The Reverse Phase High Performance Liquid Chromatography Method Biology Essay

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The reverse phase high performance liquid chromatography method was used in the estimation of Rutin. The instrument used was Agilent technologies (1200 series).Rutin drug was validated in the isocratic HPLC system with the usage of Lichrosorb-5 (RP18) column. The mobile phase used was acetonitrile: methanol: water in the concentration of 200:150:650 with 1ml of phosphoric acid was added to it. The results showed the good retention time with low flow rate of about 1ml/min. The wavelength was adjusted at 360nm which gives very sharp peak. After the chromatograms obtained various parameters were calculated such as the standard deviation, retention time, LOD, LOQ which is calculated from the regression equation and the slope from the obtained linear graph. The chromatograms were studied for the retention time and by using their mean and standard deviation were calculated. The result formed from these was that there was no much difference found in the mean values of the 5 different concentrations.

2. HPLC INTRODUCTION

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HPLC is the most widely used analytical technique because of its sensitivity, ready adaptability to accurate quantitative determination, its suitability for separation of non volatile species or thermally fragile one and its ease of automation. HPLC has been used for the separation of many materials such as amino acid, proteins nucleic acid, hydrocarbon, antibiotics, steroids, terpenoids, pesticides and the variety of inorganic and organic substances. HPLC is also known as high pressure liquid chromatography.

In HPLC the mobile phase is liquid which contains the sample. These types are often classified by separation mechanism or by the type of stationary phase. The various types are: - Partition chromatography, ion-exchange chromatograph or ion chromatography, affinity chromatography, adsorption chromatography or liquid-solid chromatography, size exclusion chromatography and chiral chromatography.

The modern HPLC apparatus have one or more glass reservoirs which contains 500mL or more solvent. The apparatus also have the provisions to remove the dissolved gases and dust from the liquid which can lead to band spreading or irreproducible flow rates. In HPLC liquid mobile phase containing the compound of interest is injected into a loop (a 20µL is commonly used). The valve switch is then rotated, which allows a sample in mobile phase to swap the sample from the loop to a column. These are packed with a suitable stationary phase in which the sample separates. The sample stream of mobile phase is delivered from a pump at a constant rate (1mL/min) and at a very high pressure to overcome the back pressure of the column (1000-2000psi). The particle size of the column material is inversely proportional to the separation efficiency. These are the separations which depends on the surface interaction and depend on the site of absorption.

High pressure is required which are directly proportional to high performance. The time required for a substance to pass through the column is retention time. Modern HPLC are equipped with valves which introduce liquids from two or more reservoirs at a ratio that can be varied continuously. The main adsorbent parameters are particle size (3-10µm), pore size (70-300AËš), particle size distribution (as narrow as possible), surface area (50-250 / g), and bonding phase density. Property of the solvent used for HPLC is purity, detector compatibility, and low viscosity, solubility of sample, chemical inertness, and reasonable price.

2.1 HPLC INSTRUMENTATION

Solvent reservoir:-

In HPLC the reservoir used is made up of glass in the form of a bottle. The simplest reservoir is 1d glass bottle fitted with a lid and a 1/8 inch diameter Teflon tube to carry the mobile phase to the pump. Teflon tubing's are used to connect to the pump. The solvent is kept under helium atmosphere. It is useful to apply vacuum for 5-10 min.

Solvent degasser:-

It is used to remove the dissolved gases and dust from the liquid. Dissolved gases can lead to irreproducible flow rates and band spreading. Air bubbles and dust interfere with the performance of the detectors. Degasser may consist of a vacuum system, distillation system and a device for heating and stirring.

Mixing Vessel:-

It is used to mix solvents in different proportions and pass through the columns.

Pumps:-

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Two types of pumps are used such as reciprocating pump and displacement pump. The pump used in HPLC must have the following properties: - pressure generation up to 6000psi, pulse free output, flow rate in range of 0.1-10mL/min, resistance to corrosion by various solvents, flow reproducibility's of 0.5% relative or better.

Reciprocating Pump:-

It consists of a small chamber in which is solvent is pumped by the motor driven system. There are two ball check valves which controls the flow of solvent into and out of a cylinder. The solvent is in direct contact with the piston due to which the pressure is transmitted to the solvent via diaphragm which in turn is hydraulically pumped by a reciprocating pump. The advantage of this pump is small internal volume (35-400µL), and high output pressure (up to 10,000psi).

Fig: Reciprocating pump

Displacement pump:-

It consists of a syringe like chamber with a plunger and is activated by screw driven mechanism. It is independent of viscosity and back pressure. They have a pulse free output. The disadvantage of this pump is that they have a limited solvent capacity (250mL).

Injectors:-

Samples in HPLC can be introduced with auto samplers and microprocessors. If the sample is liquid it can be injected directly if the sample is solid it needs to be dissolved in an appropriate solvent to avoid detector interference, column interference or loss in efficiency. The particles can be removed from the sample by centrifugation or by filtering over a 5µm filter to avoid blocking the injection devices or column. Different types of injecting devices are:-

Septum injectors:-

For injecting the sample through a rubber septum.

Rheodyne injector:-

This injector has two modes load position: - when the sample is loaded in the loop and inject mode: - when the sample is injected.

Stop flow:-

In this the sample is injected via a valve device and the flow of mobile phase is stopped.

Column:-

The types of columns mostly used in HPLC are Analytical columns and guard columns.

Analytical columns:-

Most of the lc column range from 5-25 cm long. The length is also increased by coupling two or more columns. The diameter of the column is often 3-5mm. The size of the particles in packing is 3-5µm. The analytical columns are 10-15cm long, 4.6mm in inside diameter, and packed with 5µm particles. Column of this type generates 40,000 to 70,000 plates/meter.

Guard columns:-

Guard column removes the particulate matter and contaminants from the solvent and also the sample components that bind irreversibly to the stationary phase. The guard column is introduced before the analytical column to increase the life of the analytical column. When the guard column is contaminated it is removed or discarded and is replaced by the new one.

Detectors:-

There are two basic types of detectors used in HPLC:

Bulk property detector:-Respond to a bulk property mobile phase.

Solute property detector:-Respond to the property of the solute.

Types of detectors commonly used are:-

UV detector: - It is based on the light absorption characteristics of the sample.

Fluorescence detectors: - It is used for the separation and determination of the samples that fluoresce.

Refractive index detectors: - This is a non specific low sensitivity detector.

Evaporative light scattering detector:- In this detector nitrogen is used which converts the effluent in fine mist which are then evaporated through a controlled temperature and then passes through a laser beam.

Electrochemical detector: - It is used for the detection and determination of the components in mixture containing thiols and disulfides.

2.2 Applications of HPLC

HPLC has several applications in several fields like in food analysis, environmental analysis, pharmacokinetic studies, in biological and natural compounds, in drugs etc. HPLC is used in the developing process for synthesizing chemical compounds, in control and improve process yield to evaluate product stability and monitor degradation.

In pharmaceutical it is used for so many purposes such as in identification of drug, identification and isolation of compound, in chemical separation in identification of mixture of compound and in purification of compound. HPLC is also used in the separation of many compounds and has applies to combinatorial chemistry in the field of pharmaceutical and biomedical industries. This method is also used for the separation of homologous series. This is also used for the separation of non polar species, structural isomers and compound classes such as aliphatic carbon and aliphatic alcohol. HPLC is applied in polymer analysis, analysis of surfactants, physicochemical measurements and can also be used to analyse ions of organic and inorganic species.

3. MATERIAL AND METHOD

Chromatographic system:-

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The reverse phase high performance liquid chromatography system was used to perform the chromatographic separation. The instrument used for the analysis of HPLC is Agilent technologies 1200 series. This system is isocratic which is fitted with an auto sampler. The multi detector G1365D is used with the serial no DE64255278. The column used in the system is Lichrosorb-5 RP18 with the series of 85115282. The size of the column is 150mm X 4.6mm which means the length is 150mm and the internal diameter is 4.6mm. The sample is weighed on the balance using Mettelr AC 88 delta range balance with series of 806430 and the asset number is FS13125. The syringe used is the capacity of 20µl manufactured by SGE ltd. Vacuum degasser is used which is attached to the quaternary pump. Other apparatus used are measuring cylinder, pipettes, membrane filter, spatula, beaker and volumetric flask.

The conditions maintained for this experiment are:-

Injection volume: - 20µl

Flow rate of mobile phase: - 1ml/min

Wavelength: - 360nm

RUTIN:-

Rutin is a bioflavonoid which is made up of quercetin and the disaccharide rutinose (Rhamnose and Glucose).Rutin has several health benefits such as it strengthens capillaries and useful in controlling bleeding. It has various useful properties such as antioxidant, anti-inflammatory, ant allergic, antiviral, prevention of atherogenesis, reduces excess LDL cholesterol and helps in inhibition of cancerous and pre cancerous condition.

Molecular weight: -

610.53

Molecular formula: -

C27H30O16

RUTIN

Synonyms:-

Rutoside, quercetin-3-rutinoside and sophorin.

Source:-

The main source of Rutin is fruits and vegetables. Rutin for medical use comes from various sources such as buckwheat, Japanese pagoda tree and eucalyptus macrorhyncha. Other sources are leaves of several species of eucalyptus, lime tree flowers, elder flowers, hawthorn leaves and flowers, ginkgo biloba. The Buckwheat source is rich in protein which is around 12% to 15% and contains the essential amino acid such as lysine which is 5% to 7% that are not found in many cereal crops and they also have more amount in lipids, minerals, vitamin (b) and Rutin.

Chemistry:-

Rutin consist of 50% quarcetin. Rutin is found to be a solid substance which appears pale yellow in colour and is slightly soluble in water.

Applications:-

Rutin is a bioflavonoid which reduces pain and intraocular pressure in combination with vitamin c. Rutin has ability to strengthen and regulate the permeability of parts such as the walls of blood vessels and the capillaries. It is a phenolic antioxidant that can scavenge superoxide radicals. It offers nutritional support to the circulatory system and the capillaries in the eyes. It can be used in the treatment of chronic venous insufficiency, glaucoma, hay fever, haemorrhoids, varicose veins, poor circulation, oral herpes, cirrhosis, stress, low serum calcium and for cataracts.

ACETONITRILE

Synonym: - Methyl cyanide, cyanomethane, ethylnitrile, ethanenitrile

Molecular weight

41.05

Chemical formula

CH3 CN

Appearance

Clear, colourless liquid

Odour

Ether odour

Solubility

Miscible in water

Specific gravity

0.79@15ËšC

Boiling point

81.6ËšC

Melting point

-46ËšC

Vapour density

1.4(air=1)

Acetonitrile is used in the mobile phase. It is found stable under the ordinary conditions. Acetonitrile was found incompatible with oxidising materials, chlorosulfonic acid, nitrating agents etc. It should be stored in cool dry place. It has very low viscosity. It has found wide application in pharmaceutical uses as a mobile phase. It is also used for various purposes such as in battery, in cyclic voltametry, in liquid chromatography etc.

METHANOL

Chemical formula: CH3OH

Molecular weight: 32.04

Methanol is used as a solvent for mobile phase which was found 99.9% precision pure assay by gas chromatography. The methanol used was provided by fisher scientific, UK. It has found wide applications in HPLC, UV-Vis spectroscopy. It has also been used as fuel, solvent, in waste water treatment plant etc.

PHOSPHORIC ACID

Synonym: - ortho phosphoric acid

Molecular weight

98.0

Chemical formula

H3PO4in water

Appearance

Clear

Odour

odourless

Solubility

Miscible in water

pH

1.5

Boiling point

158ËšC

Melting point

21ËšC

Phosphoric acid is used in the removal of rust, in processed food, in medical and dentistry, as electrolyte etc.

MOBILE PHASE:-

The mobile phase were prepare with the solvents such as acetonitrile, methanol, deionised water in the concentration of 200:150:650 and 1ml of phosphoric acid was added in it. The pH was adjusted to 3. The mixture was filtered and degassed by using degasser.

PREPARATION OF STANDARD STOCK SOLUTION:-

10.2mg of standard Rutin hydrate was added to 25ml ethanol in a volumetric flask. The flask was then transferred to sonicator for 15min. Then 1ml of the solution was added in 10ml of mobile phase to produce 40µg/ml. Then other concentrations were made of 60µg/ml, 80µg/ml, 100µg/ml, 120µg/ml of Rutin.

PREPARATION OF SAMPLE SOLUTION:-

Sample solution was prepared by weighing one Rutin tablet and then it is crushed in pestle and mortar. The powder was then transferred to 250ml volumetric flask. The powder was then dissolved in ethanol and then the final volume was made up to 250ml with ethanol. The mixture was then transferred to sonicator for 15min. This is done at room temperature. Then mixture was then filtered using a membrane filter and then 1ml of this extract was added to a volumetric flask of 10ml and the volume was adjusted to 10ml with the mobile phase. The final concentration was equal to 40µg/ml.

4. OPTIMIZATION OF THE HPLC METHOD

Rutin drug was injected into the HPLC system with the mobile phase concentrations of acetonitrile, methanol and phosphoric acid, the pH was maintained to 3.0 with acetic acid.

The flow rate was maintained at 1ml/min and the wavelength of 360nm gave the satisfactory results. The peaks were observed to be sharp, resolved and well defined with very minimum tailing compared to the different mobile phases.

5. EXPERIMENT

5.1 Experimental conditions:-

The mobile phase was prepared first with the concentrations of acetonitrile: methanol: water in the concentration of 200:150:650 and 1ml of phosphoric acid was added in it. The mobile phase is then connected to the system. Initially the flow rate was 0 to 0.5ml/min which gradually increased to 1ml/min when the pressure was constant. The absorbance was adjusted to the wavelength of 360nm which was required to perform the experiment and was maintained constant throughout the experiment. The system used was Agilent system in which the temperature was controlled due to the inbuilt thermostat.

5.2 Experimental procedure:-

The Agilent system was used in the experiment which is very advance and the changes can also be made in the system without any deflections in the obtained chromatograms. The HPLC system was first turned on by switching on the pumps, auto sampler and the detector. The Agilent chemstation software is used which is turned on. The experimental conditions are adjusted such as flow rate, pH, wavelength and the series of samples were prepared by the information of the sample preparation.

First after switching on the system the mobile phase was run through the system at the low rate of 1ml/min to find out if any impurity is present or not. The base line was obtained without any noise and without any interactions. When the base line was obtained the solutions concentrations prepared were introduced one by one in the system.

The concentrations prepared were allowed to run many times in the system and the time was adjusted in the system to stop the run and then proceed to next run. Every time after injecting the sample the syringe is washed continuously with the methanol which is adjusted in the system.

The experiment was repeated in the week's period to find out the stability of the drug by maintaining all the same experimental conditions.

6. VALIDATION OF HPLC

Validity of the sample is demonstrated in the laboratory using the samples or standards which are similar to the unknown sample. The preparation should follow the step by step protocol. For the preparation of the validation reports following parameters are required:-

Selectivity/Specificity

Determination

Precision and Reproducibility

Accuracy and recovery

Linearity

Range

Robustness of method

Limit of detection

Limit of quantification

Ruggedness

Selectivity/Specificity:-

The term specific refers to a method which produces a response for a single analyte only while the term selective refers to a method in which the response is for various chemical entities which can or cannot be distinguished from each other. If the response is distinguished from all other responses it is termed as selective.

Determination:-

When the analyses are performed the positive results should be compared with the negative results to confirm that the positive result is not due to the materials structurally or closely related to analyte.

Precision and Reproducibility:-

7. RESULT

The chromatograms of Rutin drug was obtained from the HPLC technique. The peak area from the chromatograms was used for the calculation of LOD, LOQ and standard deviation.

FINAL CONCENTRATION CALCULATION:-

Amount of total drug taken: - 0.0102gm

This drug is added to 25ml of ethanol

Initial concentration of drug = 0.0102/25 gm/ml

= 4.08 X 10-3 gm/ml

Concentration table:-

Sr. No

Concentration mg/ml

Concentration of drug present

in solution

1

1

4.08 X 10-5

2

1.5

6.16 X 10-5

3

2.0

8.24 X 10-5

4

2.5

10.32 X 10-5

5

3.0

12.40 X 10-5

STANDARD DEVIATION CALCULATIONS:-

Standard deviation can be calculated from the formula below. The deviations can be measured with the chromatograms obtained.

SN = Standard deviation

S = Sum of

x= value in the data set

xÌ… = mean of all values

N= number of value in the data set

Serial number

Peak area for Concentration at 1mg/100ml

Peak area for Concentration at 1mg/100ml

Peak area for Concentration at 1mg/100ml

Peak area for Concentration at 1mg/100ml

Peak area for Concentration at 1mg/100ml

1

680.488

1075.11

1393.88

1773.26

2244.16

2

681.422

1073.79

1393.11

1774.57

2247.47

3

680.953

1072.94

1392.91

1775.71

2261.54

4

681.162

1075.00

1389.16

1759.57

2219.47

5

680.46

1074.90

1387.11

1762.59

2210.43

Sum

3404.485

5371.74

6956.17

8845.7

11183.07

Mean

680.897

1074.348

1391.23

1769.14

2236.614

Standard deviation

0.1829

0.3407

1.185

1.8425

3.37

LIMIT OF DETECTION

Formula for LOD:-

LOD =3.3 X σ / Slope

LOD = 3.3 X 0.182 / 13.649

= 0.04

LIMIT OF QUANTITATION

Formula for LOQ:-

LOQ =10 X σ / Slope

LOD = 10 X 0.182 / 13.649

= 0.134

Retention time from the chromatograms:-

RETENTION TIME FOR 5 CONCENTRATION

Serial number

1

1.5

2.0

2.5

1

1.888

1.890

1.898

1.903

2

1.887

1.892

1.898

1.904

3

1.888

1.895

1.899

1.905

4

1.887

1.897

1.899

1.906

5

1.888

1.896

1.900

1.903

Mean

1.887

1.894

1.898

1.904

Standard

Deviation

0.0004

0.001

0

0.0004

8. DISCUSSION

The experiment is performed for the validation of Rutin using the RP-HPLC method. The discussions are based on the method of the validation for Rutin drug. There are various other methods which are available for the estimation of Rutin, which are available in various other methods which are available in various pharmacopoeias such as I.P, B.P and U.S.P. The other methods include the colour test for Rutin which with the heavy metals forms the coloured complexes.TLC, HPLC, UV-Vis spectrophotometric analysis is also used for the estimation of Rutin.

Rutin drug was validated in the isocratic HPLC system with the usage of Lichrosorb-5 (RP18) column. The mobile phase used was acetonitrile: methanol: water in the concentration of 200:150:650 with 1ml of phosphoric acid was added to it. The results showed the good retention time with low flow rate of about 1ml/min. The wavelength was adjusted at 360nm which gives very sharp peak.

For the experiment various concentrations were prepared and was injected into the system such as 1ml, 1.5ml, 2.0ml, 2.5ml and 3ml. The stability of the drug was determined in a week's period. The concentrations formulated were maintained in the environmental conditions for a week's time which are continuously repeated in the same way.

After the chromatograms obtained various parameters were calculated such as the standard deviation, retention time, LOD, LOQ which is calculated from the regression equation and the slope from the obtained linear graph. The chromatograms were studied for the retention time and by using their mean and standard deviation were calculated. The result formed from these was that there was no much difference found in the mean values of the 5 different concentrations.

9. CONCLUSION

The sensitive analytical RP-HPLC method was selected for the determination of Rutin. This method was selected for the estimation of Rutin drug parameters such as LOD, LOQ, linearity and retention time. The five different concentrations of 1ml, 1.5ml, 2.0ml, 2.5ml and 3ml were prepared and this experiment for a week's time was repeated with the same experimental condition. The mobile phase was prepared by using the solvents such as acetonitrile: methanol: water in the concentration of 200:150:650 with 1ml of phosphoric acid were added to it. The wavelength was maintained at 360nm. The flow rate was maintained at 1ml/min with the RP 18 column. The constant linearity was observed and the calibration co-efficient was = 0.9957. The conclusion drawn from the retention time was there was not much difference which confirmed the stability of Rutin over a week's time. After the analysis of stability study no degradation or any loss of analytes were observed over a week's time. The linearity and correlation coefficient were observed to be excellent from the rutin's calibration curve.

10. FUTURE WORK

In future the Rutin drug can be used to perform more stability studies for example more than a week's time (two weeks, three weeks, one month, two month). The various parameters such as repeatability, ruggedness can be changed and more experiments can be performed by varying these parameters. Various other changes can also be made such as in the concentration of mobile phase, in the volume of flow rate, various other columns can be used, the wavelength can be changed to find out the other properties of Rutin. The pH can also be modified.