Skoog, Holler and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing, Fort Worth, TX 1998, Ch 33.
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1 CHEM 3281 Experiment Ten Determination of Phosphate by Flow Injection Analysis Objective: The aim of the experiment is to investigate the experimental variables of FIA for a model system and then to use the system to determine the phosphate content in an unknown sample. Text Reference: Skoog, Holler and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing, Fort Worth, TX 1998, Ch 33. Introduction: In most analytical measurement systems, the signal is obtained on a discrete or batchwise basis. In certain circumstances, however, it can be advantageous to obtain analytical measurements on a continuous basis. This can allow the following advantages: 1. greater speed of analysis; 2. the ability to automate the analysis; 3. better reproducibility; 4. smaller sample volume. In automated analysis systems, solution is continuously pumped through tubes (with a peristaltic pump), the sample is injected into the stream and various reactions are carried out by mixing streams of sample and reagent, buffer, etc. The concentration of the product(s) is determined by a detector or transducer. Initially analysis in a flowing stream was done using air bubbles introduced in regular time intervals (segmented-flow analysis). This was done to prevent excess sample dispersion, to insure mixing of sample and reagents and to prevent cross-contamination. Ruzicka and Hansen (the co-inventors of FIA) found that the air bubbles were unnecessary in properly designed systems and that sample throughput could be much higher and simpler equipment could be used. (Air bubbles cause problems in most detection systems!) Flow Injection Analysis is now the method of choice in automated analysis. To use FIA knowledgeably, you must understand the principles of dispersion as they apply to FIA. Theory A rectangular "plug" of sample injected into a flowing stream will initially experience no dispersion and will assume the concentration profile shown in Fig. 1.a. If this plug passes a detector, the recorder readout of the peak would have the shape shown underneath. In practice, however, between the points of injection and detection, some dispersion will always have occurred. Physical dispersion is brought on by convection and diffusion.
2 Initially convection caused by laminar flow gives the profile in Fig 1.b. This gives rise to a parabolic head and tail and concomitant detector output. Further broadening is due to diffusion. Two types of diffusion can occur: radial and longitudinal. In narrow bore tubing, however, longitudinal diffusion is not significant and can be ignored. Radial diffusion (diffusion perpendicular to flow direction) is important in FIA and serves to insure adequate mixing and keeping the walls clean. Most FIA experiments are run under conditions where both convection and radial diffusion are contributing to dispersion. (Fig 1.c.) The dispersion, D, is given by: D = C o /C= q H o /H where: C o is the concentration of the injected sample (mol/l), C is the concentration of sample at detector (mol/l), q is a constant, H is the recorded peak height, and H o is the peak height with the cell full of undispersed colored solution. Dispersion will take place in the injection port, D i, in the tubing, D t, and the detector, D d such that the total dispersion, D total is given by: D total = D i D t D d Dispersion is classified into three categories for FIA: limited (D t = 1-3); medium (D t = 3-10); and large (D t 10). Limited dispersion, D = 1-3 : The rise of curve will have the form: C = C o ( 1 - exp {- ks}) and the fall curve C = C o exp {-ks}, where k is a system constant and s is the sample volume (ul). Here dispersion is due mostly to convection and gives rise to the classic Poisson distribution or "tailing peak" seen in the detector output in Fig 1.b. Medium Dispersion, D = 3-10: Here, a combination of convection and radial diffusion are creating the dispersion. Detector output should look like Fig 1.c. Dispersion should (1) increase linearly with pumping rate; (2) increase as the square root of traveled distance or residence time. Large Dispersion, D 10: Here, dispersion is caused by radial diffusion only and the
3 distribution is gaussian as seen in Fig.1.d. Most FIA experiments employ narrow tubing ( mm) and are conducted using medium flow rates (10-2 ml/min). Under these conditions, the dispersion is generally medium and mixing is due to radial diffusion. Sample cross-contamination is minimized by adequate time delay between injections. The delay period is determined by the extent of sample dispersion. The analytical procedure is based upon the following reactions. 7H 3 PO (NH 4 ) 6 Mo 7 O H + 7(NH 4 ) 3 PO 4-12MoO NH H 2 O (1) (NH 4 ) 3 PO 4-12MoO 3 + Ascorbic Acid blue Mo(V) complex (2) The intermediate product (NH 4 ) 3 PO 4-12MoO 3 is a yellow colored Mo(VI) complex. When this species is treated with ascorbic acid, a blue colored Mo(V) complex results and is detected spectrophotometrically. Experimental Apparatus: For the colorimetric analysis of phosphate, the flow manifold should be set up as follows: where: A-- is distilled water B-- is the acidified ammonium heptamolybdate solution ( M (NH 4 ) 6 Mo 7 O 24 and 0.2 M HNO 3 ) C --is 0.25% (w/v) ascorbic acid solution (with glycerine & Sb catalyst) A phosphate standard (~100 ug/ml) and a phosphate unknown will be provided.
4 Experimental Procedure: The experimental apparatus will be set up when you arrive to the lab. To begin, place all three of the inlet tubes into a large beaker of water and turn on the pump. Place the waste end of the line into the nearest sink. Allow the water to run through the system until all air bubbles are eliminated in the lines and detector. Preparation: 1. PUMPING SYSTEM. Set the pump speed to "4". Once you are assured that only water is exiting the waste line is at this speed, place the line in a 10 ml graduated cylinder and measure the time required to deliver 1-2 ml of water. Calculate the flow rate in ml/min (remember there are three inlet lines, so the measured flow rate at waste needs to be divided by three). Repeat this procedure for pump speeds of "1", 2, "6" and "8". The aqueous waste generated can be poured down the sink. Place the waste line into the sink. 2. DETECTOR. Open the FIALAB software. GO to Spectrometer and take a DARK SPECTRUM. Turn on the visible source and click on VOLTAGE & REPEATED SCAN. Go to SPECTROMETER SETUP. Make sure there are NO AIR BUBBLES in the detector flow cell. Lower the integration time until guassian power spectrum of tungsten lamp has the highest point about 3500 with no flattening. (Will have to switch back and forth between SPECTROMETER SETUP and SPECTROMETER ). Wavelength should be set at ~660 nm. 3. STANDARDS. Prepare standards containing 100 ppm, 50 ppm, 25 ppm, 12.5 ppm of phosphate by successive dilution of the stock solution with water. Use 50 ml volumetric flasks. (Remember C 1 V 1 = C 2 V 2 )! Obtain an UNKNOWN and record its number. 4. INJECTOR. Practice filling the 50 ul injection loop with a syringe equipped with a Luer fitting and injecting this into the sample stream. A counter clockwise rotation of the injector knob is appropriate for filling the loop; a clockwise rotation is appropriate for flushing the loop. Make sure you can fill the loop without introducing any air bubbles. System Optimization: The following study will provide you with understanding of the experimental variables affecting the dispersion and overall efficiency of FIA. Both reaction (1) & (2) are NOT instantaneous so you must optimize the system to allow enough time for the blue Mo(V) complex to form and not allow too much dispersion so the peak shape doesn t get too broad. The LINE LENGTH STUDY will show you what coil lengths to use.
5 One person must inject while the other runs the spectrometer Now place the inlet lines into their designated container A, B, or C. Be sure you get it into the correct bottle. Set the flow rate to 4 and fill all lines with the correct chemical solution. 1. LINE LENGTH STUDY : Observe the influence of tube length (in the mixing coil) on the peak shape by varying the length of tubing for each coil over the range 50 cm, 100 cm. and 200 cm. (Hold the other coil constant at 100 cm.) Be sure each inlet line is full of the correct solution, no air bubbles in the lines. You must learn how to run the spectrometer so that each run can be compared to the other runs. This most easily accomplished by forcing the X and Y axes to always have the same scale each run so that you can easily make the plots called for later on. Unfortunately this software is autoscaling both Absorbance and Time continuously which is not useful for this optimization. Plan to run the spectrometer for say 60 secs each run. Load the 100 ppm phosphate standard into the sample loop Take a REFERENCE before each run (Make the injector person check detector for air bubbles Turn on REPEATED SCAN and tell lab partner to inject. Run for 60 secs. Go to ANALYSIS mode and call up data. Your most recent data should be in TIME SERIES BY CHANNEL. Click on AXIS icon and scale the Y axis to same scale each time (say 0.8 to 0.2 absorbance units). (Can click on STYLE and deselect data point tags if desired ) Note: LOCAL MAX. and PEAK AREA are random numbers only-not functional software! Click on PRINTER icon and then click on Print on system page to obtain hardcopy. While computer person is scaling the run the injector person should turn off pump, put another coil length in place and turn pump back on to remove air bubbles. Vary coil I then coil II. By using the reaction coils of different lengths, you are allowing for longer times spans in which the reactions can occur. Determine from the data, the optimum reaction coil lengths and install them for the FLOW RATE STUDY below. Note that if the flow rate is too fast the blue product will form too late (past the detector). Adjust the flow rate appropriately if needed. FLOW RATE STUDY. With your optimized coil lengths in place, inject 50 ul samples of 100 ppm Phosphate Standard into the stream at flow rates of "2", "3", "4", and "6" and 8. Run the lowest flow rate first so you can determine how long to run the spectrometer each time. Start with 30 secs and see how long it takes for the blue peak to come through the system.
6 Load the 100 ppm phosphate standard into the sample loop Take a REFERENCE before each run (Make the injector person check detector for air bubbles Turn on REPEATED SCAN and tell lab partner to inject. Run for 30 secs or until you are sure the peak has cleared the system! Go to ANALYSIS mode and call up data. Click on AXIS icon and scale the Y axis to same scale each time (say 0.8 to 0.2 absorbance units). Click on PRINTER icon and then click on Print on that page to obtain hardcopy. While computer person is scaling the run the injector person should turn be adjusting the flow rate and loading the sample loop again. 3. THROUGHPUT STUDY: At the flow rate that provides the highest absorbance reading in the shortest amount of time, determine how rapidly it is possible to make successive injections without causing the resultant peaks to overlap. You decide how to do this experimentally! Do this by timing the interval between injections and monitoring the output on the spectrometer. The smallest time interval that will "resolve" the peaks is what you are trying to find!! Decide if you want to use PEAK AREA or PEAK HEIGHT for an analysis. Unknown Determination: Now that you understand how varying the flow rate and line length varies your output, set the optimum parameters (flow rate and coil length) for running a calibration curve and unknown. 1. Prepare a calibration curve by injecting your standards containing 100, 50, 25 and 12.5 ppm of phosphate; starting with the highest concentration. Again run the spectrometer for the same time each run and scale the Y axis post-run to be the same. Run one standard in triplicate to obtain an estimate of the error. 2. Repeatedly inject your unknown (at least three times) and measure the peak absorbance. Calculations Make the following plots: Can measure peak height and width at half-height (W 1/2 ) with a good millimeter ruler. In the real world you would run each data set into EXCELL and have spreadsheet do these calculations! 1. Peak height vs. line length for coil I and II 2. W 1/2 vs.line length. 3. Peak height vs. flow rate 4. W 1/2 vs. flow rate 5. Peak height or peak area (1/2 base x height) vs. concentrations of standards Calculate the concentration of unknown from this plot. Report unknown + error
7 Discussion: Include a discussion of the specific procedure and criteria you used in selecting the optimum parameters. The discussion section should contain answers to the following questions: 1. What type of dispersion do you appear to have at the different flow rates? Look at the shape of each curve. 2. What is the maximum number of samples per hour that you estimate could be determined by this procedure? (Assume an autosampler, rather than manual injection) Explain your answer. 3. What effect does the tube length have on peak height and peak width. Why? 4. What is the concentration of your unknown. Did you use peak height or area? Is your calibration curve linear or non-linear? What is your uncertainty in the unknown measurement and how did you obtain it? 5. Do you think flow injection analysis could be used with detectors other than a spectrophotometer? Suggest a possible alternative detector and describe a suitable analysis.
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