Color Value Methodology

Effective Date:   May 2, 2018

1.0  Equipment & Materials

1.1    Microbalance or equivalent (e.g. Mettler Toledo) with precision of at least 0.1 mg

1.2    Spectrophotometer and related software or equivalent (e.g. SpectraMax M5/SoftMax Pro) with scanning monochromator or ability to measure absorbance from emission wavelengths of 280nm, 618nm, and 620nm

1.3    pH meter and associated pH electrode with precision of at least 0.1 pH units

1.4    Magnetic stir plate with magnetic stir bar

1.5    Utensils for transferring powder (e.g. scoopula or spatula)

1.6    Pipettes and pipette aide (e.g. Drummond)

1.7    Pipetman and tips or equivalent (e.g. P-1000)

1.8    UV Cuvette semi-micro/macro or equivalent (e.g. PMMA)

1.9    100 mL Volumetric flask

1.10  Beaker or equivalent with at least 300 mL capacity

1.11  Glass bottle with cap with at least 1000 mL capacity

1.12  Conical centrifuge tubes or equivalent (e.g. Falcon)

1.13  Plastic weigh boat, weigh paper or equivalent

1.14  Paraffin film (e.g. Parafilm)

1.15  Dry wipe (e.g. cloth towel, paper towel, Kimwipe)

1.16  Aluminum Foil

2.0  Reagents

2.1    Water

2.2    100 mM sodium citrate buffer stock solution (pH 6.0)

2.3    100 mM citric acid stock solution

2.4    1M NaOH stock solution

3.0  Procedure

In duplicate:

3.1     Remove sample(s) to be analyzed from storage, cover with foil, and warm to room temperature.

3.2     In a glass bottle, make 1 L of sodium citrate buffer (10 mM) by diluting 100 ml of the 100 mM sodium citrate buffer stock solution into 900 ml of water. Secure bottle cap and invert to mix. Use the pH meter to check the pH of the 10mM buffer solution and adjust to pH 6 as necessary. If the pH requires adjustment add 100 mM citric acid or 1M NaOH for adjustment.

3.3    Tare the microbalance with the weigh boat/paper, then weigh out 300 +/- 1 mg of dried 

3.4    Add the powder to the beaker, rinsing the weigh boat/paper with ~5 mL aliquots of 10 mM sodium citrate buffer using a 10 mL pipette. Repeat the rinse until all visible blue color is gone.

3.5    Add magnetic stir bar and stir on magnetic stir plate at room temperature until powder has dissolved into the citric acid buffer. Stirring should not occur for more than 1 hour.

3.6    Pour the contents of the 300mL beaker into a 100 ml volumetric flask. Transfer the remaining colored solution from the beaker to the volumetric flask by again rinsing via 10mL pipette with 10 mM sodium citrate buffer until the beaker including the stir bar is rinsed of all visible blue color.

3.7    Swirl the volumetric flask and add additional 10 mM sodium citrate buffer via 10mL pipette until the solution is 100 mL in volume as indicated by the marking on the volumetric flask.

3.8    Using a 10 ml pipette transfer 9 ml of the sodium citrate buffer to a 15 ml conical tube.

3.9    Stretch paraffin film over the top of the 100 mL volumetric flask to seal. Place thumb over the covered opening at the top of the flask and invert the flask 5-10 times to mix.

3.10  Using the Pipetman; transfer 1 ml of the solution to the 9 ml of citrate in the tube. Rinse the tip by pipetting up and down 1-3 times. Mix by capping the tube and inverting 5-10 times.

3.11  Place 1 mL of 10 mM sodium citrate buffer into a cuvette and place in spectrophotometer.

3.12  Set spectrophotometer to measure absorbance at 280 nm, 618 nm, and 620 nm.

3.13  Blank the spectrophotometer at these wavelengths by pressing “reference” in the software.

3.14  Place 1mL of diluted solution from step 3.9 into a new cuvette. Take the absorbances using the spectrophotometer at 280 nm, 618 nm, and 620 nm in the cuvette and record.

3.15    Calculate the color value. 

3.16  If results between the duplicate color value measurements differ by more than 10% of higher value, then repeat from step 3.2.


[End of Purchase Color Value Assay]

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