Using the Spectrophotometer

The spectrophotometer uses indicator compounds to determine the concentration of the desired species. In this case we'll be measuring nitrate concentration, one of the principal contaminants in urban surface waters. Use of the Hach Test kit involves mixing a pre-measured amount (a ``pillow'') of the indicator compound with a measured amount of sample water, then measuring the absorption of light of a particular wavelength. After mixing and a delay for reaction, a color will appear (for nitrogen it is amber color), the darkness of which is measured in the spectrophotometer. The sample is introduced to the spectrophotometer via a pour-through-cell apparatus (Fig. 4.9), to minimize optical interferences from sample handling.

Figure 4.9: Pour-through cell arrangement for spectrophotometer measurements. After Hach DR/2010 Spectrophotometer Instrument Manual, p. 10, 1997.

The procedure for making a nitrate analysis via spectrophotometry is:

1. Obtain at least 50 ml of filtered sample
   1. set up a filter and funnel in the funnel holder (fold the round filter paper in half, then half again, place in funnel and spread to make open cone)
   2. place clear 250 ml sample jar (these have light blue lids) under funnel to catch filtrate
   3. pour sample into filter until water level is close to the top
   4. wait for sample to gravity drain into clear jar
   5. repeat, after second filter-full you should have enough sample to analyze for nitrate

2. Prepare spectrophotometer
   1. make sure drain hose from pour-through apparatus leads to a large-volume waste container
   2. rinse out pour-through-cell pour 25-50 ml of de-ionized water into the pour-through funnel and allow it to drain
   3. Enter the stored program number for high range nitrate nitrogen (NO3-N). Press 355 ENTER. The display will show Dial nm to 500.
   4. If needed, rotate the wavelength dial until the small display shows: 500 nm . When the correct wavelength is dialed in, the display will quickly show : ``Zero Sample'', then `` mg/L NO3-N HR'' (indicating measurement of nitrogen ``N'' reported as nitrate ``NO$_3$'' for expected high range ``HR'' of concentration)
3. Prepare sample for measurement
   1. Fill a mixing bottle (clear with white lid, has fill-lines on it for 20 and 25 mL) with 25 mL of filtered sample (top line)
   2. Put on rubber gloves, move DOWNWIND from the class
   3. Tear open one NitraVer 5 Nitrate Reagent packet, push the sides together to form a square opening along the top
   4. Add entire contents of packet to the mixing bottle, cap firmly
4. Follow measurement program:
   1. Press SHIFT (release) then TIMER, display will show 1 minute countdown
   2. Shake the cell vigorously until the timer beeps
   3. Press SHIFT then TIMER again. Spectrophotometer will begin five minute reaction period countdown
   4. while waiting, fill the pour-through funnel with filtered sample, this will be the blank against which the color of the reacted sample will be compared. The spectrophotometer reports the difference between these two as the final composition.
   5. When the timer beeps, the display will show mg/L NO3-N HR
   6. Press ZERO (this measures and sets the blank concentration). The display will show: Zeroing ? then, 0.0 mg/L NO3-N HR.
   7. Pour the reacted sample into the pour-through funnel
   8. wait until the water level in the funnel stops falling
   9. Press READ The display will show: ``Reading'' then the result in mg/L nitrate nitrogen (NO3-N) will be displayed.
   10. Record the value in your notebook.