Course Objectives Teach fundamentals of instrumental analysis Lecture: Discuss theory and background for (1) chemical/physical property measured (2) origin of chemical/physical property (3) instrument design and nature of response (4) signal processing and relationship between readout to property measured Laboratory: Provides hands-on experience in (1) relating lecture material to practical analysis (2) design and operation of a real instrument (3) measurements on range of instruments (4) example analyses to illustrate value of technique Introduction Classification of Analytical Methods Qualitative instrumental analysis is that measured property indicates presence of analyte in matrix Quantitative instrumental analysis is that magnitude of measured property is proportional to concentration of analyte in matrix Species of interest All constituents including analyte. Matrix-analyte =concomitants Often need pretreatment - chemical extraction, distillation, separation, precipitation (A) Classical: Qualitative - identification by color, indicators, boiling points, odors Quantitative - mass or volume (e.g. gravimetric, volumetric) (B) Instrumental: Qualitative - chromatography, electrophoresis and identification by measuring physical property (e.g. spectroscopy, electrode potential) Quantitative - measuring property and determining relationship to concentration (e.g. spectrophotometry, mass spectrometry) Often, same instrumental method used for qualitative and quantitative analysis Types of Instrumental Methods: Property Example Method Radiation emission Emission spectroscopy - fluorescence, phosphorescence, luminescence Radiation absorption Absorption spectroscopy - spectrophotometry, photometry, nuclear magnetic resonance, electron spin resonance Radiation scattering Turbidity, Raman Radiation refraction Refractometry, interferometry Radiation diffraction X-ray, electron Radiation rotation Polarimetry, circular dichroism Electrical potential Potentiometry Electrical charge Coulometry Electrical current Voltammetry - amperometry, polarography Electrical resistance Conductometry Mass Gravimetry Mass-to-charge ratio Mass spectrometry Rate of reaction Stopped flow, flow injection analysis Thermal Thermal gravimetry, calorimetry Radioactivity Activation, isotope dilution (Often combined with chromatographic or electrophoretic methods) EnergyAnalyte Analytical Data Stimulus (in matrix) Response encoded information Example: Spectrophotometry Instrument: spectrophotometer Stimulus: monochromatic light energy Analytical response: light absorption Transducer: photocell Data: electrical current Data processor: current meter Readout: meter scale Data Domains: way of encoding analytical response in electrical or non-electrical signals. Interdomain conversions transform information from one domain to another. Light Intensity Photocell → Current Current Meter → Scale Detector (general): device that indicates change in environment Transducer (specific): device that converts non-electrical to electrical data Sensor (specific): device that converts chemical to electrical data Non-Electrical Domains Electrical Domains Physical (light intensity, color) Current Chemical (pH) Voltage Scale Position (length) Charge Number (objects) Frequency Pulse width Phase Count Serial Parallel Time - vary with time (frequency, phase, pulse width) Analog - continuously variable magnitude (current, voltage, charge) Digital - discrete values (count, serial, parallel, number*) Digital Binary Data Advantages (1) easy to store (2) not susceptible to noise 4th bit 3rd bit 2nd bit 1st bit Hi Count Lo 2 1 0 2 2 2 Hi Serial Lo Hi 0 2 Lo Hi 1 3 separate 2 Lo signals Parallel Hi 2 Lo 2 20=1, 21=2, 22=4... Performance Characteristics: Figures of Merit How to choose an analytical method? How good is measurement? How reproducible? - Precision How close to true value? - Accuracy/Bias How small a difference can be measured? - Sensitivity What range of amounts? - Dynamic Range How much interference? - Selectivity Precision - Indeterminate or random errors i= N ∑ ()− 2 xi x − Absolute standard deviation: s = i 0 N −1 Variance: s2 s Relative standard deviation: RSD = x s Standard deviation of mean: s = m N Accuracy - Determinate errors (operator, method, instrumental) = − Bias: bias x x true Sensitivity dSignal S = c + Signal Calibration sensitivity: dc blank = + mc Signal blank (larger slope of calibration curve m, more sensitive measurement) Detection Limit Signal must be bigger than random noise of blank = + Minimum signal: Signal min Av. Signal blank ksblank From statistics k=3 or more (at 95% confidence level) Dynamic Range At detection limit we can say confidently analyte is present but cannot perform reliable quantitation Level of quantitation (LOQ): k=10 Limit of linearity (LOL): when signal is no longer proportional to concentration LOL Dynamic range: 102 to > 106 LOQ Selectivity: No analytical method is completely free from interference by concomitants. Best method is more sensitive to analyte than interfering species (interferent). = + + Matrix with species A&B: Signal mA cA m Bc B Signal blank = m B Selectivity coefficient: kB,A m A k's vary between 0 (no selectivity) and large number (very selective). Calibration methods Basis of quantitative analysis is magnitude of measured property is proportional to concentration of analyte ∝ = + Signal [x] or Signal m[x] Signal blank Signal −Signal [x]= blank m Calibration curves (working or analytical curves) Dynamic Range Instrument Response LOQ (Signal) DL LOL Slope m Signalblank [x] Example (if time): Analyte Concentration (ppm*) Absorbance 0.0 (blank) 0.05 0.9 0.15 2.0 0.24 3.1 0.33 4.1 0.42 *ppm=1 µg per L Define Variance and Covariance: ∑(x − x)2 ∑(x − x)(y − y) S = i S = i i xx N −1 xy N −1 x = 2.02 y = 0.238 2 2 2 2 2 ()2.02 +1.12 + 0.02 +1.08 + 2.08 10.828 S = = = 2.707 xx 4 4 ()−2.02 ×−0.188 +−()1.12 ×−0.088 +−()0.02 × 0.002 +... S = xy 4 0.9562 = = 0.23905 4 Sxy 0.23905 Slope: m = = = 0.0883 Sx 2.707 b = y − mx Intercept: = 0.238 − (0.0883× 2.02) = 0.0596 Calibration expression is Absorbance=0.0883[Analyte (ppm)]+0.0596 Chem466 Lecture Notes Spring, 2004 Overview of the course: Many of you will use instruments for chemical analyses in lab. settings. Some of you will go into careers (medicine, pharmacology, forensic science, environmental monitoring & remediation) which requires a working knowledge of instrumental analysis. The most widely used instrumental methods in these career paths are separation methods (gas & liquid chromatography) and mass spectrometry (often joined with chromatography in one instrument). The next tier of importance includes UV-VIS absorption spectroscopy, IR spectroscopy, fluorescence spectroscopy, and atomic analysis methods (absorption and emission). All of these methods will be covered in this course. The order of topics (see the syllabus) is designed to provide essential information as it is needed. Consequently, spectroscopic methods are covered first and separation methods second because these methods are used as detectors in separation science methods. Mass spectroscopy follows separation science methods because it is easier to discuss hyphenated methods (e.g., gas chromatography-mass spectrometry or GC-MS) after discussing the separation methods. If there is time, the section on capillary electrophoresis, an increasingly important separation method, will be covered. In order to allow discussion of many methods, there will be material in the lecture notes which will not be covered in lecture but for which you will be responsible. These will be clearly indicated. At the end of each section, there is a brief review of the important definitions, concepts and calculations on which you will be tested. A few topics are left in which will not appear in lecture or on tests. They are for your benefit. Additional reading assignments are from Skoog, Holler and Nieman, Principles of Instrumental Analysis, 5th edition. The SH&N reading assignments are NOT required; they are for the instructor’s benefit, since this course was originally tailored to that textbook. I. DEFINITIONS. Analyte - the substance being identified or quantified. Sample - the mixture containing the analyte. Also known as the matrix. Qualitative analysis - identification of the analyte. Quantitative analysis - measurement of the amount or concentration of the analyte in the sample. 1 Signal - the output of the instrument (usually a voltage or a readout). Blank Signal - the measured signal for a sample containing no analyte (the sample should be similar to a sample containing the analyte) For most instrumental methods (exception: potentiometry), the signal is linear with respect to the concentration of the analyte over a range of concentrations: S = mC + Sbl where C = conc. of analyte; S = signal of instrument; m = sensitivity; Sbl = blank signal. The units of m depend on the instrument, but include reciprocal concentration. A standard (a.k.a. control) is a sample with known conc. of analyte which is otherwise similar to composition of unknown samples. A blank is one type of a standard. The words “standards” and “blanks” often refer to the signals generated by these types of samples. The standard method of checking the above equation and defining the sensitivity of an instrumental method is to obtain a calibration curve - a plot of signal vs conc. for a set of standards. Calibration curves are often nonlinear at high and low concentrations, and linear at intermediate concentrations. The linear part of plot is the dynamic