18 CEE 453: Laboratory Research in Environmental Engineering Fall 2006 Laboratory Measurements and Procedures Introduction Measurements of masses, volumes, and preparation of chemical solutions of known composition are essential laboratory skills. The goal of this exercise is to gain familiarity with these laboratory procedures. You will use these skills repeatedly throughout the semester. Theory Many laboratory procedures require preparation of chemical solutions. Most chemical solutions are prepared on the basis of mass of solute per volume of solution (grams per liter or Moles per liter). Preparation of these chemical solutions requires the ability to accurately measure both mass and volume. Preparation of dilutions is also frequently required. Many analytical techniques require the preparation of known standards. Standards are generally prepared with concentrations similar to that of the samples being analyzed. In environmental work many of the analyses are for hazardous substances at very low concentrations (mg/L or µg/L levels). It is difficult to weigh accurately a few milligrams of a chemical with an analytical balance. Often dry chemicals are in crystalline or granular form with each crystal weighing several milligrams making it difficult to get close to the desired weight. Thus it is often easier to prepare a low concentration standard by diluting a higher concentration stock solution. For example, 100 mL of a 10 mg/L solution of NaCl could be obtained by first preparing a 1 g/L NaCl solution (100 mg in 100 mL). One mL of the 1 g/L stock solution would then be diluted to 100 mL to obtain a 10 mg/L solution. Absorption spectroscopy is one analytical technique that can be used to measure the concentration of a compound. Solutions that are colored absorb light in the visible range. The resulting color of the solution is from the light that is transmitted. According to Beer's law the attenuation of light in a chemical solution is related to the concentration and the length of the path that the light passes through. logoPbcPε⎛⎞=⎜⎟⎝⎠ 2.1 where c is the concentration of the chemical species, b is the distance the light travels through the solution, ε is a constant Po is the intensity of the incident light, and P is the intensity of the transmitted light. Absorption, A, is defined as: logoPAP⎛⎞=⎜⎟⎝⎠ 2.2 In practice Po is the intensity of light through a reference sample (such as deionized water) and thus accounts for any losses in the walls of the sample chamber. From equation 2.1 and 2.2 it may be seen that absorption is directly proportional to the concentration of the chemical species. A bcε= 2.319 Laboratory Measurements and Procedures The instrument you will use to measure absorbance is a Hewlett Packard (HP) model 8452A diode array spectrophotometer. The diode array spectrophotometer uses a broad-spectrum source of incident light from a deuterium lamp. The light passes through the sample, 1 cm path length, and is split by a grating into a spectrum of light that is measured by an array of diodes. Each diode measures a bandwidth of 2 nm with 316 diodes covering the range from 190 nm to 820 nm. The wavelengths of light and their colors are given in Table 2-1. The light path for the diode array spectrophotometer is shown in Figure 2-1. The HP 8452A spectrophotometer has a photometric range of 0.002 - 3.3 absorbance units. In practice absorbance measurements greater than 2.5 are not very meaningful as they indicate that 99.7% of the incident light at that wavelength was absorbed. Conversely, an absorbance of 0.002 means that 0.5% of the incident light at that wavelength was absorbed. When measuring samples of known concentration the Spectrophotometer software (http://ceeserver.cee.cornell.edu/mw24/Software/Spectrophotometer.htm) calculates the relationship between absorbance and concentration at a selected wavelength. The slope (m), intercept (b) and correlation coefficient (r) are calculated using equation 2.4 through 2.6. The slope of the best fit line is ()22xynxymxxn∑∑−=−∑∑∑ 2.4 The intercept of the line is b myx=− 2.5 The correlation coefficient is defined as Table 2-1. Wavelengths of light color wavelength (nm) ultra violet 190-380 violet 380-450 blue 450-490 green 490-560 yellow 560-590 orange 590-630 red 630-760 Diode ArraySpectrograph LensShutterGratingSlitSample CellSource LensDeuterium Lamp Figure 2-1. Diagram of light path in diode array spectrophotometer.20 CEE 453: Laboratory Research in Environmental Engineering Fall 2006 () ()2222xynxyrxyxynn∑∑−=⎛⎞⎛⎞⎜⎟⎜⎟−−⎜⎟⎜⎟⎝⎠⎝⎠∑∑∑∑∑ 2.6 where x is the concentration of the solute (methylene blue in this exercise), y is the absorbance, and n is the number of samples. Experimental Objectives To gain proficiency in: 1) Calibrating and using electronic balances 2) Digital pipetting 3) Preparing a solution of known concentration 4) Preparing dilutions 5) Measuring concentrations using a UV-Vis spectrophotometer Experimental Methods Mass Measurements Mass can be accurately measured with an electronic analytical balance. Perhaps because balances are so easy to use it is easy to forget that they should be calibrated on a regular basis. It is recommended that balances be calibrated once a week, after the balance has been moved, or if excessive temperature variations have occurred. In order for balances to operate correctly they also need to be level. Most balances come with a bubble level and adjustable feet. Before calibrating a balance verify that the balance is level. The environmental laboratory is equipped with balances manufactured by Denver Instruments. To calibrate the Denver Instrument balances: 1) Zero the balance by pressing the tare button. 2) Press the MENU key until "MENU #1" is displayed. 3) Press the 1 key to select Calibrate. 4) Note the preset calibration masses that can be used for calibration on the bottom of the display. 5) Place a calibration mass on the pan (handle the calibration mass using a cotton glove or tissue paper). 6) The balance will automatically calibrate. A short beep will occur and the display will read CALIBRATED for three seconds, and then return to the measurement screen. Dry chemicals can be weighed in disposable plastic "weighing boats" or other suitable containers. It is often desirable to subtract the weight of the container in which the chemical is being