Hplc Analysis Of Aloe Vera Tablets Biology Essay

Hplc Analysis Of Aloe Vera cardts Biology raiseThe project work was aimed to achieve the quantitative determination of aloin and aloe emodin in the bound of tablets by employing HPLC. The mode utilise was shock load utmost effect liquid chromatography. Calibration roll method was apply for the quantification of aloin and aloe emodin. The ready strain was the miscellany of acetonitrile and deionised irrigate in the ratio of 6040 respectively. The diligent form was pumped at 1.5 ml/minute and the analyte was quantified at the wavelength of 220 and 296nm. The tower used for insulation was kromasil 5C18. Reverse phase Isocratic run of common fanny aloin and commonplaceised aloe emodin was d champion and the measures obtained from their analysis were used to comp atomic get 18 the test example meridians. Aloe vera colax tablets make by Aloe pura laboratories were used as the test exemplification tablets which were extracted with water, metha nary(prenominal) , acetonitrile, methanol-water and acetonitrile-water. After stemma they were subjected for isocratic run in HPLC instrument and the data obtained were comp atomic return 18d with that of the standard.CHAPTER 1INTRODUCTION1.1 gateway to Aloe VeraAloes is the dried juice of the leaves of Aloe barbadensis Miller, cognize as curacoa aloes, or of Aloe perryi Baker known as Socotrine aloes, or of Aloe ferox Miller and hybrids of the species of Aloe afri lowlifea Miller and Aloe spicata Baker, known as drapery aloes belonging to the family Liliaceae. 2,3 The synonym of aloes is Aalwee, Aalwyn, Kumari, sense experience cactus, Aroe, Acibar, Babosa, etcetera 1Aloes is indigeneous to eastern and s pophern Africa and grown in Cape colony, Zanzibar and islands of Socotra. It is in similar manner cultivated in Caribbean islands, Europe and many parts of India, including unification West Himalayan region. 2All the varieties of aloe ar the major sources of anthraquinone glycosides. Th e principal spry com position 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 aerophilous hydrolysis into aloe-emodin-anthrone, little aloe-emodin and glucose. 2Along with barbaloin, aloes too contains isobarbaloin, b-barbaloin, aloe-emodin and rosins. The do drugs comparablewise contains aloetic acid, homonataloin, aloesone, chrysophanic acid, chrysamminic acid, galactouronic acid, choline, choline salicylate, saponins, mucopolysaccharides, glucosamines, hexuronic acid, coniferyl alcohol, etc. 2The amount of barbaloin in different commercial varieties varies to a large extent. Curacao aloes contain about 22 percent of barbaloin. Indian variety, in general Aloe vera contain rattling less(prenominal)(prenominal) bar (3.5 to 4 percent). Curacao aloes contains devil and half ms quantity of aloe-emodin , comp atomic moment 18d to Cape-aloe-emodin. 2The resin of aloe principally contains Aloesin. It is a type of C- glucosyl chromome. Aloesin is also responsible 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 stronger purgative action in the series of all crude drugs with anthracene glycosidal content. To riposte effect the gripping action, it is assumption along with carminatives. 2It facilitates the healing of any class of skin wound, burn, or scald even speeding recovery metre after surgery. 4It is applied topically in acne, sunburn, frostbite (it appears to celeb pasture decrease blood consort), 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 c ases of HIV and AIDS due to its immune establishment stimulant properties. 4It appears to be of help in jakescer patients (including lung discountcer) by cativating snow-clad blood cells and promoting growth of non- faecal mattercerous cells. 4Aloe also appears to work on heartburn, arthritis, and rheumatoid arthritis pain and asthma. 2, 4It also lowers the blood sugar levels in diabetics. 2, 4Other situations in which it appears to work when taken internally inclue congestion, internal worms, indigestion, stomach ulcers, colitis, hemorrhoids, liver problems much(prenominal) as cirrhosis and hepatitis, kidney infections, urinary tract infections, prostate problems, and as a general detoxifier. 2, 4CHAPTER 2HPLC2.1 HPLC Introduction and InstrumentationThe proficiency of high effect liquid chromatography is so called because of its improved performance when compargond to classical editorial chromatography. It is also called as high- insisting liquid chromatography since wed ge is used when comp atomic number 18d to classical chromatography pillar chromatography. Instead of a issue being allowed to drip with a tug downstairs gravity, it is forced with under high pressure of up to 400 atmospheres. For the insularity, identification and quantification of coalesces, this method is frequently used in biochemistry and analytic chemistry. 11, 12The information of HPLC from classical column chromatography can be attri nonwithstandinged to the development of smaller particle sizes. downhearteder particle size is important since they walk much than surface field over the conventional larger sizes. 7mid-sixties 40 to 60m1970s 10 to 20m1980s 5 to 10m1990s 1 to 3mA porous particle of 5m offers a surface bea of 100-860 sq.metres/ guanine with an median(a) 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 chromatographyHPLCSta tionary phase particle size considerable60-200mSmall3-20mtower sizeLength x int. diameterLarge0.5-5m x 0.5-5cm i.d.Small5-50cm x 1-10mm i.d.tower materialGlassMostly metalColumn packing pressureSlurry packed at low pressure practically gravitySlurry packed at high pressure 5000 psi direct pressureLow ( spirited (500 3000 psi)Flow ratesLow to very lowMedium to high(Often 3ml/min)Sample loadLow to mediocre (g/mg)Low to very low (mg)ParameterClassical column chromatographyHPLC termsLowHighDetector flow cell slewLarge 300 to 1000mlLow 2 to 10mlColumn efficiencyi.e. Resolving attitude(Low) Theoretical plates per meter(High) often 100,000Plates per meterTypes of nonmoving phases available peculiar(a) rangeWide rangeScale of operationPreparative exceed uninflected and preparative scale2.2 Types of HPLC techniques 7, 9, 10, 11, 12Based on Modes of ChromatographyThere are ii expressive styles viz. Normal phase modality and Reverse phase mode. These modes are based on the char gedity of stationary phase and liquid phase. Before explaining the modes, it is important to know the interactions, which occur surrounded by solute, stationary and expeditious phase.Polar Polar interaction or comparison is more unionized nonglacial interaction or affinity is morePolar Non diametral interaction or affinity is lessNormal phase mode In mean(prenominal) phase mode, the stationary phase (eg. Silica gel) is polar in nature and the roving phase is non-polar. In this technique, non-polar compounds travel speedy and are washd maiden. This is because of less affinity between solute and stationary phase. Polar compounds are retained for longer clock in the column because of more affinity towards stationary phase and take more quantify to be eluted from the column. This is non advantageous in pharmaceutical applications since most of the drug molecules are polar in nature and takes longer season to be eluted and detected. Hence this technique is non 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 conviction. Since most of the drugs and pharmaceuticals are polar in nature, they are non 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 replys are methanol, acetonitrile, tetrahydrofuranand water.Based on principle of legal separation surface assimilation chromatographyIon exchange chromatographyIon pair chromatographySize exclusion or Gel suffusion chromatographyAffinity chromatographyChiral phase chromatographyEach 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 statio nary phase. This principle is seen in practice phase as well as reverse phase mode, where adsorption takes place.Ion exchange chromatographyThe principle of separation is ion exchange, which is reversible exchange of practicable 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 chromatographyIn ion pair chromatography, a reverse phase column is converted temporarily into ion exchange column by employ ion matrimony agents like pentane or hexane or heptane or octane sulphonic acid sodium salt, trtramethyl or tetraethyl ammonium hydroxide, etc.Size exclusion or gel pervasion chromatographyIn this type of chromatography, a mixture of components with different molecular sizes is quarantined by using gels. The gel used acts as molecular filmdom and hence a mixture of center of attentions 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 dissemination effects.Affinity chromatographyAffinity chromatography uses the affinity of the seek with specific stationary phases. This technique is mostly used in the depicted object of Biotechnology, Microbiology, Biochemistry, etc.Chiral phase chromatography disengagement of optical i more or lessrs can be done by using chiral stationary phases. Different principles operate for different types of stationary phases and for different tests. The stationary phases used for this type of chromatography are mostly chemically bonded silica gel.Based on elution technique1. Isocratic separationIn this technique, the equivalent mobile phase faction is used doneout the process of separation. The same mark or elution strength is maintained throughout the process. In this technique, the peak breadth increases with retention time linearly according to the equation for N, the number of supposititious plates.Gradient separationIn this technique, a mobile phase combination of lower mark or elution strength is used followed by gradually change magnitude the planetary house 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 fainthearted solvent and B is the strong solvent. Weak solvent allows the solute to elute behind 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 much(prenominal) 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 normally not done, since the sample used is low. Eg. mg quant ities.2. Preparative HPLCWhere the individual fractions of pure compounds can be collected using fraction collector. The collected samples are reused eg. Separation of fewer grams of mixtures by HPLC.Based on type on analysis1. qualitative analysisWhich 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 analysisWhich is done to designate the quantity of the individual or several components in a mixture. This is done by analyse 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 stati onary 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 digests solvent delivery system mix unit, gradient controller and solvent degassingInjector manual or auto injectors observe columnsDetectorsRecorders and integratorsFig. 3 The schematic diagram of HPLC 131. Pump Solvent delivery systemThe solvents or mobile phases used mustiness 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 much(prenominal) high pressure is recommended. There are different types of pumps available. They are mechanised pumps and pneumatic pumps. A mechanical pump operates with constant flow rate and uses a sapphire piston. This type of pump is used in analytic scale. Pneumatic pumps operate with constant pressure and use highly c ompress 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 crinkly baseline caused by the pumps.2. Mixing unit, gradient controller and solvent degassingMixing 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 motionless 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 prepared by using pure solvent or mixture of solvents, i.e. solvent of same eluting power or polarity is used. But in gradien t 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(prenominal) gases are soluble in organic solvents. When solvents are pumped under high pressure, gas bubbles are formed which lead 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 followers technique. make clean filtration which can remove all air bubbles. But it is not forever and a day reliable and complete.Helium purging i.e. by passing helium through the solvent. This is very effective but helium is high-priced.Ultrasonication by using radicalsonicator, which converts ultra high frequency to mechanical vibrations. This causes the removal of air bubbles.3. Injector Manual or auto injectorsSeveral devices are avai lable 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.Rheodyne injector (Loop valve type) It is the most popular injector. This has a fixed slew 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, wh ich 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, polythene 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 50mm fraction 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 pursuit groupsC18 Octa Decyl Silane (ODS) columnC8 Octyl columnC4 Butyl columnCN Nitrile columnNH2 Amino columnFor ot her 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 demodulators available areUV detector This detector is based upon the light absorption characteristics of the sample. 2 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 one hundred ninetynm 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 light radiation emitted by almost class of compounds. The exitation wavelength and emission wavelength can be selected for each compound. This detector has more specificity and sens itivity. The disadvantage is that some compounds are not fluorescent.Conductivity detector Based upon electrical conductivity, the response is learn. 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. The airing current recorded is proportional to the immersion of the compound eluted. This is applicable when compounds have functional groups, which can be either oxidised or reduced. This is a highly nociceptive 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 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. property 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 impact 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 point of injection and appearance of peak maxima. Retention time is the time indispensable for 50% of a component to be eluted from a column. Retention time i s 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 rateSeparation broker (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 vegetable marrowKb, Ka= Partition coefficients of b and atb, ta = Retention time of substance b and aS = depends on liquid phase, column temperatureIf at that place 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 ar e closer and the separation factor is less. stoppageResolution is a measure of the extent of separation of two components and the baseline separation achieved. It can be determined by using the following regulationRs = 2 (Rt1 Rt2)/ (W1 +W2)Theoretical plate (Plate theory)A notional plate is an speculative 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, 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 metrical by using the following formulaHETP = length of the column/ number of theoretical platesHETP is accustomed by Van Deemter equationHETP = A + (B/u ) + CuWhere,A = Eddy dispersion term or multiple path diffusion which arises due to packing of thecolumn. This is unaffected by mobile phase hurrying 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 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 formulan = 16 Rt/wWhere,n = no. of theoretical platesRt = retention timew = peak width at baseRt 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 chromatograp hic columns, a value of 600/ metre is sufficient. But in HPLC, high values like 40,000 to 70,000/ metre are recommended.Asymmetry factorA 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 fecundation 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 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 ap plications, biochemical separations, Biotechnology, Food analysis, etc. In fact on that point 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 soft analysis It is nothing but identification of compound. This is done by comparing the retention time of the sample as well as the standard. at a lower place 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 peak at the half heightA1/ A2 = a (W1/ W2)Where,A1 and A2 are peak area of sample and standardW1 and W2 are weight or tightness 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 stringency 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 characteris tics 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 alike 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 celluloid melodic 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 tha t 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 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 tec hnologies 1200 series, whose model number is G1310A and the serial number is DE 629565453.2.2 Software usedThe 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 pipette usedThe pipette used was Volac ultra (made in U.K.), S. No. 29186, sticker 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 counterbalance AC 88 was used to weigh the sample drug whose Biom