TITLE: Mobile Phone Spectrophotometer Setup [Chlorophyll experiment version]
AUTHORS: Katharine Hubbard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol allows you to turn a mobile phone (smart phone) into a colorimeter/spectrophotometer. It goes through how to set up your phone and workspace, making up a dilution series, constructing a calibration curve and making measurements. </div><div class = "text-block">The protocol is designed so that it can be carried out independent of a laboratory - our undergraduate students used it as an at home experiment during the Covid-19 pandemic. </div><div class = "text-block">This version of the protocol is used to test the hypothesis "Chlorophyll content from sun leaves will be greater than shade leaves from the same plant".</div><div class = "text-block">The chlorophyll extraction requires you to have access to either 80% acetone (v/v with water) or an acetone based nail polish remover. Note that you will need to test whether your nail polish remover reacts with the plastic cuvettes or not before continuing with the experiment - some of our students found that their cuvettes dissolved using the nail polish remover directly. </div><div class = "text-block"><span>The protocol is inspired by and adapted from this video - </span><a href="https://www.youtube.com/watch?v=LzYhyXYcs0M&feature=youtu.be" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">smartphone spectrophotometer</span></a></div></div>

[STEPS]
?. [Setting up your phone]
You first need to set up your phone to act as a colorimeter - video Instructions for this section are availabile: Setting up Your Mobile Phone as a Colorimeter
?. [Setting up your phone]
Choose a well lit space to work in. This protocol works better during the day under natural light. If you are doing the experiments after dark you might want to position a lamp near your set-up so you have fewer shadows and greater contrast in your images.
?. [Setting up your phone]
Fully charge your phone! Unplug your phone from it's charger and put the charger away to reduce the risk of electrocution while handling liquids.
?. [Setting up your phone]
Install a colour picker app on your phone - you need one that gives you numerical Red Green Blue (RGB) values. I recommend 'ColorPicker' for iPhones, and 'ColorGrab' for Android.
?. [Setting up your phone]
Download the template and make the cuvette holder - you will need sellotape to fix it together.
?. [Setting up your phone]
Sellotape the cuvette holder to the table/workshop where you are going to work. Place a piece of white card behind the cuvette holder. You might want to tape this into place too.
?. [Setting up your phone]
Open the colour picker app. Position your phone so that you are pointing the colour picker pointer in the middle of your cuvette. I would recommend resting your phone against something (e.g. a mug/box) so that it stays in the same position for each of your measurements.  My set-up is shown below - note for these pictures the cuvette is filled with a tea solution not chlorophyll!
?. [Calibration curve (chlorophyll experiment)]
To test that your phone is able to measure different concentrations you will need to set up a calibration curve. For this you will construct a dilution series of different concentrations, and measure the RGB values for each concentration. You then plot this on a graph to determine if there is a relationship between concentration and RGB. The calibration curve is to test whether or not the phone is able to measure a relationship between concentration and RGB values - it is a test of your phone, not of your experimental samples. Video instructions for chlorophyll extractions and calibration curve
?. [Calibration curve (chlorophyll experiment)]
You will extract chlorophyll using nail polish remover. You need to use a brand of nail polish remover that is acetone based - read the ingredients before purchasing some! If you have access to a lab you can use 80% acetone for the extractions.
Acetone is flammable, so make sure there are no naked flames anywhere around your workspace
?. [Calibration curve (chlorophyll experiment)]
Before starting, you need to test whether your brand of nail polish remover reacts with the plastic cuvettes - brands with very high acetone content will start to dissolve the cuvettes. Put 3 mL of nail polish remover into a cuvette and leave it for 10 minutes. If the plastic stays clear you can use the nail polish remover directly.If the plastic goes cloudy or deforms you will need to dilute the nail polish remover for your experiments. We suggest an 80% dilution e.g. 40 mL nail polish remover mixed with 10 mL water.
?. [Calibration curve (chlorophyll experiment)]
Using a plastic disposable pipette, set up a dilution series of different concentrations of chlorophyll in the cuvettes. I suggest you make up cuvettes with the following concentrations. Make sure you label your cuvettes clearly at the top or on the frosted side. Note that you are making up your dilutions with nail polish remover, not water. Most nail polish removers have a coloured dye in them so you need to take this into account when preparing your dilution series. Concentration of chlorophyllVolume of chlorophyll stock solutionVolume of nail polish remover100%3 mL0 mL80%2 mL0.5 mL60%1.5 mL1 mL40%1 mL1.5 mL20%0.5 mL2 mLVolume of 10% chlorophyll stockVolume of nail polish remover10%3 mL0 mL5%1.5 mL1.5 mL0%0 mL3 mL
Concentration of chlorophyllVolume of chlorophyll stock solutionVolume of nail polish remover100%3 mL0 mL80%2 mL0.5 mL60%1.5 mL1 mL40%1 mL1.5 mL20%0.5 mL2 mLVolume of 10% chlorophyll stockVolume of nail polish remover10%3 mL0 mL5%1.5 mL1.5 mL0%0 mL3 mL
?. [Calibration curve (chlorophyll experiment)]
Place the 100% cuvette into the cuvette holder with the transparent side facing towards you. [Note that the cuvettes have a clear side and a frosted side]
?. [Calibration curve (chlorophyll experiment)]
Load up the color picker on your phone and record the RGB Values for the 100% cuvette.
?. [Calibration curve (chlorophyll experiment)]
Repeat for each of your dilutions.
?. [Calibration curve (chlorophyll experiment)]
Plot a calibration curve with concentration on the x-axis and measured RGB value on the y-axis. Mine is shown below - you will notice that there is a negative relationship between RGB and concentration. This is because on the RGB scale white = 255 and black = 0.
?. [Calibration curve (chlorophyll experiment)]
To give a positive relationship between concentration and RGB I recommend that you normalise your data as follows:normalised .value = 1-(RGB value/255)For example, if you measured R as 86, you would calculate your normalised value as:normalised.value = 1 -(86/255) = 1 - 0.337 = 0.663Alternatively, you could calculate this relative to a blank (empty) cuvette, which would be a better approximation for a spectrophotometer. This would be calculated as follows:normalised.value = 1-(sample.RGBvalue/blank.RGBvalue)For example, if you measured B as 120 for your sample, and 190 for your blank you would calculate the normalised value as:normalised.value=1-(120/190)=1-0.631= 0.368
?. [Calibration curve (chlorophyll experiment)]
Plot a second calibration curve with the normalised values. Mine is below using the blank normalisation calculation. - You will see that this normalisation has given a positive relationship between concentration and normalised RGB. Normalised RGB is now scaled so that 0 = white and 1 = black.
?. [Methodology Decisions]
Decide which colour channel you are going to use for the rest of your experiments. Pick the channel which gives the best relationship between concentration and RGB. For example, in my chlorophyll experiment I would pick the Blue channel as this has the strongest relationship between concentration and RGB value.
?. [Experimental Measurements]
Put 3 mL of nail polish remover into a fresh cuvette.
?. [Experimental Measurements]
Cut out 3 cm2 of leaf material from your chosen plant. Cut this into small pieces and place into the cuvette - push the pieces so they are fully submerged in the nail polish remover.
?. [Experimental Measurements]
Put the cuvette into the cuvette holder and measure the RGB for your chosen colour channel using the colour picker app, and record your data.
?. [Experimental Measurements]
Repeat for all replicates in your experiment. Make sure you use the same incubation time for each replicate, and try and cut the leaf material into similar sized pieces so that you are consistent between samples. If you want to perform a t-test, you will need at least 5 replicates of each sample (i.e. 5 extractions from sun leaves, 5 extractions from shade leaves).
?. [Calibration curve (chlorophyll experiment)]
Pour 20 mL of nail polish remover into a plastic falcon tube labelled 'Chlorophyll stock'
?. [Calibration curve (chlorophyll experiment)]
Get ~8 medium sized leaves (I used spinach leaves) and cut them up into small pieces (I used 0.5 cm squares). Push them into the falcon tube so they are immersed in the acetone and put the lid on.
?. [Calibration curve (chlorophyll experiment)]
Leave the Chlorophyll stock for ~30 minutes to let the chlorophyll dissolve into the acetone
?. [Calibration curve (chlorophyll experiment)]
Pipette 2.5 mL of the extract into a new 50mL tube. Add 22.5 mL of acetone so you have a total of 25 mL. Label this as ‘10% chl stock’
?. [Experimental Measurements]
Leave for 20 minutes to let the chlorophyll diffuse into the acetone.
?. [Experimental Measurements]
Transfer the acetone into a fresh cuvette, leaving the leaf material behind. You could do this by pouring the liquid into a new cuvette, or by pipetting it across.