Measurement of Potassium
in Unashed Plant Leaves 1
This procedure uses a TCA (trichloroacetic acid) extraction of the tissue to release all of the calcium.
It was found that there was no significant interference
from sodium, potassium or phosphate present in a
sample at normal physiological levels. Calcium concentration in the range of 0.5 – 3.0 μg Ca / 100mg sample is readily determined.
Solution and Standard Preparation
Deionised distilled water is used throughout to prepare the following solutions:
Organic Diluting Fluid containing 500ml isopropyl alcohol, 300 ml water, 0.2 ml Non-Ion-Ox (ex. Aloe Scientific Co., St. Louis, Mo. USA), 74.6 mg KCl and 292 mg NaCl. TCA solution for tissue extraction was 6.2% w/v and for standard preparation was 8.3% w/v and also contained 19.5 mg NaCl / 100ml.
6.25% trichloroacetic acid solution is first prepared. To do this accurately weigh out 6.25g of trichloroacetic acid and add it to a 100ml volumetric flask, then make up to the mark with deionised or milliq water. To make up 8.3% w/v TCA solution, 8.3g of TCA was dissolved in the same manner.
Primary Calcium Standard Solution was prepared by dissolving 250 mg CaCO3 in 25ml of 2M HCl and diluted to 1l to give 100 μg Ca / ml Standards and reagent blanks containing 0 – 3 μg Ca /ml were prepared by mixing 80ml of Organic Diluting Fluid, 15ml of 8.3% TCA, 0 – 3ml of Primary Calcium Solution and deionised water to a final volume of 100ml. When carrying out analysis and calibration, you will be prompted to either analyse or skip blank analysis prior to calibration and sample measurement. The Organic Diluting Fluid prepared previously should be run as the blank for this analysis.
Prepare a Slice of tissue approximately 0.5mm thick. Quickly dip the tissue slice in a calcium-free incubation solution and carefully blot on filter paper. Accurately weigh, to yield samples in the range of 100 mg wet weight.
Transfer sample to centrifuge tube, add sufficient 6.2% TCA is give a volume of 2 ml. Vigorously shake the tube 3 times over a 30 minute period.
Add a volume of 8 ml of Organic Diluting Fluid to the tube and centrifuge. The supernatant is analysed directly.
Preparation of Standard Graph
Set the flame photometer in accordance to MultiPoint/Single Ion Calibration found on page 24 of the BWB Technologies Installation and Operation Manual, to measure potassium emission. Nebulise the working standard solutions and adjust the controls until steady zero and maximum readings are obtained. Nebulise the intermediate working standard solutions and construct a graph relating raw emission data (known as RAW in BWB the flame photometer) to concentration of all the standard solutions.
1 Geyer, R.P. and Bowie, E.J., ‘The Direct Microdetermination of Tissue Calcium by Flame Photometry’, Anal. Biochem., 2, (1961), 360-369.