DESC:FIELD OF INVENTION
The present invention relates to an improved process for the extraction of a compound, called humic acids (HA), useful in agriculture, in poultry food preservation, in rubber industry and in medicines. In particular, the present invention relates to a column-based continuous elution (CCE) method for extraction of HA from solid sources of HA as vermicompost (VC).

BACKGROUND OF INVENTION AND PRIOR ART
Humic substances (HS) is a major component of natural organic matter (NOM) in soil, in geological deposits such as lignite, leonardite, peats, brown coals and shales and in water bodies like lake, ocean and their sediments. It is formed through a process called humification where microorganisms play an important role (Pramanik et al. 2009, Waste Manag. 29, 574-578). HS can be classified into four main fractions: HA, fulvic acids (FA), hymatomelanic acids and humin. HA has been known to be used in agriculture for their beneficial role in preserving the soil health, increasing seed germination rate, improving plant growth and enhancing crop yield (Mayhew 2004, Acres. 4, 34 (1&2); Zandonadi et al. 2007, Planta 225, 1583-1595). It further assists in transferring micronutrients from the soil to the plants and stimulates development of microflora population in soils (Manivannan et al. 2006, J. Environ. Biol. 30, 275-281). In many tropical countries, organic carbon status of arable soils is very low and farmers apply organic matter to agricultural fields for sustaining their productivity (Inbar et al. 1985, Acta Hort. (ISHS) 172, 75-82). HA is an important constituent of manure and compost (Huang et al. 2006, Bioresour. Technol. 97, 1834-1842; Plaza et al. 2005, Environ. Sci. Technol. 39, 7141-7146).
Composting has been known since long as a biological process for converting solid waste into a stable, humus like product for use as a soil conditioner. Vermicomposting is emerging as an important biotechnological tool for recycling various organic wastes including agricultural residues which are considered as potential renewable sources of plant nutrients. To monitor maturity of compost of pig manure and sawdust by determining HA content, Huang et al. (2006) used the alkaline protocol of International Humic Substance Society (IHSS) with little modifications. Similarly, the IHSS method has also been used to isolate HA and FA from compost of olive oil mill waste and tree cuttings for studying the acid-base properties of HA and FA.
The various components of humic matters are often known by their functional attributes based on their solubility in acid, alkali and alcohol. HA is soluble in alkali, FA is soluble in acid and alkali, hymatomelanic acids are soluble in alkali and alcohol while humin is insoluble in any of these solvents. Based on such differential solubility profiles, extraction of HS using alkaline solvents still remains one of the most efficient and widely used for the isolation of soil HA. The choice of such extractant(s) plays an extremely crucial role as it is source sensitive and has serious implications on the quality of HA.
Alkaline extraction of HS for preparation of liquid fertilizers containing HA/FA for medicinal purpose were attempted by several groups using composted/raw sources like cassava dregs, domestic garbage/ sewage, low-rank coal, oxidized coal, inflammable materials, peat, leonardite, lignite, soil, sediment, straw and manure, compost of a variety of plants, trees, vegetables, fruits and plant litters. In addition, there are protocols for extraction of HA using aqueous alkali solutions which apply mechanical shaker, magnetic stirring, ultrasound or microwave energy for solubilization of HS (Moreda-Piñeiro et al. 2004, Anal. Chim. Acta 524, 97-107; Romarís-Hortas et al. 2007, Anal. Chim. Acta 602, 202–210).
Ultrasound and microwave-assisted extraction methods of HAs and FAs have been reported in prior art (Mecozzi et al. 2002, Ultrason. Sonochem. 9, 11-18; Moreda-Piñeiro et al. 2004, Romarís-Hortas et al. 2007). Such methods are naturally expensive and limited to quantitative analysis of Ha and FA.
A multi-solution and multi-columns system which requires multiple solenoid valves and complicated software has also been reported (Zomeren et al. 2008, Anal. Bioanal. Chem. 391, 2365-2370). Further drawbacks of this system include longer extraction time, poor yield and difficulties in scaling up.
CN101666051 (A) discloses a method of producing HA and FA, namely by using biomass domestic garbage and domestic sewage and by applying a catalytic kettle, drying equipment, crushing equipment, humic materialization equipment, a methane purifier, a gas storage tank, condensation equipment and solid-liquid separation equipment.
RU2429214 discloses an alkaline extraction of HA in batches using ammonia solution from peat at 1060oC and applying hydromodulus 25200 under conditions of cavitation exposure in a rotor at 3000rpm thus reducing the time of extraction to about 10 minutes. The method follows the general protocol of IHSS, however, the quality of HA obtained by this type of harsh and undesirable conditions has not been investigated.
US 5004831 discloses a method of recovering HA from oxidised coal by mixing with aqueous alkali in the temperature range 100-180oC under sufficient pressure to prevent evaporation of the water, and maintaining the elevated temperature for a time sufficient to extract a substantial amount of the available HA from the material.
US2012279266 discloses a method for extracting HS substances (to be used as liquid fertilizers) from the liquid component in the organic compost mixture, which was separated by means of a separator, such as a centrifuge, belt press, filter press, or membrane press by heating at 170oF to 220oF using water.
Depending upon the source materials of HA and their intended applications, different solid-liquid separation protocols like belt pressing, filter pressing, semi-permeable membrane dialysis, centrifugation, freeze drying, etc. were used for separation of HS by several inventors using alkaline extraction method (Lee and Kim, 2011 (KR20110063010); Shanlin, 2010 (CN101666051); Yuanbo et al., 2012 (CN102558573); Tiannan and Weimin, 2012 (CN 201110250034)).
Although preparation times of the HS formulation was relatively short but most of them applied high temperature for the extraction procedure. In addition, none of these prior art sought to provide better quality HA.
Evidently most of these methods of extraction of HS are time, resource and energy intensive. Apart from being expensive, methods involving ultrasonicators and microwaves are difficult to scale up, complex and cumbersome. Further none of the prior art literature indicates or even suggests HA extraction directly from indigenous sources such as VC.
Therefore, there remains a need in the art for simple, expeditious and cost effective process for extraction of HA.
Accordingly, the present invention provides a novel process involving column-based continuous elution (CCE) for extraction of HA. Such method is not only more efficient and faster than prior art methods, such as IHSS, but also has better yield.

OBJECTS OF THE INVENTION
It is an object of the present invention to provide a novel, expeditious yet simple process for extraction of HA.
It is another object of the present invention to provide an improved process of extraction of HA from any source containing HS such as natural organic matter from soil, compost, vermicompost, geological deposits such as lignite, leonardite, peats, brown coals and shales and like matter containing humic substances using a column-based continuous elution (CCE) method.
It is a further object of the present invention to provide a process employing CCE where the solid phase is preferably vermicompost or intended source material and the mobile phase is an alkali.
It is yet another object of the present invention to retain the molecular integrity of HA without involving addition of inert gases.
It is yet another object of the present invention to provide a high-purity-grade HA using common, inexpensive chemicals and equipment’s.
It is yet another object of the present invention to provide an economical, labour and energy efficient process.
It is another object of the present invention to provide a process for extraction of HA for use in agriculture, which is ecological and environment friendly.

SUMMARY OF THE INVENTION
According to one aspect of present invention, there is provided a process for the extraction of humic acids from a humic acids source, wherein said process employs a column-based continuous elution, said process comprising steps of:
a) treating said source with an acid for decalcification followed by neutralization using an alkali;
b) Mixing the treated source resulting from the step (a) with an extractant/mobile phase forming a slurry of said humic acids source;
c) treating slurry resulting from the step (b) through the column-based continuous elution method, wherein said slurry is the solid phase and an alkali is the extractant/mobile phase, to obtain an elute;
d) treating said elute resulting from the step (c) with an acid for flocculation of said humic acids; and
e) performing washes of the humic acids flocculate resulting from step (d) with a salt solution, an acid and water until said flocculate is free of chloride ions followed by a further wash to obtain said humic acids.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Figure 1: Schemes for alkaline extraction of humic acids from vermicompost.
Figure 2: Humic substance solubilization efficiencies from vermicompost by different modes using 0.2 N NaOH.
Figure 3: Flow-rate dependence of extraction yield of humic acids from vermicompost by CCE method.
Figure 4: Auto-degradation study of HA by spectophotometric method.
Figure 5: TOF/ESI-MS (in positive mode) spectra of humic acids extracted from vermicompost.
Figure 6: UV-VIS spectrum of humic acids.

DETAILED DESCRIPTION OF THE INVENTION
In the present invention a novel and improved process for extraction of humic acid (HA) from humic substances (HS) has been disclosed, wherein the said process comprises column-based continuous elution (CCE) resulting in an expeditious, cost-efficient and convenient process compared to the prior art methods.
According to the present invention, the process comprises column-based continuous elution where the solid phase is a solid source of HA as compost, vermicompost, geological deposits such as lignite, leonardite, peats, brown coals and shales and such like matter containing HS and preferably vermicompost and the mobile phase is an alkali. Vermicompost is an abundant source of HA thereby rendering the method economical and efficient. For the purposes of the present invention, the inventors prepared the vermicompost using standard methods.
The present invention follows a scheme as represented in Figure 1 which shows extractions from vermicompost by conventional (….….), modified (????) IHSS and continuous column elution (--) methods.
In the present invention, HA has been extracted from vermicomposted agricultural crop residues and weeds by employing a CCE method, which is newly developed. It was surprisingly found that this novel and simple method has better extraction efficiency compared to the IHSS method and other prior art methods.
In the present invention the preferred acid for declacification of the source in step (a) is hydrochloric acid (HCl).
In the present invention the alkalis for neutralizing and extraction as carried out in steps (b) and (c) can be selected from potassium hydroxide (KOH) and sodium hydroxide (NaOH). The more preferred alkali being sodium hydroxide (NaOH).
In the present invention the preferred acid for flocculation in step (d) is hydrochloric acid (HCl).
In the present invention the preferred salt, for washing the HA flocculate in step (e) is sodium chloride (NaCl) or potassium chloride (KCl) or the like.
In the present invention the preferred acid for wash the HA flocculate in step (e) is hydrochloric acid (HCl)..
In the present invention, the further wash of the HA flocculate is carried out with ethanol-water or methanol- water or ethyl acetate-water.
In the present embodiment, the invention has been described with respect to the process of extraction of HA from solid source including natural organic matter from soil, compost, vermicompost, geological deposits such as lignite, leonardite, peats, brown coals and shales, however, such description should not be considered as restricting the scope of the present invention. Further it would be possible for a person skilled in the art to practice the present extraction process considering other sources as well without departing from the scope of the present invention.
In this embodiment the present invention provides a process for the extraction of humic acids from vermicompost, wherein said process employs a CCE method, said process comprising steps of:
a) treating said vermicompost with HCl for decalcification followed by neutralization using NaOH;
b) mixing the treated vermicompost resulting from the step (a) with NaOH forming a slurry of said vermicompost, which forms the solid phase;
c) treating slurry resulting from the step (b) through the chromatography column employing with NaOH (mobile phase) to obtain the elute;
d) treating said elute resulting from the step (c) with HCl for flocculation of said humic acids; and
e) performing washes of the humic acids flocculate resulting from step (d) with NaCl, HCl and water until said flocculate is free of chloride ions followed by a further wash with ethanol-water to obtain highly pure humic acids.
Further it was surprisingly found that the yield of HA was about 100% when the present invention was carried out at ambient temperature using 0.2 M NaOH for 48 hours, thereby providing highly pure HA. The oxidative degradation of HA during extraction was limited by the CCE method without using an inert gas atmosphere. Upon characterizing HA, it showed presence of aliphatic as well as aromatic characters corroborating their microbial origin.
The present inventors compared the efficiency of the present process with that of IHSS method in terms of yield obtained by the extraction methods and in spike recovery test of pre-purified HA. In addition, characterization of the HA by elemental composition, studying molecular integrity during extraction by TOF/ES-MS as well as spectrophotometrically validates the newly developed method, which has been exemplified in Figures 4 and 5 hereinbelow.
In Figure 2, the HS solubilization efficiencies from VC by different modes (magnetic stirring, continuous mechanical stirring or ultrasonication) using 0.2 N NaOH have been represented. Decalcified and neutralized VCwas treated at 1:10 (w/v) by different modes. Small aliquots were collected at different times of solubilization, filtered and absorbance of the filtrate was measured at 1:20 dilution at 400 nm. Lines are representative of 2-3 sets of independent experiments.
The present inventors prepared a slurry of 5 g VC after decalcification and neutralization were packed in glass column (9 x1cm) fitted with pinch cock to adjust the flow rate and elutes were collected at different times for flocculation of HA. The flow-rate dependence of extraction yield of HA from VC by CCE method is represented in Figure 3. Data presented are mean±SD from three or more independent experiments.
The comparative tests which were carried out between the present process and modified IHSS has been represented in Figures 4 and 5. Figure 4 represents an auto-degradation study of HA by spectophotometric method. HA extracted at different times of extraction by modified IHSS method using 0.1 M, 0.2 M, 0.4 M NaOH and by CCE method using 0.2 M NaOH were redissolved in phosphate buffer, pH 7.4. Absorbances at 400 nm and 600 nm were determined and ratio of the absorbances (E4/E6) was plotted against time of aliquot collection for extraction. Data presented are mean ± SD from three or more independent experiments. Figure 5 illustrates the TOF/ES mass spectrometry data of the product obtained by performing the present process and the modified IHSS process. The molecular sizes are calculated manually using two adjacent lines of charge states at the most populated m/z values Mann et al. 1989, Anal. Chem. 61, 1702-1708. Majority of HA are of molecular sizes 53.76 kDa by the modified IHSS method and 77.16 kDa by the CCE protocol and there is minimum degradation in the latter method as can be seen from the mass spectra exemplified in Figure 5A. Some population of HA of molecular size 20.42 kDa are present in the mass spectra of HA extracted by CCE method, which is an artifact of determination as it occurred repeatedly at almost similar positions. As it can be observed the molecular size of HA obtained by the CCE method is significantly larger than the modified IHSS method by a margin of about 15-20% since substantial percentage of HA is extracted at the beginning of extraction.
The below examples are intended for the purposes of illustration only and are not intended to limit the scope of the present teachings.

EXAMPLES
Example 1: Preparation of VC
VC was prepared from a mixture of agricultural crop residues and weeds conditioned with cow-dung (70-30; w/w) using earthworm (Eisenia fetida @ 10 nos kg-1) using standard methods. The matured VC was separated from earthworms and other extraneous substances on the 60th day by sieving and stored in a cool and dark place.
Example 2: Extraction of HA by conventional and modified IHSS methods
HA from the VC were extracted following the conventional IHSS method (Swift, 1996) and with some modifications as outlined in Fig. 1. In brief, ground 5-10 g VC was treated with 1 N HCl (1:10 w/v) for 1 h for decalcification and then neutralized with 1 N NaOH. The neutralized mass was treated for 1.5 – 2 h with the extractant at 1: 10 (w/v) for solubilization of HS facilitated by either occasional manual shaking, magnetic stirring, continuous mechanical shaking (@rpm 30) or ultrasonication (at 42 kHz for 5 min with 2 min intervals) and allowed to settle for 10 – 20 min before filtering through careful decantation over glass wool placed on a cone of ordinary filter paper in a funnel. The residue was retreated with the extractant three to four times before overnight incubation (10 – 12 h) followed by one more washing. The filtrate pooled from several batches were treated with 1 N HCl for flocculation of HA, kept overnight and filtered. The precipitate of HA was washed with cold 0.5 N NaCl followed by HCl and HF (1% each in water) and cold water until freed of Cl- (checked using AgNO3) and finally with 1:1 ethanol-water. The total yield was determined gravimetrically and expressed as mg of HA per g dry weight of VC or percentage of extractable HA (EHA). In the conventional method, filtration was performed after incubation of the neutralized VC with the extractant for 24 h.
Example 3: Extraction of HA by the CCE method
Pretreatment of the VC for decalcification, post-treatment of elute for flocculation of HA and washing of HA are depicted in Fig. 1. The decalcified and neutralized VC (dry weight 5g) was made into slurry with the appropriate extractant and packed into a one-side open glass column (>9 x 1 cm) and eluted under gravity with extractants at 12, 25 and 60 ml/h flow rates.
Example 4: Determination of the value of EHA
The HA which was extracted consecutively for 4 days without interruption in the modified IHSS method and/or for 48 h without pause in elution by the CCE method was considered EHA. Statistical analysis was done by t- test for the means at 95% confidence level.
Example 5: Recovery test
Previously purified HA were spiked at 60 and 105 mg/g with the fresh and untreated VC and extraction was performed following the modified IHSS method and CCE method. Percentage of recovery was calculated by comparing the actual yield of HA obtained with respect to the total amount of HA to be extracted which is the sum of mean of EHA and the spiked amount of HA. It is to be stated that all the extractions and recovery tests were performed at ambient temperatures which varied from 25 to 35°C.
Example 6: Study of degradation of HA during extraction
Relative molecular weights of HA was determined spectrophotometrically at different times of extraction by reading the absorbances at 400 nm to 600 nm (Tan and Giddens 1972, Geoderma 8, 221-229). In brief, aliquots of solubilized HS at different times during extractions were collected, HA was flocculated, filtered, washed and then vacuum dried. A part of the HA was redissolved with 50 mM phosphate buffer, pH 7.6, and absorbances at 400 nm and 600 nm were noted and their ratios (E4/E6) calculated. Other portion of the HA from the aliquots was analysed by TOF/ES-MS spectrometry in the positive mode.

Prior art method
Example 7: Preparation of VC
Like other organic wastes, a mixture of agricultural crop residue and weeds conditioned with cow-dung has been successfully converted to VC in this study following the reported method of Chattopadhyay (2005) using Eisenia fetida. High technology dependant methods for producing hygienic, stable and safe compost (Schaub and Leonard 1996, Trends Food Sci. Technol. 7, 263-268; Tiannan and Weimin, 2012 (CN 201110250034)) were not selected in this study to minimize requirement of resources and infrastructure which is a matter of concern in developing small scale industries in a developing country like India. The odourless and granular VC was collected on 60th day of incubation, because after this period microbial activity is reported to decline and degradation of HS also starts (Bansal and Kapoor 2000, Bioresour. Technol. 73, 95-98; Inbar et al. 1990, Soil Sci. Soc. Am. J. 54, 1316-1323).
Example 8: Effect of solubilization modes and repeated batch treatment on extraction of HA by IHSS method
Rate of solubilization of HS from VC in 0.2 M NaOH, as monitored by recording the absorbances of the aliquots collected at different times by magnetic stirring, continuous mechanical stirring or ultrasonication for solubilization (Fig. 2) was faster than applying occasional manual shaking or incubation (not shown in the Fig. 2 due to broad differences in time scale; solubilization by incubation in 24 h was found to be almost equal to that by occasional manual shaking in 2 h). However, filtration of solubilized HS solution after 30 min of treatment with 0.2 M NaOH took 16.7±4.2, 33.5±8.3, 71.7±15.2 and >120 min and after 1 h of treatment took 27.7±7.3 min, 1 h 25 ±10 min, >120 min and >120 min by applying occasional manual shaking, continuous mechanical shaking, magnetic stirring and occasional ultrasonication, respectively. There are reports on extraction of HA from soil or compost by applying either mechanical shaker, magnetic stirring, ultrasound or microwave energy (Moreda-Piñeiro et al., 2004; Romarís-Hortas et al., 2007; Siddiqui et al. 2009, Int. J. Agric. Biol. 11, 448-452). In all those studies, centrifugation was applied to separate the dissolved HS from the soil residues. In the present study, as the separation of solubilized HS from mineral matrix by filtration after occasional brief manual shaking took approximately the same time as a normal centrifugation step, expensive equipments like centrifuge, shaker and ultrasonic bath were not used which have minimized requirement of infrastructure and consumption of energy associated with this invention.
The extraction of HA using 0.2 M NaOH by the conventional protocol was extended up to 4 days (data for up to 48 h are given in Table 1), but substantial amount of HA (~ 20% of EHA) remained unextracted. This is in agreement with the reports that extraction of HA from soil/peat by conventional IHSS method takes longer time (Romarís-Hortas et al., 2007; van Zomeren and Comans 2007, Environ. Sci. Technol. 41, 6755-6761; van Zomeren et al., 2008 Anal. Bioanal. Chem. 391, 2365-2370). Modification of the IHSS method by applying occasional brief manual shaking and repeated batch-wise treatments (Fig. 1) was found to be more efficient in terms of yields compared to the single step extraction by overnight incubation as done in the conventional IHSS method (Table 1).
The repeated batch-wise treatment with 0.2 M NaOH resulted in significantly higher yields in the modified IHSS method compared to the conventional IHSS method possibly due to effective solubilization. Similar improvement in extraction yield by repeated batch treatment was also reported by several other groups (Romarís-Hortas et al., 2007; van Zomeren and Comans 2007).
It is to be stated that extraction of HA from VC was little faster at 1:20 (w/v) VC to extractant ratio, but considering half volume of the extractant to be required at the 1:10 (w/v) ratio, the most suitable ratio was chosen to be the latter which is also recommended by the IHSS for other source materials.

RESULTS OF COMPARATIVE STUDIES
Table 1: Yields of HA extracted from VC by conventional , modified IHSS and CCE methods using different extractants at 24h and 48h

Comparison of yields of HA by the developed CCE method vis-à-vis conventional and modified IHSS methods
In the CCE protocol developed in this study, solubilization of HA from the decalcified and neutralized vermicomposted organic waste was accomplished in a column by continuous elution using 0.2 M NaOH (Fig. 1). As can be seen from Table 1, in 24 h, 74.7% of the EHA were extracted by applying the CCE method using 0.2 M NaOH as compared to 59.4% by the modified IHSS method and 47.5% by the conventional IHSS method; the yields in 48 hours were 97.9, 76.0 and 63.3%, respectively. The yields obtained by the CCE method were significantly higher (evaluated by t-test for the means at 95% confidence level) compared to the yields obtained by the IHSS methods at both the points of time during extraction. Complete extraction (100% EHA) by the modified IHSS method was achieved in 4 days. It may be noted here that there was no significant variation (evaluated by t-test for the means at 95% confidence level) in the yields obtained by the CCE method in 48 h and by the modified IHSS method in 4 days. In addition, after these time period by the respective methods, there was no elution/extraction of HS as checked by reading the absorbances of the respective solutions. Therefore, that yield has been considered as the EHA.
Comparison of extraction efficiency of the modified IHSS method and CCE method by the spike recovery test
Recovery test was conducted to compare the extraction efficiency of the CCE method with the modified IHSS method using previously purified HA spiked with VC. Since there was no standard reference available to substitute the VC, the untreated VC was taken as the reference source material to ensure similar matrix environment for the spiked HA. At both the spiked amounts tried, percentages of HA recovered by the CCE method were higher as compared to those recovered by the modified IHSS method at 24 and 48 h (Table 2). Though the base level HA concentration was substantial (which is EHA itself), the increase in the percentage of recovery at the higher percentage of spiked amount of HA may be due to supramolecular nature of HA (Piccolo 2002 Adv. Agron. 75, 57-134; Sutton and Sposito, 2005; van Zomeren and Comans, 2007). The differences in the spike recovery between 24 and 48 h was narrower in the CCE method as compared to the IHSS method and converged to 100% in 48 h by the former method indicating better extraction efficiency of this method as compared to the modified IHSS method.

Table 2

Table 3 provides the results of elemental analysis of HA extracted from vermicomposted organic waste extracted by the present invention (CCE method) with the modified IHSS method. The percentages of C, H, N and molar ratios of H/C and N/C were found to be within the range for HA reported in the prior art. Thus there was no difference in composition.

Table 4 provides the FT-IR spectra of the HA extracted from vermicompost by the present invention (CCE method) with the modified IHSS method showed similar absorptions.

Advantages of the CCE method
Based on the afore-discussed comparative studies, it is evident that the present invention provides an improved extraction method which has quantitative yields of almost 100% HA in 48 hours at ambient temperature. Continuous elution of HS from VC and subsequent flocculation by adding HCl preserved the molecular integrity of HA without requiring an inert atmosphere. The CCE method can be operated overnight without manning which was one of the major advantages of this extraction protocol. The extraction time mentioned in the IHSS methods were only the times required for solubilization excluding the time required for separation of solubilized HS. Whereas, in the CCE method, no additional steps for separation of solubilized HS was required and thus effective extraction time was also significantly shorter.
The present method is thus easy to scale up in an energy and cost efficient manner.
,CLAIMS:1) A process for the extraction of humic acids from a humic acids source, wherein said process is based on a column-based continuous elution, said process comprising steps of:
a) treating said source with an acid for decalcification followed by neutralization using an alkali;
b) mixing the treated source resulting from the step (a) with an extractant/mobile phase forming a slurry of said humic acid source;
c) treating slurry resulting from the step (b) through the column-based continuous elution , wherein said slurry is the solid phase and an alkali is the extractant/mobile phase, to obtain an elute;
d) treating said elute resulting from the step (c) with an acid for flocculation of said humic acids; and
e) performing washes of the humic acids flocculate resulting from step (d) with a salt solution, an acid, and water until said flocculate is free of chloride ions followed by a further wash to obtain said humic acids.
2. The process as claimed in claim 1, wherein said humic acids source is a solid source.
3. The process as claimed in claim 2, wherein said solid source is natural organic matter from soil, compost, vermicompost, geological deposits such as lignite, leonardite, peats, brown coals and shales and like matter containing humic substances.
4. The process as claimed in claim 3, wherein said vermicompost is prepared from a mixture of agricultural crop residues, weeds conditioned with cow dung, earthworm and like.
5. The process as claimed in claim 1, wherein said acid is hydrochloric acid.
6. The process as claimed in claim 1, wherein said alkali is selected from potassium hydroxide and sodium hydroxide.
7. The process as claimed in claim 6 wherein said alkali is sodium hydroxide.
8. The process as claimed in claim 7, wherein said alkali is present in a concentration between 0.1M and 0.2M.
9. The process as claimed in claim 1, wherein said salt solution is selected from potassium chloride, sodium chloride and the like.
10. The process as claimed in claim 1, wherein said further wash is carried out with ethanol-water, methanol-water or ethylacetate-water.