Upper left: typical dark blue fecal coliform colonies on a membrane filter after incubation on M-FC agar. Lower center (purple): M-FC agar before

Transcription

1 KIMIA ANALISA 3 SKS Prof. Dr. Heru Setyawan Jurusan Teknik Kimia FTI ITS

2 The Language of Analytical Chemistry

3 Analysis, Determination, and Measurement Analysis: A process that provides chemical or physical information about the constituents in the sample or the sample itself. Analytes: The constituents of interest in a sample. Matrix: All other constituents in a sample except for the analytes. Determination: An analysis of a sample to find the identity, concentration, or properties of the analyte. Measurement: An experimental determination of an analyte s chemical or physical properties.

4 Analysis, Determination, and Measurement In 1974, the federal government enacted the Safe Drinking Water Act to ensure the safety of public drinking water supplies. To comply this act municipalities regularly monitor their drinking water supply for substantially harmful substances. One such substance is coliform bacteria. Municipal water departments collect and analyze samples from their water supply. To determine the concentration of coliform bacteria, a portion of water is passed through a membrane filter. The filter is placed in a dish containing a nutrient broth and incubated. At the end of the incubation period the number of coliform bacterial colonies is measured by counting (see figure). Thus, municipal water departments analyze samples of water to determine the concentration of coliform bacteria by measuring the number of bacterial colonies that form during a specified period of incubation. Upper left: typical dark blue fecal coliform colonies on a membrane filter after incubation on M-FC agar. Lower center (purple): M-FC agar before use. Upper right (blue): a control plate streaked with an E.coli culture.

5 Techniques, Methods, Procedures, and Protocols Technique: A chemical or physical principle that can be used to analyze a sample. Method: A means for analyzing a sample for a specific analyte in a specific matrix. Procedure: Written directions outlining how to analyze a sample. Protocol: A set of written guidelines for analyzing a sample specified by an agency.

6 Techniques Graphite furnace atomic absorption spectroscopy Methods Pb in Water Pb in Soil Pb in Blood Procedures APHA ASTM Protocols EPA Chart showing hierarchical relationship among a technique, methods using that technique, and procedures and protocols for one method. Abbreviations: APHA = American Public Health Association ASTM = American Society for Testing Materials EPA = Environmental Protection Agency

7 Classifying Analytical Techniques If a technique responds to the absolute amount of analyte in the sample, the signal due to the analyte S A S A = kn A Since cylinder 2 contains twice as many moles of Cu 2+ as cylinder 1, analyzing the contents of cylinder 2 gives signal that is twice that of cylinder 1. If a technique responds to the relative amount of analyte in the sample, the signal Graduated cylinders containing 0.01 M Cu(NO 3 ) 2. (a) Cylinder 1 contains 10 ml, or mol, of Cu 2+. (b) Cylinder 2 contains 20 ml, or mol, of Cu 2+. due to the analyte S A S A = kc A Since the solutions in both cylinders have the same concentration of Cu 2+, their analysis yields identical signals.

8 Classifying Analytical Techniques Total analysis techniques A technique in which the signal is proportional to the absolute amount of analyte; also called classical techniques. Mass, volume, and charge are the most common signals for total analysis techniques, and the responding techniques are gravimetry, titrimetry, and coulometry. Concentration techniques A technique in which the signal is proportional to the analyte s concentration; also called instrumental technique, e.g., spectroscopy, potentiometry, and voltammetry. The relationship between the signal and the analyte is a theoretical function that depends on experimental conditions and the instrumentation used to measure the signal, so the value of k must be determined experimentally.

9 Validation involves determining: selectivity linearity accuracy precision sensitivity range limit of detection limit of quantitation ruggedness/robustness Standard reference materials (SRMs) best for determining accuracy. General process for evaluation/validation of methodology.

10 Selecting an Analytical Method Accuracy A measure of the agreement between an experimental result and its expected value. obtained result expected result % Error = 100 expected result Analytical methods may be divided into three groups based on the magnitude of their relative errors: < 1% : highly accuracy Between 1% and 5% : moderately accuratcy > 5% : low accuracy In general, total analysis methods produce results of high accuracy, and concentration methods range from high to low accuracy. Precision An indication of the reproducibility of a measurement or result. Depends on those factors affecting the relationship between the signal and the analyte. Of particular importance are the uncertainty in measuring the signal and the ease of handling sampling reproducibly.

11 Selecting an Analytical Method (a) (b) Two determinations of the concentration of K + in serum, showing the effect of precision. The data in (a) are less scattered and, therefore, more precise than the data in (b).

12 Selecting an Analytical Method Sensitivity A measure of a method s ability to distinguish between two samples; reported as the change in signal per unit change in the amount of analyte (k). If S A is the smallest increment in signal that can be measured, then the smallest difference in the amount of analyte that can be detected is n C Detection limit A A = = S k S k A A (total analysis method) (concentration analysis method) A statistical statement about the smallest amount of analyte that can be determined with confidence.

13 Selecting an Analytical Method Selectivity A measure of a method s freedom from interferences as defined by the method s selectivity coefficient. S samp = S A + S I = k A n A + k I n I S samp = S A + S I = k A C A + k I n I Selectivity coefficient (total analysis method) (concentration analysis method) A measure of a method s sensitivity for an interferent relative to that for the analyte (K A,I ) K = A,I k k I A

14 Selecting an Analytical Method 1. A method for the analysis of Ca 2+ in water suffers from an interference in the presence of Zn 2+. When the concentration of Ca 2+ is 100 times greater than that of Zn 2+, an analysis for Ca 2+ gives a relative error +0.5%. What is the selectivity coefficient for this method? 2. Barnett and colleagues developed a new method for determining the concentration of codeine during its extraction from poppy plants. As part of their study they determined the method s response to codeine relative to that for several potential interferents. For example, the authors found that the method s signal for 6- methoxycodeine was 6 (arbitrary units) when that for an equimolar solution of codeine was 40. a) What is the value for the selectivity coefficient K A,I when 6- methoxycodeine is the interferent and codeine is the analyte? b) If the concentration of codeine is to be determined with an accuracy of ±0.50%, what is the maximum relative concentration of 6-methoxycodeine (i.e., [6-methoxycodeine]/[codeine]) that can be present?

15 Selecting an Analytical Method Robust A method that can be applied to analytes in a wide variety of matrices is considered robust. Rugged A method that is insensitive to changes in experimental conditions is considered rugged.

16 Selecting an Analytical Method Scale of Operation Three limitations of particular importance: The amount of sample available for the analysis. The concentration of analyte in the sample. The absolute amount of analyte needed to obtain a measurable signal.

17 Selecting an Analytical Method -log(% analyte as %w/w) Ultratrace Trace Minor Major Scale of operation for analytical methods % 10-9 % 10-8 % 10-7 % 10-6 % 10-5 % 10-4 % 10-3 % 10-2 % 0.1% 1% 10% 100% 1 g sample, 1% analyte 0.1 g sample, 10% analyte 0.01 g sample, 100% analyte Macro Meso Micro Ultramicro -log(weight of sample) g mg µg ng

18 Selecting an Analytical Method Equipment, Time, and Cost Analytical methods can be compared in terms of their need for equipment the time required to complete an analysis the cost per sample. Methods relying on instrumentation are equipment-intensive intensive and may require significant operator training. The graphite furnace atomic absorption spectroscopic method for determining lead levels in water requires a significant capital investment in the instrument and experienced operator to obtain reliable results. Other methods such as titrimetry, require only simple equipment and reagents and can be learned quickly. The time needed to complete an analysis for a single sample is often fairly similar from method to method. This is somewhat misleading because much of this time is spent preparing the solutions and equipment needed for the analysis. Once the solutions and equipment are in place, the number of samples that can be analyzed per hour differs substantially from method to method. The cost of an analysis is determined by many factors, including the cost of necessary equipment and reagents, the cost of hiring analysts, and the number of samples that can be processed per hour. In general, methods relying on instruments cost more per sample than other methods.

19 Calibration and Standardization Calibration The process of ensuring that the signal measured by a piece of equipment or an instrument is correct. For example: balances are calibrated using a standard weight whose mass can be traced to the internationally accepted platinum-iridium iridium prototype kilogram. Standardization The process of establishing the relationship between the amount of analyte and a method s signal. For a total analysis method, standardization is usually defined by the stoichiometry of the chemical reactions responsible for the signal. For a concentration method, the relationship between the signal and the analyte s concentration is a theoretical function that cannot be calculated without experimental measurements. To standardize method, the value of k is determined by measuring the signal for one or more standards, each containing a known concentration of analyte. When several standards with different concentrations of analyte are used, the result is best viewed visually by plotting S meas versus the concentration of analyte in the standards. Such plot is known as a calibration curve.

20 Problems 1. When working with a solid sample, it often is necessary to bring the analyte into solution by dissolving the sample in a suitable solvent. Any solid impurities that remain are removed by filtration before continuing with the analysis. In a typical total analysis method, the procedure might read After dissolving the sample in a beaker, remove any solid impurities by passing the solution containing the analyte through filter paper, collecting the solution in a clean Erlenmeyer flask. Rinse the beaker with several small portions of solvent, passing these rinsings through the filter paper, and collecting them in the same Erlenmeyer flask. Finally, rinse the filter paper with several portions of solvent, collecting the rinsings in the same Erlenmeyer flask. For a typical concentration method, however, the procedure might state After dissolving the sample in a beaker, remove any solid impurities by filtering a portion of the solution containing the analyte. Collect and discard the first several milliliters of solution before collecting a sample of approximately 5 ml for further analysis. Explain why these two procedures are different.

21 Problems 2. A certain concentration method works best when the analyte s concentration is approximately 10 ppb. a. If the sampling volume for the method is 0.5 ml, about what mass of analyte is being measured? b. If the analyte is present at 10% w/v, how would you prepare the sample for analysis? c. Repeat for the case in which the analyte is present at 10% w/w. d. Based on your results, comment on the suitability of this method for the analysis of a major analyte. 3. An analyst needs to evaluate the potential effect of an interferent, I, on the quantitative analysis for an analyte, A. She begins by measuring the signal for a sample in which the interferent is absent and the analyte is present with a concentration of 15 ppm, obtaining an average signal of 23.3 (arbitrary units). When analyzing a sample in which the analyte is absent and the interferent is present with a concentration of 25 ppm, she obtains an average signal of a. What is the analyte s sensitivity? b. What is the interferent s sensitivity? c. What is the value of the selectivity coefficient? d. Is the method more selective for the analyte or the interferent? e. What is the maximum concentration of interferent relative to that of the analyte (i.e., [interferent]/[analyte]), if the error in the analysis is to be less than 1%?

22 Problems 4. A sample was analyzed to determine the concentration of an analyte. Under the conditions of the analysis, the sensitivity is 17.2 ppm -1. What is the analyte s concentration if S meas is 35.2 and S reag is 0.6? 5. A method for the analysis of Ca2+ in water suffers from an interference in the presence of Zn2+. When the concentration of Ca2+ is 50 times greater than that of Zn2+, an analysis for Ca2+ gives a relative error of -2.0%. What is the value of the selectivity coefficient for this method? 6. The quantitative analysis for reduced glutathione in blood is complicated by the presence of many potential interferents. In one study, when analyzing a solution of 10-ppb glutathione and 1.5-ppb ascorbic acid, the signal was 5.43 times greater than that obtained for the analysis of 10-ppb glutathione. What is the selectivity coefficient for this analysis? The same study found that when analyzing a solution of 350-ppb methionine and 10-ppb glutathione the signal was times less than that obtained for the analysis of 10 ppb- glutathione. What is the selectivity coefficient for this analysis? In what way do these interferents behave differently? 7. Oungpipat and Alexander described a new method for determining the concentration of glycolic acid (GA) in a variety of samples, including physiological fluids such as urine. In the presence of only GA, the signal is given as S samp,1 = k GA C GA and in the presence of both glycolic acid and ascorbic acid (AA), the signal is S samp,2 = k GA C GA + k AA C AA

23 Problems When the concentration of glycolic acid is M and the concentration of ascorbic acid is M, the ratio of the signals was found to be S samp,2 /S samp,1 = 1.44 a. Using the ratio of the two signals, determine the value of the selectivity ratio K GA,AA = k AA /k GA b. Is the method more selective toward glycolic acid or ascorbic acid? c. If the concentration of ascorbic acid is M, what is the smallest concentration of glycolic acid that can be determined such that the error introduced by failing to account for the signal from ascorbic acid is less than 1%?