DETERMINATION OF ANIONIC CONTENT
REAGENTS:1. 0.004M Hyamine Solution.
2. Chloroform
3. Methylene Blue Indicator solution.
PROCEDURE:1. Weigh about 2.0-3.0 gm of sample in Beaker Add in it 0.5 to 1.0
gm of nonionic surfactant and 2-3 ml of methanol.(In case of water
dispersible surfactant) Mix well and add 100 ml of distilled water.
2. Dilute this solution to 500ml Volumetric flask.
3. Take 10 ml of above diluted solution by pipette in measuring
cylinder . Add to it 25ml methylene blue indicator,15ml chloroform
shake well.
4. Titrate against 0.004M Hyamine solution till color of the two layers
matches perfectly.
5. Note the Burette Reading.
CALCULATIONS:B.R. X 5 X mol. wt. of Anionic X Molarity of Hyamine
Active Matter =
Weight of Sample.
B.R. = Burette Reading.
SDBS (Mol. Wt.= 349)
DETERMINATION OF NON-IONIC CONTENT
(SPREADFAST)
Apparatus:1.
2.
3.
4.
Petri dish.
Pin
Desicator
Laboratory Oven
Method:1. Weigh an empty Petri dish along with a pin.
2. Weigh accurately about 2-2.5 gms of the material into Petri
dish.(diameter 9.6 cm, height 1.5 cm.)
3. Spread the sample in the Petri dish with the pin.
4. Keep it in oven at 105-110 oC for 2 hrs.
5. Remove Petri dish and spread the sample again and keep in
the oven for further 30 min.
6. Remove the Petri dish from the oven and then keep it in a desicator
for 30 min. Weigh the dish till constant weight is obtained.
Calculation:Wd x 100
% Active matter =
Ws
Wd = weight of the sample after drying
Ws = weight of the sample before drying.
Humic Acid Testing Method (HUMIGEL)
Procedure for determining humic acid content
.
Extraction Solution
In a 2l volumetric with 500ml of DI H2), add 80g NaOH, 8
gm(0.02 M) DTPA, 40ml ethanol and bring to volume.
.
Standard Stock Preparation (1000 ppm):
Dry Humic Acid (Aldrich) for 4 hours @ 105o C. Weigh out 0.1075
g of Humic Acid and dilute to 100ml with extraction solution.
Shake for 1 hour.
.
Standard Preparation
Take 5ml of Humic Acid Standard Stock Solution and dilute to
100ml with DI H20 to make 50 ppm standard solution. Take 10ml
and dilute to 100mlL to make 100 ppm standard. Take 20ml and
dilute to 100ml to make 200 ppm standard.
.
Sample Preparation
Weigh out 2g of dry sample and add sufficient extraction solution
to dissolve entire sample. Mix sample well. Centrifuge an aliquot of
the sample to remove any particulates. Dilute sample as necessary
so it falls within the range of the standard curve.
.
Test Procedure
With Spectrophotometer set at 450 nm, set up standard curve with
the 50, 100 and 200 ppm standards, using water as a blank. Read
your sample and calculate concentration from the standard curve.
.
Calculations
(ppm Humic Acid from standard curve) X (dilution factor)
= ppm Humic Acid
% Humic Acid = ppm Humic Acid / 10,000
DTPA = Diethylenetriamine Penta Acetic Acid
Determination of Brassinolide content by HPLC
PRINCIPLE:- Brassinolide is dissolved in acetonitrile and analyzed by HPLC method at
222 nm.
APPARATUS
High Performance Liquid Chromatograph
A HPLC unit equipped with UV detector and Software. It is operated with following
parameters.
1) Column:- C-18, 25 cm x 4.6 mm SS,5 micron particle size or equivalent.
2) Flow Rate:- 0.8 to 1 ml/min. approx.
3) Detector:- Ultraviolet (UV).
4)Wavelength:- 222 nm
5)Mobile Phase:- Acetonitrile : water (62:38) v/v
6)Retention Time:- 7-8 min.(Approx.)
Micro syringe:- 1 ml Capacity.
Reagents:Reference Standard Brassinolide:- of purity 95 %. Min.
Acetonitrile:- HPLC grade
Water:- HPLC grade
Volumetric flask;- 1 ml & 10 ml
Pipettes:- Graduated.
Preparation of Reference Standard Solution:Weigh accurately 1mg of standard Brassinolide of known purity into 10 ml volumetric
flask and dissolve in Acetonitrile and dilute up to the mark. Pipette out 1 ml of this stock
solution into 100 ml volumetric flask and make up the volume with Acetonitrile.
Preparation of sample solution:Weigh accurately sample equivalent to 1 mg of BR content into 10 ml volumetric flask.
Add about 7 ml of acetonitrile and shake for 5 min. and make up the volume with
acetonitrile. Pipette out 1 ml of solution into a 100 ml volumetric flask and make up the
volume with acetonitrile.
Estimation:- Inject 20 micro liter of standard brassinolide And sample solution resp. to
get area of two consecutive injections should not vary more than 2%. From HPLC
chromatogram calculate Brassinolide content as follows:
Brassinolide content
Percent by mass = A1/A2 x m2/m1 x P
Where
A1= peak area of Brassinolide in sample solution
A2= peak area of Brassinolide in reference standard solution
m1= mass, in gram of the sample taken for the test.
m2= mass in gram of the reference standard Brassinolide.
P = Purity of reference standard Brassinolide.