European Journal of Biotechnology and Bioscience

European Journal of Biotechnology and Bioscience

Online ISSN: 2321-9122, Impact Factor: RJIF 5.44
www.biosciencejournals.com
Volume 5; Issue 3, May 2017; Page No. 06-09

By using RP-HPLC Technique, Quantitative and Qualitative analysis of Gallic acid from Industrial waste
1
Dr. N Lokeswari, 2 Dr. Lenin Kumar B
1
Assistant Professor, Dept. of Biotechnology, Dr. B.R. Ambedkar University, Etcherla, Srikakulam, Andhra Pradesh, India
2
Dept. of Biotechnology, Dr. B.R. Ambedkar University, Etcherla, Srikakulam, Andhra Pradesh, India

Abstract
RP- HPLC is a simple, precise and simple technique which was used for qualitative assurance of Gallic acid from testa of
Anacardium occidentale L., UV detection at 271nm was developed and validated. Separation was performed on a Phenomenax
column (150 × 4.6 mm, 5 μm pore size) by using mobile phase, 80:20 ratiog of acetonitrile and water containing 0.01% v/v ortho
phosphoric acid for the present study. For the separation and identification of gallic acid direct injection method has been
performed in the HPLC technique. Studies, which activated the accurateness of the method, 99.63% recovery percentage was
showed for this acid.

Keywords: tannin, gallic acid, anacardium occidentale l, testa

1. Introduction 2. Plant material

All most in all plants gallic acid is present. Gallic acid is Cashew (Anacardium occidentale L.) testa were acclimated as
beginning in about all plants. A powerful well known substrate in the solid state fermentation. Tannins from testa
antioxidant, Gallic acid is found in variety of foods and herbs. were analysed.
Herbs such as blueberiies, walnuts, flax seed, tea and apples
all contain gallic acid. It is also present in sumac, gallnuts, Fermentation and isolation of tannase
hazel, watercress, oak bark and other plants. Gallic acid For one week at thirty degree centigrade for week, A. Niger
(3,4,5-trihydroxy benzoic acid) is a phenolic compound and changed into grown of changed czapek’s dox medium. Spores
finds application in various fields. In the higher plants, the had been calm beneath antibacterial action utility Tween
sysnthesis of gallic acid is still an open question. An α- eighty. The organized spore suspension changed into adjusted
oxidation of 3,4,5-trihydroxycinnamic acid and, by to 107 spores/ml. 250ml Erlenmeyer flasks containing 50g of
hydroxylation of 3,4-dixydroxycinnamic (protocatechuic) cashew testa, 3g tannic acid and KH2PO4 5g. NH4NO3 10g,
acid, and (c) by direct dehydration of 3-dehydroshikimic acid, MgSO47H2O 1g, CaCl26H2O 0.1g, MnCl26H2O 0.02g,
an intermediate compound of the shikimate pathway is the NaMo O4 2H2O 0.01g, FeSO47H2O 0.125g, turned into
three pathways were proposed for accumulation of gallic acid. adjusted to pH 5.5 with 100mM NaOH and incubated at thirty
An antibacterial agent in combination with sulfonamide is a degree centigrade for 2 days with three milliliters of organized
lot of important use in for accomplishment trimethoprim spore suspension inoculum. The brewed biomass alloyed very
(TMP), Gallic acid is a poly-phenolic compound, which is well observed via centrifugation at ten thousand revolutions
obtained from the hydrolysis of natural plant poly-phenols and according to minute to abolish the mycelia mass, after
used as a reductant. It has been used historically to yield blue incubation time.
ink as its reduction of iron chloride produces a blue
precipitate. By the enzymatic hydrolysis of tannic acid, Gallic Extraction and Purification
acid (3, 4, 5-hydroxy benzoic acid) is produced at the Crude enzyme was a far from the brewed amount at 4°C for
industrial scale level. Anti-malarial drug Trimethoprim is 20 min, by centrifugation at ten thousand revolutions per
manufactured by using Gallic acid. An antioxidant propyl minute. The crude extract was precipitated with 80% of
gallate, used in food industry was also manufactured by using Ammonium sulphate and collected the accelerate by
gallic acid. Gallic acid is also used to produce Pyrogallal, centrifugation at ten thousand revolutions per minute for 20
which is used in leather and hair, staining fur and also a min at 4°C. After overnight dialysis was done with precipitate
photographic developer. Further, gallic acid possesses against 0.5M citrate buffer with pH=5 at 4°C. Fractions were
numerous biological activities such as antiviral, antibacterial, collected through ion exchange Chromatography (DEAE
anti- poptotic and analgesic activities. Several different cellulose anion exchange column 1×20cm) for dialyzed
biological backdrop and bartering applications, gallic acid is sample. The tannase action was estimated by fractions.
an admixture of abundant absorption to both biologic and
actinic industries. Present paper reports, a simple, accelerated Tannase activity
and absolute acclivity HPLC method with an economical At thirty degree centigrade for five min. pre-incubate Citrate
mobile phase for quantification and qualification of gallic buffer with pH=5.0, 0.67% methanolic rhodanine and
acid. 0.5mol/L KOH had been needed for this assay. An aliquot of

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0.5 mL turned into mixed with 0.3 mL of methanolic solvent extraction. Ethyl acetate was added to the reaction
rhodanine solution and incubated. After that, 0.2 mL of KOH mixture in the ratio of 1:1 in separating funnel, mixed
solution had been added and incubated again. Finally, four mL vigorously and left for 10 min to form two immiscible clear
of distilled water had been brought to the reaction mixture and phase. The solvent was evaporated in rotary vacuum
incubated at 30 °C for 10 min and the absorbance turned into evaporator at 750C and 100rpm respectively. By using HPLC
examine at 520 nm. The amount of gallic acid released gallic acid was analysied with Phenomenax column parameter
throughout hydrolysis of tannic acid represents the tannase 150 × 4.6 mm and 5μm particle and UV detector were used for
interest. Gallic acid turned into measured with the rhodanine analysis. Methanol, acetic acid and deionized water in the ratio
response. One unit of enzyme interest is described as 1μ mole of 15:5:80 used as mobile phase respectively. Isocratic run at
of gallic acid launched according to minute inside the assay 1ml/min flow rate for 10 min run time was setted. For the
conditions. detection of the product 271 nm wavelength was used. By
using Sigma Aldrich Analytica grade gallic acid three
Gallic acid estimation standards were prepared of 25, 50 and 75ppm. By using
By using methanolic rhodanin, Gallic acid content was appropriate gallic acid standard solution, gallic acid content
assayed by spectrophotometric method. was calculated. In the spectra or chromatograms were
produced there is no interferences due to other ingredients and
Instrument and chromatographic conditions excipients was detected.
Analysis were performed on a HPLC of Shimadzu LC-10AT
model. Separation was carried out using a Phenomenax 3. Results and Analysis
cavalcade (150 × 4.6 mm i.d., 5 μm pore size). The column In the present study, a simple, precise, authentic and
was maintained at 27°C throughout the assay and detection accelerated reverse phase HPLC method has been developed
was carried at 271 nm. Acetonitrile and water containing and accurate for the assurance of gallic acid. Fig 1 and Fig 2
0.01% v/v ortho phosphoric acid in the ratio of 80: 20 shows the actinic structures of the gallic acid and tannic acid
respectively was used as mobile phase.. Data accretion was respectively. The accurateness of the method was bent by
done with LC Solutions adaptation 1.2 software. The artful the accretion of gallic acid by the adjustment of
chromatographic altitude had ahead been optimized to accepted accession. The accurateness of the adjustment was
accomplish the best resolution and peak shape. Apprehension arrested by intercepting arrangement ambit (which is plotted
was performed at λ=271nm accepting breeze amount 1ml/min. between the area under curve on y- axis and concentration of
The archetypal chromatogram for accepted and sample is standard solutions on x-axis) with the sample area under curve
shown in Fig. 2 & 3. which is obtained when injecting the 80 μg/mL, 90 μg/mL and
100 μg/mL standard solutions. The allotment accretion for
Preparation of standard solution gallic acid was begin to be 98.7 %, 99.36 % and 99.63 %
In a series of 10 cm3 standard volumetric flasks varying appropriately which was apparent in the Table 3.
amounts of Gallic acid were taken and make up upto to the
mark by using methanol as diluent.

Calibration Curve
Appropriate dilutions were made by taking 0.2 mL, 0.4 mL,
0.6 mL, 0.8 mL, 1 mL and 1.2 mL of the standard solution of
gallic acid and then making up the volume up to 10 mL with
mobile phase resulting in concentrations of 20 μg/mL, 40
μg/mL, 60 μg/mL, 80 μg/mL and 100 μg/mL of gallic acid
respectively, from the standard solutions. A calibration curve
was plotted from the peak areas obtained.

High Performance Liquid Chromatography (HPLC)

The Sample was loaded at a concentration of 1 mg/ml to the
HPLC (Shimadzu). Sample was diluted in Acetonitrile:Water
(50:50). Injection volume was 50μl. Absorbance was Fig 1: Production of Tannase by solid state fermentation
monitored at 271 nm and flow rate maintained at 1 ml/min.
The sample was applied to Phenomenax column (150 × 4.6 The crude tannase was precipitated by ammonium sulphate
mm. 5μm pore size) and eluted with a linear gradient of precipitation the action was 28 U/g/min. After dialysis the
acetonitrile containing 0.1 % TFA (trifluroacetic acid). The specific activity activity of 36 U/g/min was obtained. The
sample and standard gallic acid were run identically. sample was added purified through DEAE-Sephadex G-100
chromatography and the eluted fractions, which showed 43
Purity analysis U/g/min.
By using ethyl acetate, Gallic acid was concentrated by

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Fig 2: HPLC Chromatogram of the standard gallic acid

Table 1

Table 1 and Table 2 represents the retention time and peak gallic acid 2871053 shows in the table 1, Retention time is
areas of the HPLC chromatograms. Peak area of the standard 1.82 min.

Fig 3: Chromatogram shows peak curve of the extracted gallic acid sample

Table 2

Table 3: Recovery abstraction of the gallic acid by HPLC method.

Conc (µgm/mL) Peak area Recovery (µgm/mL) % Recovery
80 2140817 78.96 98.7
90 2783109 89.42 99.36
100 2869098 99.63 99.63

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4. Conclusion
The RP-HPLC method was acclimated for assurance of Gallic
acid from the testa of Anacardium Occidentale L. The after-
effects announce that the adjustment is awful precise. As the
proposed method is awful accurate, careful and absolute
appropriately can be acclimated for a accepted superior
ascendancy assay of Gallic acid. The method is aswell fast and
requires about 20 minute for analysis.

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