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.
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. 
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. 
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. 
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. 
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. 
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. 
The resin of aloe principally contains Aloesin. It is a type of C- glucosyl chromome. Aloesin is also responsible for purgative action of aloes. 
Fig. 1 Fig. 2
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Aloin  Aloe emodin 
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. 
It facilitates the healing of any kind of skin wound, burn, or scald – even speeding recovery time after surgery. 
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. 
It appears to be of help in cancer patients (including lung cancer) by cativating white blood cells and promoting growth of non- cancerous cells. 
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]
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. 
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 
Classical column chromatography
Stationary phase – particle size
Length x int. diameter
0.5-5m x 0.5-5cm i.d.
5-50cm x 1-10mm i.d.
Column packing pressure
Slurry packed at low pressure – often gravity
Slurry packed at high pressure >5000 psi
Low (<20 psi)
High (500 – 3000 psi)
Low to very low
Medium to high
Low to medium (g/mg)
Low to very low (mg)
Classical column chromatography
Detector flow cell volume
Large – 300 to 1000ml
Low – 2 to 10ml
i.e. Resolving power
Theoretical plates per meter
(High) often >100,000
Plates per meter
Types of stationary phases available
Scale of operation
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
Based on principle of separation
Ion exchange chromatography
Ion pair chromatography
Size exclusion or Gel permeation chromatography
Chiral phase chromatography
Each of the above technique is described in brief as follows:
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 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.
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
Recorders and integrators
Fig. 3 The schematic diagram of HPLC 
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.
These are present to control the flow rate of solvent and back pressure.
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.
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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.
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)
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 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
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
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.
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)
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.
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.
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 Biom
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