged species.

Because their electrophoretic motion is in the same direction of electroosmotic flow, cations are first eluted.

However, neutral charges move at the same speed that the buffer solution and are thus eluted first.

Because their electrophorectic flow opposes the electroosmotic flow, anions must be eluted first.

Capillary electrophoresis separation would result in a mixture of paracetamol, salicylic acids, and caffeine. The first step would be to remove caffeine, then paracetamol and finally, salicylic acid.

Peak 1 is the peak of caffeine, peak 2, paracetamol and peak 3, respectively.

Pka for caffeine is 0.52 (5).

The Pka for paracetamol is 9.78, while the Pka for salicylic acid (2.98 (6)).

Because caffeine is a weakly acid solution, it is neutral at PH 9. Therefore, it is eluted first.

At PH 9, paracetamol is 50% ionized to form anionic substances. Salicylic acid, on the other hand, is fully ionized and produces anionic species.

Paracetamol, caffeine and salicylic acids can be reversed by changing their buffer PH.

Low PH will cause caffeine to be protonated and produce its conjugate acid (4).

The presence of a nitrogen atom within a molecule acts as a proton receiver, causing the protonation of caffeine.

Paracetamol’s structure also contains a nitrogen atom. This makes it a proton acceptor. It is protonated under acidic conditions in order to form anionic substances.

Because caffeine has a very low Pka value, very little of its molecule is protonated to make its conjugate acid ((5)).

Salicylic acid, on the other hand will not react to acidic conditions because it has a Pka of 2.98.

Salicylic acid, which is neutral at low Ph values, will be eluted the first.

Paracetamol, which is neutral at PH 2, will be eluted first.

A small amount of caffeine, however, will be protonated and form anionic species. This will be eluted next (6).

Two distinct advantages are offered by capillary electrophoresis over RP-HPLC.

First, capillary electrophoresis provides a higher resolution than RPHPLC (8).

The nature of flow and the velocity of the mobile phase will affect the resolution.

Capillary electrophoresis is made easier by the smaller diameter capillary tubes. This reduces temperature differential and lateral diffusion.

The buffer solution’s velocity is therefore constant.

Capillary electrophoresis also has a significantly lower band widening than RP-HPLC (9).

Laminar flow is possible in RP-HPLC because the mobile phase is pumped under high pressure.

Flat flow occurs because electroosmotic flow has a velocity independent of pressure and capillary diameter.

RP-HPLC has a lower velocity at the interface of the tube walls and the mobile phases, which results in a velocity profile with bulging at the center that causes band widening.

Capillary electrophoresis has a higher selectivity than RP-HPLC (10).

Capillary electrophoresis can be adjusted to ensure a separation of components.

However, capillary electrophoresis is less robust than RP-HPLC (11).

Capillary electrophoresis is a method that separates substances based on their charges. This limits the types of analytes that can be analysed by this method.

RP-HPLC, on the other hand can be used to analyze multiple types of analytes depending on the type of detector.

Capillary electrophoresis cannot achieve this level of robustness.

PD MiniTrap G-10 uses a gel filtration chromatographic method to separate molecules based upon differences in molecular sizes (12).

Sephadex G-10 makes up the column of PD MiniTrap G-10. This allows for rapid separation of molecules with larger molecular dimensions from those with smaller ones.

The chromatographic column elutes first molecules whose sizes exceed the pores of the Sephadex matrix.

The pores of the Sephadex matrix allow molecules smaller than their sizes to penetrate the pores at different levels.

They are eluted at different times according to their size, with larger molecules starting first (13).

The PD MiniTrap G-10 can be used for buffer exchange and cleaning up biological samples (13).

To remove radioactive labels or dyes from biological samples, such as small proteins, oligosaccharides and peptides are removed.

Because contaminants have larger pores than the Sephadex matrix, they are removed from the Sephadex matrix during separation (14).

The biological samples, on the other hand penetrate the pores and are eluted according to their size.

The buffer molecules penetrate the Sephadex matrix, while the buffer molecules elute the buffer from impurities.

Therefore, the buffer is eluted from a column without impurities or contaminants.

PD MiniTrap G-10 has significant advantages over ion exchange resins (13).

The device is fast in removing carbohydrates, proteins, and propeptides.

Gel filtration makes the device more efficient at removing contaminants.

PD MiniTrap G-10 also has a greater desalting capability than ion exchange.

The device can also be used with smaller volumes, usually 100 microliters to 1 ml.

4 a. Benzyl penicillin can be stable at PH 6 to 6.8 and below 4 (15).

Hydrolysis of the lactam-ring is the main cause of penicillin instability (16).

Temperature and PH are key factors in hydrolysis and subsequent penicillin instability.

Above PH 6.8, the carbonyl-group of benzyl penicillin is subject to necleophilic attack from the hydroxyl Ion, resulting in stable penicilloic Acid.

Hydrolysis is performed on benzyl penicillin below PH 3.

The protonation of the nitrogen atom is followed by the nucleophilic attack on the acryl carbon.

The lactam ring then opens, resulting in the destabilization the thiazole rings.

Destabilized thiazole rings also undergo ring opening, which is acid catalyzed and forms penicillanic acids which are unstable.

Penicillanic acid is formed under acidic conditions, which causes the instability in benzyl penicillin (17).

Temperature, on the other hand affects the rate at which benzyl penicillin is hydrolyzed.

The rate of hydrolysis at low temperatures (below 4°C) is very slow.

Hydrolysis rates increase with increasing temperatures above 4.

Higher temperatures also initiate the oxidation benzyl penicillin.

Oxidation refers to the addition of oxygen at the nitrogen.

Modifying the polaramide side chain to increase the stability of benzylpenicillin will make it more resistant to acid-catalyzed hydrogenlysis.

First, substituting an electro withdrawing compound at the alpha position in benzylpenicillin (18) can improve the stability.

There are three types of electron withdrawing groups that can substitute for benzyl penicillin: amino, phenoxy and halo.

Substitution of an electron withdrawal group in the structure benzyl penicillin stabilizes it by decreasing the chance of acid catalyzed hydrogenlysis.

Substitution of electron withdrawing group results in decreased nucleophilicity at the amide carbonyloxy atom (19). This increases stability.

The amide group is less vulnerable to nucleophilic attacks, and protonation is prevented.

Amino benzyl penicillin is more stable than benzyl.

To improve benzyl penicillin’s chemical stability, another structural modification is possible. This would be the incorporation of an acidic substituent (20).

Incorporating the highlighted groups into the side chain would decrease the chance of the benzyl rings opening up and increase chemical stability.

The chemical stability of benzylpenicillin can also be improved by adding potassium or sodium to the structure.

Hydrolysis is more difficult for potassium and sodium benzyl penicillin.

Refer to

Skoog (D.), Holler (F.), and Niemen (T. Principles of Instrumental Analysis (5th).

Buszewski (B.), Dziubakiewicz (E.) and Szumski (M. Principles and Practice of Electromigration Techniques.

Wallingford R., and Ewing A. Capillary Elektrophoresis.

Journal of Advanced Chromatography 29(1) (2013): 1-67.

Nishi, H. Enantiomer seperation of basic drugs using capillary electrophoresis.

Journal of Chromatography 735 (2016): 345-351.

Cains, D. Physicochemical characteristics of drugs: Essentials in Pharmaceutical Chemistry (2nd).

London: Pharmaceutical Press. 2012.

Thomson, L., Veening H., and Timothy G. Capillary Electrophoresis at the Undergraduate Instrumental Analysis Laboratory. Determination Common Analgesic Formulations.

Journal of Chemical Education, 7(9) 2013, 1117-1121.

Altria K. Capillary Electrophoresis guidebook: Principles and Operation.

Li, B. Capillary Electrophoresis.

Principles, Practice, and Applications.

Journal of Chromatography 52(8) (2012): 395.

Landers, J.P. Handbook for capillary electrophoresis with associated chromatographic methods.

New York: CRC Press. 2013.

Marina, M., Rios A., Varcalcel T. Analysis by Capillary Electrophoresis.

PD MiniTrap G-10.

Capillary electrophoresis for the determination of drug-related impurities.

Journal of Chromatography 735 (2016): 43-56.

Clarke, T. Penicillin Chemistry.

London: Princeton University Press 2012.

K. Frirk. Understanding the chemical basis for drug stability and degradation.

Journal of Pharmaceutical Chemistry, 5(3) (2014): 78-98.

Joseph, K., Ma H., Hadzija B.

Basic Physical Pharmacy.

New York: Jones and Bartlett Publishers. 2012.

Kadam, S., and Bothara K. Principles in Medical Chemistry.

Alexander, M., and Corrigan A.

Infusion Nursing.

A Evidence-Based Approach.

New York: Infusion Nursing Society. 2009.

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