USING THE TECHNIQUE OF THREE PHASE PARTITIONING (TTP) TO

ANALYSE AND OBSERVE CHANGES OF LEGUME SEED PROTEIN WITH

THE ADDITION OF INCREASING % CONCENTRATIONS OF
AMMONIUM SULPHATE.

ABSTRACT
The three-phase partitioning technique was used to analyse legume seed proteins. Three
phases of t butanol, legume seed protein and acetate buffer was seen at the end after the
addition of ammonium sulphate (NH2)4SO4 and centrifugation.
The legume seed protein concentration was measured at 280nm with a range of additions
of ammonium sulphate at % concentrations of 20%, 30% and 40%.
The absorbance measurements at 260nm were also recorded in table 1 and using the ratio
of A280/A260 under the principle of the Wartburg Christian correcting ratio for proteins. The
ratio of a pure protein is 1,7. The ratios of proteins for 20%, 30% and 40% were 1.7, 1.4 and
1.6 respectively.
The protein concentration was calculated using the Beer-Lambert law using an extinction
coefficient of 1.25 and an absorbance measured at 280nm. The protein concentrations
measured for 20%, 30% and 40% were 7.28 mg/ml, 9.68 mg/ml and 17.84 mg/ml
respectively.
This shows that as an increased amount of ammonium sulphate is added to sample, a larger
amount of protein concentration is being precipitated out of solution.

RESULTS
Table 1: Values of absorbance at A260 and A280 measured of samples containing (NH2)4SO4
with masses added and ranging % concentrations from 20%-40% with a dilution factor of
100 as well as the corresponding Legume seed protein concentrations and Wartburg
Christian Correction ratio. Centrifuged samples of protein, acetate buffer and t butanol
containing a range of (NH2)4SO4 % concentrations at 20%, 30% and 40% with particular
masses were analysed using a spectrophotometer to obtain absorbance readings at 260nm
and 280nm. Using the absorbances at 260nm and 280nm, the Wartburg Christian correction
ratio was calculated using A280nm/A260nm to show the purity of the protein. A pure protein
has a value of 1.7. The protein concentration of these samples was calculated using the
Beer-Lambert law with A280 values and an extinction coefficient of 1.25 and thereafter
multiplied by 100 considering the dilution since only 20μl of the sample was used. (
Legend)
Diluted sample Mass of A260 A280 Legume seed protein Wartburg Christian Correction
containing % of (NH2)4SO4 Concentrations using Ratio (280nm/260nm)
(NH2)4SO4 added (g) A280 (mg/ml)
20 1.25 0.055 0.091 7.28 1.7
30 1.88 0.087 0.121 9.68 1.4
40 2.50 0.143 0.223 17.84 1.6

Calculations: Protein concentration using the Beer-Lambert Law.

A=ε×c×l
Where,

A = absorbance (280nm)

ε = absorption coefficient (1,25)

c = concentration of protein

l = path length of cuvette (1cm)

20%: A = ε × c × l
: 0.091 = 1.25 × c × 1
c = 0.0728 mg/ml × 100 (because of dilution)
= 7.28 mg/ml
30%: A = ε × c × l
: 0.121 = 1.25 × c × 1
c = 0.0968 mg/ml × 100 (because of dilution)
= 9.68 mg/ml
40%: A = ε × c × l
: 0.223 = 1.25 × c × 1
c = 0.1784 mg/ml × 100 (because of dilution)
= 17.84 mg/ml
RESULTS WRITTEN SECTION:
In table 1 the centrifuged samples of legume seed protein, acetate buffer, t butanol and a
range of % concentrations of (NH2)4SO4 were diluted 1/100 times and then analysed by
measuring the absorbance of the samples using a spectrophotometer at 260nm and 280nm.
For the sample labelled 20%, 1.25g of (NH2)4SO4 was added and centrifuged. The
absorbance reading of the protein layer at 260nm and 280nm was 0.055 and 0.091
respectively. The Wartburg Christian correction ratio was calculated to be 1.7 which is
extremely accurate in terms of its purity being a protein, as pure proteins have a ratio of 1.7.
The legume seed protein concentration was calculated using the Beer-Lambert law at
280nm with an extinction coefficient of 1.25 and absorbance of 0.091. Because the sample
was diluted as only 20μl was used, the protein concentration was calculated to be 7.28
mg/ml.
For the sample labelled 30%, 1.88g of (NH2)4SO4 was added and centrifuged. The
absorbance reading of the protein layer at 260nm and 280nm was 0.087 and 0.121
respectively. The Wartburg Christian correction ratio was calculated to be 1.4 which differs
slightly from the preferred ratio of 1.7 for a pure protein. Considering the 1/100 dilution the
legume seed protein concentration using the absorbance value of 0.121 at 280nm was
calculated to be 9.68 mg/ml using the Beer-Lambert law. The extinction coefficient used was
1.25.
For the sample labelled 30%, 1.88g of (NH2)4SO4 was added and centrifuged. The
absorbance reading of the protein layer at 260nm and 280nm was 0.143 and 0.223
respectively. The Wartburg Christian correction ratio was calculated to be 1.6 which is close
to the preferred ratio of 1.7 for a pure protein. Considering the 1/100 dilution the legume
seed protein concentration using the absorbance value of 0.223 at 280nm was calculated to
be 17.84 mg/ml using the Beer-Lambert law. The extinction coefficient used was 1.25.
In table 1, it can be seen that as more (NH2)4SO4 was added to the sample, the protein
concentration was calculated to be a higher amount. As the concentration of (NH 2)4SO4 was
increased, the protein concentration is increased. This can be seen for 20%, 30% and 40%
with protein concentrations of 7.28 mg/ml, 9.68 mg/ml and 17.84 mg/ml respectively. It can
be deduced that as more (NH2)4SO4 was added to the sample it caused more protein to
precipitate out of solution and hence a higher concentration.
DISCUSSION
 In this experiment, the process of three phase partitioning was used to ultimately
get 3 phases of t butanol, legume seed protein and acetate buffer and then examine
the concentration of legume seed protein present in different concentration ranges
of (NH2)4SO4 at 20%, 30% and 40%.
 Samples of 20g of lentils soaked in 70ml od ABB solution was provided. The samples
were homogenized using a mortar and pestle, acid washed sand and a little bit of
buffer.
 The homogenate was filtered through cheesecloth and was then centrifuged in 15ml
centrifuge tubes (3 tubes were used) at 1000 x g for 5 minutes. 5ml aliquots were
separated into 3 centrifuge tubes each labelled 20, 30 and 40.
 Ammonium sulphate was added to the 3 tubes and the masses added are listed in
table 1. These made up the % concentrations of 20%, 30% and 40%.
 Ammonium sulphate was added so that proteins could be precipitated out of
solution when centrifugation at 100 x g at 5 minutes occurred. The different
concentrations of ammonium sulphate added indicated the amount of proteins that
would be eventually precipitated out.
 As seen in table 1, the above statement is proven as calculations using the Beer-
Lambert law, extinction coefficient of 1,25 and absorbance values measured at
280nm shows that as the amount of ammonium sulphate added is increased, the
protein concentration increases accordingly.
 This proves that ammonium sulphate is an agent that can precipitate proteins out of
solution and more protein can be precipitated out if lager concentrations of
ammonium sulphate is used.
 The absorbance values at A260 were also measured so that the purity of the protein
could be known using the Wartburg Christian correction ratio as seen in table 1.
 T butanol was used in this experiment because it is miscible in water but can form a
different phase and be hydrated when the addition of a salt like ammonium sulphate
is added.
 Another experiment that can be compared to this technique is the experiment on
fractional precipitation (salting out) where ammonium sulphate was also used to
precipitate proteins.
 Alternate ways to analyse the data would be to use factors such as pH, temperature
and solubility of proteins.