The Prolonged Retention Of Chlorhexidine Biology Essay

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A great difficulty for the eradication of microbial infection in the oral cavity is dilution and rapid elimination of applied drug due to flushing of saliva. Therefore the drug delivery system in which drug is incorporated plays a major role and it is to be considered that delivery system should have prolonged retention of drug in the oral cavity.

To solve the problem of prolonged retention of drug in oral cavity, we have chosen the controlled release as the drug delivery system for the CHLORHEXIDINE. Chlorhexidine is used widely in clinical dental practise as an antiseptic. Chlorhexidine reduces the adhesion of microbes to oral mucosal cells.

The prolonged retention of Chlorhexidine can be achieved by controlled release drug delivery. For the controlled release of Chlorhexidine we used Zinc phosphate cement as carrier.

Controlled release:

The basic principle involved in controlled drug delivery systems is to modify the biopharmaceutic, pharmacokinetic and pharmacodynamic properties of drug in such a way that its utility is maximized through reduction in side effects and cure or control of condition in the shortest possible time, by using smallest quantity of drug, administered by the most suitable route.

Mechanisms of controlled release:

1. Dissolution:



2. Diffusion:



3. Combination of both dissolution & diffusion.

4. Osmotic pressure controlled system


It is a process in which drug molecules diffuse from higher concentration to lower concentration until equilibrium is attained.


Optimum drug therapeutic drug concentration is maintained in blood or cell.

Better patient acceptance and compliance.

Reduction in fluctuations at plasma drug levels.

Enhancement of activity duration for short half-life drugs.

Frequent dosing, wastage of drug and side effects are reduced.

Predictable and reproducible release rates for extended periods of time.



Poor in vitro-in vivo correlation.

Less systemic availability compared to immediate release conventional dosage forms.

Possibility of dose dumping.

High formulation cost.

Reduced potential for dose adjustment.

In case of toxicity, poisoning or hypersensitivity reactions recovery of drug is difficult.


Chlorhexidine is a final irrigant prior to endodontic obturation. Chlorhexidine has the ability to restrict bacterial access and penetration into the dentinal tubules. It has immense range of substantivity (the ability of any oral antimicrobial agent to carry on its therapeutic activity for a prolonged time.), as it possess good anti microbial property and has the ability to adhere to root canal dentin.

According to Lindblade R. M. (2010), it does not affect the immediate or the post cementation bonding. When certain irrigants like NaOCl which is when used with EDTA resulted in reducing the immediate composite bond strength towards the root dentin or pulp chamber, Chlorhexidine did not show any of these negative effects rather it improved the immediate bond strength slightly with almost all posts/cements. It inhibits the collagen degrading enzymes, matrix metalloproteases etc and improves the durability of composite adhesive bonding to dentin.

Journal of Dentistry, Article in Press, Corrected Proof - Note to users

doi:10.1016/j.jdent.2010.06.011 | How to Cite or Link Using DOI

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Effect of Chlorhexidine on initial adhesion of fiber-reinforced post to root canal

Ritva M. Lindblada, c,

Received 22 February 2010; pic

Structure of Chlorhexidine

N', N'''''-hexane-1, 6-diylbis [N-(4-chlorophenyl) (imidodicarbonimidic diamide)]


Chlorhexidine Diacetate, Chlorhexidine gluconate, Chlorhexidine Hydrochloride

Physical Properties:

Molecular formula


Molecular weight


CAS Registry Number


Physical state

White to pale yellow powder



Melting point




Solubility in Water

Slightly soluble


Stable under ordinary conditions

Trade Names: Dyna-hex2, Hibatine, Hibaclens


Chlorhexidine has antibacterial effect. It is adsorbed on to the organism's cell wall thus, breaks up the integrity of the cell membrane causing the leakage of intracellular components. "#tabview=tab0"&HYPERLINK "#tabview=tab0"objCls=Drug#tabview=tab0


Chlorhexidine is used as mouth wash.

Used as irrigation solution.

Ref: British pharmacopeia, volume-3, 2007, 2411, 2413.

Used to relieve the pain in the treatment of mouth sorus.

Bacteriostatic spectrum of Chlorhexidine shows wide spectrum of activity against gram +ve bacteria.

Ref: t.d.hennessy, Journal of periodontal research, vol 8 issue 12 pg no 61-67,


Some desquamation and soreness in oral mucosa is observed with the prolonged Chlorhexidine rinsing.

Use of Chlorhexidine has shown a side effect of plaque formation and gingival conditions.

Ref: European journal of oral science, leiv florta, Gunnar rolla, Jens wearhaug. Vol 79, issue 2, pages 119-125, 2007.


Dental cement:

Material that produces mechanical effect on hardening. The material is prepared by mixing a powder and liquid.

In zinc phosphate cement the powder consists of Zinc oxide and Magnesium oxide, liquid consists of phosphoric acid and water.

Reasons for using zinc phosphate as cement:

In dentistry zinc phosphate is used as cement due to its various properties such as:

More retentiveness

Setting time

High compressive strength

High solubility



Chemical formula


Molecular weight


CAS number


Physical state

White powder

Melting point





HPLC is the most versatile and universal type of analytical procedure. This technique is a powerful tool in analysis which uses highly improved form of column packing with smaller particle size, sample forced with high pressure using physically stable pumping system and highly sensitive detectors.


A liquid mobile phase is pumped under pressure through a stainless steel column containing particles of stationery phase with a diameter of 3-10 µm. The analyte is loaded onto the head of the column via loop valve and separation of a mixture occurs according to the relative lengths of time spent by its components in the stationary phase. It should be noted that all components in the stationary phase spend more or less the same time in the mobile phase in order to exit the column. Monitoring of the column effluent is carried out with a variety of detectors.

HPLC Separation Modes:

Depending on the characteristics of chemical compounds such as polarity, molecular size, electrical charge, chirality etc different separation modes have been used. The techniques include:



Ion exchange and ion pair

Size exclusion

Chiral and affinity chromatography

Based on the polar nature of the analyte, two separation techniques such as Normal Phase and Reverse Phase Chromatography are used. There are certain atoms and molecules which attribute to the polar nature of the compounds. The location of these polar functional groups imparts its ability of chromatographic retention. A separation is created based on the relative attraction of each compound in the mixture towards stationary and mobile phase.

Normal Phase Chromatography consists of a polar stationary phase having pores large enough to accommodate and attracts the polar particles of the mixture. The mobile phase being non polar elutes away the less polar or non polar components.

Reverse Phase chromatography is the most commonly used analysis strategy in which the stationary phase made of silica is derivatised with long hydrocarbon chains to make it non polar. The mobile phase is non polar and carries the mixture. The polar molecules move faster and longer with mobile phase. The non polar components will slow their way down the column because of attractive force like vanderwaals and dispersion force towards the hydrocarbon chain.

The Silica gets covalently bonded to the hydrocarbons containing C1 - C18 carbons, yielding a thermally and hydrolytically stable stationary phase. It imparts properties to the column as hydrophobic surface, small diameter, spherical particle size distribution etc. The larger the particle size, the broader the peaks become. The relative chromatographic retention of the components of a mixture due to the binding of some components to the stationary phase or the elution in the mobile phase depends on the polarity. The components get separated from each other by giving its characteristic retention time and the corresponding peak. The Chromatogram is obtained by plotting the retention time against the peak area.


The commonly used method is UV spectrometer, as many organic molecules absorb UV light of various wave lengths. The amount of light absorbed depend on the amount of a particular analyte under investigation. The sensitivity of detectors is of significant importance, for the better resolution of peaks. For obtaining accurate chromatographic profile, the detector sampling volume should be small.


The solvent is mixed to attain the required gradient using the pumps and force the solvent through the column. Isocratic conditions where the solvent conditions are held constant, which is a very common in quality control analysis to confirm the identity of compounds.

Buffering Agents:

The variation in PH can affect the hydrophobic nature of the analyte. The buffering agents like sodium phosphate are used to control the ph variations. It also helps to neutralise any residual charge on the silica packing of stationary phase. These agents neutralise the charges on the analyte by acting as ion pairing agents.

Retention Time:

Retention time is the time taken by the analyte to elute down the column. It is measured as the time taken for the display to form the maximum peak height from the time of injection of sample. The different components show different retention time which depends on the pressure used, which internally affecting the flow rate of the solvent. The retention time also depends on the column material, its particle size, solvent composition, temperature.

T. Hanai (1999), "HPLC, a Practical Guide" ISBN- 0-85404-515-5, published by: The royal society of Chemistry, Cambridge, UK. Page -1-10.

Reverse Phase HPLC Basics for LC/MS

An Ion Source Tutorial


Andrew Guzzetta

This Tutorial was first published July 22nd, 2001

Read important laboratory safety notice at bottom of page before proceeding


Accurate and precise quantitative analysis of pharmaceutical products is done by using the combination of HPLC and UV/Visible detectors.

Partition coefficients and pKa values of drugs can be determined.

Determination of drug protein binding.

Pure drug substances stability monitoring in formulations, with quantification of degradation products.


Readily automated.

Limited sample introduction ensures quantitative precision.

Easy handling.

HPLC has most intensive development in recent years, leading to development in columns, detectors and software control.

Availability of variety of columns and detectors helps in the selection of method which can be readily adjusted.

Less risk of sample degradation because heating is not required.


Organic solvents wastage is more, which more expensive to dispose off.

Prior to analysis drugs have to be extracted from the formulation.

Compounds without chromophore cannot be detected.

Requirement of inexpensive and reliable detectors.

Ref: David G Watson, pharmaceutical analysis, a textbook for pharmacy students and pharmaceutical chemists, Second edition, Elsevier Churchill Livingston publications.


Chemicals used:


Zinc phosphate


Sodium phosphate monobasic


Ortho-Phosphoric acid.

Equipments used:

pH meter


HPLC Instrument

Agilent 1200

Mode of Separation

Reverse Phase Chromatography


Serial No

Stationary phase

Pore size

Column dimensions



Hypersil C18


150* 4.6 mm

Mobile phase

Acetonitrile: Phosphate Buffer (45:55)


UV-Visible Spectrometer





Preparation of sample:

In our experiments we used commercial Zinc phosphate cement (Kent dental, Gillingham, Kent, UK) at powder liquid ratio of 3.6:1. Chlorhexidine (Aldrich, Poole, Dorset, UK) was used as active drug.

1.8g of Zinc phosphate cement and Chlorhexidine were weighed and placed on a ceramic tile and were mixed using a stainless steel spatula for uniform distribution of Chlorhexidine in zinc phosphate. Then 0.5 ml of liquid was added to the powder mixture and mixed to form a smooth paste.

Then freshly prepared pastes were placed in the silicone rubber moulds to form discs. Three samples were prepared for each concentration of Chlorhexidine. Three sets of samples were prepared containing 1, 2 and 5% Chlorhexidine by mass. Now moulds are placed in between two glass slides and are clamped. Moulds are allowed to dry in incubator for 10 minutes at 370C. Dried discs were carefully removed from rubber moulds, and were placed in an individual centrifuge tubes of capacity 50 ml, and 5ml deionised water was added to each tube. Then centrifuge tubes were kept in incubator for curing at 370C for 1hour.

Chlorhexidine release was then determined from each sample at an intervals of 1, 2, 3, 4, 24hrs and 1, 2, 3, 4 weeks, respectively by using HPLC (Agilant 1200 series).


Percentage of Chlorhexidine

Zinc oxide powder (gm)

Phosphoric acid (ml)

Chlorhexidine required (gm)

Deionised water (ml)



















Preparation of buffer solution:

9.5984g of sodium phosphate monobasic was weighed and added to 1000ml of deionised water with 5ml of Triethylamine in it and dissolved. After complete dissolution of sodium phosphate monobasic, pH of the solution was adjusted to 3.0 using pH meter by adding diluted 85% Ortho-Phosphoric acid.

Preparation of mobile phase:

Mobile phase is a mixture of 2 solvents, Acetonitrile and Buffer solution in the ratio of 45:55v/v. Degassing of mobile phase is done every time using it on HPLC, to avoid air bubbles.

Preparation of solvent:

Solvent consists of Acetonitrile and Formic acid 1% in the ratio of 20:80 v/v.

Preparation of standard stock solution:

50mg of Chlorhexidine was weighed and transferred to a 50ml volumetric flask.

Small amount of solvent was added and Chlorhexidine was dissolved.

Now volume was made up to 50ml with solvent.

From the standard stock solutions different dilution solutions were prepared, by using the following formula:



C1= Concentration of stock solution

C2= Concentration of required dilution

V1= Volume of unknown concentration to be calculated.

V2= Volume of volumetric flask in which dilution is made.

Volume of stock solution (ml)

Volume of volumetric flask for makeup (ml)

Concentration (µg/ml)



















Experimental procedure:

First mobile phase was degassed and connected to pump. A bottle for collecting waste was arranged. Now the HPLC equipment (Agilent 1200 series) is turned on by switching on the detector and isocratic pump, then instrument online icon on screen is clicked to get the connection with the instrument.

All the experimental conditions were setup and a directory was created. Now the pump turned on and flow rate was setup to 0.5 ml and was increased to 1.5 ml gradually till the constant base line was achieved.

Then solvent was injected to check whether any impurities are present or not. After getting a straight base line without any noise, samples were injected analyse. Before running the sample, sample information was entered. A stop time of 4 minutes was setup.

First the dilution samples were run, then other samples. Every time before injecting sample, syringe should be washed cleanly to avoid impurities. Every sample was run for every concentration, and release profiles were recorded and then the mean release was calculated. Then graphs of peak area against time were plotted.