Hplc Analysis Of Aloe Vera Tablets Biology Essay

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The project work was aimed to achieve the quantitative determination of aloin and aloe emodin in the form of tablets by employing HPLC. The method used was reverse phase high performance liquid chromatography. Calibration curve method was used for the quantification of aloin and aloe emodin. The mobile phase was the mixture of acetonitrile and deionised water in the ratio of 60:40 respectively. The mobile phase was pumped at 1.5 ml/minute and the analyte was quantified at the wavelength of 220 and 296nm. The column used for separation was kromasil 5C18. Reverse phase Isocratic run of standard aloin and standard aloe emodin was done and the peaks obtained from their analysis were used to compare the test sample peaks. Aloe vera colax tablets manufactured by Aloe pura laboratories were used as the test sample tablets which were extracted with water, methanol, acetonitrile, methanol-water and acetonitrile-water. After extraction they were subjected for isocratic run in HPLC instrument and the data obtained were compared with that of the standard.

CHAPTER 1

INTRODUCTION

1.1 Introduction to Aloe Vera

Aloes is the dried juice of the leaves of Aloe barbadensis Miller, known as Curacao aloes, or of Aloe perryi Baker known as Socotrine aloes, or of Aloe ferox Miller and hybrids of the species of Aloe africana Miller and Aloe spicata Baker, known as Cape aloes belonging to the family Liliaceae. [2,3] The synonym of aloes is Aalwee, Aalwyn, Kumari, Star cactus, Aroe, Acibar, Babosa, etc. [1]

Aloes is indigeneous to eastern and southern Africa and grown in Cape colony, Zanzibar and islands of Socotra. It is also cultivated in Caribbean islands, Europe and many parts of India, including North West Himalayan region. [2]

All the varieties of aloe are the major sources of anthraquinone glycosides. The principal active composition of aloe is aloin, which is a mixture of glucosides, among which barbaloin is the chief constituent. It is chemically aloe-emodin anthrone C-10 glucoside and is water-soluble. [2]

Barbaloin is a C- glycoside and it is not hydrolysed by heating with dilute acids or alkalies. Ferric chloride decomposes barbaloin by oxidative hydrolysis into aloe-emodin-anthrone, little aloe-emodin and glucose. [2]

Along with barbaloin, aloes also contains isobarbaloin, b-barbaloin, aloe-emodin and resins. The drug also contains aloetic acid, homonataloin, aloesone, chrysophanic acid, chrysamminic acid, galactouronic acid, choline, choline salicylate, saponins, mucopolysaccharides, glucosamines, hexuronic acid, coniferyl alcohol, etc. [2]

The amount of barbaloin in different commercial varieties varies to a large extent. Curacao aloes contain about 22 percent of barbaloin. Indian variety, generally Aloe vera contain very less quantity (3.5 to 4 percent). Curacao aloes contains two and half times quantity of aloe-emodin , compared to Cape-aloe-emodin. [2]

The resin of aloe principally contains Aloesin. It is a type of C- glucosyl chromome. Aloesin is also responsible for purgative action of aloes. [2]

Fig. 1 Fig. 2

Aloin [5] Aloe emodin [6]

1.2 Uses of Aloe Vera:

Aloes is used as purgative. Its effect is mainly on colon. It has a stronger purgative action in the series of all crude drugs with anthracene glycosidal content. To counter effect the gripping action, it is given along with carminatives. [2]

It facilitates the healing of any kind of skin wound, burn, or scald - even speeding recovery time after surgery. [4]

It is applied topically in acne, sunburn, frostbite (it appears to prevent decreased blood flow), shingles, screening out x-ray radiation, psoriasis, preventing scarring, rosacea, warts, wrinkles from aging, and eczema. [2, 4]

It also seems to help prevent opportunistic infections in cases of HIV and AIDS due to its immune system stimulant properties. [4]

It appears to be of help in cancer patients (including lung cancer) by cativating white blood cells and promoting growth of non- cancerous cells. [4]

Aloe also appears to work on heartburn, arthritis, and rheumatism pain and asthma. [2, 4]

It also lowers the blood sugar levels in diabetics. [2, 4]

Other situations in which it appears to work when taken internally inclue congestion, internal worms, indigestion, stomach ulcers, colitis, hemorrhoids, liver problems such as cirrhosis and hepatitis, kidney infections, urinary tract infections, prostate problems, and as a general detoxifier. [2, 4]

CHAPTER 2

HPLC

2.1 HPLC: Introduction and Instrumentation

The technique of high performance liquid chromatography is so called because of its improved performance when compared to classical column chromatography. It is also called as high-pressure liquid chromatography since pressure is used when compared to classical column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressure of up to 400 atmospheres. For the separation, identification and quantification of compounds, this method is frequently used in biochemistry and analytical chemistry. [11, 12]

The development of HPLC from classical column chromatography can be attributed to the development of smaller particle sizes. Smaller particle size is important since they offer more surface area over the conventional larger sizes. [7]

1960's - 40 to 60m

1970's - 10 to 20m

1980's - 5 to 10m

1990's - 1 to 3m

A porous particle of 5m offers a surface area of 100-860 sq.metres/gram with an average of 400 sq.metres/gram. These offer very high plate counts upto 100,000/metre.

Table 1: Comparison of classical column chromatography with HPLC [7]

Parameter

Classical column chromatography

HPLC

Stationary phase - particle size

Large

60-200m

Small

3-20m

Column size

Length x int. diameter

Large

0.5-5m x 0.5-5cm i.d.

Small

5-50cm x 1-10mm i.d.

Column material

Glass

Mostly metal

Column packing pressure

Slurry packed at low pressure - often gravity

Slurry packed at high pressure >5000 psi

Operating pressure

Low (<20 psi)

High (500 - 3000 psi)

Flow rates

Low to very low

Medium to high

(Often >3ml/min)

Sample load

Low to medium (g/mg)

Low to very low (mg)

Parameter

Classical column chromatography

HPLC

Cost

Low

High

Detector flow cell volume

Large - 300 to 1000ml

Low - 2 to 10ml

Column efficiency

i.e. Resolving power

(Low) <500

Theoretical plates per meter

(High) often >100,000

Plates per meter

Types of stationary phases available

Limited range

Wide range

Scale of operation

Preparative scale

Analytical and preparative scale

2.2 Types of HPLC techniques [7, 9, 10, 11, 12]

Based on Modes of Chromatography

There are two modes viz. Normal phase mode and Reverse phase mode. These modes are based on the polarity of stationary phase and mobile phase. Before explaining the modes, it is important to know the interactions, which occur between solute, stationary and mobile phase.

Polar - Polar - interaction or affinity is more

Nonpolar - Nonpolar - interaction or affinity is more

Polar - Nonpolar - interaction or affinity is less

Normal phase mode: In normal phase mode, the stationary phase (eg. Silica gel) is polar in nature and the mobile phase is non-polar. In this technique, non-polar compounds travel faster and are eluted first. This is because of less affinity between solute and stationary phase. Polar compounds are retained for longer time in the column because of more affinity towards stationary phase and take more time to be eluted from the column. This is not advantageous in pharmaceutical applications since most of the drug molecules are polar in nature and takes longer time to be eluted and detected. Hence this technique is not widely used in pharmacy.

Reverse phase mode: In reverse phase technique, a non-polar stationary phase is used. The mobile phase is polar in nature. Hence polar components get eluted first and non-polar compounds are retained for a longer time. Since most of the drugs and pharmaceuticals are polar in nature, they are not retained for a longer time and eluted faster, which is advantageous. Different columns used are ODS (Octadecyl silane) or C18, C8, C4, etc.

Common reverse phase solvents are methanol, acetonitrile, tetrahydrofuran

and water.

Based on principle of separation

Adsorption chromatography

Ion exchange chromatography

Ion pair chromatography

Size exclusion or Gel permeation chromatography

Affinity chromatography

Chiral phase chromatography

Each of the above technique is described in brief as follows:

Adsorption chromatography:

The principle of separation is adsorption. Separation of components takes place because of the difference in affinity of compounds towards stationary phase. This principle is seen in normal phase as well as reverse phase mode, where adsorption takes place.

Ion exchange chromatography:

The principle of separation is ion exchange, which is reversible exchange of functional groups. In ion exchange chromatography, an ion exchange resin is used to separate a mixture of similar charged ions. For cations, a cation exchange resin is used. For anions, an anion exchange resin is used.

Ion pair chromatography:

In ion pair chromatography, a reverse phase column is converted temporarily into ion exchange column by using ion pairing agents like pentane or hexane or heptane or octane sulphonic acid sodium salt, trtramethyl or tetraethyl ammonium hydroxide, etc.

Size exclusion or gel permeation chromatography:

In this type of chromatography, a mixture of components with different molecular sizes is separated by using gels. The gel used acts as molecular sieve and hence a mixture of substances with different molecular sizes is separated. Soft gels like agarose , dextran or polyacrylamide are used. Semi rigid gels like polystyrene, alkyl dextran in non-aqueous medium are also used. The mechanism of separation is by steric and diffusion effects.

Affinity chromatography:

Affinity chromatography uses the affinity of the sample with specific stationary phases. This technique is mostly used in the field of Biotechnology, Microbiology, Biochemistry, etc.

Chiral phase chromatography:

Separation of optical isomers can be done by using chiral stationary phases. Different principles operate for different types of stationary phases and for different samples. The stationary phases used for this type of chromatography are mostly chemically bonded silica gel.

Based on elution technique

1. Isocratic separation:

In this technique, the same mobile phase combination is used throughout the process of separation. The same polarity or elution strength is maintained throughout the process. In this technique, the peak width increases with retention time linearly according to the equation for N, the number of theoretical plates.

Gradient separation:

In this technique, a mobile phase combination of lower polarity or elution strength is used followed by gradually increasing the polarity or elution strength. One example is a gradient starting at 10% acetonitrile and ending at 90% acetonitrile after 25 minutes. The two components of the mobile phase are termed as "A" and "B". Where A is the weak solvent and B is the strong solvent. Weak solvent allows the solute to elute slowly while strong solvent rapidly elutes the solutes from the column. A is usually water where as B is an organic solvent which is miscible with water such as acetonitrile, methanol, THF or isopropanol.

Based on scale of operation

1. Analytical HPLC:

Where only analysis of the samples are done. Recovery of the samples for reusing is normally not done, since the sample used is low. Eg. mg quantities.

2. Preparative HPLC:

Where the individual fractions of pure compounds can be collected using fraction collector. The collected samples are reused eg. Separation of few grams of mixtures by HPLC.

Based on type on analysis

1. Qualitative analysis:

Which is used to identify the compound, detect the presence of impurities, to find out the number of components, etc. This is done by using retention time values.

2. Quantitative analysis:

Which is done to determine the quantity of the individual or several components in a mixture. This is done by comparing the peak area of the standard and sample.

2.3 Principle of separation in HPLC: [7, 9]

The principle of separation in normal phase and reverse phase mode is adsorption. When a mixture of components is introduced in to a HPLC column, they travel according to their relative affinities towards the stationary phase. The component, which has more affinity towards the adsorbant, travels slower. The component, which has less affinity towards the stationary phase, travels faster. Since no two components have the same affinity towards the stationary phase, the components are separated.

2.4 Instrumental Requirements [7, 9, 10, 12]

Pumps - solvent delivery system

Mixing unit, gradient controller and solvent degassing

Injector - Manual or auto injectors

Guard columns

Detectors

Recorders and integrators

Fig. 3 The schematic diagram of HPLC [13]

1. Pump - Solvent delivery system

The solvents or mobile phases used must be passed through the column at high pressure at about 1000 to 3000 psi. This is because as the particle size of stationary phase is few m (5 10m), the resistance to the flow of solvent is high. Hence such high pressure is recommended. There are different types of pumps available. They are mechanical pumps and pneumatic pumps. A mechanical pump operates with constant flow rate and uses a sapphire piston. This type of pump is used in analytical scale. Pneumatic pumps operate with constant pressure and use highly compressed gas. The solvents used must be of high purity, preferably HPLC grade and filtered through 0.45m filter.

Check valves:

These are present to control the flow rate of solvent and back pressure.

Pulse dampners:

These are used to dampen the pulses observed from the wavy baseline caused by the pumps.

2. Mixing unit, gradient controller and solvent degassing

Mixing unit is used to mix solvents in different proportions and pass through the column. There are two types of mixing units. They are low pressure mixing chamber, which uses helium for degassing solvents. High pressure mixing chamber does not require helium for degassing solvents. Mixing of solvents is done either with a static mixer, which is packed with beads, or dynamic mixer, which uses magnetic stirrer and operates under high pressure.

Gradient controller

In an isocratic separation, mobile phase is prepared by using pure solvent or mixture of solvents, i.e. solvent of same eluting power or polarity is used. But in gradient elution technique, the polarity of the solvent is gradually increased and hence the solvent composition has to be changed. Hence a gradient controller is used when two or more solvent pumps are used for such separations.

Solvent degassing

Several gases are soluble in organic solvents. When solvents are pumped under high pressure, gas bubbles are formed which will interfere with the separation process, steady baseline and the shape of the peak. Hence degassing of the solvent is important. This can be done by using any one of the following technique.

Vacuum filtration - which can remove all air bubbles. But it is not always reliable and complete.

Helium purging - i.e. by passing helium through the solvent. This is very effective but helium is expensive.

Ultrasonication - by using ultrasonicator, which converts ultra high frequency to mechanical vibrations. This causes the removal of air bubbles.

3. Injector - Manual or auto injectors

Several devices are available either for manual or auto injection of the sample. Different devices are:

Septum injectors - for injecting the sample through a rubber septum. This is not common, since the septum has to withstand high pressure.

Stop flow (on line) - in which the flow of mobile phase is stopped for a while and the sample is injected through a valve device.

Rheodyne injector (Loop valve type) - It is the most popular injector. This has a fixed volume loop like 20ml or 50ml or more. Injector has two modes, i.e. load position when the sample is loaded in the loop and inject mode, when the sample is injected.

4. Guard column

Guard column has very small quantity of adsorbent and improves the life of the analytical column. It also acts as a prefilter to remove particulate matter, if any, and other material. Guard column has the same material as that of analytical column. Guard column does not contribute to any separation.

5. Analytical columns

Analytical column is the most important part of HPLC technique, which decides the efficiency of separation. There are several stationary phases available depending upon the technique or mode of separation used.

Column material: The columns are made up of stainless steel, glass, polyethylene and PEEK (Poly ether ether ketone). Most widely used are stainless steel, which can withstand high pressure. Latest ones are PEEK columns.

Column length: Varies from 5cm to 30cm

Column diameter: Ranges from 2mm to 50mm

Particle size: From 1m to 20m

Particle nature: Spherical, uniform sized, porous materials are used.

Surface area: 1 gram of stationary phase provides surface area ranging from 100 - 860 sq.m with an average of 400 sq.m.

Functional group: the functional group present in stationary phase depends on the type of chromatographic separation. In normal phase mode it contains the silanol groups (hydroxy group). In reverse phase mode it contains the following groups:

C18 - Octa Decyl Silane (ODS) column

C8 - Octyl column

C4 - Butyl column

CN - Nitrile column

NH2 - Amino column

For other modes of chromatography, ion exchange columns, gel columns, chiral columns, affinity chromatographic columns, etc. are available.

6. Detectors [7,9,10]

Detectors used depend upon the property of the compounds to be separated. Different detectors available are

UV detector: This detector is based upon the light absorption characteristics of the sample. Two types of this detector are available. One is the fixed wavelength detector, which operates at 254nm where most drug compounds absorb. The other is the variable wavelength detector, which can be operated from 190nm to 600nm.

Refractive index detector: This is a non-specific or universal detector. This is not much used for analytical applications because of low sensitivity and specificity.

Flourimetric detector: This detector is based on the fluorescent radiation emitted by some class of compounds. The exitation wavelength and emission wavelength can be selected for each compound. This detector has more specificity and sensitivity. The disadvantage is that some compounds are not fluorescent.

Conductivity detector: Based upon electrical conductivity, the response is recorded. This detector is used when the sample has conducting ions like anions and cations.

Amperometric detector: This detector is based on the reduction or oxidation of the compounds when a potential is applied. The diffusion current recorded is proportional to the concentration of the compound eluted. This is applicable when compounds have functional groups, which can be either oxidised or reduced. This is a highly sensitive detector.

Photodiode array detector (PDA detector): This is a recent one, which is similar to UV detector, which operates from 190 - 600nm. Radiations of all wavelengths fall on the detector simultaneously. The resulting spectrum is a 3-D or three-dimensional plot of Response Vs Time Vs Wavelength. The advantage is that the wavelength need not be selected, but the detector detects the responses of all the compounds.

7. Recorders and integrators

Recorders: They are used to record the responses obtained from detectors after amplification, if necessary. They record the baseline and all the peaks obtained, with respect to time. Retention time for all the peaks can be found out from such recordings, but the area of individual peaks cannot be known.

Integrators: Integrators are improved version of recorders with some data processing capabilities. They can record the individual peaks with retention time, height, and width of peaks, peak area, percentage of area, etc. Integrators provide more information on peaks than recorders. Now a days computers and printers are used for recording and processing the obtained data and for controlling several operations.

2.5 Parameters used in HPLC [7, 9, 10]

Retention time (Rt):

Retention time is the difference in the time between the point of injection and appearance of peak maxima. Retention time is the time required for 50% of a component to be eluted from a column. Retention time is measured in minutes or seconds. Retention time is also proportional to the distance moved on a chart paper, which can be measured in cm or mm.

Retention volume (Vr):

Retention volume is the volume of mobile phase required to elute 50% of the component from the column. It is the product of retention time and flow rate.

Retention volume = Retention time x flow rate

Separation factor (S):

Separation factor is the ratio of partition co-efficient of the two components to be separated. It can be expressed and determined by using the following equation:

S = Kb/ Ka = K'a/ K'b = (tb - t0)/ (ta - t0)

Where,

t0 = Retention time of unretained substance

Kb, Ka= Partition coefficients of b and a

tb, ta = Retention time of substance b and a

S = depends on liquid phase, column temperature

If there is more difference in partition coefficient between two compounds, the peaks are far apart and the separation factor is more. If the partition coefficients of two compounds are similar, then the peaks are closer and the separation factor is less.

Resolution:

Resolution is a measure of the extent of separation of two components and the baseline separation achieved. It can be determined by using the following formula:

Rs = 2 (Rt1 - Rt2)/ (W1 +W2)

Theoretical plate (Plate theory):

A theoretical plate is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium. A theoretical plate can also be called as a functional unit of the column.

HETP - Height Equivalent to a Theoritical Plate [18, 7]

A theoretical plate can be of any height, which decides the efficiency of separation. If HETP is less, the column is more efficient. If HETP is more, the column is less efficient. HETP can be calculated by using the following formula:

HETP = length of the column/ number of theoretical plates

HETP is given by Van Deemter equation

HETP = A + (B/u ) + Cu

Where,

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the

column. This is unaffected by mobile phase velocity or flow rate. This can be

minimised by uniformity in packing.

B = Longitudinal diffusion term or molecular diffusion which depends on flow rate.

C = Effect of mass transfer which depends on flow rate.

u = Flow rate or velocity of the mobile phase.

A column is efficient only when HETP is minimum. Hence an ideal flow rate corresponding to the minimum value of HETP is used.

Efficiency (No. of theoretical plates):

The number of theoretical plates expresses efficiency of a column. It can be determined by using the formula:

n = 16 Rt²/w²

Where,

n = no. of theoretical plates

Rt = retention time

w = peak width at base

Rt and w are measured in common units (mm or cm or minutes or seconds) and are proportional to the distances marked on chart paper.

If the number of theoretical plates is high, the column is said to be highly efficient. If the number of theoretical plates is low, the column is said to be less efficient. For gas chromatographic columns, a value of 600/ metre is sufficient. But in HPLC, high values like 40,000 to 70,000/ metre are recommended.

Asymmetry factor:

A chromatographic peak should be symmetrical about its centre and said to follow Gaussian distribution. In such cases, the peak will be like an isosceles triangle. But in practice, due to some factors, the peak is not symmetrical and shows tailing or fronting.

Fronting is due to saturation of stationary phase and can be avoided by using less quantity of sample.

Tailing is due to more active adsorption sites and can be eliminated by support pre-treatment, more polar mobile phased increasing the amount of liquid phase.

Asymmetry factor (0.95 to 1.05) can be calculated by using the formula:

AF = b/a (b and a calculated at 5% or 10% of the peak height)

2.6 Applications of HPLC

HPLC is being more widely used in several fields. Apart from its use in Pharmaceutical field, it is used in Chemical and Petrochemical industry, Environmental applications, Forensic applications, Biochemical separations, Biotechnology, Food analysis, etc. In fact there is no field where HPLC is not being used. It is a versatile and sensitive technique, which can be used in several ways. Some of them are listed below:

Qualitative analysis: It is nothing but identification of compound. This is done by comparing the retention time of the sample as well as the standard. Under identical conditions, the retention time of the standard and the sample are same. If there is a deviation, then they are not the same compound.

Checking the purity of the compound: By comparing the chromatogram of the standard and that of the sample, the purity of the compound can be inferred. If additional peaks are obtained, impurities are present and hence the compound is not pure. From the percentage area of the peaks obtained, the percentage purity can also be known.

Presence of impurities: This can be seen by the presence of additional peaks when compared with a reference standard or reference material. The percentage of impurities may also be calculated from peak areas.

Quantitative analysis: The quantity of a component can be determined by several methods like

a. Direct comparison method

By injecting a sample and standard separately and comparing their peak areas, the quantity of the sample can be determined.

Area of the peak = peak height x width of peak at the half height

A1/ A2 = a (W1/ W2)

Where,

A1 and A2 are peak area of sample and standard

W1 and W2 are weight or concentration of sample and standard

a is the response factor

b. Calibration curve method:

In calibration curve method, series of standards are used to determine their peak areas. A calibration curve of peak area Vs concentration of the drug is plotted. From the peak area of the unknown sample, by intrapolation, the concentration of the sample can be determined. This method has the advantage that errors, if any, are minimised.

Internal standard method:

In this method, a compound with similar retention characteristics is used. A known concentration of the internal standard is added to the sample solution whose concentration is not known. The chromatogram is recorded and their peak areas are determined. By using formula, the concentration of unknown solution is determined.

Multicomponent analysis or Determination of mixture of drugs: Similar to the quantification of a single drug, multicomponent analysis can be done easily. The quantity of each component is determined by using any one of the above methods. Marketed formulations, which contain several drugs, can be determined quantitatively for each component.

Isolation and identification of drugs or metabolites in urine, plasma, serum, etc. can be carried out.

Isolation and identification of mixture of components of natural or synthetic origin.

Biopharmaceutical and Pharmacokinetic studies.

Stability studies.

Purification of some compounds of natural or synthetic origin on preparative scale.

2.7 Limitations: [7, 10]

The limitations of HPLC are that drugs have to be extracted from their formulations prior to analysis and large amounts of organic solvent waste are generated which are expensive to dispose off.

CHAPTER 3

Experimental Selection

3.1 Aim of Project:

The aim of this project was to carry out the quantitative determination of the active pharmaceutical ingredient aloin and aloe-emodin in the given Aloe Vera Colax tablets, manufactured by Aloe Pura laboratories and to compare the results with the given standard aloin and aloe-emodin. The technique used for analysis was reverse phase High Performance Liquid Chromatography method. The analysis was performed using standard calibration curve generated at 220 and 296nm wavelength.

3.2 Chromatographic equipment and conditions:

All the chromatographic equipments and conditions, which were used to perform HPLC in a laboratory environment under simulated GLP compliance conditions, are listed below.

3.2.1 HPLC system 5 (used for isocratic elution):

This system is manufactured by Agilent technologies 1200 series, whose model number is G1310A and the serial number is DE 62956545

3.2.2 Software used:

The software used was Microsoft windows XP, Pentium D whose product number is G 2175 BA, revision code is B. 03. 01 and its registration number is CL1CE8DB0F

3.2.3 Column used:

The column used was Kromasil 5C18 whose test number is 9203- 10344

3.2.4 Pipette used:

The pipette used was Volac ultra (made in U.K.), S. No. 29186, Model: R680/ F, 0-1000 mL and Volac ultra (made in U.K.), S.No. 29185, Model: R680/ F, 500-5000 mL.

3.2.5 Analytical Balance:

Mettler balance AC 88 was used to weigh the sample drug whose Biomax number is 57729, EA number is 12874, serial number is 806430 and asset code is 9995773/999.

3.2.6 Glassware:

Volumetric flasks (10 ml, 20 ml), beaker 100 ml, measuring cylinders (500 ml, 50 ml, 20 ml and 10 ml) and funnel were used.

3.2.7 Filter Paper:

Millipore filter paper manufactured by Fischer scientific, type HV and size 0.45 mm was used.

3.2.8 Millipore filter holder:

Part 4, made of pyrex brand glassware, approx. volume 250 ml was used.

3.2.9 Ultrasonicator:

The ultrasonicator used was made in USA whose type number is 6440AE, part number is CDC0010 and serial number is T42327.

3.3 Chemicals:

All the chemicals and solvents used were of HPLC grade.

3.3.1 Methanol:

HPLC grade methanol manufactured by Fischer scientific having the batch number 0801868 was used.

3.3.2 Acetonitrile:

HPLC grade acetonitrile manufactured by Fischer scientific having the batch number 0809411 was used.

3.3.3 Water: Deionised water was used to make the mobile phase.

3.4 Mobile phase:

The mobile phase used was acetonitrile and deionised water in the ratio of Acetonitrile : water = 60:40.

3.5 Test sample Tablets:

Aloe Vera Colax, Colon cleanse tablets, manufactured by Aloe Pura Laboraories, Yorkshire, U.K. having the batch number 2078 and whose expiry date was 07/2011 was used for analysis.

3.6 Preparation of mobile phase:

1000 ml of mobile phase was prepared in the ratio of 60:40 i.e for every 60 ml of acetonitrile, 40 ml of deionised water was taken. The mobile phase was mixed thoroughly and then it was degassed using a degassing unit so that the mobile phase was free from the air bubbles.

3.7 Preparation of standard stock solution and its dilution:

3.7.1 Aloe emodin:

3.5 mg of accurately weighed standard aloe emodin was taken and diluted with 1.75 ml of methanol to get a concentration of 2mg/ml. This stock solution was further diluted to make up a series of 0.2mg/ml, 0.1mg/ml and 0.05mg/ml of aloe emodin.

Table2: Dilution of standard stock solutions

Standard stock solution

Concentration

1 ml of 2 mg/ml + 10 ml methanol

0.2 mg/ml

1 ml of 0.2 mg/ml + 1 ml methanol

0.1 mg/ml

0.5 ml of 0.2 mg/ml + 1.5 ml methanol

0.05 mg/ml

3.7.2 Aloin:

6.5 mg of accurately weighed standard aloin was taken and diluted with 3.25 ml of methanol to get a concentration of 2mg/ml. This stock solution was further diluted to make up a series of 0.2mg/ml, 0.1mg/ml and 0.05mg/ml of aloin.

Table3: Dilution of standard stock solutions

Standard stock solution

Concentration

1 ml of 2 mg/ml + 10 ml of methanol

0.2 mg/ml

1ml of 0.2 mg/ml + 1 ml methanol

0.1 mg/ml

0.5 ml of 0.2 mg/ml + 1.5 ml methanol

0.05 mg/ml

3.8 Method for extraction of Aloe sample:

10 tablets were weighed accurately and crushed into fine powder using mortar and pestle. 5 volumetric flasks of 25 ml capacity were labelled properly and set in a row. In each volumetric flask, 1 gm of the powdered tablet was added. The volume was made up to the mark with water, methanol, acetonitrile, methanol-water (12.5 : 12.5 ml) and acetonitrile-water (12.5 : 12.5 ml) respectively. The volumetric flasks were kept in a sonic bath for 30 minutes, for the powder to dissolve properly. After cooling they were filtered using filter paper and further used for analysis.

3.9 Isocratic run of std. Aloin, std. Aloe emodin and test sample:

2 ml of each concentration prepared for aloin and aloe emodin were taken in a 2ml glass vial and their isocratic run was recorded in HPLC instrumentation. Duplicate reading of each concentration was recorded for accuracy and calibration curve. Similarly isocratic run for each test sample was also recorded. The injection volume was 20ml in each case and the flow rate was 1.5 ml/minute.

CHAPTER 4

Results and discussion:

The results obtained from the HPLC analysis of aloin and aloe emodin are recorded and statistically evaluated in detail. The graph was plotted in Microsoft excel for accuracy.

Table4: HPLC results of std. Aloin at 220nm

Concentration

Sample no.

Peak area

Average peak area

0.05 mg/ml

1

2

1368.55

1377.56

1373.05

0.1 mg/ml

1

2

1816.66

3228.99

2522.83

0.2 mg/ml

1

2

3702.96

5402.29

4552.63

Fig. 4 Calibration graph for std. Aloin

The graph is plotted in microsoft excel for accuracy by taking concentration on x-axis and the average peak area on y-axis and it was observed for linearity or for a best fit line. This graph was developed in a precise and accurate manner with standard equation of line y = mx + c and a regression analysis of 0.9998 which is the most recommended one for plotting a calibration curve. In the equation y = mx + c, m is the slope of the curve and c is the intercept of the curve.

The graph shown in Fig 4 is obtained from the data given in Table 4. The graph is linear with a correlation coefficient value of R² = 0.999, goodness of fit of linear regression was judged from the value of R². R² is the value between 0 and 1. If R² is near to 0, that means no linear relationship. If R² is near to 1, a linear relationship does exist.

Table 5: HPLC results for standard aloe emodin at 220nm

Concentration

Sample No.

Peak area

Average peak area

0.05 mg/ml

1

2

4261.19

4259.90

4260.54

0.1 mg/ml

1

2

8600.76

8617.05

8608.90

0.2 mg/ml

1

2

12534.04

12522.74

12528.39

Fig 5: Calibration graph for std. Aloe-emodin

The graph shown in fig. 5 is obtained from the data given in table 5. Since R² = 0.9523 which means that R² is near to one and hence a linear relationship does exist.

CHAPTER 5

Conclusion:

HPLC is a powerful tool for analysis. In this project HPLC technique was used because it gives better resolution. For natural product like Aloe Vera this is the best technique as it gives better separation and analysis of the components present in it which would be difficult to separate by other techniques. HPLC is a rapid and simple method for analysis and also we get better results when compared to other techniques.

In this project 220 and 296nm wavelengths were used, however 220nm gave better result when compared to 296nm. The peak for standard aloin was generated at 2.6 minutes interval and the peak for standard Aloe-emodin was generated at an interval of 4.1 minutes. Both standard aloin and aloe-emodin gave good result. However when the test sample tablets were extracted and analysed, neither aloin nor aloe-emodin could be detected in the sample tablets. This was confirmed because no peaks were observed at the same interval as that of standard aloin or standard aloe-emodin.

There were many other peaks generated with the sample tablet extraction. But those peaks are the result of other constituents present in aloe vera.

CHAPTER 6

FUTURE WORK

In this project neither aloin nor aloe emodin could be detected by reverse phase isocratic HPLC method. Since the concentration of aloin or aloe emodin is also not known in the test sample tablets, it would be better if the test sample with known concentrations of aloin and aloe emodin are used.

Various literature can be reviewed in order to find out some other methods for the extraction of the test sample tablets. Increasing the polarity of the mobile phase can also give better result. Different wavelengths should also be tried to get reliable result.

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