Hplc Analysis Of Aloe Vera Tablets Biology Essay

Hplc Analysis Of Aloe Vera Tablets Biology EssayThe project take a leak was aimed to achieve the quantitative determination of aloin and aloe emodin in the spirt of tablets by employing HPLC. The method used was reverse leg high performance bland chromatography. Calib proportionalityn curve method was used for the quantification of aloin and aloe emodin. The ready grade was the change of acetonitrile and deionised water in the ratio of 6040 respectively. The lively degree was pumped at 1.5 ml/minute and the analyte was quantified at the wavelength of 220 and 296nm. The editorial used for insularity was kromasil 5C18. Reverse phase Isocratic run of standard aloin and standard aloe emodin was done and the broadsheets obtained from their abstract were used to compargon the test warning bloom of youths. Aloe vera colax tablets manufactured by Aloe pura laboratories were used as the test take tablets which were extracted with water, methanol, acetonitrile, methanol-water a nd acetonitrile-water. After extraction they were subjected for isocratic run in HPLC instrument and the data obtained were comp bed with that of the standard.CHAPTER 1INTRODUCTION1.1 Introduction to Aloe VeraAloes is the dried juice of the leaves of Aloe barbadensis Miller, cognise as Curacao aloes, or of Aloe perryi Baker known as Socotrine aloes, or of Aloe ferox Miller and hybrids of the species of Aloe afri seata Miller and Aloe spicata Baker, known as Cape aloes belonging to the family Liliaceae. 2,3 The equivalent word of aloes is Aalwee, Aalwyn, Kumari, Star cactus, Aroe, Acibar, Babosa, etc. 1Aloes is indigeneous to eastern and s give awayhern Africa and grown in Cape colony, Zanzibar and islands of Socotra. It is as well as cultivated in Caribbean islands, Europe and m each part of India, including North West Himalayan region. 2All 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. 2Barbaloin is a C- glycoside and it is non hydrolysed by heating with dilute acids or alkalies. Ferric chloride decomposes barbaloin by oxidative hydrolysis into aloe-emodin-anthrone, little aloe-emodin and glucose. 2Along with barbaloin, aloes likewise 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. 2The tot up 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 actually little quantity (3.5 to 4 percent). Curacao aloes contains two and half eras quantity of aloe-emodin , co mpared to Cape-aloe-emodin. 2The resin of aloe principally contains Aloesin. It is a type of C- glucosyl chromome. Aloesin is also creditworthy for purgative action of aloes. 2Fig. 1 Fig. 2Aloin 5 Aloe emodin 61.2 Uses of Aloe VeraAloes is used as purgative. Its effect is mainly on colon. It has a concentrateder purgative action in the serial of all crude drugs with anthracene glycosidal content. To counter effect the gripping action, it is given along with carminatives. 2It facilitates the healing of any kind of skin wound, burn, or scald even hotfoot recovery snip after surgery. 4It is applied topically in acne, sunburn, frostbite (it appears to prevent decreased blood menses), shingles, screening out x-ray radiation, psoriasis, preventing scarring, rosacea, warts, wrinkles from aging, and eczema. 2, 4It also seems to help prevent opportunistic infections in cases of HIV and AIDS due to its immune system stimulant properties. 4It appears to be of help in crappercer patients (including lung green goddesscer) by cativating pureness blood kiosks and promoting growth of non- cancerous cells. 4Aloe also appears to work on heartburn, arthritis, and rheumatism pain and asthma. 2, 4It also lowers the blood sugar levels in diabetics. 2, 4Other situations in which it appears to work when taken intragrouply 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, 4CHAPTER 2HPLC2.1 HPLC Introduction and InstrumentationThe technique of high performance liquid chromatography is so called because of its improved performance when compared to classical newspaper column chromatography. It is also called as high-pressure liquid chromatography since pressure is used when compared to classical column chromatography. Instead of a dissolving agent being allowed to drip finished a column to a low er place gravity, it is forced finished under high pressure of up to 400 atmospheres. For the separation, identification and quantification of compounds, this method is frequently used in biochemistry and analytic chemistry. 11, 12The development of HPLC from classical column chromatography can be attributed to the development of smaller particle coats. Smaller particle size is important since they offer much surface area over the conventional large sizes. 71960s 40 to 60m1970s 10 to 20m1980s 5 to 10m1990s 1 to 3mA porous particle of 5m offers a surface area of 100-860 sq.metres/ gramme with an intermediate of 400 sq.metres/gram. These offer very high plate counts upto 100,000/metre.Table 1 Comparison of classical column chromatography with HPLC 7ParameterClassical column chromatographyHPLC nonmoving phase particle sizeLarge60-200mSmall3-20m pillar sizeLength x int. diameterLarge0.5-5m x 0.5-5cm i.d.Small5-50cm x 1-10mm i.d.Column materialGlassMostly metalColumn packing p ressureSlurry packed at low pressure often gravitySlurry packed at high pressure 5000 psiOperating pressureLow (High (500 3000 psi)Flow ratesLow to very lowMedium to high(Often 3ml/min) savour loadLow to medium (g/mg)Low to very low (mg)ParameterClassical column chromatographyHPLCCostLowHighDetector flow cell volumeLarge 300 to 1000mlLow 2 to 10mlColumn efficiencyi.e. Resolving power(Low) Theoretical plates per meter(High) often 100,000Plates per meterTypes of stationary phases procurableLimited rangeWide rangeScale of operationPreparative scaleAnalytical and preparative scale2.2 Types of HPLC techniques 7, 9, 10, 11, 12Based on Modes of ChromatographyThere are two modes viz. Normal phase mode and Reverse phase mode. These modes are comprise on the sign of the zodiac of stationary phase and mobile phase. in the beginning explaining the modes, it is important to know the interactions, which occur between solute, stationary and mobile phase.Polar Polar interaction or affini ty is moreNonpolar Nonpolar interaction or affinity is morePolar Nonpolar interaction or affinity is lessNormal phase mode In modal(prenominal) 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. thusly 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, tetrahydrofuranand water.Based on principle of separationAdsorption chromatographyIon substitution chromatographyIon p breeze chromatographySize exclusion or Gel permeation chromatographyAffinity chromatographyChiral phase chromatography each(prenominal) of the above technique is described in brief as followsAdsorption chromatographyThe principle of separation is adsorption. withdrawal 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 vary chromatographyThe principle of separation is ion flip, which is reversible exchange of functional groups. In ion exchange chromat ography, 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 chromatographyIn ion pair chromatography, a reverse phase column is converted temporarily into ion exchange column by utilise ion mating agents like pentane or hexane or heptane or octane sulphonic acid sodium salt, trtramethyl or tetraethyl ammonium hydroxide, etc.Size exclusion or gel permeation chromatographyIn this type of chromatography, a mixture of components with different molecular sizes is separated by exploitation 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 chromatographyAffinity chromatography us es the affinity of the prototype with specific stationary phases. This technique is mostly used in the line of crossings of Biotechnology, Microbiology, Biochemistry, etc.Chiral phase chromatographySeparation 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 technique1. Isocratic separationIn 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 largeness increases with retention time linearly according to the equation for N, the phone rate of theoretical plates.Gradient separationIn this technique, a mobile phase combination of lower polarity or elution strength is used followed by gradually increasing the po larity or elution strength. One example is a gradient starting at 10% acetonitrile and shutdown 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 operation1. Analytical HPLCWhere only analysis of the samples are done. Recovery of the samples for reusing is ordinarily not done, since the sample used is low. Eg. mg quantities.2. Preparative HPLCWhere the individual(a) 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 analysis1. Qualitative analysisWhich is used to identify the compound, detect the pres ence of impurities, to find out the number of components, etc. This is done by using retention time values.2. three-figure analysisWhich is done to determine the quantity of the individual or several(prenominal) components in a mixture. This is done by canvas the peak area of the standard and sample.2.3 Principle of separation in HPLC 7, 9The 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, 12Pumps solvent delivery system motley unit, gradient controller and solvent de catalystsingInjector Manual or auto injectorsGuard columnsDetectorsRecorders and integratorsFig. 3 The schematic diagram of HPLC 131. Pump Solvent delivery systemThe 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 robotlike 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 valvesThese are present to control the flow rate of solvent and back pressure.Pulse dampnersThese are used to dampen the pulses observed from the wavy baseline caused by the pumps.2. Mixing uni t, gradient controller and solvent degassingMixing unit is used to mix solvents in different proportions and pass through the column. There are two types of potpourri 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 both with a static mixer, which is packed with beads, or dynamic mixer, which uses magnetic stirrer and operates under high pressure.Gradient controllerIn an isocratic separation, mobile phase is hustling by using pure solvent or mixture of solvents, i.e. solvent of same eluting power or polarity is used. solely 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 degassingSeveral gases are soluble in organic solvents. When solvents are pumped u nder 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 injectorsSeveral devices are available either for manual or auto injection of the sample. Different devices areSeptum 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.Rheody ne 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 columnGuard 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 columnsAnalytical 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 30cmColumn diameter Ranges from 2mm to 50mmParticle size From 1m to 20mParticle 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 groupsC18 Octa Decyl Silane (ODS) columnC8 Octyl columnC4 Butyl columnCN Nitrile columnNH2 Amino columnFor other modes of chromatography, ion exchange columns, gel columns, chiral columns, affinity chromatographic columns, etc. are available.6. Detectors 7,9,10Detectors used depend upon the property of the compounds to be separated. Different sensors available areUV sensing element This detector is based upon the light absorption characteristics of t he 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 decline or oxidation of the compounds when a potential is applied. Th e 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 flow 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 integratorsRecorders 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. retentiveness time for all the peaks can be found out from such preserves, 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, 10Retention time (Rt)Retention time is the difference in the time between the rouse 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 mensurable in minutes or seconds. Retention time is also proportional to the distance moved on a chart paper, which can be careful 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 rateSeparation 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 equationS = Kb/ Ka = Ka/ Kb = (tb t0)/ (ta t0)Where,t0 = Retention time of unretained substanceKb, Ka= Partition coefficients of b and atb, ta = Retention time of substance b and aS = depends on liquid phase, column temperatureIf 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.ResolutionResolution is a measure of the extent of separation of two components and the baseline separation achieved. It can be determined by using the following formulaRs = 2 (Rt1 Rt2)/ (W1 +W2)Theoretical plate (Plate theory)A theoretical plate is an imaginary or supposititious 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, 7A 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 formulaHETP = length of the column/ number of theoretical platesHETP is given by Van Deemter equationHETP = A + (B/u ) + CuWhere,A = Eddy diffusion term or multiple pathway diffusion which arises due to packing of thecolumn. This is unaffected by mobile phase velocity or flow rate. This can beminimised 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 a n ideal flow rate synonymic 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 formulan = 16 Rt/wWhere,n = no. of theoretical platesRt = retention timew = peak width at baseRt and w are metric 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.un equilibrize factorA chromatographic peak should be symmetrical about its shopping mall 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 symmetri cal and shows tailing or fronting.Fronting is due to saturation of stationary phase and can be avoided by using less quantity of sample. go after 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 formulaAF = b/a (b and a calculated at 5% or 10% of the peak height)2.6 Applications of HPLCHPLC 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 belowQualitative analysis It is nothing but identification of compound. This is done by comparing the retention time of the sample a s 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 likea. Direct comparison methodBy 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 pea k at the half heightA1/ A2 = a (W1/ W2)Where,A1 and A2 are peak area of sample and standardW1 and W2 are weight or concentration of sample and standarda is the response factorb. Calibration curve methodIn 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 methodIn 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 railway line.Biopharmaceutical and Pharmacokinetic studies.Stability studies.Purification of some compounds of natural or synthetic origin on preparative scale.2.7 Limitations 7, 10The 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 ostracize off.CHAPTER 3Experimental Selection3.1 Aim of ProjectThe 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 conditionsAll 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 629565453.2.2 Software usedThe computer 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 CL1CE8DB0F3.2.3 Column usedThe column used was Kromasil 5C18 whose test number is 9203- 103443.2.4 Pipet te usedThe 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 BalanceMettler balance AC 88 was used to weigh the sample drug whose Biom