Spectrophotometry –long name easy procedure

Our first indoor lab assignment was conducted on September 13, 2010. The objective of this lab was to use the spectrophotometry technique to determine the phosphate concentrations for water samples collected from three different marine/aquatic environments.

Phosphate is an essential but limiting nutrient required for marine and aquatic plant/algae growth. Phosphate occurs naturally in the environment, usually in sediments and rocks. However, surface runoff (water) mainly caused by rainwater that is unable to infiltrate the soil, creates a way for phosphate as well as other nutrients to enter marine/aquatic systems. High concentrations of phosphate usually result in over-enrichment (eutrophication) of the marine/aquatic system, causing the phenomenon called algal blooms. These blooms use up so much oxygen that fish and others species die. However, when phosphate concentrations are low there is very little productivity (algal growth) and the environment is considered nutrient poor (oligotrophic).

In order to assess what phosphate concentrations may be associated with varying marine/aquatic environments, water samples for this assignment were collected from the following areas: 1) a brackish water creek surrounded by marshlands that flows along the backside of Savannah State University (SSU), 2) a phytoplankton tank that is a part of a controlled biofuel study being conducted by a fellow graduate student, and 3) the effluent (outflow) of a wastewater treatment plant that receives high mineral and nutrient loads.




Collecting water sample from Phyto-tank



The Spectrophotometer

The technique used to determine the phosphate concentrations for each water sample was called spectrophotometry. This technique utilizes light absorption to determine the concentration of particles in solution. The instrument used for this technique was the spectrophotometer: a very sensitive by precise apparatus. Our first step was to calibrate the spectrophotometer with 10, 15, 20, 30, 50 and 100 µL standards of a known phosphate concentration (50 mg L-1 PO43-), using the molybbdate blue complex phosphate determination. This determination tells us how much and how well light pastes through a sample, therefore, the amount of light absorbed by the sample is equal to its concentration. For analysis, this determination required preparing a concentrated mixed phosphate reagent and a color developing solution, which were placed in 1-cm cuvettes. 2 mL of each standard was placed in the 1 cm cuvettes and 250 µL of concentrated mixed phosphate reagent and 100 µL of the color developing solution was added.. Each standard was placed one at a time in the spectrophotometer, and the phosphate concentrations were recorded.

Cuvettes containing water sample

This completed our calibration process. Our next step was to prepare the water samples collected from the three different sites. The preparation technique for these samples was exactly the same as for the standards: 2 mL of each sample was placed in 1 cm cuvettes and 250 µL of concentrated mixed phosphate reagent and 100 µL of the color developing solution was added. Each water sample was placed one at a time in the spectrophotometer, and the phosphate concentrations were recorded.

Courtney preparing samples for processing

The phosphate concentration data collected from the standards was used to construct a calibration curve, and the phosphate concentration data collected from the water samples were compared to this calibration curve. The unit used to represent concentration was absorption (arb), because that the amount of light absorbed by the sample was a reflection of its concentration. The major finding was that that wastewater effluent had the greatest concentration of phosphate with 0.291 arb, followed by the estuary with 0.022 arb and the phyto tank with 0.004 arb. These observations could lead to the hypothesis that high phosphate concentration may be associated with high nutrient loads. Hence, the result from the water sample collected from the wastewater treatment plant was as expected; it had the highest levels of nutrients. While the photosynthetic activity occurring in the phytoplankton tank, which was full of algae using up the nutrients, may have resulted in it having the lowest phosphate concentration.

Water Sample              Absorbance (Arb.)


Macrotank (estuary)    0.022

Wastewater (effluent)  0.291

Phyto tank                  0.004___________
Results table showing the water samples arb ratios.