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1. IO Rodeo A Educational Colorimeter Kit User Manual amp Lab Activities Table of Contents Introduction Educational Colorimeter kit contents Electronics Parts Colorimeter Hardware Enclosure Parts laser cut acrylic Optional TuxCase kit Assembly of the Educational Colorimeter Assembly of the colorimeter Assembly of Arduino onto base plate Optional Assembly of Arduino with TuxCase Upgrading from original colorimeter hardware to the new design Programming the Arduino with the Educational Colorimeter firmware Educational Colorimeter software data collection and analysis Colorimeter basic program Colorimeter plotting program Colorimeter concentration program Lab 1 Introduction to Colorimetry Background and Objectives Materials Methods Sample Data Lab 2 Beer s Law and Molar Extinction Coefficient Background and Objectives Materials Methods Step 1 Prepare 1 mM stock of dyes Step 2 Preparation of standard curve Step 3 Measure absorbance with the colorimeter and plot data Sample Data Lab 3 Ammonia and nitrate measurements Background and Objectives Materials Chemicals and solutions Methods Day 0 Setting up the experiment and initial sample collection Day 2 Prepare solutions Day 3 Prepare ammonia standard curve Day 4 Prepare nitrate standard curve Day 5 Measure nitrate and ammonia in samples Sample Data Introduction Colorimeters are analytical devices commonly used in science labs to measure th
2. Description Description Vendor TuxCase flat head mounting screws 4 40 flat head machine screw 3 16 long mcmaster carr 97124A125 Arduino mounting screws 4 40 round machine screws 5 16 long mcmaster carr 91773A107 4 Nylon standoff Unthreaded Spacer 1 8 long mcmaster carr 94639A610 4 Nylon washer self retaining washer mcmaster carr 91755A205 Enclosure Parts laser cut acrylic Base Plate Top Plate Green holes Arduino Red holes TuxCase LED Mount Sensor Mount FR CDE Io LL I DEN TOP CD CH OC g O Li CH a i O L L LI L pi BOTTOM KENE Outer Slider Inner Slider Clear acrylic cover O IO Rodeo O Colorimeter A Divider Wall Side Wall 2 Optional TuxCase kit The optional colorimeter TuxCASE kit can be used as a protective enclosure for the Arduino protecting the electronics boards from liquid spills in the lab when using the colorimeter The aluminum enclosure is the same as the TuxCASE for Arduino The main difference is the top the clear acrylic top has been modified to include a cutout for the header on the colorimeter shield A black acrylic spacer is included to raise the height of the clear top TuxCASE is designed and manufactured by Tux Lab For additional information on the TuxCASE manufacturing procedure and supporting documentation visit the Tux Lab project page TuxCase kit contents e Aluminum TuxCase for Arduino U
3. Place the clear cover on the enclosure Secure all the parts in place with the last four enclosure screws from Bag A You have now completed assembly of the colorimeter In the next section you will mount the Arduino onto the base plate If you will be using a TuxCase enclosure for the Arduino skip down to the Instructions starting on Page 20 16 Assembly of Arduino onto base plate Step 1 To mount the Arduino Uno take the bag of screws and plastic standoffs from Bag C You do not need the 4 flat head screws Place one of the screws through one of the Arduino mount holes On the other side place a plastic standoff and washer The washer will hold the standoff in place while you work Repeat for the remaining 3 mount holes OTYPING PLATF vi OPEN PROTOTY e ARDUINO L en ai BAGEN ay ies FYENS Step 2 Place the Arduino onto the base plate and line up the screws from the above step with the holes in the Arduino holes in the base plate Screw them down into the base plate Note the screws will go partly into the base plate not all the way through 17 Step 3 Mount the colorimeter shield onto the Arduino board There is only one orientation possible Note that the shield pins are labelled on the silkscreen white text to match the label on the corresponding header on the Arduino board Step 4 Connect the ribbon cable to both the enclosure and the colorimeter shield We have found it easier to conne
4. You can also choose to include the aluminum TuxCase Arduino enclosure Upgrade 1 no TuxCase e New base plate e 6x colorimeter enclosure screws see hardware Bag A These longer screws replace the original screws that mount the 6 standoffs to the acrylic base e 4 x Arduino screws 4 x nylon standoffs and 4 x nylon washers see hardware Bag C Upgrade 2 with TuxCase e New base plate e 6x colorimeter enclosure screws as in hardware Bag A These longer screws replace the original screws that mount the 6 standoffs to the acrylic base 4 x TuxCase screws as in hardware Bag C TuxCase kit includes hardware 4 x 1 4 long 18 8 stainless screws for mounting Arduino into the TuxCase and 4 x 1 2 long 18 8 stainless screws for securing the clear acrylic top Step 1 Start by unscrewing and taking apart your colorimeter Remove the standoffs and set aside the old base plate Step 2 With the new base plate and enclosure screws follow the assembly steps in the User Manual to re assemble the colorimeter Step 3 Follow the steps for either mounting the Arduino kit 1 or mounting the TuxCase kit 2 21 Programming the Arduino with the Educational Colorimeter firmware Download the Educational Colorimeter firmware from_www iorodeo com software colorimeter_ onto your computer Unzip the downloaded file to a Known location After unzipping you should see an colorimeter firmware folder containing the different files used by t
5. Flip over the base plate and place a rubber bumper on each corner Step 5 To begin assembly of the colorimeter electronics you will use the hardware in Bag B Mount the Colorimeter LED Board onto the LED mount Make sure that the placement of the board is close to the bottom edge of the LED Mount as shown in the image below Secure in place with four PCB screws and four PCB nuts iO Rodeo inc Ui 11 Step 6 Mount the Colorimeter Sensor Board onto the sensor mount passing the plastic 10 pin connector through the rectangular cutout left and center images Before securing in place ensure the correct orientation of the PCB as shown in the lower right image Secure in place with the last four PCB screws and nuts Inc ve an A a shi aA sate A a A m as yo Rodeo r hb ter SENSO Be glorime Me l Step 7 From Bag B take one of the black plastic 4 pin connectors and the black pre crimped wire Locate the triangle marker on the housing which denotes Pin 1 Carefully push the black wire into the Pin 1 slot of the connector until it clicks into place Next insert the red wire into the Pin 2 slot of the connector followed by the green wire Pin 3 slot and finally the blue wire Pin 4 slot Make sure all wires are held firmly in place by gently pulling on them Do not put the connector on the other end of the wires at this point 12 Step 8 Connect the assembled wires to the LED board image below so th
6. object will appear green Complementary colors Green and red are complementary colors as shown on the color wheel below A solution that absorbs mainly red light appears green and vice versa Yellow Orange Orange Red Green Violet Blue Green Violet Blue Blue Violet The objective of this lab is to build a colorimeter from electronic mechanical and software components and use it to investigate how different colored solutions absorb different wavelengths of light 29 Materials Educational Colorimeter kit Diluted food dyes We have used FD amp C red 40 blue 1 and yellow 5 food dyes which can be found in most grocery stores Dilute these dyes 10 fold before using in the lab The green food dye is a mixture of the blue and yellow dyes 5 x cuvettes part of the Educational Colorimeter kit 1 mL fixed volume pipette Water Methods This lab uses the Educational Colorimeter Basic program Before starting the lab download the software and review the operation of this program details online and in your User s Manual 1 Assemble the Educational Colorimeter enclosure connect it to the Arduino board using the colorimeter shield and connect the board to the computer ensuring that it is running the colorimeter firmware Use the instructions provided in the User s Manual This will take the majority of the lab to complete Fill five cuvettes with 1 mL of water Add 1 drop of diluted food dye a
7. the LED and the light sensor When the Educational Colorimeter is operating the RGB LED illuminates one side of the sample in the cuvette using one of three different wavelengths of light 625 nm red 528 nm true green and 470 nm blue On the opposite side the light passing through the sample also passes through a slit on an inner wall of the enclosure and falls on the light sensor Absorbance Abs of the sample is determined by comparing the intensity of incident light I to the intensity of light after it has passed through the sample La I bs og The Educational Colorimeter uses eight 8 digital signals for normal operation The provided Arduino shield connects 8 digital input output DIO pins of the Arduino microcontroller board with the light sensor and RGB LED FRE FEE BR en n n l E e a ek ia DER ER EF RR RR FE EF EF ia RER RR ER ER RR REE kon Arduino DIO 4 50 sensor output DIO 5 51 sensor output DIO 6 52 sensor photodiode type DIO 7 53 sensor photodiode type DIO 8 FREQ LED DIO 9 Blue LED DIO 10 Green LED DIO 11 Red LED Educational Colorimeter kit contents Each Educational Colorimeter Kit contains the electronics boards hardware and parts listed below The parts are described in more detail on the next 3 pages All of the kit parts are shipped in a 1 6 L storage container After assembly the colorimeter fits back into the container for safe storage between uses Note that th
8. Oxidation of 5 aminosalicylate is carried out in the presence of a catalyst nitroferricyanide which results in the formation of indosalicylate a blue colored compound The blue color is masked by the yellow color from excess nitroprusside yielding a green colored solution that absorbs light at 650 nm The intensity of the color is directly proportional to the ammonia concentration in the sample 2 1 Ammonia compounds are initially combined with hypochlorite to form monochloramine 37 2 Monochloramine reacts with salicylate to form 5 aminosalicylate Nitrate Enzyme based assay The assay for measuring nitrate is a 2 step process First nitrate in the sample is converted to nitrite enzymatically using nitrate reductase NR In the second step nitrite is measured using Greiss test In the Greiss test sulfanilamide reagent is converted to a diazonium salt by nitrite The diazonium salt is then reacted with the reagent NED N 1 napthylethylene diamine dihydrochloride to form a colored azo dye which has a purple magenta color that is measured at 520 550 nm using the green LED Step 1 Nitrate reductase NOz NADH H NO NAD H20 Step 2 Griess reaction HANO S Developed by Peter Griess in 1879 this standard test is widely used to detect nitrites 38 Materials e Labware Beakers volumetric flasks graduated pipettes test tubes solution storage bottles microfuge tubes Store bought aquar
9. Sigma cat 198269 Wavelength 522 nm Mol Wt 879 86 FD amp C Dye Blue 1 Sigma cat 861146 Wavelength 628 nm Mol Wt 792 85 Sunset Yellow Sigma cat 465224 Wavelength 482 nm Mol Wt 452 37 S ONa CS O H FD amp C Dye Yellow 6 HO 33 Materials The following list of materials is required for this lab Assembled Educational Colorimeter kit from Lab 1 Powdered food dyes erythrosin B erioglaucine and sunset yellow Analytical scale 3 x 250 mL volumetric flasks 15 x test tubes gt 5 mL 1 mL fixed volume pipette 16 x cuvettes Water Methods This lab uses the Educational Colorimeter Plotting program Before starting the lab download the software and review the operation of this program details online and in your User s Manual Step 1 Prepare 1 mM stock of dyes e Erythrosin B FW 879 86 e g 0 218 gin 250 mL distilled water e Erioglaucine FW 792 85 e g 0 198 gin 250 mL distilled water e Sunset Yellow FW 452 37 e g 0 113 gin 250 mL distilled water Step 2 Preparation of standard curve 1 Dilute the 1 mM stock solutions as shown in Table 1 using a 250 mL volumetric flask Label these flasks working stock 2 For each of the 3 dyes prepare a series of standard curve dilutions as shown in Table 2 using the test tubes Label tubes 1 5 for each dye Step 3 Measure absorbance with the colorimeter and plot data 1 Launch the colorimeter plotting program Calibrate the dev
10. at the black wire is connected to the pin on the PCB labelled GND the red wire to the pin labelled Red the green wire to the pin labelled Green and the blue wire to the pin labelled Blue Thread the free ends of the four colored wires through the round hole in the divider wall Step 9 Take the second 4 pin connector and as before insert the pre crimped wires Remember to first locate the triangular marker on the connector and then insert the black wire Into the slot corresponding to Pin 1 followed by the red Pin 2 slot green Pin 3 slot and blue Pin 4 slot wires Finally after ensuring that the cables are properly attached connect this to the Colorimeter Sensor Board using the same pin orientation as before black wire to GND pin red wire to Red pin etc 13 Step 10 Place the parts assembled in the previous step on the base plate taking note of the orientation The sensor PCB and the divider wall should be on the side of the cuvette holder with the U shaped cutout left image The cables may need to be adjusted to fit into the enclosure Step 11 Take the two side walls and insert them on the base plate so that all the corresponding tabs and slots match 14 Step 12 Place the top plate on the enclosure ensuring that the tabs fit in the slots as shown in the Image below Step 13 Lay the outer slider and inner slider onto the top plate Orient the parts as shown in the images below 15 Step 14
11. bel one as Gravel and another as Gravel Bacteria e Take a scoop 25 50 g of aquarium gravel from the store bought bag Place in the beaker labelled Gravel e To obtain nitrification bacteria remove an equivalent amount of gravel from an established aquarium as your source of nitrification bacteria Place in the beaker labelled Gravel Bacteria e Transfer 1 5 mL of sample to a clean microfuge tube for ammonia measurements Transfer 1 0 mL of sample to a clean microfuge tube for nitrate measurements Label these two samples as T O since they are taken at Day O of the experiment Store them at 20 C e Repeat sampling every 24 hours for the following 4 days of the experiment and label them T 1 T 4 respectively You will collect a total of 10 samples 5 for ammonia and 5 for nitrate measurements 40 Day 2 Prepare solutions Prepare the following solutions for making the ammonia and nitrate measurements A list of the chemicals can be found in the Appendix online 1 Hypochlorite solution e Place 1 ml of bleach into a 100 mL volumetric flask and fill with 70 mL of DI water e Add 0 5 grams of NaOH and mix until dissolved e Fill flask to the 100 mL mark 2 Salicylate Catalyst solution e Place 10 g of sodium salicylate into a 100 mL volumetric flask and fill with 70 mL of DI water until dissolved e Add 0 04 grams of sodium nitroferricyanide and mix until dissolv
12. channel Transfer the contents of Tube M1 0 ppm nitrate into a cuvette and use it for calibration Before removing the calibration sample from the enclosure click Measure Absorbance value should be 0 00 In the cell next to the measurement enter the concentration value in UM which in this case Is 0 0 Transfer the contents back into the test tube It is good practice to rinse the cuvette with distilled water between samples Transfer the next solution Tube M2 into the cuvette place it inside the enclosure and click Measure In the cell next to the measurement enter the concentration value in uM Repeat steps 11 13 for the remaining samples After completing all measurements and entering all concentration values click Plot to graph your data Using the options menu go to Options gt Export choose a filename for storing your sample curve eg studentl nitrate sc and click OK 45 TO Fig 3 Image of cuvettes with nitrate standard curve in duplicate Figure 1 k O Ae BE slope 0 001 Figure 4 Sample nitrate standard curve 46 Day 5 Measure nitrate and ammonia in samples Take your last sample from the experiment T 4 Remove from the freezer the samples collected on previous days T O T 3 and thaw Process all 10 samples 5 for nitrate and 5 for ammonia measurements the same way as you did for generating the standard curves on Days 3 a
13. ct the cable to the colorimeter first and then to the Arduino second CG i tun i 18 Optional Assembly of Arduino with TuxCase Step 1 To first mount the TuxCase onto the base plate you will need the 4 flat head screws from Bag C Place the TuxCase on the base plate and line up the four corner holes with the holes on the base plate Fasten in place with the 4 flat head screws Step 2 Place the Arduino inside the TuxCase as shown in the Images below and fasten into the case using the 4 shorter 1 4 screws that come with the TuxCase kit 19 Step 3 Mount the colorimeter shield onto the Arduino board There is only one orientation possible Note that the shield pins are labelled on the silkscreen white text to match the label on the corresponding header on the Arduino board Place the black acrylic spacer and the clear top onto the enclosure Using the remaining 4 screws from the TuxCase kit secure the top in place at the four corners Step 4 Connect the ribbon cable to both the enclosure and the colorimeter shield We have found it easier to connect the cable to the colorimeter first and then to the Arduino second 20 Upgrading from original colorimeter hardware to the new design For current users of the original colorimeter design shown opposite upgrading to the new single piece design is very easy and only requires one additional piece of acrylic the base plate and some extra hardware
14. e concentration of a solution from its light absorbing properties Colorimeters are extremely useful and flexible lab instruments for a wide range of science education labs Typically these devices are used for e Color and absorbance Investigate the relationship between the color of a substance and absorption of light at different wavelengths e Beer s Law Investigate the relationship between concentration and absorbance using colored solutions eg food dye copper sulfate Calculate molar extinction coefficient e Nitrogen cycle Quantify the breakdown oxidation of ammonia into nitrite and nitrate by nitrification bacteria e Water quality Measure several water parameters such as turbidity chlorine content pH water hardness phosphate content and more e Population growth Measure the turbidity of a microbial culture over time which serves as an indicator of population growth e Enzyme kinetics Measure the activity of an enzyme over time even under different environmental conditions temperature pH inhibitors substrate How the Educational Colorimeter works The Educational Colorimeter essentially consists of an RGB LED and a color sensor in a light tight enclosure which is connected to an Arduino via a colorimeter shield EI 10 Rodeo Inc E IO Rodeo Inc QColorimeter Sensor Board v2 0 To Arduina A cuvette holder in the center of the light tight enclosure properly positions the sample between
15. e Arduino Uno is not listed as a kit component but is required The Arduino Uno can be purchased pre programmed with the kit 10 Rodeo Inc o gaus 10 Rodeo Colorimeter shield v2 1 Colorimeter LED board Colorimeter sensor board and Colorimeter shield for the macro Arduino 3 bags of Hardware ribbon cable for connecting Arduino to the colorimeter and a Philips mini screwdriver 11 black laser cut acrylic parts and a clear laser cut engraved acrylic cover Electronics Parts Below is a list of parts that are used to make the 3 electronics boards Note that the electronics are already assembled with the kit This component listing is for informational purposes only ey oem oun Dou FON Ooo o fowm ooo ooo amore e Colorimeter Hardware Bag A Enclosure hardware Description Vendor Enclosure standoffs 4 40 hex standoffs 1 34 long mcmaster carr 91780A038 Cuvette standoffs 4 40 hex standoffs 1 4 long mcmaster carr 91780A034 Enclosure screws 4 40 machine screws 12 long mcmaster carr 91249A111 Rubber bumpers Digikey SJ5012 0 ND Bag B Electronics hardware lemer lee Don PCB screws 3 mcmaster carr 91249A108 ia machine screws 8 long 8 PCBNUW Nuts PCB Nuts memaster carr memaster carr 96537A120 Colored pre crimped wires SR female female wire Pololu 1806 2 Connector Connector housing Connector housing Polou am l C Arduino and TuxCase hardware on
16. ed Add 0 5 grams NaOH to adjust the pH to the 12 0 range Fill flask to the 100 mL mark Transfer solution into a dark airtight glass bottle for maximum longevity Due to limited storage life prepare fresh solutions weekly 3 25 mM EDTA e Dissolve 9 3g EDTA in 1L of distilled water 4 Phosphate buffer 25 mM KH2PO4 0 025 mM EDTA pH 7 5 e Dissolve 3 75 g of potassium phosphate KH2PO4 and 1 4 g potassium hydroxide KOH in 800 mL of distilled water in a LL volumetric flask e Add 1 mL of 25 mM EDTA and fill to the mark 5 2 units mL nitrate reductase e Add 1 mL of NECI proprietary enzyme diluent to 2 units of freeze dried enzyme and reconstitute following the instructions supplied with the enzyme 6 1 mg mL NADH e Dissolve 0 1 g of NADH FW 709 4 in 100 mL distilled water Aliquot and store unused NADH in the freezer 7 1 sulfanilamide solution e Weigh out 0 15g of sulfanilamide into a small amber bottle Add 15 mL of 3M HCI 8 0 02 NED e Weigh out 0 02 g of NED into an amber bottle Add 100 mL of distilled water 41 Day 3 Prepare ammonia standard curve Ki Transfer 20 mL of the 10 ppm ammonia standard solution to a 100 mL volumetric flask Fill flask to the 100 mL mark with distilled water and invert several times to mix Label flask as 2 0 ppm ammonia Label nine large test tubes 1 9 Pipette the indicated volumes of 2 0 ppm ammonia and distilled water into the test tubes as shown in the Table below T
17. er plot of the data 25 slope 0 020 Serial Port 0 5 red green blue white men an ion uM ss Absorbance 0 4 1 0 000 0 000 a 2 2 210 0 050 CSS 3 4 420 0 100 0 3 pwan a 4 6 630 0 140 z pn 8 54 8 840 0 190 E 6 11 100 0 220 w end Z 13 300 0 270 B 15 500 0 320 viel 9 17 700 0 370 0 1 aa 10 19 900 0 400 21 22 100 0 450 0 05 i0 15 20 25 concentration uM 00 Ba 7 Save data at any point after calibration you can save the data in the table toa standard raw text file Using the File gt Save menu item at the top of the program window 8 Export standard curves once you have your standard curve you can export it for later use with the concentration program using the Options gt Export Test Solution menu item at the top of the program window After exporting this file will be automatically available for use with the concentration program Se File Options Import Test Solution Edit Test Solutions 5 red ae 2 135 217 879 3 13 682 4 14 791 Calibrate green Clear blue white Concentration 0 09 0 97 0 62 0 72 Ctri l Colorimeter Plot Disconnect Absorbance Measure Plot Other features Users can also clear data and load import previously saved data files 26 Colorimeter concentration program This program measures the concentration of an unknown solution Instructions for using this s
18. he device b Click on Calibrate The program will display a value of 1 00 for Transmittance and 0 00 for Absorbance on all color channels 23 Transmittance Absorbance red 1 000 red 0 000 green 1 000 green 0 000 blue 1 000 blue 0 000 white 1 000 white 0 000 Samples 1000 red green blue white Serial Port dev ttyACMO 4 Select LED initially all of the color channels are selected You can deselect one or more of them at any time after calibration The program will display values only for the selected color channels 5 Measure and plot data place the cuvette containing your sample inside the enclosure and click Measure The Transmittance and Absorbance text areas will display the measurements corresponding to the selected color channels In addition a second window will automatically open displaying a bar graph of the measurements If the bar graph is not needed for your measurements uncheck the Plot checkbox sl TI colorimeter Basic Absorbanck Plot 200 4 BR Transmittance Absorbance red 0 041 red 1 387 green 0 770 green 0 114 blue 0 296 blue 0 529 white 0 273 white 0 564 vw D VI EE Samples 1000 ki red green f blue white 2 a 2 Sal ou ase tn es We RS 9 Plot 24 Colorimeter plotting program This program allows users to generate calibration curves that are typically used to find the concentration of a solution Instructions for using this soft
19. he firmware Connect your Arduino board to the computer start the Arduino IDE installation instructions available at http arduino cc en Guide HomePage and open the main firmware file firmware pde This file should compile without needing to download additional libraries After selecting the Arduino board model and the serial port it is using under Tools menu of the Arduino IDE upload the firmware to the board Educational Colorimeter software data collection and analysis Download the software for your choice of Operating System Windows Mac or Linux from www iorodeo com software colorimeter The files are provided as precompiled binaries so that they can be launched immediately after download by double clicking any of the 3 program files For the more adventurous users the source files are available at http bitoucket org iorodeo colorimeter The software suite we have developed for use with the Educational Colorimeter consists of 3 different programs After download unzip the colorimeter_software_suite zip file onto a Known location in your computer 1 Note If you received a pre programmed Arduino with your colorimeter kit then you can skip this step More detailed instructions for using the Arduino IDE can be found at http arduino cc en Guide Environment 22 Colorimeter basic program This program reports the Transmittance and Absorbance measured by the colorimeter for the wavelength s of ligh
20. ice with a cuvette containing water as described in Lab 1 2 Starting with erythrosin B measure the absorbance for each standard curve solution with the appropriate color channel and enter the corresponding concentration in the program 3 Once all the samples are measured click on the Plot button Repeat measurements for erioglaucine and sunset yellow Record values for the slope in Table 3 To determine which color channel to use measure absorbance at all 3 wavelengths as described in Lab 1 34 Table 1 Preparation of working solutions Dye Volume of 1 mM stock Concentration of Color channel working stock wavelength Erythrosin B 1 mL in 250 mL 4 00 uM Green 528 nm 2 5 mL in 250 mL 10 00 UM Red 625 nm Sunset Yellow 10 mL in 250 mL 40 00 uM Blue 470 nm Table 2 Preparation of standard curves Tube Volume of Erythrosin B Erioglaucine Sunset Yellow a Dem stock ni im amHO oam ooo ou Iouw o baang foom dow fom banne fom ome fom Table 3 Molar extinction coefficient Plotted Slope Molar extinction coefficient Reported value UM vs Abs M cm Sigma spec sheets Erythrosin B 0 056 56 000 at 528 nm 2 82 500 524 528 nm Erioglaucine 0 098 98 000 at 625 nm 2 80 000 627 637 nm Sunset Yellow 0 020 20 000 at 470 nm 2 20 000 479 485 nm 35 Sample Data Fig 1 Image of cuvettes with 3 different food dye standard curves OO BE slope 0 093 Fig 2 Sample data Er
21. ioglaucine standard curve 36 Lab 3 Ammonia and nitrate measurements Background and Objectives Nitrification bacteria play an important role in the nitrogen cycle oxidizing ammonia first to nitrite and finally to nitrate Nitrification in nature is the result of actions of two groups of organisms 1 Nitrosomonas bacteria ammonia oxidizing bacteria convert ammonia to nitrite NH3 O gt NO 3H Ze 2 Nitrifying bacteria nitrite oxidizing bacteria convert nitrite to nitrate NO H20 NO 2H 2e The objective of this lab is to monitor the levels of ammonia and nitrate over the course of 4 days in the presence of nitrification bacteria Nitrification bacteria are widespread in soil and water and are found in highest numbers where considerable amounts of ammonia are present In this lab substrate gravel from an established aquarium will be used as the source of nitrification bacteria Colorimetric tests Previously in Labs 1 and 2 we used food dyes which are already colored However ammonia and nitrate are colorless in water In this lab we will use colorimetric assays which yield a color only in the presence of ammonia or nitrate Ammonia Salicylate test The ammonia salicylate method involves a three step reaction sequence The first reaction step involves the conversion of ammonia to monochloroamine by the addition of chlorine The monochloroamine then reacts with salicylate to form 5 aminosalicylate
22. ium gravel unused Gravel from an established and healthy aquarium Chemicals and solutions Chemical Vendor Cat Approx Cost Sodium hydroxide Sodium hydroxide Carolina Biologicals Carolina Biologicals 889425 mm 25 Sodium salicylate 52679 1006 35 00 Sodium nitroferricyanide 228 10 5G 30 20 6 Sodium common household bleach available 2 00 hypochloride from most grocery and hardware stores NECI Nitrate Elimination Company 800302 39 00 Cem Units NADH Sigma 43420 100MG 100MG ena 90 EDTA Carolina Biologicals 861780 16 25 Potassium phosphate Sigma P5655 100G 21 50 KH2PO Potassium hydroxide Carolina Biologicals Carolina Biologicals 883485 5 25 25 Sufanitamide Pepe GE 3M Hydrochloric acid Carolina Biologicals 867861 6 75 NED Sigma 33461 5G 38 50 N 1 naphthylethylenedi amine dihydrochloride Pame freres O 39 Methods This lab requires the use of both the Educational Colorimeter Plotting and Concentration programs Using the Export functionality of the former standard curves are generated for use In the latter Before starting the lab download the software and review the operation of these programs Day 0 Setting up the experiment and initial sample collection e Transfer 1 mL of a 1 000 ppm ammonia stock to a 500 mL volumetric flask Bring up to the mark using distilled water This is your 2 ppm ammonia stock e Divide the 2 ppm solution into two beakers and la
23. nd 4 Don t forget to also process a distilled water sample for calibration Once you have the samples ready for measurement open the Educational Colorimeter Concentration program Starting with ammonia select the standard curve generated on Day 3 from the drop down list eg student1 ammonia sc Note that the Red color channel will be automatically selected Calibrate the colorimeter with the O UM ammonia control and measure all of your ammonia samples Label the corresponding measurements as Day 0 Day 4 Once you have finished click the Plot button Save the displayed graph Repeat steps above for nitrate Select the standard curve generated on Day 4 from the drop down list Note the Green color channel will be automatically selected The two bar graphs should follow a linear trend as a function of time See sample data below 47 Sample Data A Ammonia concentration uM B Nitrate Concentration uM 200 As BE OO tr MO 160 100 140 En LI E E pun E ei mm z OG d o Mi an 20 ZU z Day Dar 1 Day 2 Cam 3 Day 4 ai Day Day 1 Day Day 3 Day a Garaplas Camas Fig 5 Final experimental data showing ammonia decreasing and nitrate increasing as a result of nitrification bacteria Table Ammonia and nitrate concentration in the control sample no nitrification bacteria 48
24. no e Black acrylic spacer e Clear acrylic cover with connector cutout e 4x long 18 8 stainless 4 40 round phillips machine screws for mounting Arduino into the case e 4x long 18 8 stainless 4 40 round phillips machine screws for securing the clear top cover Assembly of the Educational Colorimeter Before starting e Review the guides on the previous pages to easily identify the laser cut parts highlighted in bold below e Peel off the protective backing from the laser cut acrylic parts This will be either brown paper backing clear and Lo black or blue plastic on most of the 4 black acrylic parts e Sort and identify the hardware parts Assembly of the colorimeter Step 1 To begin assembly of the enclosure you will use the hardware in Bag A After removing the paper backing from the base plate place two of the enclosure screws through the two center holes Secure in place with tape Flip the base plate over and place one of the cuvette holders on the base plate with the U shaped cutout facing the two rectangular slots in the center of the base plate Screw the two cuvette standoffs in place Step 2 Mount the second cuvette holder onto the standoffs with two of the enclosure screws with the same orientation as the previous cuvette holder 10 Step 3 Keeping the same orientation of the base plate place one enclosure standoff on each corner and secure in place with an enclosure screw Step 4
25. oftware are as follows 1 Launch software double click on the concentration program icon which is inside the previously downloaded colorimeter software suite folder 2 Establish connection enter the serial port corresponding to your device in the program window and click on the Connect button 3 Select standard curve from the drop down menu select the standard curve corresponding to the solution you are going to be measuring The menu contains both default and user generated standard curves Optionally the user may enter the coefficient and select the measurement LED manually Colorimeter Measurement File Options Serial Port Disconnect manually specify ma nat ee D efault Nitrite D Test Solution Ammonia U Nitrite U lt U ser generated Calibrate Connected Mode Stopped 4 Calibrate sensor follow the calibration procedure described for the basic program 21 5 Measure samples place the cuvette containing your sample inside the enclosure and click Measure The measured concentration values are displayed in these table The user can edit the sample label Colorimeter Measurement ne x File Options Serial Port dev ttyACMO l Disconnect Test Solution Ammonia U Coefficient 7692 4 LED Sample Concentration uM 1 Sample 1 0 81 2 Sample 2 0 21 Calibrate Clear Measure Plot 6 Deleting data even though the entries for Concentration a
26. pproximately 10 uL to individual cuvettes This will yield one cuvette with red dye one with blue one with green and one with yellow One cuvette should be left with just water for calibration Mix the solutions by pipetting them up and down several times Launch the colorimeter basic program on your computer Place the cuvette with water in the colorimeter and click on calibrate The display should now read 0 00 Absorbance and 1 00 Transmittance across all 4 color channel readings Place the cuvette with red dye in the colorimeter and click on measure Ensure that all color channels Red Green and Blue are selected in the program Save the plot for later use if required Repeat for the yellow blue and green food dyes 30 Sample Data A Red Dye B Green Dye eg E Brcptrbapen Absorbance n5 0 0 mo i reg gren DLC Win LED C Blue Dye D Yellow Dye ear hanor Man a a Gon i dal gren DL wri Ot LED 31 Lab 2 Beer s Law and Molar Extinction Coefficient Background and Objectives Colorimeters and spectrophotometers measure absorbance of light of a specific wavelength by a solution Absorbance values can be used to determine the concentration of a chemical or biological molecule in a solution using the Beer Lambert Law also known as Beer s Law Beer s Law states that absorbance of a sample depends on the molar concentration light path length in centimeters l and molar extinc
27. re non editable you can select any number of rows right click anywhere on your selection and select Delete to remove those entries from the table image above Note that the labels Sample column can be edited at any point 7 Plotting data if at least one Concentration value exists on the table the data can be plotted by clicking the Plot button On a separate window a bar graph will display the data in the same order as it is entered in the table The bars will be labeled accordingly using the entries on the Sample column File Options Serial Port lev Test Solution Ammonia U ZOO As B amp G Disconnect 0 8 0 6 Sample Concentration uM S 1 Sample 1 0 81 2 Sample 2 0 21 S 0 4 TTT E i 4 Sample 4 0 26 0 2 0 0 Calibrate Clear Measure Plot Sample 1 Sample 2 Sample 3 Sample 4 Samples Connected Mode Stopped 28 Lab 1 Introduction to Colorimetry Background and Objectives The visible light spectrum consists of a range of frequencies each of which corresponds to a specific color Any visible light that strikes an object and becomes reflected or transmitted to our eyes will contribute to the color appearance of that object In the same way the color of a solution is a direct result of the wavelengths of light absorbed by the solution So if a solution absorbs all of the frequencies of visible light except for the frequency associated with green light then the
28. rve eg student1 ammonia sc and click OK slope 0 008 Absorbance red led 0 20 40 60 80 100 120 140 160 Concentration Fig 2 Sample ammonia standard curve 43 Day 4 Prepare nitrate standard curve 1 Remove the NADH and nitrate reductase prepared in Step 2 from the freezer Thaw the NADH In a test tube prepare a 10 x master mix of enzyme NADH and phosphate buffer as shown in the Table below rene nari 2 Units mL Nitrate 10 uL 0 1 mL reductase 2 Label nine tubes M1 M2 M9 To each tube transfer 1 mL of the master mix 3 To prepare the standard curve samples label nine test tubes 1 9 Pipette the indicated volumes of 10 ppm nitrate standard and distilled water into these test tubes as shown the Table below Tube N Conc NO3 NO3 Volume of 10 ppm Volume of distilled ppm Conc Conc nitrate mL water mL ppm HM KEN CA CO foo om C2 fees oor foo ee C ars fem fare n on cs feo fes for ka ERE REC cr fro fom lows ln e fs fam Jas o f o oo fe fw e if on in 44 10 11 12 13 14 15 16 Add 50 uL of each standard curve sample to the corresponding tube containing the master mix eg from tube 1 to tube M1 etc Mix thoroughly and incubate for 20 30 minutes Add 500 uL of 1 sulfanimide to each tube and mix Add 500 uL of 0 02 NED to each tube and mix Launch the Educational Colorimeter Plotting program and select the Green color
29. t selected by the user Instructions for using this software are as follows 1 2 Launch software double click on the colorimeter basic program icon which can be found inside the previously downloaded colorimeter_software_ suite folder Establish connection enter the serial port corresponding to your device in the program window and click on the Connect button in the lower left hand side Link DGG colorimeter RE Samples Serial Port dev ttyACMO Connect 3 Calibrate sensor once connected to the hardware you need to first take a calibration blank measurement to enable the Measure feature of the program This needs to be done at least once when you first start to use the program but it can be done additionally at any time while taking measurements Typically a calibration measurement is carried out with a solution which does not contain any of the color you are interested in measuring It is usually the liquid you used to dissolve or dilute your colored solution In many cases this liquid is water As an example if you are measuring the absorbance of blue food dye you will use water to calibrate the color sensor However if you are carrying out a colorimetric assay such as the ammonia assay in Lab 3 then you will calibrate against the assay solution developed with distilled water In all cases the steps are the same a Place a cuvette with water or other blank solution into t
30. tion coefficient for the dissolved substance at the specified wavelength A Beer Lambert Law Abs ecl An example of a Beer s Law plot concentration versus absorbance is shown below The slope of the graph absorbance over concentration Abs c equals the molar absorptivity coefficient x LL The objective of this lab is to calculate the molar extinction coefficients of three different dyes from their Beer s Law plot 1 0 Abs 0 40 0 8 Incident light o 0 6 V gt 2 O 2 toatl Slope x l 0 2 0 05 20 40 60 80 101 Concentration 20 Food dyes are used to color a variety of food products such as sweets cereal and sports drinks and are often used in high school and undergraduate labs The 3 dyes used in this lab were chosen as they absorb in the range of the colorimeter LED wavelengths Path length distance that light travels through the solution is determined by the cuvette that the sample is placed in Most colorimeters and spectrophotometers including the one in this kit use cuvettes with a path length of 1 cm Molar extinction coefficient is a measure of how strongly a substance absorbs light at a particular wavelength and is usually represented by the unit M cm or L mol cm For example Sigman and Wheeler 2004 J Chemical Education 81 10 1475 1478 Henary and Russell 2007 J Chemical Education 84 3 480 482 32 Erythrosin B FD amp C Dye Red 5
31. ube N Conc NH3 NH Conc Volume of 2 0 ppm Volume of distilled ppm Conc uM ammonia mL water mL ppm 10 11 12 13 14 Transfer 1 mL of each sample to be tested into a new test tube Note we recommend doing the measurements in triplicate Add 250 uL of hypochlorite solution and mix Add 250 uL of salicylate catalyst solution and mix Let tubes stand for 5 10 mins to develop color Launch the Educational Colorimeter Plotting program and select the Red color channel Transfer the contents of Tube 1 0 0 uM ammonia into a cuvette and use it for calibration Before removing the calibration sample from the enclosure click Measure Absorbance value should be 0 00 In the cell next to the measurement enter the concentration value in uM which in this case is 0 0 Transfer the contents back into the test tube It is good practice to rinse the cuvette with distilled water between samples Transfer the next solution Tube 2 into the cuvette place it inside the enclosure and click Measure In the cell next to the measurement enter the concentration value in uM Repeat steps 11 13 for the remaining samples 42 15 After completing all measurements and entering all concentration values click Plot to graph your data The points should roughly follow a linear trend see sample graphs below 16 Using the file menu go to File gt Export choose a filename for storing your sample cu
32. ware are as follows 1 Launch software double click on the Educational Colorimeter Plotting program icon which is inside the previously downloaded colorimeter software suite folder 2 Establish connection enter the serial port corresponding to your device in the program window and click on the Connect button 3 Select LED choose which LED to use from the options in the upper bar Unlike the basic program users can only select one LED for the measurements D 6 colorimeter Plot Slammer File Help Serial Port dev ttyACMO _ Connect red green blue white 4 Calibrate sensor follow the calibration procedure described for the basic program Note that in this case you should also measure the calibration solution ie sample with 0 00 Concentration for including the zero data Concentration Absorbance in your standard curve 5 Measure samples place the cuvette containing your sample inside the enclosure and click Measure The value for the absorbance measurement will be displayed in the first row of the table You can either enter the concentration value or skip ahead to the next measurement Note that whereas the Concentration values are editable the Absorbance measurements are not as they correspond to readings from the sensor 6 Plot data at any point after calibration you can plot the data in the table by clicking Plot A second window will automatically open and display a scatt

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