Spectrophotometric Analysis

Example Lab Report

Spectrophotometric Analysis

CEE 341 Fluid Mechanics for Civil Engineers

Prepared by: Dr. Laminar

For: Civil Engineers of Arizona State University

January 29, 1998

Table of Contents

List of Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 3 List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 3 1.0 Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 4 2.0 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 4 3.0 Anticipated Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 5 4.0 Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 5 5.0 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 6 5.1 Working Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 6 5.2 Sample Description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 7 5.3 Sample Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 7 6.0 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 8 7.0 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 10 8.0 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 12 9.0 Critique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 13 10.0 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 14 10.1 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 14 10.2 Sample Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 14 10.3 Original Data Sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 14

List of Tables

Table 1: Measured Absorbance by Spectrophotometric Analysis . . . . . . . . . . . . . . . . . . . . . page 8 Table 2: Analytical Phosphate Concentration in g/L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 9 Table 3: Statistical Reduction of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 10

List of Figures

Figure 1 : Spectrophotometric Analysis Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 6 Figure 2 : Standard Curve for Phosphate Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 9

1.0 Objective

Spectrophotometric analysis for determining the amount of an inorganic compound in solution involves a reaction between an organic reagent and an analyte to form a colored complex. The reaction can be used to determine analyte concentrations assuming the color intensity and absorbance is proportional to the analyte concentration, the complex is stable, and the reagent does not significantly react with other constituents thereby causing interferences. A spectrophotometer is the specific device which measures the absorption of a monochromatic light beam by a sample and added reagent. The objective of this laboratory exercise is to become familiar with a typical spectrophotometric analysis and to examine the effect of an interfering substance. The inorganic analyte being considered in this particular analysis is phosphate and the interfering substance is arsenic.

2.0 Theory

The first portion of a spectrophotometric analysis consists of preparing six standard solutions, each with a known phosphate concentration. By measuring the absorbance of each standard and added reagent with a spectrophotometer and plotting its value relative to the known phosphate concentration, a standard phosphate curve can be developed [Standard Methods, 1992]. The second portion of this type of experimental analysis involves measuring the absorbance water samples and reagent having unknown phosphate concentrations. These samples typically consist of canal water filtered through acid-washed GF/C filters, canal water stored without filtration for one day and then filtered through nonwashed GF/C filters, filtered canal water with 1.0 mg/L of arsenic added, and a distilled water blank. The standard curve can be used with the measured absorbance to determine the unknown phosphate concentration in each sample. This specific method of analysis is often referred to as the ascorbic acid method for determining phosphate concentrations.

Beer's law can be described by the following expression [Standard Methods, 1992]:

A = k2C

(1)

where k2 is Beer's proportionality constant, A is absorbance, and C is concentration. According

to Beer's Law, the relationship between absorption and concentration is linear.

A statistical analysis is used to evaluate consistency and performance in spectrophotometric analysis. The standard deviation, a common measure of variability, is evaluated using the following expression given [Hogg and Ledolter, 1987]:

sx =

--------(--X----i---?-----X----A---V---G----) n?1

(2)

Where Xi is the computed concentration value, XAVG is the mean concentration value, n is the

number of samples analyzed, and sx is the standard deviation.

The coefficient of variation, which is used to normalize the standard deviation to the mean, is determined by:

CV = s---x----?----1---0---0--

(3)

X AVG

A large coefficient of variation indicates widely scattered or varied results, and thus precision has

been somewhat sacrificed in the analysis.

3.0 Anticipated Results

The following is a short list of anticipated results and trends which will be confirmed by this laboratory exercise:

? An intensely colored solution of sample and reagent should yield a higher absorbance and, in turn, a higher phosphate concentration.

? Since arsenates also react with the molybdate reagent, the presence of arsenic will most likely cause distinct interferences in the determination of phosphate concentrations.

? The phosphate concentration in stored samples should be higher than those that were not stored due to the formation and persistence of orthophosphates over time.

? Beer's Law is expected to be observed within the working range since the standard curve will be approximated as a straight line.

? The detection limit is expected to be approximately at the lower end of the working range.

4.0 Apparatus

A schematic of the apparatus used for this laboratory exercise is shown in Table 1 on page 6, if one uses their imagination. The device on the left is seated at a desk, while the device on the right

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