Calibration Curve Procedure:

Material and Procedure Recommendations:

The calibration curve is done every 6 months, when changing chemicals or spectrophotometer bulb.

Do visual inventory ahead of time to ensure that you have everything needed.

What you need to get started:

Chemicals:

  1. Sodium hydroxide 1% weight volume

  2. Chromotropic acid powder

  3. 37 % Formaldehyde

  4. 1.0 M Sodium sulfite

  5. 7.0 buffer solution

  6. 10.0 buffer solution

  7. Sulfuric acid

  8. Distilled water

  9. 0.1 N hydrochloric acid

Equipment

  1. Spectrophotometer

  2. Vortex mixer

  3. Magnetic stirrer

  4. pH meter

  5. Test tube heating block

  6. Scales

Glassware

  1. 1-400ml beaker

  2. 2-150ml beakers

  3. 7-100ml beakers

  4. 2- impinger tubes

  5. 1-50ml pipette

  6. 1-20ml pipette

  7. 25 test tubes

  8. 1-1000ml volumetric flask

  9. 4-100ml volumetric flasks

  10. 125ml burette with stand

  11. 1 spectrophotometer test tube

Accessories

  1. weigh boats

  2. kemwipe towels

  3. pipette filler dispenser

  4. impinger storage rack

  5. test tube storage rack

  6. magnetic stirring bars

  7. 1-100-1000ug adjustable volume pipette with tips

  8. 1-6.0ml repipet jr. dispenser

  9. Parafilm

  10. Scissors

  11. 1 centigrade thermometer

It is a good idea to clean glassware needed ahead of time as instructed by your trainer.

Turn on needed equipment (items 1&5), to allow it to warm up while you are mixing your chemicals.

 

Gather items listed below for mixing chemical solutions.

Chromotropic acid

Dissolve .10 grams of Chromotropic acid powder in a 100ml volumetric flask and dilute to mark with distilled water. After acid is dissolved transfer solution to 50ml glass beaker and cover with Parafilm. Store in dark place until ready to use.

1.0m sodium sulfite – made only for calibration curve.

Dissolve 12.67 grams (12.60/ACS assay % = grams) you get the ACS assay from the label on the bottle, in a 100ml volumetric flask and dilute to mark with distilled water. Shake well and recheck volume to mark. Cover with Parafilm until ready to use.

1% Sodium Hydroxide (Naoh)

Dissolve 1 gram of sodium hydroxide pellets in a 100ml volumetric flask and dilute to mark with distilled water. Cover with Parafilm until ready to use.

Solution "A"

Fill 1000ml volumetric half way with distilled water. Pipette 2.7ml of 37% formaldehyde solution into flask. Dilute to mark with distilled water. (Mix well) After you have diluted to mark on flask insert magnetic stirring bar and cover with Parafilm until ready to use. You can set this on the magnetic stirrer until ready to use.

Solution "B"

Dissolve 1-gram sodium hydroxide pellets and 1 ml of solution "A" in a 100-ml volumetric flask and dilute to mark. Cover with parafilm until ready to use.

Setup of pH meter

Calibrate your pH meter using pH 7.0 and 10.0 buffer solutions per instructions in the pH meter operator’s manual. Always rinse and dry the probe off between the different buffer solutions using distilled water. Wipe the probe off with kim wipes. You will need to use the manual until you are comfortable with calibrating the equipment. Use the centigrade thermometer to determine the temperature of the buffer solutions that you are using to calibrate the meter with to ensure accuracy. Do not rely on the chart recorder in the room for your temperature.

Standardization of formaldehyde standard solution "A" (1.0 mg/ml)

The titration you will do will determine the concentration of HCHO in solution A.; this is very important so repeat it at least 3 times.

Items Needed

Fill the burette for titrating with 0 .1 N HCL ACID. Check the flow in a spare waste beaker to ensure it is not stopped up. Pour the Sodium Sulfite solution into 1 of the 150-ml beakers. Place the solution on the magnetic stirrer and immerse pH electrode into the solution. Check the pH of the sodium sulfite solution and adjust to 9.5 using 0.1 N Hydrochloric acid. (Only a few drops are needed and this may not be necessary as the pH should be close to 9.5 anyway. If the pH is lower than 9.5 you can raise it by adding a few drops of Sodium Hydroxide. If the pH is 9.5 + 0.2, note the pH and titrate back to that pH. (9.5)

Label the remaining beakers 1,2,3. Insert into each beaker a magnetic stirring bar. This will be needed during the titration of the solutions.

Pipet 20ml of the Sodium Sulfite that you have just adjusted the pH into the three beakers marked 1,2,3. Pipet a 50ml aliquot of Solution A into the three beakers containing the 20 ml solution of sodium sulfite.

Place solution 1 on magnetic stirrer and titrate to a pH of 9.5 using the Hydrochloric acid. This should take about 17 to 19 ml., if not something is wrong and you will have to start over with clean glassware. Repeat for #2 & #3 beakers and record results on the calibration curve data sheet.

Solution "A" and the sodium sulfite will stay stable during the time you are performing the calibration curve. Overnight storage of these chemicals in a refrigerator is recommended, just be sure before you use them that they are at room temperature and stirred before use. It will take a couple of hours for them to equalize with the room temperature.

Calculate the concentration of formaldehyde in solution a:

Ug HCHO/ml = V x N x 30.03 (ug per milliequivalent)
                                                    50 (ml)

V = ML OF 0.1 N HCL average of three samples used for titration.

N = normality of the HCL, found on the bottle.

Record this value on your spreadsheet.

Calibration Curve

Prepare formaldehyde Solution B by diluting 1 ml of Solution A and 1 gram of Sodium Hydroxide to 100 ml in a volumetric flask using distilled water. This solution should be stable for a week.

Check your dilution with a gravimetric determination. 1.00 ml is equal to 1.00 gram; therefore 100.00 ml is equal to 100.00 grams.

The concentration of formaldehyde in Solution B will be calculated using this formula:

Ug HCHO/ml = Concentration in A ( mg/ml) x 1000x1
                                                   100

Record this value. Solution B can be stored for repeated use.

Prepare a 1-% Sodium Hydroxide solution by dissolving 1 gram of Sodium Hydroxide pellets in a 100ml volumetric flask and diluting to the mark with distilled water.

Note: During this hazardous operation please follow recommended safety procedures. The solution becomes extremely hot during the addition of sulfuric acid. If acid is not added slowly, some loss of sample could occur due to splattering.

Label 21 screw capped test tubes 1 through 7 and a,b,c.

Pipet the following volumes, it is very important the solutions are added in this order:

                                                1% Sodium Hydroxide

                                                                                        Solution B

                                                                                        1% Chromatropic Acid

                                                                                         Sulfuric Acid

Test Tube      Sodium Hydroxide Solution B     Chromatropic     Sulfuric   

1a,1b,1c

4.0ml

0

0.1ml

6.0ml

2a,2b,2c

3.9ml

0.1ml

0.1ml

6.0ml

3a,3b,3c

3.8ml

0.2ml

0.1ml

6.0ml

4a,4b,4c

3.7ml

0.3ml

0.1ml

6.0ml

5a,5b,5c

3.6ml

0.4ml

0.1ml

6.0ml

6a,6b,6c

3.5ml

0.5ml

0.1ml

6.0ml

7a,7b,7c

3.4ml

0.6ml

0.1ml

6.0ml

Cap all test tubes after adding all the solutions and place in the test tube rack.

Slowly and gently, agitate test tubes to effect mixing. Mixing is complete when there is no sign of stratification. Rapid mixing will cause heating and a pressure increase, which will cause the test tube to break. When mixing is complete, places capped test tube in heat block for 15 minutes + 2 minutes.

Remove from heat and vent each tube, to release pressure. Allow tubes to cool to room temperature.

Reading the absorbencies of the tubes

Standardize (zero) the spectrophotometer on distilled water at 580 NM in accordance with the instruments operating instructions. This gives an indication that the instrument is in working order. It is further recommended that the reagent blanks (tubes 1a, 1b, 1c) be read against water because an absorbance above 0.1 for the reagent blank indicates contamination of the reagent blank or improper solution preparation.

Note: When transferring the solution to the cuvette small bubbles may appear rising through the cuvette. Do not make absorbance reading until the solution is clear.

If the reagent blanks are <0.100, read each and record the values. Zero the spectrophotometer on the lowest blank. If the absorbance for the reagent blank compared to water is >0.100, repeat the entire standardization procedure.

Read and record absorbances at 580 NM of the other solution prepared (tubes 2 thru 7).

When stepping up to a higher number, use a small amount of the solution of the first tube to rinse out the spectrophotometer tube. (Do not use distilled water). Continue reading absorbancies of all tubes and record on cal curve data sheet as you go.

Plot absorbances against micrograms of formaldehyde in the color developed solution. The amount of formaldehyde (micrograms) is based upon the standardization carried out on solution A.

How to calculate this value:

Amount of solution B added (ml) x concentration of B = ug HCHO.

When preparing to calculate the calibration curve, you may use the average values from your analysis. The values of your least square regression equation are as important as the linearity of your result (r and r2 values) Using this method, values of the slope should be about 0.05. The intercept should be between 0.001 and –0.01. The R-square value should be .998. Please refer to the dynamic microchamber operator’s manual for their suggestion on the labs that plot formaldehyde against absorbance.

After you have completed your curve you are ready to run some impingers on the calibration set. Refer to the instructions in the Grademark Training Manual for instructions for this process.

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