Chem 551 :Instrumental Methods of Analysis Ralph Allen

Instrumental Analysis There is much more than the Instrument You are the analyst

Why are you taking this class? What do you want to learn? What analytical techniques do you want to study?

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You don’t need a course to tell you how to run an instrument ä They are all different and change ä Most of you won’t be analysts ä We will talk about experimental design ä Learn about the choices available and the basics of techniques

Analytical Chemistry ä art of recognizing different substances & determining their constituents, takes a prominent position among the applications of science, since the questions it enables us to answer arise wherever chemical processes are present. ä 1894 Wilhelm Ostwald

Questions to ask??? ä Why? Is sample representative ä What is host matrix? ä Impurities to be measured and approximate concentrations ä Range of quantities expected ä Precision & accuracy required

More things to ask…. ä Where is analysis to be conducted ä How many samples (per day & total) ä How soon are results needed ä Are there standards (analytical & QC) ä Long term reliability ä Form of answer required ä Special facilities available

The Analytical Approach ä Identify the problem….what do you want to know ä What instrumental methods can provide necessary results ä Which method is best ä What do the results mean

What you want to learn ä analytical process and skills ä tools for research ä solve practical problems ä medical uses (including DNA) ä how instruments work and general concepts ä environmental and forensic applications ä new advances

Techniques ä mass spectrometry ä NMR ä spectroscopy (UV, IR, AA) ä chromatography (GC, HPLC) ä measure radioactivity, crystallography, PCR, gas phase analysis

Off flavor cake mix (10%) ä Send it off for analysis ä Do simple extractions ä Separation and identification by GC/MS ä Over 100 peaks but problem was in a valley between peaks (compare) ä Iodocresol at ppt ä Eliminate iodized salt that reacted with food coloring (creosol=methyl phenol)

Pan Am % of the “Maid of the Sea” recovered 16,000 pieces of property recovered

Reason to understand how an instrument works ä What results can be obtained ä What kind of materials can be characterized ä Where can errors arise

Design of instrumentation to probe a material ä Signal Generation-sample excitation ä Input transducer-detection of analytical signal ä Signal modifier-separation of signals or amplification ä Output transducer-translation & interpretation

Characterization of Properties ä chemical state ä structure ä orientation ä interactions ä general properties

Molecular Methods ä macro Vs micro ä pure samples Vs mixtures ä qualitative Vs quantitative ä surface Vs bulk ä large molecules (polymers, biomolecules)

Molecular Spectroscopy IR, UV-Vis, MS, NMR ä What are interactions with radiation ä Means of excitation (light sources) ä Separation of signals (dispersion) ä Detection (heat, excitation, ionization) ä Interpretation (qualitative easier than quantitative)

Elemental Analysis ä bulk, micro, contamination (matrix) ä matrix effects ä qualitative Vs quantitative ä complete or specific element ä chemical state

Extreme trace elemental analysis ä Direct instrumental determination - multi- element - direct excitation---should be least expensive ä These are relative physical methods requiring appropriate standards & systematic errors like spectral interferences occur ä NAA, XRF, sputtered neutral MS

Extreme trace elemental analysis ä Multi-stage procedures --- sample separation and preparation before quantitation ä Standards are less of a problem ä Time consuming & subject to losses or contamination ä Chromatography coupled with analysis

Comparing Methods ä Detection limits ä Dynamic range ä Interferences ä Generality ä Simplicity

Your ideas ä cost ä sensitivity ä accuracy/precision ä time ä compatibility ä conditions ä availability

Statistics are no substitute for judgment ä Common sense put into a mathematical form ä Analysis of results - accuracy & precision ä Elimination of errors ä Detection limits - signal to noise ä Chemometrics - what do the results mean

There is a difference - you need both

Errors in Analytical Measurements ä Determinant - unidirectional errors ascribable to a definite cause ä Indeterminate - uncertainties from unknown or uncontrollable factors - generally random - noise

Systematic errors - sources ä Inhomogeneity - handling & storage ä Contamination - sampling to reagents ä Adsorption on surface or volatilization ä Unwanted or incomplete chemical reactions ä Matrix effects on generation of analytical signal ä Incorrect standards or calibration

Recognition of systematic errors ä Reproducibility gives NO information on accuracy (high std. dev. hints at problems) ä Make comparisons with other methods ä Check standard reference materials (available from NIST) ä Run blanks (be sure background is small and reproducible)

Errors in Analytical Measurements ä Determinant - unidirectional errors ascribable to a definite cause ä Indeterminate - uncertainties from unknown or uncontrollable factors - generally random - noise

Gaussian Distribution ä Random fluctuations ä Bell shaped curve ä Mean and standard deviation ä 1sigma 68.3%, 2sigma 95.5%, 3sigma 99.7% ä Absolute Vs Relative standard deviation ä Accuracy and its relationship to the measured mean

Limit of detection ä signal - output measured as difference between sample and blank (averages) ä noise - std dev of the fluctuations of the instrument output with a blank ä S/N = 3 for limit of detection ä S/N = 10 for limit of quantitation

Sources of Noise ä Environmental - 60 Hz electrical, vibrational (shield) ä Johnson (thermal) noise - random fluctuations in charge carriers (cool) ä Shot noise - pulses ä 1/f (flicker) noise - important at low frequencies

Noise Reduction ä Avoid (cool, shield, etc.) ä Electronically filter ä Average ä Mathematical smoothing ä Fourier transform

Single channel scanning ä 3 objects each measured 3 times (averaged to reduce noise) ä Balance requires 9 measurements ä Monochromator - broad band source to dispersive device and then wavelengths are selected one at a time ä Increase intensity by scanning slower or increasing bandpass

Multidetector Spectrometer ä Get 3 balances and measure all 3 samples simultaneously on separate balances ä Can make measurements in 1/3 time or measure 3 times as much (noise is random and proportional to square root of number of measurements) ä Use of diode arrays instead of slits

Signal Transformation ä Double pan balance - mesure multiple objects simultaneously & measure linear combinations ä y(1)=X(1) + X(2) ä y(2)=X(1) + X(3) ä y(3)=X(2) + X(3) ä 3 equations & 3 unknowns (each object measured twice in half the time)

Hadamard multiplexing (transform) ä Use one detector and replace the slit with a mask of slits at certain locations (n)- some are open & others closed (2n-1 slits in mask with just more than half open) ä For n=3 a mask of (1 is open) can be slid to give 110, 101, 011 ä Linear equations improve S/N

Fourier advantage ä Put all weights on 2 pan balance at the same time ä Change what is measured (not weights but angle of pointer showing difference in the 2 pans) ä Z(1)=X(1) + X(2) - X(3) ä Z(2)=X(1) - X(2) + X(3) ä Z(3)= -X(1) + X(2) + X(3)

h(t) = a cos 2 pi freq. x time ä sum = cos(2pi((f1+f2)/2)t ä beat or difference = cos(2pi((f1-f2)/2)t ä 5104-sine-wa

Fourier transform - beat frequency (time domain) ä We can sample the time domain at N equally spaced time intervals ä Represent each measurement in terms of a series of frequencies ä Decoding procedure to decode N algebraic equations ä Fourier transform requires a computer

An analytical checklist ä Have the analytical tasks and goals been defined? ä Have issues of sampling been defined?(eg. size, homogeneity, composites) ä Are there facilities for sample storage (custody) available and is there a means of identification and retreival)

Checklist 2 ä Is pretreatment (eg. extraction, dissolution) necessary? (facilities, equipment, reagents) ä Is the sample analyzed representative? (mixing, weighing, size) ä Are the instruments appropriate for the required measurements? (sensitivity, sample state)

Checklist 3 ä What is the time required for each analysis? ä What expertise is needed to prepare, analyze, and interpret? ä How is data captured, calculated, presented, and stored for future comparisons? ä Are there appropriate quality controls? ä Define time line for tasks and analysis and then calculate overall costs

Attenuated Internal Reflection ä Surface analysis ä Limited by 75% energy loss