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Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006


                Recovery of Chemicals from Pressmud –
                       A Sugar Industry Waste
                                                 N. Partha and V Sivasubramanian
                                                                .
                                       Department of Chemical Engineering, Anna University, Chennai 600 025




                      Paper received : 11.4.06                                              Revised paper accepted: 19.7.06



Indian sugarcane crop cultivation forms an important part of                  industry has been equally impressive. The distilleries
the Indian agricultural economy. Production of sugar has                      in their turn have led to the increased availability of
shown a phenomenal increase in the last 65 years. One of                      effluent, commonly termed as ‘spent wash’. The
the byproducts of sugar industry is pressmud, a solid residue,
                                                                              major waste products from sugar processing and
obtained from sugarcane juice before crystallization of sugar.
                                                                              alcohol manufacture are:
Generally pressmud is used as manure in India. The aim of
the present investigation is to recover protein, sugar and wax                1.    Cane residues left in the field after cane
from pressmud. The amount of protein is estimated to be                             harvesting.
3.3%. The percentage of sugar is about 0.8%. Extraction of
                                                                              2.    Bagasse from the cane crushing operation.
wax by solvent has resulted in a recovery of about 12%.
                                                                              3.    Pressmud from the cane juice filtration.
                                                                              4.    Spent wash from distilleries.



T
         he two main sources of sucrose are the                                    Bagasse finds profitable use in the paper
        sugarcane and the sugar beet. Sugarcane                               industry for the manufacture of wrapping and
        is tall grass having a number of bamboo-like                          packing. Certain mills also manufacture paper by
stems, which grows to a height of 4 meters or more.                           employing suitable blends of conventional pulp with
The sugar content in sugarcane is about 10 to 15 per                          that obtained from bagasse. Its use is encouraged by
cent. As regards the sugar beet, the juice of the beet                        the government by providing subsidies to the
contains sugar identical to that found in cane. The                           manufacturers. Bagasse is also extensively used as a
sugar beet is found to contain 5 to 6 per cent of                             convenient source of fuel in mill boilers, often
sucrose. In improved varieties, a yield of 13 to 17                           resulting in availability of excess energy. The other
percent is common and in this respect it compares                             cane residues could probably double the amount of
favourably with sugarcane. [1]                                                energy availability. Table 2 describes the number of
                                                                              factories in operation, their crushing capacity, area,
Indian Scenario
                                                                              yield, cane and sugar production since the inception
Cane crop cultivation and production of sugar has
                                                                              of sugar industry and their contribution to the Indian
shown a phenomenal increase in India over the last
                                                                              economy.
60 years, since the industry started in an organized
manner [2]. The detailed figures are given in Table
1.                                                                                         Table 2 : Factories in Operation
     The increase in sugarcane and sugar production
                                                                               Sugarcane                           From 36 million tons in
has inevitably resulted in increased quantity of waste                                                             1930 to 296 million
products. It is well known that molasses has found                                                                 tonnes in 1999.
very useful applications in the manufacture of                                 Yield per hectare                   From 31 tons in 1930 to
                                                                                                                   72.5 tonnes in1999.
ethanol and potable spirits. The growth of this
                                                                               Area under cultivation              From 1.2 million
              Table 1 : Cane Crop Cultivation                                                                      hectares in 1930 to 4.1
                                                                                                                   million hectares in
 Sugarcane                      From 35M tonnes in 1930 to                                                         1999.
                                296M tonnes in 1999.                           No. of sugar industries             550
 Yield/hectare                  From 31 tonnes/hectare in 1930                 Total cane production               220 million tonnes per
                                to 72.5 tonnes/hectare in 1999.                                                    year.
 Area under cultivation         From 1.2M hectares in 1930 to                  Total cane available fro crushing   135 million tonnes per
                                4.1M hectares in 1999.                                                             year.
 Sugar production               From 0.1M tonnes in 1930 to                    Total sugar production              13.5 million tonnes per
                                15.5M tonnes in 1999.                                                              year.

160
Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006

Pressmud                                                       free from all pathogens, harmful bacteria, weeds
Pressmud from the sugar mills is a very useful source          and seeds. Fertilizer is free flowing, easy to
of fertilizer as well as some chemicals. The major             handle, to pack and transport. The biocompost
use that has recently been developed in India is in            contains 25-30% organic carbon, 1.2-2.0%
biocomposting (usually trade named as Bioearth)                nitrogen, 1.5-2.0% phosphorous and 2.5-3.0%
where it is treated with the spent wash from the               potash.
distillery [3]. The composition of pressmud is given            The shortcoming of this operation is the limited
in Table 3. Its usefulness as fertilizer is based on the   period of operation of sugar plants. The pressmud
nutrient content of the mud and the spent wash as          has to be stored in large open areas and large lagoons
shown below:                                               are to be set up to store the spent wash. However,
     Pressmud – N- 1.15 to 3.0%; P- 0.60 to 3.50%          the long-term effects of application of this fertilizer
     and K- 0.30 to 1.80%.                                 remain to be studied.
     Spent wash – N-2630 mg/l; P- 201 mg/l and             Chemicals from Pressmud
     K- 222 mg/l.                                          Pressmud can be a useful source of chemicals. Work
     The bio-compost is produced by spraying spent         has been done at the laboratories of the Anna
wash on stacks of pressmud called windrows. The            University at Chennai to recover the chemicals. It is
period required to produce usable fertilizer varies        to be noted that the total quantity of pressmud from
with the process used. A typical 45-day process            the sugar industry in India could be as much as 35,000
includes the following steps:                              tonnes per day. [4]
1. Formation of windrows and reduction of moisture         Experimental
   content from 70% to 50% in five days.                   Recovery of Protein
2. Inoculation of microbial culture.                       Protein being soluble in water, double distilled water
3. Spraying of spent wash and homogenization of            was used to separate it from pressmud. Protein was
   windrows for 30 days.                                   separated from aqueous phase by precipitation with
4. Maturation period of 10 days to reduce moisture         ammonium salts. A Hitachi, U-2000, double beam
   to 30%.                                                 UV visible spectrophotometer was used to measure
The benefits that accrue are:                              the absorbance. Estimation of protein was done by
1.   Very low power requirement.                           Lowry’s method using sodium carbonate, sodium
2.   Zero discharge to inland water resourses and          potassium tartarate and ammonium salts.
     freedom from river or ground water pollution.         Recovery of Sugar
3.   Organic fertilizer produced is rich in micro-         Sugar left in the aqueous phase after the recovery of
     nutrients and can reduce the requirement of           proteins was recovered by evaporation. A Hitachi,
     chemical fertilizers. It also provides bacteria for   U-2000, double beam UV visible spectrophotometer
     nitrogen fixing, solubilisation of phosphates,        was used to measure the absorbance of the sugar
     humus that will keep the soil healthy and             solution using anthrone reagent.
     develop the self-reclamation cycle.                   Recovery of Wax
     Since biocomposting is accompanied by a rise in       Several solvents such as benzene, toluene and hexane
     temperature, chances are that the fertilizer is       were tried for recovery of wax from the solids left
                                                           behind after the recovery of proteins and sugar. Crude
                                                           wax was separated from the wax-laden solvent by
          Table 3 : Composition of Pressmud
                                                           deep freezing.
           Crude wax                 5-14%                 Results
           Fiber                     15-30%                Effect of pH on Protein Recovery
           Crude Protein             5-15%
                                                           The percentage recovery of protein, sugar and wax
           Sugar                     5-15%
                                                           from pressmud is given in Table 4. The amount of
           SiO                       4-10%                 protein present in pressmud was about 3.3%. The
           CaO                       1-4%                  percentage recoveries of protein at different pH levels
           PO                        1-3%                  are shown in Table 5. From the table, it was found
           MgO                       0.5-1.5%              that percentage recovery of protein increased as pH
           Total ash                 9-10%                 of the pressmud solution increased. The maximum
                                                           amount of protein, i.e., 3.3% was recovered at a pH
                                                                                                                          161
Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006

            Table 4 : Recoeries from Pressmud
                                                                                      3

                Protein                       3.3 %

                Sugar                         0.8 %                                   2




                                                                              E (g)
                Wax                           12 %


        Table 5 : Percentage Recovery of Protein                                      1

                                                                                                                           0 . 1 m m p a r t i c le
                   pH                   %protein                                                                           0 . 5 m m p a r t i c le

                   5.5                       2.59                                     0
                                                                                             0       100    200     300       400                 500
                   6.0                       2.62                                                            Time (min)
                   7.0                       3.00                             Fig. 1 : Extraction of Wax by Solvent Toluene
                   8.0                       3.13
                                                                                         2
                   8.5                       3.30



of 8.5. The sugar left after the separation of proteins



                                                                                 E (g)
was recovered by evaporation. The percentage of                                          1
sugar was about 0.8% in pressmud.
Model for Wax Recovery                                                                                                     0.1 mm particle

The wax recovered from pressmud was about 12%.                                                                             0.5 mm particle
                                                                                         0
It had a melting point of 79°C and a density of 0.96.
                                                                                             0       100    200     300        400                500
The following mathematical expression was used to                                                            Time (min)
model the recovery of wax [5] :                                                           Fig. 2 : Extraction of Wax by Solvent
                                                                                                   Toluene and Benzene
                      E=Emax θ/(θ+k)                              ... 1
                                                                                             4

Where, Emax and k are characteristic constants for
                                                                                             3
given system, q represents the time of extraction and
E represents the weight of wax. Fig. 1-3 show the
                                                                                  E (g)




                                                                                             2
recovery of wax in weight (g) as time proceeds using
the solvents toluene, toluene and benzene (50%) and
                                                                                             1
hexane, respectively. The maximum recovery of wax                                                                          0.1 mm particle
(0.1 mm particle size) was 2.7, 1.58 and 2.9 using                                                                         0.5 mm particle
                                                                                             0
toluene, toluene and benzene (50%) and hexane
                                                                                                 0    100    200     300         400                  500
respectively. The maximum recovery of wax (0.5 mm                                                             Time (min)
particle size) was 1.2, 1.64 and 2.78 using toluene,
                                                                               Fig. 3 :Extraction of Wax by Solvent Hexane
toluene and benzene (50%) and hexane respectively.
The maximum quantity of wax was recovered by
                                                                          Conclusion
using hexane as solvent. The results are reported in
Table 6.                                                                  In conclusion, one could positively derive wealth from
                                                                          the sugarcane and sugar industry wastes. Studies are
                  Table 6 : Emax and Values                               also done to model the Indian sugar industry with
                                                                          precise mathematical approaches and to forecast its
  SYSTEM                                Emax (g)        K (min)           performance in future. The amount of protein was
  Toluene (0.1 mm)                        6.67        0.17
                                                                          estimated to be 3.3%. The percentage of sugar was
                                                                          about 0.8%. Extraction of wax by solvent resulted in
  Toluene (0.5 mm)                        3.33        0.17
                                                                          a recovery of about 12%.
  Toluene + Benzene (0.1 mm)              4.00        0.56
  Toluene + Benzene (0.5 mm)              2.50        0.25                Acknowledgement

  Hexane (0.1 mm)                         7.23         0.8                The authors acknowledge with thanks the South Indian Sugar
                                                                          Research Foundation for providing pressmud and other sample
  Hexane (0.5 mm)                         3.87        0.79
                                                                          materials for carrying out this investigation.

162
Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006

References                                                      4.   Partha, N., and Krishnan, M.R.V.,: Pressmud for
1.   Hogot, E.,: Handbook of Cane Sugar Engineering, 3rd             Chemicals/fine chemicals, Kisan World.27, 11:35, (2000).
     completely revised ed., Elsevier Publishing Company, NY,   5.   Jacobs, M.B., and Scheflan, L.,: Chemical Analysis of
     U.S.A., (1986).                                                 Industrial Solvents, Interscience publishers, New York,
2.   Mathur, R.B.L.,: Handbook of Cane Sugar Technology, 1 st        U.S.A., (1953).
     print, Oxford and IBH Publishing Company, New Delhi,       Nomenclature
     India, (1975).                                             Emax Characteristic constant for given system
3.   Padmanabhan, P Rakkiyappan, P and Alexander, K.C.,:
                     .,              .,                         k     Characteristic constant for given system
     Enrichment of Pressmud by Microorganisms, Sugarcane        q     Time of extraction, min
     Breeding Institute Extension, Publication No.39, (1993).   E     Weight of wax, g



APPENDIX

Estimation of Protein                                           Estimation of Sugar
Estimation of protein was done by Lowry’s method.               For estimation of sugar, anthrone reagent was used.
The principle behind the method is the aromatic                 The concentration of sugar was estimated with the
amino acids, tyrosine and trytophan present in the              help of a spectrophotometer.
protein reacts with Folin-Phenol reagent (contains
                                                                Estimation of Wax
phosphomolybdic acid and tungstate) and produce a
dark blue colour. The reagents prepared were sodium             After the removal of proteins and sugar from the
carbonate solution, sodium potassium tartarate                  aqueous solutions, the remaining wax was repeatedly
solution and Folin-Phenol reagent. Standard protein             extracted with solvent hexane. Crude wax was
solution was pipetted out into clean test tubes.                separated from the wax laden solvent by chilling. The
Reagents were mixed and the blue colour at 650 nm               resulting wax was dried and weighed. The melting
was read in a spectrophotometer. From the standard              point of wax was found to be 79°C and specific gravity
graph, the concentration of unknown was obtained.               0.96.




                                                                                                                                163

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Recovery of Valuable Chemicals from Sugar Industry Waste

  • 1. Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006 Recovery of Chemicals from Pressmud – A Sugar Industry Waste N. Partha and V Sivasubramanian . Department of Chemical Engineering, Anna University, Chennai 600 025 Paper received : 11.4.06 Revised paper accepted: 19.7.06 Indian sugarcane crop cultivation forms an important part of industry has been equally impressive. The distilleries the Indian agricultural economy. Production of sugar has in their turn have led to the increased availability of shown a phenomenal increase in the last 65 years. One of effluent, commonly termed as ‘spent wash’. The the byproducts of sugar industry is pressmud, a solid residue, major waste products from sugar processing and obtained from sugarcane juice before crystallization of sugar. alcohol manufacture are: Generally pressmud is used as manure in India. The aim of the present investigation is to recover protein, sugar and wax 1. Cane residues left in the field after cane from pressmud. The amount of protein is estimated to be harvesting. 3.3%. The percentage of sugar is about 0.8%. Extraction of 2. Bagasse from the cane crushing operation. wax by solvent has resulted in a recovery of about 12%. 3. Pressmud from the cane juice filtration. 4. Spent wash from distilleries. T he two main sources of sucrose are the Bagasse finds profitable use in the paper sugarcane and the sugar beet. Sugarcane industry for the manufacture of wrapping and is tall grass having a number of bamboo-like packing. Certain mills also manufacture paper by stems, which grows to a height of 4 meters or more. employing suitable blends of conventional pulp with The sugar content in sugarcane is about 10 to 15 per that obtained from bagasse. Its use is encouraged by cent. As regards the sugar beet, the juice of the beet the government by providing subsidies to the contains sugar identical to that found in cane. The manufacturers. Bagasse is also extensively used as a sugar beet is found to contain 5 to 6 per cent of convenient source of fuel in mill boilers, often sucrose. In improved varieties, a yield of 13 to 17 resulting in availability of excess energy. The other percent is common and in this respect it compares cane residues could probably double the amount of favourably with sugarcane. [1] energy availability. Table 2 describes the number of factories in operation, their crushing capacity, area, Indian Scenario yield, cane and sugar production since the inception Cane crop cultivation and production of sugar has of sugar industry and their contribution to the Indian shown a phenomenal increase in India over the last economy. 60 years, since the industry started in an organized manner [2]. The detailed figures are given in Table 1. Table 2 : Factories in Operation The increase in sugarcane and sugar production Sugarcane From 36 million tons in has inevitably resulted in increased quantity of waste 1930 to 296 million products. It is well known that molasses has found tonnes in 1999. very useful applications in the manufacture of Yield per hectare From 31 tons in 1930 to 72.5 tonnes in1999. ethanol and potable spirits. The growth of this Area under cultivation From 1.2 million Table 1 : Cane Crop Cultivation hectares in 1930 to 4.1 million hectares in Sugarcane From 35M tonnes in 1930 to 1999. 296M tonnes in 1999. No. of sugar industries 550 Yield/hectare From 31 tonnes/hectare in 1930 Total cane production 220 million tonnes per to 72.5 tonnes/hectare in 1999. year. Area under cultivation From 1.2M hectares in 1930 to Total cane available fro crushing 135 million tonnes per 4.1M hectares in 1999. year. Sugar production From 0.1M tonnes in 1930 to Total sugar production 13.5 million tonnes per 15.5M tonnes in 1999. year. 160
  • 2. Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006 Pressmud free from all pathogens, harmful bacteria, weeds Pressmud from the sugar mills is a very useful source and seeds. Fertilizer is free flowing, easy to of fertilizer as well as some chemicals. The major handle, to pack and transport. The biocompost use that has recently been developed in India is in contains 25-30% organic carbon, 1.2-2.0% biocomposting (usually trade named as Bioearth) nitrogen, 1.5-2.0% phosphorous and 2.5-3.0% where it is treated with the spent wash from the potash. distillery [3]. The composition of pressmud is given The shortcoming of this operation is the limited in Table 3. Its usefulness as fertilizer is based on the period of operation of sugar plants. The pressmud nutrient content of the mud and the spent wash as has to be stored in large open areas and large lagoons shown below: are to be set up to store the spent wash. However, Pressmud – N- 1.15 to 3.0%; P- 0.60 to 3.50% the long-term effects of application of this fertilizer and K- 0.30 to 1.80%. remain to be studied. Spent wash – N-2630 mg/l; P- 201 mg/l and Chemicals from Pressmud K- 222 mg/l. Pressmud can be a useful source of chemicals. Work The bio-compost is produced by spraying spent has been done at the laboratories of the Anna wash on stacks of pressmud called windrows. The University at Chennai to recover the chemicals. It is period required to produce usable fertilizer varies to be noted that the total quantity of pressmud from with the process used. A typical 45-day process the sugar industry in India could be as much as 35,000 includes the following steps: tonnes per day. [4] 1. Formation of windrows and reduction of moisture Experimental content from 70% to 50% in five days. Recovery of Protein 2. Inoculation of microbial culture. Protein being soluble in water, double distilled water 3. Spraying of spent wash and homogenization of was used to separate it from pressmud. Protein was windrows for 30 days. separated from aqueous phase by precipitation with 4. Maturation period of 10 days to reduce moisture ammonium salts. A Hitachi, U-2000, double beam to 30%. UV visible spectrophotometer was used to measure The benefits that accrue are: the absorbance. Estimation of protein was done by 1. Very low power requirement. Lowry’s method using sodium carbonate, sodium 2. Zero discharge to inland water resourses and potassium tartarate and ammonium salts. freedom from river or ground water pollution. Recovery of Sugar 3. Organic fertilizer produced is rich in micro- Sugar left in the aqueous phase after the recovery of nutrients and can reduce the requirement of proteins was recovered by evaporation. A Hitachi, chemical fertilizers. It also provides bacteria for U-2000, double beam UV visible spectrophotometer nitrogen fixing, solubilisation of phosphates, was used to measure the absorbance of the sugar humus that will keep the soil healthy and solution using anthrone reagent. develop the self-reclamation cycle. Recovery of Wax Since biocomposting is accompanied by a rise in Several solvents such as benzene, toluene and hexane temperature, chances are that the fertilizer is were tried for recovery of wax from the solids left behind after the recovery of proteins and sugar. Crude wax was separated from the wax-laden solvent by Table 3 : Composition of Pressmud deep freezing. Crude wax 5-14% Results Fiber 15-30% Effect of pH on Protein Recovery Crude Protein 5-15% The percentage recovery of protein, sugar and wax Sugar 5-15% from pressmud is given in Table 4. The amount of SiO 4-10% protein present in pressmud was about 3.3%. The CaO 1-4% percentage recoveries of protein at different pH levels PO 1-3% are shown in Table 5. From the table, it was found MgO 0.5-1.5% that percentage recovery of protein increased as pH Total ash 9-10% of the pressmud solution increased. The maximum amount of protein, i.e., 3.3% was recovered at a pH 161
  • 3. Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006 Table 4 : Recoeries from Pressmud 3 Protein 3.3 % Sugar 0.8 % 2 E (g) Wax 12 % Table 5 : Percentage Recovery of Protein 1 0 . 1 m m p a r t i c le pH %protein 0 . 5 m m p a r t i c le 5.5 2.59 0 0 100 200 300 400 500 6.0 2.62 Time (min) 7.0 3.00 Fig. 1 : Extraction of Wax by Solvent Toluene 8.0 3.13 2 8.5 3.30 of 8.5. The sugar left after the separation of proteins E (g) was recovered by evaporation. The percentage of 1 sugar was about 0.8% in pressmud. Model for Wax Recovery 0.1 mm particle The wax recovered from pressmud was about 12%. 0.5 mm particle 0 It had a melting point of 79°C and a density of 0.96. 0 100 200 300 400 500 The following mathematical expression was used to Time (min) model the recovery of wax [5] : Fig. 2 : Extraction of Wax by Solvent Toluene and Benzene E=Emax θ/(θ+k) ... 1 4 Where, Emax and k are characteristic constants for 3 given system, q represents the time of extraction and E represents the weight of wax. Fig. 1-3 show the E (g) 2 recovery of wax in weight (g) as time proceeds using the solvents toluene, toluene and benzene (50%) and 1 hexane, respectively. The maximum recovery of wax 0.1 mm particle (0.1 mm particle size) was 2.7, 1.58 and 2.9 using 0.5 mm particle 0 toluene, toluene and benzene (50%) and hexane 0 100 200 300 400 500 respectively. The maximum recovery of wax (0.5 mm Time (min) particle size) was 1.2, 1.64 and 2.78 using toluene, Fig. 3 :Extraction of Wax by Solvent Hexane toluene and benzene (50%) and hexane respectively. The maximum quantity of wax was recovered by Conclusion using hexane as solvent. The results are reported in Table 6. In conclusion, one could positively derive wealth from the sugarcane and sugar industry wastes. Studies are Table 6 : Emax and Values also done to model the Indian sugar industry with precise mathematical approaches and to forecast its SYSTEM Emax (g) K (min) performance in future. The amount of protein was Toluene (0.1 mm) 6.67 0.17 estimated to be 3.3%. The percentage of sugar was about 0.8%. Extraction of wax by solvent resulted in Toluene (0.5 mm) 3.33 0.17 a recovery of about 12%. Toluene + Benzene (0.1 mm) 4.00 0.56 Toluene + Benzene (0.5 mm) 2.50 0.25 Acknowledgement Hexane (0.1 mm) 7.23 0.8 The authors acknowledge with thanks the South Indian Sugar Research Foundation for providing pressmud and other sample Hexane (0.5 mm) 3.87 0.79 materials for carrying out this investigation. 162
  • 4. Indian Chemical Engr. Section A, Vol. 48, No.3, July-Sept. 2006 References 4. Partha, N., and Krishnan, M.R.V.,: Pressmud for 1. Hogot, E.,: Handbook of Cane Sugar Engineering, 3rd Chemicals/fine chemicals, Kisan World.27, 11:35, (2000). completely revised ed., Elsevier Publishing Company, NY, 5. Jacobs, M.B., and Scheflan, L.,: Chemical Analysis of U.S.A., (1986). Industrial Solvents, Interscience publishers, New York, 2. Mathur, R.B.L.,: Handbook of Cane Sugar Technology, 1 st U.S.A., (1953). print, Oxford and IBH Publishing Company, New Delhi, Nomenclature India, (1975). Emax Characteristic constant for given system 3. Padmanabhan, P Rakkiyappan, P and Alexander, K.C.,: ., ., k Characteristic constant for given system Enrichment of Pressmud by Microorganisms, Sugarcane q Time of extraction, min Breeding Institute Extension, Publication No.39, (1993). E Weight of wax, g APPENDIX Estimation of Protein Estimation of Sugar Estimation of protein was done by Lowry’s method. For estimation of sugar, anthrone reagent was used. The principle behind the method is the aromatic The concentration of sugar was estimated with the amino acids, tyrosine and trytophan present in the help of a spectrophotometer. protein reacts with Folin-Phenol reagent (contains Estimation of Wax phosphomolybdic acid and tungstate) and produce a dark blue colour. The reagents prepared were sodium After the removal of proteins and sugar from the carbonate solution, sodium potassium tartarate aqueous solutions, the remaining wax was repeatedly solution and Folin-Phenol reagent. Standard protein extracted with solvent hexane. Crude wax was solution was pipetted out into clean test tubes. separated from the wax laden solvent by chilling. The Reagents were mixed and the blue colour at 650 nm resulting wax was dried and weighed. The melting was read in a spectrophotometer. From the standard point of wax was found to be 79°C and specific gravity graph, the concentration of unknown was obtained. 0.96. 163