TITLE: Flame Photometry Protocol
AUTHORS: Mariam Awlia

[STEPS]
?. [Preparation of 1% nitric acid]
If stock is 70% nitric acid, pour  of MilliQ-water for every  of nitric acid. (x 21.4 for 1.5L).If stock is 69% nitric acid, pour  of MilliQ-water for every of nitric acid. (x 21.8 for 1.5L)Pour  nitric acid to  MilliQ-water.
Work in the fume hood.Wear lab coat, eye protection, gloves and lower screen in front of face.Always add acid to water slowly, never water to acid.
For 250 samples (250*5=1.25L), 1.5L of 1% nitric acid should be sufficient.
69 ml
1 ml
68 ml
1 ml
21.8 ml
1483.4 ml
?. [Preparation of dilutions]
Use 2 salt and 2 control Eppendorfs to test the dilutions and standards before doing the rest of the samples.Flip the falcon tube to remove condensation, before pipetting.
Prepare dilutions in fresh MilliQ-water and make sure to be extremely precise in all measurements.Prepare replicates for each sample, just in case.
Suggested dilutions:    Salt 1:20 (1.90ml H2O + 100μL sample)     Control 1:5 (1.6ml H2O + 400μL sample)Suggested standards: Salt 500μM NaCl, 100μM KCl                        Control 10-100μM NaCl, 1000μM KCl
Make sure to avoid carrying over any plant-fibre to the dilutions.
?. [Calibration]
Shake the standard solution bottle before use, then add around  to a beaker.
10 ml
?. [Calibration]
Set system to dual and peak only. Put in blank solution, press “Blank”, wait till “100” is flashing, then remove blank solution.
?. [Calibration]
Put in selected standard solution and press “Cal” for Na and K and when “100” flashes, press “Cal” again.
?. [Calibration]
Press “Measure” and wait till it blinks to measure Blank (should be 0±2) and Standard (should be 100±2).
?. [Calibration]
Start measuring Eppendorf samples and wipe the tube with tissue when changing solutions.
Correct measurements for Salt (20 - 120) and Control (10 - 20)
?. [Calibration]
Run standard as test every 12-24 Samples
When switching between standard solutions, press “Blank” for 3 seconds to restart calibration.
?. [Shut down and clean-up]
Make sure the running solution is still under the U-tube, so the flame photometer doesn’t dry out.
?. [Shut down and clean-up]
Turn gas valve off and wait until the error E61 is displayed and flame is out, then switch off and close air.Dispose of solution and plant material down sink with excess water.
?. [Preparation of samples]
Put all samples in  falcon tubes and determine fresh/dry weight of plant material.
10 ml
?. [Preparation of samples]
Add  of 1% nitric acid to  tubes of samples and close lids tightly.
3 ml
10 ml
?. [Preparation of samples]
Put in oven at  overnight.
70 °C
?. [Preparation of samples]
After , shake gently without breaking the rosette to cover all in nitric acid.
?. [Preparation of samples]
Remove samples and let them cool down on the bench before diluting.
?. [Operation of flame photometer]
Fill two beakers with fresh MilliQ-water; the running solution and blank solution.
?. [Operation of flame photometer]
Put the running solution under the U-tube and add water with droplet to the connecting tube.
?. [Operation of flame photometer]
Open “air” on the main valve and check that liquid is being drawn into the machine and that waste liquid is flowing out into waste bucket.
?. [Operation of flame photometer]
Open gas bottle by turning the valve all the way, then a half-turn back.
?. [Operation of flame photometer]
Switch flame photometer and check that the flame has been lit, and then close the hatch.
?. [Operation of flame photometer]
Turn computer on to record the measurements manually. Leave the running solution under the U-tube and let it run for    to  before use.
?. [Operation of flame photometer]
Check that the U-tube doesn’t have any air bubbles; wiggle it around if necessary.
?. [Operation of flame photometer]
Check waste container and dispose of solution down the sink.
?. [Operation of flame photometer]
Check at the back of instrument that pressure is approx. 11 psi.