CHEM*3440

Chemical Instrumentation Analytical III

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The Analysis Concept

PROBE
Electrons, photons, atoms, molecules, ions, heat

Sample For Analysis

RESPONSE
Heat, ions, molecules, atoms, photons, electrons

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Some Experiments
Emission of Radiation Absorption of Radiation Scattering of Radiation Diffraction of Radiation Polarization of Radiation Mass Mass-to-Charge Ratio Thermal Properties Electrical Properties Radioactivity Emission spectroscopy (X-ray, UV-visible, IR), Fluorescence, Phosphorescence Spectrophotometry (X-ray, UV-visible, IR), Photoacoustic spectroscopy, NMR, ESR, EXAFS, NEXAFS Raman spectroscopy, turbidimetry X-ray diffraction (XRD), Electron diffraction Polarimetry, Optical Rotatory Dispersion (ORD), Circular Dichroism (CD) Gravimetry, Quartz Crystal Nanobalance Mass Spectrometry Thermal Gravimetric Analysis, Differential Thermal Analysis Differential Scanning Calorimtery (TGA, DTA, DSC) Potentiometry, Coulometry, Voltammetry, Differential Capacitance, Polarography Neutron Activation, Isotope Enrichment
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Instruments move data across domains

Controls the applied probe. Measures the systems response. Instrument Encodes Data Transformations The Analysts The Physical and Domain Chemical Domain
Scale Physical and Chemical Domain Position Number Non-electrical Domains

Current

Charge Electrical Domains

Power

Voltage

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Domain Conversion
An analyst seeks to measure the physical or chemical properties of a
system. An instrument creates an electrical signal which represents this datum. As analytical chemists, we must understand and quantify this representation (Exact? Interferences? Specificity?) Data proceeds through the instrument where different transducers convert the signal from one domain to another. As analytical chemists, we must understand these transducers and this conversion process (Speed? Sensitivity? Impedance?) The analysis of an instruments behaviour proceeds by characterizing it as a sequence of data domain converters which can each be analyzed separately. It is possible to construct highly integrated instruments where this separation is murky or completely lost, but not in CHEM3440

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Transducers and sensors

All modern instrumentation employs data conversion between at least three domains and often more. Each domain transformation is accomplished by a transducer, or sensor. Input Transducer converts data from a non-electrical domain to an electrical domain. Sensor a species-specific transducer (SHC, page 9) Output Transducer converts data from an electrical domain to a non-electrical domain.
A thermocouple generates a specific voltage at a certain temperature. It is a temperature-to-voltage input transducer. A glass electrode produces a potential (voltage) that is related specifically to the [H+] in solution. It is a sensor. A stepper motor running a pen on a recorder moves the pen in response to a current flow. It is a current-to-position output transducer.
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Example: The Thermocouple

A temperature-to-voltage transducer. Connect two dissimilar metal wires appropriately (A-B)-(B-A). The thermoelectric effect produces a voltage difference between the ends of the wires whose magnitude depends upon the temperature difference between the two junctions. V Metal 1 Metal 2

Treference

Tmeasure

In modern instruments, the Reference junction has often been replaced by an integrated circuit that provides a voltage that is constant, thus simulating the effect of a thermocouple junction at a fixed temperature (usually 0 oC). e.g. Analog Devices AD594 As a side note, progress in the physical sciences is often dependent upon progress in the measurement sciences. So the take-home message is !

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Thermocouples - 1
Voltage magnitude depends upon temperature difference (Tm - Tr). The relationship is called the transfer function. Tr is almost always chosen to be 0 C. The transfer function for a thermocouple is often used from a table. However, it is often useful to automate the conversion which requires an analytic function. For a thermocouple, the transfer function is generally written as

f (Tm Tr ) = A(Tm Tr )+ B(Tm Tr ) +

C (Tm Tr )

In most cases for well-chosen materials, B and C are quite small and are safely ignored, leaving us with a linear transfer function in a small range of T. For the chromel/alumel (K-type) thermocouple, A has the value of about 4 x 10-5 V/ C. This usually requires the use of a voltage amplifier to get easily measured voltages (we will see this later : OpAmps are an analytical chemists friend!)
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Thermocouples - 2
A measurement instrument is created when the voltage of the thermocouple transducer is amplified and turned into a current (or voltage) which subsequently turns a meter dial to report the temperature as viewed by the analysts eye.

Amplifier V-I

I Scale position

3
Physical and Chemical Domain Current

Scale Position

Thermocouple T-V

Number Charge

Hmm 37 C

Power

Voltage

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Transfer Functions
Each transducer has its own transfer function. The thermocouple has the (almost) linear function described earlier. V = f1(T). Although linear relationships are easy to manipulate, any analytical function can be used. The amplifier generally needs to increase the size of the signal, since it is usually in the millivolt range. This would be another linear relationship. I = f2(V) = f2(f1(T)) The needle on the meter will deflect a certain amount for a given current the passes through the coil. This relationship is another transfer function. D = f3(I) = f3(f2(f1(T))) The convolution of these functions, describes how a change in temperature will give a rise to a change in needle deflection. You could define an overall transfer function that directly relates the deflection, D, as a function of T. In effect, this is what the paper scale on the back of the meter dial does!
When is this a good idea ? When is this a bad idea ?
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Example: The pH Meter

A similar situation arises when we want to monitor and record the hydrogen ion activity (pH) of a solution and how it changes with time. The first sensor is a pair of electrodes; one at a fixed pH and the other sampling the unknown solution. Together they produce a voltage difference. This is amplified and turned into a current to drive the pen displacement motor on a chart recorder, permanently recording the changes in pH with time.

Amplifier V-I

Recorder

pH

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Reference Standards
All measurement devices involve a difference detector and a reference standard. The magnitude of the signal generated arises from the difference between the sample under test and the reference standard.

Qo = (Qm - Qr) +Qr = Qm Q m - Qr Qm

Quantity to be measured Difference Detector

Qr

Qr
Quantity of reference standard

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Example: Double Pan Balance

Unknown mass to be measured

Difference Detector

System for adjusting a collection of standard weights

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Balances - 1
A collection of reference masses are added to the pan until the needle points to zero. This is called a null detector. The result depends upon how small is the smallest reference mass. This defines the units sensitivity. Smallest increments also dictate the reproducibility of a measurement. This is called the precision. Only get a good result if the standard masses are correctly calibrated and are not dirty or worn. This affects the measurements accuracy. When magnitude of difference in null detector is not considered (only sign is needed to tell operator which way to adjust reference weights to get closer to the null) then it is called a comparator. We will see that this is an often used type of null detector.

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Balance2
The mechanical balance has a series of precise counterweights that are used to approximately balance off most of the total mass present on the pan (ie the sample being weighed). The null detector is calibrated. A final almost null condition provides a measure of the last fractional mass needed for balance.
What is the logic behind this two-step weighing process?

Top Loading Mechanical Balance:

Top Loading Electronic Balance: The complete measurement is made by measuring the deformation of a mechanical element (often a piezo-electric ceramic) under the weight of the sample.
What material science constraints does this impose on the balance design?

Bathroom Scale:

Manufacturer has calibrated the springs against a set of standard masses to make the entire measurement to be done by the markings of the off-null detector. The reference standard is remote, instrument is subject to non-linearities, calibration drifts, and potentially large errors. Especially my scales at home, which are very wrong, always.
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Signal
Every analytical procedure depends upon a signal which is derived from the output of the difference detector. Every analytical instrument has a non-zero output, even when no difference is present at the inputs to the detector. This non-zero output is called the background or baseline. This background is often slowly varying in time. This changing background is called drift.

The analytical signal is the difference between the output amplitude and the expected baseline at the same moment in time. Quite often, one of the analytical challenges is to predict the baseline with sufficient certainty. This is especially true for remote instrumentation (think Mars rovers, or Polar research stations, or high-altitude weather probes)

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Noise
There are other variations in the output signal level; they can occur at all frequencies and constitute an unwanted random or almost random time-dependent changes in the output. These variations are collectively called noise. Noise is measured in the same units as the signal. This can be current, voltage, or power. Two of the most common measures of noise are: Peak-to-Peak
5 4 3 2 1 0 -1 0 -2 -3 -4 -5 2 4 6 8 10 12 14 16 18 20

Vp-p

RMS : the root-(of the) mean- (of the) square of the difference between the signal and its average value
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Signal-to-Noise
The determination of the magnitude of the analytical signal level requires measuring the difference between the background and the sample signal. This measure is blurred by the presence of noise. One has to account for both the signal level and the noise level in arriving at this measure. Because of this, the important quantity is not the signal level alone nor is it the noise level alone; rather it is the ratio of the two that dictates the measurability of the signal level. This is the signal-to-noise ratio or simply S/N.

S N

mean standard deviation

x s

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Performance Characteristics
A performance characteristic is used to describe a general property of analytical techniques that permit their comparisons so that a user can evaluate it for its applicability in a given situation.

Precision Sensitivity Quantitation Limit Dynamic Range

Accuracy Detection Limit Linearity Limit Selectivity

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Figure of Merit
A figure of merit is a number which has been derived experimentally for a given analytical instrument or technique that permits an evaluation or comparison of the technique to assess its applicability to a particular analysis problem. Performance characteristics are measured in terms of figures of merit. Some Important Figures of Merit

Mean

x s2 s RSD sm

xi
N

Variance

(x i x )
N 1 s2 s x s N

Standard Deviation Relative Standard Deviation

Standard Deviation of the Mean

The N-1 denominator for the Variance is perhaps confusing. We use this when we have taken a sample of data from a larger population. If we had the entire set of data values (rare in measurement science) then you could use N, but when using such large data sets, the difference between N and N-1 is usually negligible.

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Precision
Mutual agreement of replicate measurements.
The standard deviation and the variance are the most common measurements of a set of datas precision for a series of replicate measurements. It is presumed to be the result of random errors.
But what are random errors?

Although it is difficult for us to work with any other hypothesis, it is often very difficult for us to prove that we are truly operating with random errors. Causal factors Temporal sequences Hysteresis Specific noise sources in the environment For example, consider (1) a quantitative study of the arrival times for students to CHEM3440 in the morning, and (2) a similar study for the arrival time of the professor.

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Accuracy
Errors in Accuracy arise from the presence of determinate errors, or nonrandom errors. This shifts the measured mean value of a set of measurements away from the true value and is referred to as the error of the mean. There are (at least) three basic types of such errors: Instrumental: something wrong with the instrument (batteries low, temperature effects the circuitry, calibration errors, etc. Personal: judgment errors, reading the meter from the wrong angle, lack of careful technique. Method: often a result of non-ideal chemical behaviour; slow reactions, contaminants, instability of reagents, loss of analyte by adsorption. Must use guaranteed standards (NIST).

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Parallax of Viewing Angles

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Sensitivity
Refers to a techniques ability to detect changes in the signal property.
How much does the signal change for a change in the measured variable? Two factors dictate a techniques sensitivity: 1. 2. Slope of calibration curve (i.e. the nature of the Transfer Function). Precision or reproducibility of measurement (i.e. the properties of the machine)
High Precision = High Sensitivity High Sensitivity

Low Sensitivity

Low Precision = Low Sensitivity

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Calibration and Analytical Sensitivity

Slope of calibration curve (most curves are linear or are transformed to a linear form, purely for convenience of the operator).
signal

S = m C + Sbl
slope

Blank signal (y-intercept)

Concentration, or abundance

m is the calibration sensitivity (e.g. volts per kg).

precision. To incorporate precision, try
Analytical sensitivity factor

m is not the best figure of merit as it has no measure of the

= m/s
Standard deviation of measurement Slope of calibration response

This definition is not affected by amplification. Increases in gain lead to increases m and s by similar factors, so the ratio is unaffected. Independent of measurement units but may depend upon concentration since s can vary with concentration.
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Limits of Detection and Quantification

LOD : This is the smallest amount of analyte that can be reliably detected; it simply determines if the analyte is present or not. Depends upon signal/noise ratio. Analysis signal must be larger than blank signal. How much larger? What
does reliably really mean?
Standard deviation on blank signal

Sd = Sblank + k sblank
Minimum distinguishable analytical signal Mean blank signal

Usually taken to be 3

LOQ : To determine How much of the analyte is present? requires a larger

signal than the LOD. The widely accepted level at which the analyte can be quantified is TEN times the standard deviation.

Sq = Sblank + 10 sblank
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Signal Relationships

Mean Background Signal Level

Detection Limit

Quantitation Limit

Distribution of blank measurements

3 sbl

10 sbl

Measured Signal Level

0

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Linearity Limit
As the concentration or intensity increases, at some point, every detector stops responding linearly. In conventional (old school) devices, this identifies the upper limit of concentration to which the technique can be successfully applied. Its origin can be electrical (e.g. the amplifier cannot produce a larger output voltage) or mechanical (e.g. the balance arm deforms under this load) in nature.
Note : The limits of linearity do not necessarily correspond to the limits of reproducibility. With increased use of smart instruments, there is a tendency to increase the useful limits towards those imposed by the reproducibility of the device, and to use more sophisticated Transfer Functions to describe the non-linear regimes of operation.

Instrument response

Linearity Limit

Concentration

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Dynamic Range
This is the region between the Quantitation Limit (LOQ -Limit of Quantitation) and the Linearity Limit (LOL - Limit of Linearity). This is the range over which the technique is useful. To be viewed as a worthwhile, a technique should have a dynamic range of at least two orders of magnitude (SHC). Many techniques have a dynamic range of five to six orders of magnitude.

Two orders of magnitude appears to be quite an arbitrary Figure of Merit.

To be useful, a technique has to perform as required over the required sample and instrument conditions.

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Selectivity
In most analyses, we look for a signal that comes from a specific analyte or class of analytes. In every analysis, we obtain a signal that has a contribution from everything that is present in the sample. We need to minimize contributions from other species and be certain that they are negligible, or else account for their contribution by determining their selectivity coefficient.

Stotal = R ai + R kij aj(i/j)

Total Signal Signal of Analyte species i Activity of each species

Signal of other species j when in the presence of species i

Selectivity Coefficient for the detection j when trying to detect I.

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Calibration Curves: What do they tell us?

Consider the analysis of, for instance, the UV/Vis spectra of an acqueous Co2+ sample (strong absorption band at ~500 nm).
Concentration (ppm) 0.00 2.13 5.63 10.29 15.33 21.71 Average Signal 0.0363 0.111 0.268 0.503 0.715 1.016 Replicants 25 5 5 5 5 5 Standard Deviation 0.0037 0.0091 0.0108 0.0103 0.0129 0.0141

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Calibration Curve - 1
1. 2. Use a Least Squares approach to find the parameters for this curve and plot the curve. Use Excel (or similar) spreadsheet. 1. 2. Trendline LINEST function
1.2 1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25 Concentration (ppm) y = 0.0456x + 0.0231 R2 = 0.9992

m = 0.0456 b = 0.0231 m is slope of line and encodes how the instrument responds to sample concentration. b is the y-intercept. It is the magnitude of the blank that will be subtracted from every measurement.

m=

xi yi

xi yi
b=

N ( xi )2 xi2 N

yi m xi
N N

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Calibration Curve - 2
What is the standard deviation for b and m?

xi yi 2 x y yi ) i i ( N yi2 2 Use the following equations. N ( x ) xi2 N i sr = N2 1 sm = sr ( xi )2 2 xi N

Part of the output from LINEST in Excel or

sb = sr

xi2 2 N xi2 ( xi )
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Calibration Curve - 3
What is the calibration sensitivity for the transfer function? This is simply m, the slope of the least squares fit: m=0.0456 What is the analytical sensitivity? This value changes with concentration so we obtain a value of g for each data point in the plot.
Concentration
2.13 5.63 10.29 15.33 21.71

Analytical Sensitivity
5.0 4.2 4.4 3.5 3.2

m i = si

What is the LOD (Limit of Detection)?

L.O.D. =

3 sblank 3 0.0037 = = 0.24 ppm m 0.0456

What is the LOQ (Limit of Quantitation)?

L.O.Q. =

10 s blank 10 0.0037 = = 0.81 ppm m 0.0456

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Calibration Curve - 4
What is the concentration of an unknown sample when the mean signal measured for 5 replicated measurments is 0.447?

cx =

0.447 b 0.447 0.0231 = = 9.3 ppm m 0.0456

What is the standard deviation of this measurement? (M = 5).

s 1 1 sx = r + + m M N

(cx y ) = 0.16 ppm ( xi )2 2 2 m x

2

What are the 95% confidence limits for this measurement? recall that we made 5 measurements for this unknown. From Students ttable, t = 2.78 for this number of measurements at this confidence level.

t s (2.78)(0.16) = = 0.20 ppm N 5

Cx = 9.3 0.2 ppm with 95% confidence

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Standard Addition
Most samples are not clean; there are a many other components to the matrix that may interfere either chemically or physically. Recreating the exact environment to use the calibration curve method can be difficult and even impossible. Standard Addition method is a good attempt around that. 1. Take an aliquot of the sample into a volumetric flask, add any needed additional components (pH buffer, complexing agent, etc.), and dilute to the final volume. 1 Take another volumetric flask and introduce an identical aliquot and same treatments. However, in addition, introduce a volume of a known standard solution of the analyte. Then dilute to volume. 2

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Standard Addition - 2
Measure the signal (S1 and S2) for both. The signal depends upon the concentration of the target analyte, which is simply diluted to the final volume, Vt. The experiment has some response factor k that relates the concentration to the signal amplitude. We can write

S1 = k

Vx c Vt x

V V S2 = k x cx + s cs Vt Vt

From these two measurements, we can solve for the unknown concentration, and obtain the expression

cx =

S1 Vs c (S2 S1 ) Vx s

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Standard Addition - 3
The previous scheme only required two measurements. Better results are made by spiking several samples with varying volumes of added standard. 1. 2. Form one sample with the unknown as before. Form a series of samples with unknown and increasing volumes of added standard (perhaps 5 total samples).

These 5 samples are measured, each giving its own signal Si. These data can be graphed as signal against added standard volume. The slope and intercept can be determined and used to calculate the unknown concentration.

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Standard Addition - 4
Consider the following data. The volume of the unknown and each standard increment is 5.00 mL. The standard has a concentration of 8.7 ppm.
Standard Addition Method
1.2

Added Volume (mL) 0.00 5.00 10.00 15.00 20.00 25.00

Signal (arbitrary) 0.251

1

y = 0.035x + 0.2548 R2 = 0.9994

0.422 0.617 0.785 0.957 1.121

0.8 0.6 0.4 0.2 0 0 5 10 15 20 25

Volume of Added Standard (mL)

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Standard Addition - 5
A least squares analysis gives the slope and intercept (m and b) for this straight line. The equations solve to give an expression for the unknown concentration as b 0.2548

cx =

m Vx

cs =

(0.035)(5.00 )

8.7 = 12.7 ppm

With the equations given previously, we can find the error in m and b and assuming that those errors dominate, we can find the error in our result as
2 2 2 2 sm + sb = c 0.00045 + 0.00018 sc = cx x m b 0.03498 0.2548 = 12.7 0.0129 = 0.16 ppm

Use Students t-table for 95% and 5 samples. We would report the final answer as cx = 12.7 0.20 ppm at 95% confidence

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Standard Addition - 6
A graphical solution can be easily obtained. Extrapolate the curve back to the x-axis (in the negative-x region). This x-intercept represents the volume of standard solution which has the same amount of analyte as the unknown solution.
Standard Addition Method

-V0,s cs = Vx cx cx = -(V0,s/Vx) cs = -(-7.28/5.00) 8.7 ppm =12.7 ppm

1.2 1 0.8 0.6 0.4 0.2 0 -10 -0.2

y = 0.035x + 0.2548 R2 = 0.9994

V0,s = -7.28 ml

-5

10

15

20

25

Volume of Added Standard (ml)

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Internal Standard
In this experiment, a substance different from the analyte is added in equal amounts to all unknowns and calibration standards. We measure both the analyte signal and internal standard signal for all samples and calculates the ratio of analyte signal to internal standard signal and plots this ratio against the standard analyte concentration, forming a calibration curve. The rest proceeds as with any calibration curve. This procedure can correct for many matrix interferences. The major problem is finding the right internal standard and adding it in a reproducible manner for all standards and unknowns. Best procedure is when internal standard is an isotope of the analyte. Then all interfering chemistry is identical. But must not occur naturally and the two species must be measurable and distinguishable.

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