ADVANCED ANALYTICAL LAB TECH (Lecture) CHM 4130-0001 · 2016-08-19 · ADVANCED ANALYTICAL LAB TECH...

ADVANCED ANALYTICAL LAB TECH (Lecture) CHM 4130-0001

Spring 2015 Professor Andres D. Campiglia

Textbook: “Principles of Instrumental Analysis” Skoog, Holler and Crouch, 5th Edition, 6th Edition or newest Edition

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INTRODUCTION • A common challenge faced by an analytical chemist is the determination of target species in

complex samples • Complex sample: sample with numerous species. Example of complex samples: physiological

fluids (blood, urine, saliva), environmental samples (air, water, soil), etc. • Target species is the species of interest. It is also called analyte. Example: benzo[a]pyrene in

soil sample, PSA (prostate specific antigen) in physiological fluid, etc. • Some possibilities: Analyte: Other species = concomitants:

Analyte is the main component in the sample with

only two types of species

Analyte is not the main component in the sample

but sample contains

only two types of species

Analyte is not the main component in the sample

and sample contains

several types of species

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Sample Collection Sample = Matrix

Sample Preparation: Clean-up

and/or Pre-concentration

Analytical Sample

Qualitative and Quantitative Analysis

Statistical Analysis of Data

General Scheme

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Quantitative and Qualitative Analysis

• Classical and instrumental methods • Classical methods = wet-chemical methods Analyte separation: precipitation, extraction or distillation Qualitative analysis: chemical reactions yielding products of characteristic colors boiling or melting points solubility in a series of solvents odors, optical activities or refractive indexes Quantitative analysis: Gravimetric or volumetric analysis • Main disadvantages of classical methods:

Time consuming Numerous manual steps, which make them prone to indeterminate (random)

errors

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Instrumental Methods

• Most instrumental methods require a source of excitation to stimulate a measurable response from the analyte. See Figure 1-1.

• The first six entries in Table 1-1 involve interactions of the analyte with electromagnetic radiation.

• The first characteristic response involves radiant energy produced by the analyte. • The next five properties involve changes in electromagnetic radiation brought about

by its interaction with the sample. • Four electrical properties and miscellaneous properties follow. • The name of the corresponding instrumental method is given in the second column of

Table 1-1.

Evaluation of Analytical Data (Appendix One)

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• Analytical chemists may be presented with two types of problems 1) Provide a qualitative answer Example: Does this distilled water contain any Boron? Is this soil sample contaminated with polycyclic aromatic hydrocarbons (PAH)? 2) Provide a quantitative answer Example: How much lead is in this water sample? This steel sample contains traces of chromium, tungsten and manganese; how much of each

one? • Often, both types of questions are answered with quantitative methods Example: B, Pb, Cr, W, Mn in H2O: AAS or AES PAH in H2O: HPLC • In cases where a positive answer is obtained, the analyst will give the answer in terms of

analyte concentration Example: This water sample contain 1 mg/mL of B Most certainly, if the analyst repeats the experiment with the same sample using the same

method he/she will find a different result Why? Because of inherent experimental errors

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Random and systematic errors

• Example: Four students (A-D) each perform an analysis in which exactly 10.00mL of exactly 0.1M sodium hydroxide is titrated with exactly 10.00mL of exactly 0.1M hydrochloric acid. Each student performs five replicate titrations with the results shown in the following table

Student Results (mL)

A 10.08, 10.11, 10.09, 10.10, 10,12

B 9.88, 10,14, 10.02, 9.80, 10.21

C 10.19, 9.79, 9.69, 10.05, 9.78

D 10.04, 9.98, 10.02, 9.97, 10.04

A Results are all very close to each other (10.08-10.12) = highly reproducible

All the results are too high (they are all higher than 10.00, which is the theoretical value)

Two separate types of errors have occurred with this student: Random errors: these cause the individual results to fall on both sides of the average value (10.10mL)

Systematic errors: these cause all the results to be in error in the same sense (too high)

Random errors affect the reproducibility of an experiment or precision Systematic errors affect the proximity of the experimental value to the theoretical value or accuracy

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B The average of the five results (10.01mL) is very close to the theoretical value = data is accurate,

without substantial systematic error The spread of the results is very large (9.80 – 10.21) = data is imprecise, with the presence of

substantial random errors

Comparing A and B A: precise and inaccurate

B: poor precision and accurate

Random and systematic errors can occur independently of one another

C His work is neither precise (range 9.69 – 10.19mL) nor accurate (average = 9.90mL)

D

Precise results (9.97 – 10.04mL) and accurate (average = 10.01mL)

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Distinction between random and systematic errors, and precision and accuracy

Student Results

A

Precise but inaccurate

B

Accurate but imprecise

C

Inaccurate and imprecise

D

Accurate and precise

Terms used to describe accuracy and precision of a set of replicate data

• Accuracy (systematic errors): absolute error or relative error #1 • Precision (random errors): standard deviation, variance or coefficient of variation

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Random Errors • Whenever analytical measurements are

repeated on the same sample, a distribution of data similar to that in Table a1-1 is obtained.

• The variations among the individual results are due to the presence of random (indeterminate) errors.

• The data can be organized into equal-sized, adjacent groups or cells, as shown in Table a1-2.

• Figure a1-1A shows the histogram of the data, i.e. the relative frequency of occurrence of results in each cell.

• As the number of measurements increases, the histogram approaches the shape of the continuous curve shown as plot B in Figure a1-1.

• Plot B shows a Gaussian curve, or normal error curve, which applies to an infinitely large set of data.

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Table a1-1

Table a1-2

Figure a1-1

Systematic Errors and the Gaussian Curve • Systematic errors have a definite value

and an assignable cause and are of the same magnitude for replicate measurements made in the same way.

• Systematic errors lead to bias in measurement results.

• Figure a1-2 shows the frequency distribution of replicate measurements in the analysis of identical samples by two methods that have random errors of identical size.

• Method A has no bias so that the mean (mA) corresponds to the true value. Method B has a bias that is given by:

bias = mB – mA

• The analyst should be able to identify systematic errors and remove them from the method of analysis.

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Figure a1-2

Statistical Treatment of Random Errors

• Random errors can not be completely eliminated from experiments. • Statistical treatment of random errors provide the means to evaluate their

contribution to final results. • Definition of some terms: Population Mean (m) #2 Sample Mean #3 Population Standard Deviation (s) and Population Variance (s2) #4 Sample Standard Deviation (s) and Sample Variance (s2) #5 Relative Standard Deviation (RSD) and Coefficient of Variation (CV) #6

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The Normal Error Law • In Gaussian statistics, the results of replicate

measurements arising from indeterminate (random) errors distribute according to the normal error law, which states that the fraction of a population of observations, dN/N, whose values lie in the region x to (x+dx) is given by:

#7 • The two plots in Figure a1-3a are plots of the

equation above. The standard deviation for the data in curve B is twice that for the data in curve A.

• (x – m) is the absolute deviation of the individual values of x from the mean.

• Figure a1-3b plots the deviations from the mean in terms of the variable z:

z = x – m / s

when x – m = s z = 1 x – m = 2s z = 2 x – m = 3s z = 3 and so forth. • The distribution of dN/N in terms of the single variable z

is given by: #8 15

Figure a1-3a

Figure a1-3b

Characteristic Properties of the Normal Error Curve • Zero deviation from the mean occurring with

maximum frequency. • Symmetrical distribution of positive and

negative deviations about this maximum • Exponential decrease in frequency as the

magnitude of the deviation increases. Thus, small random errors are much more common than large random errors.

• The area under the curve in figure a1-3b is the integral of equation #8, which is given by:

#9 • The fraction of the population between any

specified limits is given by the area under the curve between these limits.

Examples: -1 z 1 DN/N = 0.683 = 68.3% of a

population of data lie within 1s.

-2 z 2 DN/N = 0.954 = 95.4% of a population of data lie within 2s.

-3 z 3 DN/N = 0.997 = 99.7% of a population of data lie within 3s.

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Figure a1-3b

Confidence Intervals Keep in mind the following: • The equation for the Normal or Gaussian distribution is derived for a set of infinite measurements

(N = ∞). • Derivation assumes no systematic errors. • The infinite number of measurements is called the population. • The mean obtained with an infinite number of measurements is the true value (m). • The true value has a standard deviation denoted by s. • In practical situations the number of measurements is far from infinite. For a finite number of

measurements, the set of results is called the sample. • The mean (x) obtained with a sample is an estimate of the true (m) value and its standard

deviation (s) is an estimate of s. • In other words: when N → ∞ ; x → m and s → s. • In most of the situations encountered in chemical analysis, the true value of the mean (m) can not

be determined because a huge number of measurements (N = infinite) would be required. • The best we can do is to establish an interval surrounding an experimentally determined mean (x)

within which the population mean (m) is expected to lie with certain degree of probability. This interval is known as the confidence interval.

• Example: assume an analysis for potassium gave concentration with the following confidence interval: 7.25 0.15% K. The significance of this result is the following: 17

7.25 7.25 + 0.15 = 7.40 7.25 - 0.15 = 7.10

99% probability that m is within this

Interval of experimental results.

Calculation of Confidence Intervals

• When the value of s is known: • The general expression of the confidence interval

(CI) of the true mean of a set of measurements is obtained via the following equation:

#10 • Values of z at various confidence levels are given

in Table a1-3. • Note the following: a) For the same number of repetitions (N =

constant) and as the probability increases, the size of the confidence interval increases with the value of z.

b) For the same probability (z = constant), the size of the confidence interval decreases as the number of repetitions increases.

• When the value of s is unknown: • When the number of repetitions is far from infinite

(N 30), the confidence interval is calculated via the following equation:

#11 • Table a1-5 summaries t values for various levels

of probability. • With the t value the confidence interval follows

the same trend as the one observed with the z value. 18

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CALIBRATION OF INSTRUMENTAL METHODS (Chapter 1)

• Unless a correlation between the analyte response and the analyte concentration is somehow established by the analyst, Instrumentation by itself does not provide concentrations.

• A very important part of all analytical procedures is the calibration and standardization process. • Calibration determines the relationship between the analytical response and the analyte

concentration. • Calibration is usually accomplished with the use of chemical standards. • Two types of chemical standards: External standard is prepared separately from the sample. Internal standard is added to the sample itself. • External standards are used when there is no interference effects from matrix components

(concomitants)

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The Calibration Curve Method

• The calibration curve method used when there is no interference effects from matrix components (concomitants).

• Experimental procedure for the calibration curve method:

a) Several standards of known concentrations of analyte are introduced into the instrument and the instrumental response is recorded.

b) The instrumental response is obtained with a blank.

Blank = all the components of the analytical sample - analyte

Analytical sample = is the sample presented to the instrument. In many cases, the analytical sample is different than the original sample.

• The signal intensity is plotted as a function of analyte concentration. If the relationship between signal and analyte concentration is linear, the perfect calibration curve should look as the one in the figure.

• The unknown concentration can then be obtained by interpolation.

Analyte Concentration*

Signal Signal Average

Zero I0,1; I0,2; I0,3 I0 ± s0

C1 I1,1; I1,2; I1,3 I1 ± s1

C2 I2,1; I2,2; I2,3 I2 ± s2

C3 I3,1; I3,2; I3,3 I3 ± s3

C4 I4,1; I4,2; I4,3 I4 ± s4

O = data points from external standards ● = data from unknown

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• The general behavior of experimental data is “far from ideal” • This type of plot generates several questions: a) Is the calibration graph linear? b) What is the best straight line through these points? c) When the calibration plot is used for the analysis of a test sample, what are the

errors and the confidence limits for the determined concentration?

You need to consider the following:

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Is the calibration graph linear?

• There are two ways of checking for linearity which are complementary rather than exclusive: Correlation coefficient Graphically

• Correlation coefficient • The correlation coefficient (r) is given by the equation: #12 • r can take values in the range –1 r 1

r = +1 describes perfect positive correlation, i.e. all the experimental points lie on a straight line of positive slope

r = -1 describes perfect negative correlation, i.e. all the experimental points lie on a straight line of negative slope

r = 0 describes no linear correlation between y and x

y

x

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Example of calculation of r: Standard aqueous solutions of fluorescein are examined in a fluorescence spectrometer, and yield the following fluorescence intensities (in arbitrary units): Fluorescence intensity: 2.1 5.0 9.0 12.6 17.3 21.0 24.7 Concentration, pg/mL: 0 2 4 6 8 10 12

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• Keep in mind that the correlation coefficient equation will always generate an r value even if the

data are patently non-linear in character, i. e. experience shows that even quite poor-looking calibration plots give very high r values

Lesson of this example: Calibration curve must always be physically plotted On graph paper or computer monitor, otherwise a

straight-line relationship might wrongly be deduced from the calculation of r

This example is a remainder that r = 0 does not mean That y and x are entirely unrelated; it only means that they

are not linearly related

Correlation coefficients are simple to calculate but they can lead to serious misinterpretation

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• Graphically • Plot the data in a “x” versus “y” plot keeping always in mind the following convention: Analytical response = y Concentration of external standard = x • Visually, determine the linear dynamic range (LDR):

• Calculate the correlation coefficient of the LDR excluding the data points that do not

belong to the LDR. Include only the data points that belong to the LDR.

• Although you already know the graph is linear, the correlation coefficient gives you a

quantitative measure on how well the data points fit a straight line.

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What is the best straight line through these points?

• What points? The points that belong to the LDR. The points that you used to calculate the correlation coefficient.

• The mathematical expression that describes a straight line can be represented as: y = mx + b Where m is the slope and b is the intercept.

y

x b

Blank signal

Experimental data points

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Random Errors in Concentrations Obtained with the Calibration Curve Method • The least squares method assumes that any deviation of

the individual points from the straight line arises from error in the measurement, i.e. only from the variation in the instrumental signal. In other words, the error in concentration is considered negligible in comparison to the instrumental signal.

• The difference between any given experimental value of the signal and the corresponding signal fitted in the best straight line by the least-squares method is called the residual. The concept is shown in figure a1-6.

• The least-squares method minimizes the residuals for all the experimental points to provide the best straight line within a given set of experimental data.

• The slope (m) of the best straight line is given by: #13 • The intercept of the best straight line is given by: #14 • The standard deviation of the slope (sm ) is given by: #15 • The standard deviation of the intercept (sb) is given by: #16 • The standard deviation of a concentration (sc) is given by: #17 • Confidence interval is given by: #18

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Standard-Addition Methods

• Standard addition methods are particularly useful for analyzing complex samples in which the likelihood of matrix effects is substantial.

• One of the most common standard addition procedures involves adding one or more increments of a standard solution to sample aliquots containing identical volumes.

• Each solution is then diluted to a fixed volume before measurement. • Assume that several aliquots Vx of the unknown solution with an unknown concentration Cx are

transferred to volumetric flasks having a volume Vt. • To each of these flasks is added a variable volume Vs of a standard solution of the analyte

having a known concentration Cs. • Suitable reagents are added and each solution is diluted to volume. • Instrument measurements are then made on each of these solutions and corrected for any

blanks response to yield a net instrument response S. • Assuming that the blank-corrected instrument response is proportional to analyte concentration: S = kVsCs / Vt + kVxCx/Vt

Where k is a proportionality constant. • A plot of S as a function of Vs is a straight line where the slope m and the intercept b are given

by: m = kCs/Vt and b = kVxCx/Vt

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Volumetric flask 1 with volume Vt





Add the same volume of solution (Vx) of unknown concentration (Cx) of to the five volumetric flasks

Volumetric flask 1 with

analyte mass = CxVx


analyte mass = CxVx


analyte mass = CxVx


analyte mass = CxVx


analyte mass = CxVx

To each of these flasks is added a variable volume Vs’ of a standard solution of the analyte having a known concentration Cs


analyte mass = CxVx


analyte mass = CxVx


analyte mass = CxVx


analyte mass = CxVx


analyte mass = CxVx

No standard addition

Standard Volume = Vs’ Analyte mass = CsVs’

Standard Volume = 2Vs’ Analyte mass = 2CsVs’

Standard Volume=3Vs’ Analyte mass = 3CsVs’

Standard Volume=4Vs’ Analyte mass = 4CsVs’

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analyte mass = CxVx + 0CsVs’


analyte mass = CxVx + CsVs’







Suitable reagents are added and each solution is diluted to volume


CxVx + 0CsVs’ +

reagents + solvent


CxVx + CsVs’ +

reagents + solvent


CxVx + 2CsVs’ +

reagents + solvent


CxVx + 3CsVs’ + reagent + solvent


CxVx + 4CsVs’ + reagent + solvent

General formula that represents analyte concentration (CA) in any given volumetric flask: CA = CxVx + nCsVs’ / Vt where n = 0, 1, 2, 3, 4, …

If we make nVs’ = Vs CA = CxVx + CsVs / Vt; where Vs is the variable volume of standard addition

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Instrument measurements are then made on each of these solutions and corrected for any blanks response to yield a net instrument response S


CxVx + CsVs +

reagents + solvent


CxVx + CsVs +

reagents + solvent


CxVx + CsVs +

reagents + solvent


CxVx + CsVs +

reagent + solvent


CxVx + CsVs +

reagent + solvent

S0 - Sblank S1 - Sblank S2 - Sblank S3 - Sblank S4 - Sblank

Assuming that the blank-corrected instrument response is proportional to analyte concentration: S = kVsCs / Vt + kVxCx/Vt ; where k is a proportionality constant

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• The analytical signal is plotted vs. the volume of standard added to the volumetric flasks:

Best straight line obtained by the

least square method Y = mx + b

Vs

m = kCs / Vt and b = kVxCx / Vt

b / m = kVxCx/Vt = VxCx KCs/Vt Cs

Cx = bCs / mVx or

Volume of standard equivalent to the

amount of analyte present in the

unknown sample.

S

• The standard deviation in concentration is given by: #19 • The standard deviation in volume is given by the following equation: #20

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The standard addition is a powerful calibration method that is often used improperly due to failure to understand the assumptions involved

• There must be a good blank measurement so that there is no contribution to the measured analytical signal from other species

• The calibration curve for the analyte in the sample matrix must be linear Note: The multiple addition procedure provides a check of this assumption • Analyte interferences must increase or decrease the analytical signal from the original analyte in

the sample and the analyte added in the addition by the same constant fraction independent of analyte concentration

Curve a is obtained with a proper blank measurement while in curve b the blank measurement does not

compensate for a blank interference, so all analytical signals are too high by a fixed amount, and the value

of Cx determined is too high

The standard addition procedure provides a check on recovery and the presence of analyte interferences. The slope of a standard additions plot can be compared to the slope of a conventional calibration curve. If the slopes are different, the recovery of the analyte is incomplete or analyte interferences in the sample

matrix affect the slope

Exercise 1-11- Modified Exactly 5.00 mL aliquots of a solution containing phenobarbital were measured into 50.00 mL volumetric flasks and made basic with KOH. The following volumes of standard solution of phenobarbital containing 2.000 mg/mL of phenobarbital were then introduced to each flask and the mixture was diluted to volume: 0.000, 0.500, 1.00, 1.50, and 2.00 mL. The fluorescence of each of these solutions was measured with a fluorimeter, which gave values of 0.326, 4.80, 6.41, 8.02, and 9.56 counts per second (cps), respectively. The blank signal was equal to 1.00 cps. a) Come up with the composition of a suitable blank. b) Plot the data. c) Using the plot from (b), calculate the concentration of phenobarbital in the

unknown. d) Derive a least-squares equation for the data. e) Find the concentration of phenobarbital from the equation (d). f) Calculate a standard deviation for the concentration obtained in (d).

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Example 1-1

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Analytical Figures of Merit (AFOM)

• Linear Dynamic Range (LDR) • Limit of Quantitation • Limit of Detection • Sensitivity • Analytical Sensitivity • Relative Standard Deviation

Linear Dynamic Range (LDR) or Dynamic Range, Limit of Quantitation (LOQ) and Limit of Detection

• The LDR of an analytical method extends from the lowest concentration at which quantitative measurements can be made (limit of quantitation, LOQ) to the concentration at which the calibration curve departs from linearity (limit of linearity, LOL).

• LOQ is the signal equivalent to 10 times the

standard deviation of the blank.

• LDRs are usually expressed in terms of orders of magnitude.

• The limit od detection (LOD) is defined as the minimum concentration or mass of analyte that can be detected at a known confidence level.

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Sensitivity and Selectivity

• Sensitivity is the ability of a method to discriminate between small differences in analyte concentrations.

• Two parameters limit sensitivity: the slope of the calibration curve and the

precision of measurements. • There are two figures of merit for sensitivity: calibration sensitivity and

analytical sensitivity. • Selectivity of an analytical method refers to the ability of the method to

accurately determine the analyte in the presence of concomitants. • Concomitants can cause spectral interference, chemical interference or

both.

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Introduction to Spectroscopy (Chapter 6)

Before even attempting to understand spectrochemical analysis, one needs to have a basic understanding of electromagnetic radiation and radiation and

matter interactions

Sample Electromagnetic

radiation Spectrum

How is electromagnetic radiation generated? How is electromagnetic radiation detected?

How is a spectrum generated? Before even attempting to understand spectrochemical analysis, one needs to

have a basic understanding of instrumentation

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Electromagnetic radiation Electromagnetic radiation can be described by two models: the wave model and the particle model. The two models are not exclusive but, rather, complementary. Wave properties of electromagnetic (EM) radiation An EM wave can be represented as electric and magnetic fields that undergo in-phase sinusoidall oscillations at right angles to each other and to the direction of propagation. Both fields can be represented as vectors perpendicular to each other and to the direction of propagation The term plane-polarized implies that all oscillations of either the electric or the magnetic fields lie within a single plane The term monochromatic implies that the EM radiation is composed only by one wavelength

Wave of EM radiation Polarizer

Plane-polarized EM radiation

Plane-polarized EM radiation

Wavelength selector

Monochromatic plane-polarized

radiation

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Representation of a beam of monochromatic, plane-polarized radiation:

Interaction of the electric field and the magnetic field of an EM wave on an orbit about the nucleus

We will focus our discussion on the electric field of the EM wave

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Velocity = wavelength x frequency

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Some important properties of EM waves

• One of the most interesting characteristics of light is that no matter what its color or frequency, the wavelength times the frequency is a constant that we know as the speed of light.

• The frequency of the EM radiation is determined

by the source and remains constant, i.e. it does not depend on the medium of propagation (air, water, vacuum, glass, etc.).

• The velocity of propagation of a wave depends

on the composition of a medium. If the velocity of propagation depends on the medium, so does the wavelength.

• Refractive index

• Refraction of radiation

• Reflection of radiation

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The Electromagnetic Spectrum

Interactions of Radiation with Matter

Absorption and emission spectra

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Absorption of Radiation

• Example: Atomic absorption spectroscopy • Instrumentation

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Emission of Radiation

• Example: Atomic Emission Spectroscopy • Instrumentation

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Spectral differences between atoms and molecules Absorption of radiation

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Spectral differences between atoms and molecules Emission of radiation

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6-22

6-19

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Multiplicity of an electronic state

• The multiplicity (M) of an electronic state is related to the total spin quantum number, i.e.:

M = 2S + 1 • The total spin quantum number is equal to: S = ∑si, where si = +1/2 or -1/2. • Molecules in the ground state have a total spin quantum number equal to zero. • As a consequence, the multiplicity of the ground state is equal to 1. • Electronic states with multiplicity equal to 1 are called “singlet” states and denoted by

“S”. • When one of the two electrons of opposite spins, belonging to a molecular orbital of a

molecule in the ground state, is promoted to a molecular orbital of higher energy, its spin is - in general - unchanged.

• Because the total spin quantum number does not change, the multiplicity of the ground and excited state do not change and the transition is called a singlet-singlet transition:

S0 S1; S0 S2; S1 S2; etc. S0 denotes the ground state. • A molecule in the singlet state may undergo a state where the promoted electron has

changed its spin; because there are then two electrons with parallel spins, the total spin quantum number is 1 and the multiplicity is 3. Such a state is called a triplet state (T1, T2, T3, etc.) and the transition is called singlet-triplet transition.

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Example of Electronic Transitions in a Molecule

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Energy levels of molecular orbitals in formaldehyde HOMO: Highest Occupied Molecular Orbital

LUMO: Lowest Unoccupied Molecular Orbital

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Photoluminescence Phenomena

• S = Singlet electronic states S0 = fundamental electronic state = ground state S1 = first singlet excited state S2 = second singlet excited state • T = Triplet electronic states T2 = second triplet excited state T1 = first triplet excited state • Vibrational levels are associated with each

electronic state • Rotational levels are not represented in the

diagram • Radiationless processes: IC = internal conversion ISC = intersystem crossing VR = vibrational relaxation • Emitting processes: Fluorescence Phosphorescence • Delayed (“Late”) Fluorescence: not represented

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Optical Spectroscopy Methods are based on the Absorption of Radiation

Absorption of radiation is related to Transmittance

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(a) Molecular absorption spectroscopy (b) Molecular fluorescence spectroscopy (c) Atomic emission spectroscopy

Chapter 7

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Excitation sources

• Conventional sources • Laser sources

b) Temporal behavior Continuous

Pulsed

a) Spectral output Continuum sources

Line sources

Continuum plus line

• Conventional Sources a) Spectral output b) Temporal behavior

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Continuum sources

• Emit a continuum spectrum over a broadband wavelength region • There are two types: Incandescent lamps See examples a and b They are basically a resistive material heated electrically. As current

passes through the resistance, temperature raises and part of the energy is dissipated in the form of light

Arc discharge lamps See examples c to e These lamps are quartz or glass envelopes containing a gas that, upon

plasma formation, emit radiation

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Incandescent lamps

• The spectral radiance of an incandescent lamp follows a theoretical model known as blackbody • A blackbody absorbs all radiant energy upon it regardless wavelength. I has unity spectral

absorbance at all wavelengths and emits as much energy as it absorbs

• The spectral radiance of a blackbody is given by the following equation: where: Bl

b = spectral radiance of the blackbody in W. cm-2.sr-1.nm-1

h = Plank’s constant = 6.626 x 10-34 Js k = Boltzmann’s constant = 1.380 x 10-23 J K-1

c = 3 x 108 m.s-1

T = temperature (K) l = wavelength of emission (nm) c1 = 2hc2 = 1.190 x 1016 W.nm4.cm-2.sr-1

c2 = hc/k = 1.438 x 107 nm K

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• From the equations we note that the spectral radiance

at a given wavelength depends only on temperature • The figure shows a plot of Bl

b vs. l at several temperatures. Note the wavelength shift to the blue and the increase in the spectral radiance as the temperature increases

• The wavelength of maximum radiance (lm) is obtained by differentiating the spectral radiance equation with respect to wavelength and setting the derivative equal to zero

where: l is in nm and T is in K

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• To describe the spectral radiance of real sources, often called gray bodies, the spectral radiance equation of the blackbody model needs to be modified to account for non-idealities

where: e (l) = spectral emissivity of the source Tw (l) = transmission factor of the lamp window • 0 < e (l) < 1 e (l) is the ratio of the spectral radiance of the real

source to that of the blackbody at a specific wavelength and temperature

#Tw (l) accounts for reflective losses and absorptions at

the window surface of the lamp if the lamp is enclosed in an envelope such as quartz or glass. In cases where there is no envelope,

Tw (l) = 1 • The figure compares the total emittance of a

blackbody and a tungsten lamp

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Arc lamps

• Arc lamps consist of a sealed glass or quartz envelope filled with a gas

• The two electrodes (cathode and anode)

initiate a discharge in the envelope that is maintained by a dc or ac current

• The discharge creates electrons at the

cathode that are accelerated towards the anode

• Collisions between the electrons and gas cause ionization of gas molecules

• Positive ions are accelerated towards the cathode. Upon collision with the cathode, positive ions produce secondary electrons

• Secondary electrons collide with gaseous molecules to maintain ionization in the tube. A plasma is created that emits radiation

Gas

AC or DC

Cathode (-)

Anode (+)

= electron

Anode (+)

Cathode (-)

= positive ion

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• Hydrogen and deuterium lamps provide strong spectral continua in the UV spectral region

• At wavelengths longer than 370 nm, the spectra from hydrogen and deuterium are no longer continua

• In the visible region, the spectral radiance of hydrogen or deuterium lamps is rather weak

• Spectrophotometers that operate in the UV and visible regions often require two sources: a hydrogen or deuterium lamp is used for the UV region (200 – 350 nm), and a tungsten-filament lamp is used for the visible region (350 nm)

• Molecular fluorescence and phosphorescence spectrometry require high-intensity, continuum sources of UV and visible radiation. The Xe arc lamp is the most common source for molecular luminescence spectrometry

• The Xe-Hg lamp is an example of a continuum + line source. The spectral radiance is high, exceeding 1 W cm-2 nm-1 sr-1. Because of the presence of atomic lines, this source is not very useful for molecular absorption spectroscopy, where it is desirable to have a flat spectral distribution. It is an excellent source for photoluminescence spectroscopy, mainly if a Hg line corresponds to the wavelength of excitation of the compound of interest

Spectral irradiance of a D2 lamp

(a) Spectral radiance of a Xe lamp (b) Spectral radiance of a Xe-Hg lamp

68

Note the following: a) Nernst glower and Globar are IR sources. Their intensity is much weaker than UV and visible sources b) Tungsten lamps emit stronger in the visible than H2 or D2 lamps c) Xe lamps emit strongly in the ultraviolet and visible regions

The following table summaries several characteristics of common continuum sources

69

Wavelength selectors • There are three types: filters, monochromators and spectrographs Filters • There are four common types: band-pass or absorption filters, cut-off filters, density filters

and interference or Fabry-Perot filters • Band-pass, cut-off and density filters are based on the absorption of radiation

• Absorbing materials can be colored glasses, crystals, solutions, thin films, etc. • Wavelength selection is usually achieved with absorption or interference filters.

Incident Radiation

Io

Transmitted Radiation

I

Filter made of absorbing material

A = - log T = - log I/Io A = absorbance

T = transmittance

70

Band-pass filters • Band-pass filters are characterized by plots of

their spectral transmittance vs. wavelength. The characteristics of interest are shown in the first figure:

lm = wavelength of maximum transmittance Tm

Dl = full-width at half maximum (FWHM) • lm and Dl depend on the absorbing material,

as shown in the second and third figure • Typical values of the FWHM range from 30 to

250 nm

• Examples of their use in analytical spectroscopy a) photometers

Transmittance of Corning glass filters. The

numerical designations are the product numbers for specific filters.

71

Interference filters

• The first figure shows a Fabry-Perot interference filter

• The thin dielectric is a material of low refractive index such as quartz, CaF2, MgF2, ZnS, ThF4, or sapphire

• The filter is constructed in such a way that the rays from most wavelengths that strike the filter suffer destructive interference, while only rays within a small wavelength band experience constructive interference and are passed

• The condition for constructive interference is

where: d = dielectric thickness h = refractive index of dielectric material q = angle of incidence n = order = 1,2,3,… l = transmitted wavelength

(a) Schematic cross-section of interference filter (b) Schematics to show conditions for constructive

interference

72

Echellette Grating

nl = d(sini + sinr)

74

Dispersion Angular dispersion: dr/dl = n/dcosr Linear dispersion: D = dy/dl = f (dr/dl) Reciprocal linear dispersion: D-1 = dl/dy = (1/f)(dl/dr) D-1 = dcosr/nf Units of D-1 in UV-Vis nm/mm or Å/mm At small angles of diffraction (r<20°): cosr ≈ 1 D-1 = d/nf For small angles of diffraction the linear dispersion of a monochormator is constant.

75

Resolving Power of a Monochromator

• It describes the limit of a monochromator to separate adjacent images that have a slight difference in wavelengths

R = l / Dl Where: l = average wavelength of the two images Dl = difference between the two wavelengths R = l / Dl = nN Where: n = diffraction order N = number of grating blazes illuminated by

radiation from the entrance slit • Better resolution is a characteristic of longer

gratings, smaller blaze spacing's, and higher diffraction orders

• Typical R values for bench-top UV-Vis monochromators = 103 to 104

Light Gathering Power of Monochromators • Also known as the f-number (F) of a

monochromator • It provides a measure of the ability of a

monochromator to collect the radiation that emerges from the entrance slit

• F = f/d where: f = focal length of the collimating mirror

(or lens) and d is the diameter • The light-gathering power of an optical device

increases as the inverse square of the f-number

=> f/2 lens gathers 4x more light than f/4 lens

76

• Echelle gratings are used in Echelle monochromators

• Echelle monochromators contain two dispersing elements arranged in series

Echelle Grating

r ≈ i = b nl = 2dsinb

D-1 = dcosb / nf

High dispersion is achieved by making the angle b small and the order of diffraction n large

77

• For the same focal length, the linear dispersion and resolution are an order of magnitude greater for the echelle

• The light-gathering power of the echelle is also somehow superior

• Echelle gratings are very common in Atomic Emission Spectroscopy

78

Fig. 7-24

• Bandwidth: span of monochromator settings needed to move

the image of the entrance slit across the exit slit • Effective bandwidth or spectral bandpass or

spectral slit width: one-half of the bandwidth when the two slits

(entrance and exit) are identical Dleff = wD-1

79

Choice of Slits • Most monochromators are

equipped with variable silts so that the effective bandwidth can be changed.

• The use of the minimal slit width is desirable when narrow absorption or emission bands must be resolved.

• The available rediant power decreases significantly when the slits are narrowed:

Molecular spectroscopy (I a w2) Atomic spectroscopy (I a w)

• Wider slit widths may be used for quantitative analysis rather than for qualitative analysis.

80

Optical transducers

• The purpose of an optical transducer is to convert radiant power into an electrical signal

• Optical transducers fall into two categories: a) Thermal detectors b) Photon detectors • Thermal detectors sense the change in temperature that is produced by the

absorption of incident radiation

• Photon detectors respond to incident photon arrival rates rather than to photon

energies

81

Photon detector characteristics • The following characteristics are useful to compare photon detectors: a) Responsivity b) Linear range c) Spectral responsivity • Responsivity and linear range

• Spectral responsivity

82

Vacuum phototube

• The cathode is a photosensitive material such as Cs3Sb or AgOCs.

• When photons of a certain energy strike the photosensitive material, electrons are ejected from the cathode.

• Only photons with greater than threshold energy yield photoelectrons with sufficient kinetic energy to escape the photocathode.

• The electrons ejected from the photocathode are attracted by the anode, which is positively biased in relation to the cathode.

• The electron flux leaving the cathode is known as the cathodic current.

• Only a fraction of cathodic electrons are collected at the anode.

• The electrons arriving at the anode create the anodic current that is measured with an external circuit.

83

Photomultiplier Tube • The PMT contains a photosensitive cathode

(photocathode) and a collection anode • The cathode and the anode are separated by

several electrodes (dynodes) that provide electron multiplication or gain

• The cathode is biased negative with respect to the anode (the potential difference is anywhere from 400 to 2500V)

• A photoelectron ejected by the photocathode strikes the first dynode and releases 2 to 5 secondary electrons

• Each secondary electron is accelerated by the electric field between the first and second dynode with sufficient energy to release another 2 to 5 electrons in the second dynode

• Since each dynode down the chain is biased 100V more positive than the preceding dynode, this multiplication process continues until the pulse of electrons reach the anode

84

• P-n junction

• A photodiode detector is a p-n junction

operating in a reverse-bias mode • Absorption of EM radiation by the p-n

junction causes covalent bonds to brake forming hole-electron pairs that populate the depletion region

• Since the number of hole-electron pairs in the depletion region increases with the arrival of photons, the limiting current is proportional to the radiant power of incident radiation

• The voltage drop across a load resistor is measured and the voltage is proportional to the arrival of photons ate the depletion zone

Photodiodes

85 85

N-semiconductor

Silicon (IVA) is doped with trace quantities of

P (VA). The free electron of P is free to

move along the Si lattice.

P-semiconductor

Silicon (IVA) is doped with trace quantities of B (IIIA). The lack of an

electron creates a hole that electrons

can migrate from Si to B. Electron migration

creates positive charges.

- - - - - - - - -

- - - - - - - - - -

+ + + + + + + + + + + + +

86 86

The photodiode is a p-n junction under reverse bias

Negative terminal

Positive terminal

87 87

PN junction reverse biased: Initially, there is a very small amount of current because of the minority of carriers

crossing the junction. The combination of electrons and holes creates a depletion region with negligible

charge. The depletion region acts as a barrier for further attempts of charges crossing the

barrier. High resistance to current flow.

88 88

Forward Biased Conduction

When the p-n junction is forward biased, the electrons in then-type material combine with the holes in the p-type material, making possible a

continuous forward current through the junction. Low resistance to current flow.

89

Linear photodiode array (LPDA)

• LPDA is a linear arrangement of several small silicon photodiodes

• The typical dimension of each element (pixel) is 2.5 by 0.025 mm

• The number of transducers in a chip ranges from 64 to 4096, with 1024 being perhaps the most widely used

• The LPDA is operated in the charge storage mode. The charge accumulated by each element is given by:

Where: q = accumulated charge (C) E = incident radiation (W) R(l) = responsivity (A.W-1) t = integration time (s) • Note: longer integration times accumulate

more charge and provide better signal-to-noise ratios

90

How does it work?

• The LPDA is placed at the focal plane of the exit slit of a monochromator (spectrograph) • Photodiodes located at different positions in the linear array are exposed to different

wavelengths • Each pixel, or set of pixels, collects the intensity at a specific wavelength • The computer screen plots intensity versus wavelength. The wavelength corresponding to each

pixel is known because the multi-channel system is calibrated taking into consideration the optical parameters of the spectrograph and the dimensions of the LPDA

• Obtaining a scan with the LPDA corresponds to the process of sequential pixel interrogation. See figure

• To interrogate the entire array takes a period of microseconds. The longer the interrogation time, the longer it takes to obtain a scan

91

• The first figure shows the stored signal charge as a function of light exposure for a Silicon LPDA at a wavelength of 575 nm

• The charge accumulates linearly until

reaching the saturation charge where the corresponding exposure is the saturation exposure

• The second figure shows the

responsivity of the LPDA. The responsivity is defined as the ratio between the saturation charge and the saturation exposure

• LPDA can not match the performance PMT with respect to sensitivity, dynamic range and signal-to-noise ratio

92 92 92

Charge-coupled device (CCD)

• CCDs are silicon-based semiconductor chips bearing two dimensional matrixes of photodiodes or pixels

• The pixels are arranged in rows and columns. Rows run horizontally and columns vertically. See figure

• A typical CCD chip may comprise 256 rows and 1024 columns

• When the CCD is placed at the focal plane of a spectrograph, the position of a pixel (or several pixels) corresponds to the position of a projected wavelength image. The charge collected by that pixel (or set of pixels) corresponds to the intensity of radiation at that wavelength

93

TYPES OF OPTICAL INSTRUMENTS • It is a rarity that all the optical information produced in a spectrochemical measurement is useful

or desirable. Sorting out the desirable optical information is a major step in a spectrochemical measurement.

• The vast majority of optical spectroscopic techniques select the desired information based on its wavelength. Wavelength selection in spectrochemical measurements can be based on absorption or interference filters, spatial dispersion of wavelengths or interferometry.

• Wavelength selectors which disperse the spectral components of the optical signal spatially are the most common. Some of the major configurations are shown in the figure below:

=> The entrance slit defines the area of the source of radiation that is viewed. => The dispersive element can be a prism which spatially separate wavelengths by refraction or a

grating that disperses light based on diffraction. => The image transfer system produces an image of the entrance slit on the focal plane. • The name of the wavelength selector depends on the arrangement of apertures or slits in the focal

plane where the spectrum is dispersed: => Spectrograph => Monochromator => Polychromator

94

• Spectroscope: is an optical instrument used for the visual identification of atomic emission lines. It uses a spectrograph with a viewing screen for observing the spectrum in the focal plane.

• Colorimeter: is an instrument for absorption measurements in which the human eye serves as the detector using one or more color-comparison standards.

• Photometer: consists of a source, a filter, a photoelectric transducer, and a signal processor and readout system.

• Fluorometer or fluorimeter: photometers designed for fluorescence measurements.

• Spectrometer: is an instrument that provides information about intensity of radiation as a function of wavelength or frequency. Dispersing unit can be a monochromator, a spectrograph or a polychromator.

• Spectrophotometer: is a spectrometer equipped with one or more exit slits and photoelectric transducers.

• Spectrofluorometer or spectrofluorimeter: spectrophotometer for fluorescence analysis.

• Single-channel and multi-channel spectrometers

The name of the instrument depends on both the wavelength selector and the type of detection system

UV-Vis Absorption Spectrophotometer for molecular spectroscopy

Fig. 13-19

95

Phototubes, PMT and photodiodes are used in single channel spectrometers for both atomic and molecular spectroscopy

Single-channel system for UV-Vis molecular absorption spectroscopy

Single-channel system for Atomic Absorption Spectroscopy (AAS)

Fig. 13-13a

Fig. 9-13a

96

LPDA, CCD and CID are used in multiple-channel spectrometers for both atomic and molecular spectroscopy

Multi-channel system for UV-Vis molecular absorption spectroscopy

Multi-channel system for

Atomic Emission Spectroscopy (AES)

Fig. 13-14

Fig. 10-07

97

INTRODUCTION TO OPTICAL ATOMIC SPECTROSCOPY

• Atomic spectroscopy techniques: Optical spectrometry Mass spectrometry X-Ray spectrometry • Optical spectrometry: Elements in the sample are atomized

before analysis. • Atomization: Elements present in the sample are

converted to gaseous atoms or elementary ions. It occurs in the atomizer (see Table 8-1).

• Optical spectroscopy techniques: Atomic Absorption Spectroscopy (AAS) Atomic Emission spectroscopy (AES) Atomic Fluorescence Spectroscopy (AFS)

98

Optical Atomic Spectra • Figure 8-1a shows the energy level diagram for sodium. • A value of zero electron volts (eV) is arbitrarily assigned to

orbital 3s. • The scale extends up to 5.14eV, the energy required to

remove the single 3s electron to produce a sodium ion. 5.14eV is the ionization energy.

• A horizontal line represents the energy of and atomic orbital. • “p” orbitals are split into two levels which differ slightly in

energy: 3s → 3p: l = 5896Å or 5890Å 3s → 4p: l = 3303Å or 3302Å 3s → 5p: l = 2853.0Å or 2852.8Å • There are similar differences in the d and f orbitals, but their

magnitudes are usually so small that are undetectable, thus only a single level is shown for orbitals d.

Spin-orbit coupling

99

Multiplicity: number of possible orientations of the resultant spin angular momentum = 2S +1

100

Atomic Line Widths

• Widths of atomic lines are quite important in atomic spectroscopy.

• Narrow lines in atomic and emission spectra reduce the possibility of interference due to overlapping lines.

• Atomic absorption and emission lines consists of a symmetric distribution of wavelengths that centers on a mean wavelength (l0) which is the wavelength of maximum absorption or maximum intensity for emitted radiation.

• The energy associated with l0 is equal to the exact energy difference between two quantum states responsible for absorption or emission.

• A transition between two discrete, single-valued energy states should be a line with line-width equal to zero.

• However, several phenomena cause line broadening in such a way that all atomic lines have finite widths.

• Line width or effective line width (Dl1/2) of an atomic absorption or emission line is defined as its width in wavelength units when measured at one half the maximum signal.

Sources of broadening: (1) Uncertainty effect (2) Doppler effect (3) Pressure effects due to collisions (4) Electric and magnetic field effects

101

Uncertainty Effect

• It results from the uncertainty principle postulated in 1927 by Werner Heisenberg.

• One of several ways of formulating the

Heisenberg uncertainty principle is shown in the following equation:

Dt x DE = h • The meaning in words of this equation is as

follows: if the energy E of a particle or system of particles – photons, electrons, neutrons or protons – is measured for an exactly known period of time Dt, then this energy is uncertain by at least h/ Dt.

• Therefore, the energy of a particle can be

known with zero uncertainty only if it is observed for an infinite period of time.

• For finite periods, the energy measurement

can never be more precise then h/ Dt. • The lifetime of a ground state is typically long,

but the lifetimes of excited states are generally short, typically 10-7 to 10-8 seconds.

• Line widths due to uncertainty broadening are

called natural line widths and are generally 10-5nm or 10-4Å.

Note: Dl = Dl1/2

102

Doppler Effect

• In a collection of atoms in a hot environment, such as an atomizer, atomic motions occur in every direction.

• The magnitude of the Doppler shift increases with the velocity at which the emitting or absorbing species approaches or recedes the detector.

• For relatively low velocities, the relationship between the Doppler shift (Dl) and the velocity (v) of an approaching or receding atom is given by:

Dl / l0 = v / c Where l0 is the wavelength of an un-shifted

line of a sample of an element at rest relative to the transducer, and c is the speed of light.

• Emitting atom moving: (a) towards a photon detector, the detector sees wave crests more often and detect radiation of higher frequency; (b) away from the detector, the detector sees wave crests less frequently and detects radiation at lower frequency.

• The result is an statistical distribution of frequencies and thus a broadening of spectral lines.

Dl

103

Pressure Effects Due to Collisions

• Pressure or collisional broadening is caused by collisions of the emitting or absorbing species with other atoms or ions in the heated medium.

• These collisions produce small changes in energy levels and hence a range of absorbed or emitted wavelengths.

• These collisions produce broadening that is two to three orders of magnitude grater than the natural line widths.

• Example: Hollow-cathode lamps (HCL): • Pressure in these lamps is kept really low

to minimize collisional broadening. • Glass tube is filled with neon or argon at

a pressure of 1 to 5 torr.

lA lE

lA’ lE’

Ene

rgy

(eV

)

Atom 1 Atom 2

E1

E2

E1

E2

104

The Effect of Temperature on Atomic Spectra

• Temperature in the atomizer has a profound effect on the ratio between the number of excited an unexcited atomic particles.

• The magnitude of this effect is calculated with the Boltzmann distribution equation:

Nj / N0 = (gj / g0) x [exp(-Ej/kT)] where: - Nj and N0 are the number of atoms in the excited state

and ground state, respectively - k is the Boltzmann’s constant - T is absolute temperature (K) - Ej is the energy difference between the excited and the

ground state. - gi and g0 are statistical factors called statistical weights

determined by the number of states having equal energy at each quantum level.

• Example shows that a temperature fluctuation of only 10K results in a 4% increase in the number of excited sodium atoms.

• A corresponding increase in emitted power by the two lines would result.

• An analytical method based on the measurement of emission requires close control of atomization temperature.

105

Band and Continuum Spectra Associated with Atomic Spectra

• When atomic line spectra are generated, both band and continuum radiation are usually produced as well.

• Molecular bands often appear as a result of molecular species in the atomizer. Molecular species can be associated or not to the element of interest. For instance, the molecular bands shown in the figure can be used to determine Ca.

• Continuum radiation appears as a result of thermal radiation from hot particulate matter in the atomization medium.

• Molecular bands and continuum radiation are a potential source of interference that must be minimized by proper choice of wavelength, by background correction, or by change in atomization conditions.

Molecular flame emission and flame absorption spectra for CaOH

Background emission spectra from an ICP. The upper recording was taken favoring continuum and band emission while the lower recording was taken under conditions minimizing continuum and band emission.

106

Sample Introduction Methods

• Achilles’ heel of Atomic Spectroscopy because in many cases limits the accuracy, the precision and the limits of detection of analytical method.

• Primary purpose is to transfer a reproducible and representative portion of a sample into one of the atomizers presented in Table 8-1.

• Table 8-2 lists the common sample introduction methods for Atomic Spectroscopy and the type of samples to which each method is applicable.

• Atomizers “fit” into two classes: continuous and discrete atomizers.

• Continuous atomizers: flames and plasmas. Samples are introduced in a steady manner.

• Discrete Atomizers: electro-thermal atomizers. Sample introduction is discontinuous and made with a syringe or an auto-sampler.

Discontinuous

Con

tinuo

us

107

Nebulizers

• Direct nebulization is the most common method of sample introduction with continuous atomizers. The solution is converted into a spray by the nebulizer.

• Types of nebulizers: (a) Concentric tube: most common nebulizer. It

consists of a concentric-tube in which the liquid sample is drawn through a capillary tube by a high-pressure stream of gas flowing around the tip of the tube.

(b) Cross-flow: the high pressure gas flows across a capillary tip at right angles. It provides independent control of gas and sample flows.

(c) Fritted disk: the sample solution is pumped onto a fritted surface through which a carrier gas flows. It provides a much finer aerosol than a and b.

(d) Babington: it consists of a hollow sphere in which a high pressure gas is pumped through a small orifice in the sphere’s surface of the sphere. It is less subject to clogging than a-c. It is useful for samples that have a high salt content or for slurries with a significant particulate content.

Continuous Atomizer

108

Electro-thermal Vaporizers Discrete Atomizer

• Sample introduction in discrete atomizers is typically made manually with the aid of a syringe.

• The steps that convert the liquid solution into a vapor of free atoms are the same as those in continuous atomizers.

• The most common type of discrete atomizer is the electro-thermal atomizer.

• An electro-thermal atomizer is a small furnace tube heated by passing a current through it from a programmable power supply.

• The furnace is heated in stages. The dry and ash step removes water and organic or volatile inorganic matter, respectively.

• The atomization step produces a pulse of atomic vapor that is probed by the radiation beam from the hollow-cathode lamp (HCL).

109

Free-Atom Formation in

Atomizers

• Desolvation and Volatilization : desolvation leaves a dry aerosol of molten or solid particles. The solid or molten particle remaining after desolvation is volatilized (vaporized) to obtain free atoms. The efficiency of desolvation and volatilization depends on a number of factors: atomizer temperature, composition of analytical sample (nature and concentration of analyte, solvent and concomitants) and size distribution. In the case of nebulizers, it also depends on the nebulizer design, aerosol trajectories and resident times of the particles.

• Dissociation and Ionization: in the vapor phase, the analyte can exist as free atoms, molecules or ions. In localized regions of the atomizer, molecules, free atoms and ions co-exist in equilibrium.

• Dissociation of molecular species: molecular formation reduces the concentration of free atoms and thus degrades the detection limits. The dissociation constant for a molecular species (MX) into its components (MX <=> M + X) can be written as: Kd = nM.nX / nMX, where n is the number density (number of species per cm3). For a diatomic molecule:

logKd = 20.274 + 3/2log MMMX/MMX + log ZMZX/ZMX + 3/2(logT) – 5040Ed/T where Mi is the molecular or atomic weight of species i, Zi is the partition function of species i, Ed is the

dissociation energy in eV, and T is the temperature in K. Note: The final term in this equation describes most of the temperature dependence: small values of Ed and high temperatures lead to large values of Kd and thus high degrees of dissociation.

• Ionization: it can also be consider an equilibrium process: M <=> M+ + e-. The ionization constant can be written as: Ki = n M+ .ne / nM, where ne is the number density of free electrons. The ionization constant can be obtained from:

logKi = 15.684 + logZM+ /ZM + 3/2(logT) – 5040Eion/T where Eion is the ionization energy in eV. Note: The final term in this equation describes most of the

temperature dependence: small values of Eion and high temperatures favor the formation of ions.

Continuous Atomizer

Discrete Atomizer

110

Atomic Absorption (AAS) and Atomic Fluorescence (AFS) Spectrometry

• The two most common methods of sample atomization encountered in AAS and AFS are flame and electro-thermal atomization.

• Flame atomization: A solution of the sample is nebulized by a flow of gaseous oxidant, mixed with a gaseous fuel and carried into the flame where atomization occurs.

Oxidant (g)

Fuel (g)

Carrier (g)

Note: • If the gas flow rate does not exceed the burning velocity, the flame propagates back into the burner, giving flashback. • The flame is stable where the flow velocity and the burning velocity are equal. • Higher flow rates than the maximum burning velocity cause the flame to blow off.

111

Flame Structure

• Primary combustion zone: Thermal equilibrium is not achieved in this zone and thus it is rarely used for flame spectroscopy.

• Inter-zonal area: Free atoms are prevalent in this area. It is the most widely used part of the flame for spectroscopy.

• Secondary combustion zone: products of the inner core are converted to stable molecular oxides that are then dispersed to the surroundings.

• Maximum temperature: it is located in the flame about 2.5cm above the primary combustion zone.

• Note: It is important to focus the same part of the flame on the optical beam for all calibrations and analytical measurements.

• Optimization: of optical beam position within the flame prior to analysis provides the best signal – to – noise ratio (S/N). It depends on element and it is critical for limits of detection.

Increased number of Mg atoms produced

by the longer exposure to the heat

of the flame.

Secondary combustion zone: oxidation of Mg

occurs. Oxide particles do not absorb at the

observation wavelength.

Ag is not easily oxidized so a continuous increase

in absorbance is observed.

Cr forms very stable oxides that do not absorb

at this observation wavelength.

112

AA Spectrometer

Commercial Flame Atomizers

• Typical commercial laminar-flow burner. • The aerosol formed by the flow of oxidant is

mixed with fuel and passes a series of baffles that remove all but the finest solution droplets.

• The baffles cause most of the sample to collect in the bottom of the mixing where it drains to waste container.

• The aerosol, oxidant, and fuel are then burned in a slotted burner to provide a 5- to 10-cm high flame.

• The quiet flame and relatively long-path length minimizes noise and maximizes absorption. These features result in reproducibility and sensitivity improvements for AAS.

Excitation source

Flame

Monochromator

Detector

113

Commercial Electro-thermal Atomizers Cylindrical graphite tube

where atomization occurs. Dimensions:

about 5cm long and 1cm internal diameter.

This tube is interchangeable.

Cylindrical graphite electrical contacts. These

contacts are held in a water-cooled metal

housing.

L’vov platform. Made of graphite, sample is evaporated

and ashed on this platform. Temperature on the platform does not change as fast as it changes in the walls of the

furnace. Atomization occurs in an environment where

temperature does not change so fast, which improves

reproducibility of measurements.

Facilitates furnace cleaning, which reduces memory effects.

Longitudinal (b) and transversal (c) furnace heating. Transversal mode is preferred because it provides a uniform

temperature profile along the entire length of the tube and optical path.

Output signal

114

Flame Atomizers versus Electro-thermal Atomizers

• Advantages of Flame Atomizers • Better reproducibility of measurements RSD: Flame ≈ 1% Electro-thermal ≈ 5% - 10% • Much faster analysis times than electro-

thermal atomizers • Wider linear dynamic ranges, up to 2 orders

of magnitude wider than electro-thermal atomizers.

• Advantages of Electro-thermal Atomizers • Smaller sample volumes (0.5mL to 10mL of

sample) than flame atomizers. • Better absolute limits of detection (ALOD ≈

10-11 to 10-13 g of analyte) than flame atomizers

Note: ALOD = [Sample Volume] x LOD

Electro-thermal atomization is the method of choice when flame atomization provides inadequate limits of detection or sample availability is limited.

115

Specialized Atomization Techniques

• Glow-Discharge Atomization • Cold-Vapor Atomization • Hydride Atomization

• Hydride Atomization • It provides a method for introducing samples

containing arsenic (As), antimony (Sb), tin (Sn), bismuth (Bi) and lead (Pb) into the atomizer as a gas.

• This procedure improves limits of detection 10 to 100x.

• Their determination at low levels is very important because of their high toxicity.

• Volatile hydrides are generated by adding an acidified aqueous solution of the sample to a small volume of a 1% aqueous solution of sodium borohydride contained in a glass vessel. A typical reaction is:

3BH4-(aq) + 3H+(aq) + 4H3AsO4(aq) →

3H3BO3(aq) + 4AsH3(g) + 3H2O (l) • The volatile hydride is swept into the atomization

chamber by an inert gas. • The chamber is usually a silica tube heated to several

hundred degrees in a furnace or in a flame where atomization takes place.

116

Atomic Absorption Instrumentation

• Main components: (1) Radiation source (2) Sample holder = atomizer (3) Wavelength selector (4) Detector (5) Signal processor and readout

• Radiation source: • Why not using a broadband source with a

monochromator for excitation? • The emission profile of the source should have a

narrower effective bandwidth than the absorption line of the element of interest.

• HCL and electrodeless discharge lamps (EDL) satisfy this condition.

• Disadvantage over broadband source/monochromator: One source per element.

HCL EDL

EDL provide intensities one to two orders of magnitude better than HCL. EDL are only available for ≈ fifteen elements. Particularly useful for Se, As, Cd and Sb because it provides better LOD.

117

Source Modulation

• The output of the source is modulated so its intensity fluctuates at a constant frequency.

• The detector receives two types of signal, an alternating signal from the source and a continuous signal from the flame.

• A high-pass RC filter is then used to remove the continuous signal and pass the alternating signal for amplification.

• Source modulation can be done with a chopper or rotating disk or a power supply.

Emission from the sample + emission from the flame Monochromator is able to eliminate flame interference based on wavelength separation. However, when the wavelength of

interference is the same as the analyte wavelength the monochromator is unable to eliminate interference.

H2 - O2

C2H2-N2O

C2H2 – O2

High-pass filter

Choppers

118

Spectrophotometers for AAS

• Single beam and double beam instruments. • Double beam instruments provide the advantage

of correcting for source fluctuations. In this arrangement, however, the reference beam does not pass through the flame and, therefore, it does not correct for loss of radiant power due to absorption or scattering by the flame itself.

• Loss of radiant power due to absorption or scattering in the flame could have different sources:

(a) fuel and oxidant mixture alone (b) concomitants in sample matrix (c) all of the above • When the source of loss of radiant power is only

due to the fuel and the oxidant of the flame the solution is simple: make blank measurements and correct analytical data. This type of correction should be done with both types of spectrophotometers, i.e. single and double beam.

119

Loss of radiant power due to absorption or scattering from concomitants in the sample matrix

• Example of spectral matrix interference due to absorption: presence of CaOH in the analysis of Barium.

Solution: raise the temperature of the flame. Higher temperatures will decompose CaOH and remove its potential interference.

• Example of spectral interference due to scattering by products of atomization: most often encountered when particles with diameters greater than the absorption wavelength are formed in the flame:

a) Concentrated solutions containing Ti, Zr, and W. These elements form refractory oxides.

b) Organic species or when organic solvents are used to dissolve the sample. Incomplete combustion of the organic matter leaves carbonaceous particles.

• Methods for correcting spectral interference: (1) The two-line correction method (2) The continuum source correction method (3) Zeeman background correction method

F

HCL l0

F > l0

Scattering

120

Zeeman Effect

• It takes place when an atomic vapor is exposed to a strong magnetic field (≈10KG).

• It consists of a splitting of electronic energy levels, which leads to formation of several absorption lines for each electronic transition.

• The simplest splitting pattern is observed with singlet transitions, which leads to a central (p) line and two equally spaced (s) satellite lines (≈ 0.01nm).

• The central line corresponds to the original wavelength. It has an absorbance that is twice the absorbance of the satellite lines. Both s lines have the same intensity.

No Yes

Ene

rgy

No A

l

Yes A

l

p

s- s

121

Background Correction Based on the Zeeman Effect

• The behavior of p and s lines is different with respect to plane polarized radiation: p lines absorb plane polarized radiation parallel to the external magnetic field (II). s lines absorb plane polarized radiation perpendicular (90°) to the external magnetic field.

122

Ionization Equilibria • Ionization of atoms and molecules in atomizers

can be represented by the equilibrium: M <=> M+ + e- • The equilibrium constant for this reaction is: K = [M+] [e-] / [M] • The ionization of a metal will be strongly

influenced by the presence of other ionizable metals in the flame:

B <=> B+ + e- • The ionization of M will be decreased by the

mass-action effect of the electrons formed from B.

• B can then act as an ionization suppressor. • Ionization suppressors are often added to the

flame to improve sensitivity of analysis. • Ionization suppressors are commonly used with

higher temperature flames such as N2O-acetylene.

• The concentration of ionization suppressor needs to be controlled to avoid primary inner filter effects, i.e. absorption of excitation radiation.

123

Atomic Fluorescence Spectroscopy (AFS)

• There are five basic types of fluorescence: resonance fluorescence, direct-line fluorescence, stepwise-line fluorescence, sensitized fluorescence and multi-photon fluorescence. The figure shows an energetic diagram level for resonance fluorescence.

• In all cases, the basic instrumentation is the same. EDL are the best excitation sources for AFS. • The advantage of AFS over AAS is that it provides better limits of detection for several elements.

Ene

rgy

124

Atomic Emission Spectrometry

• The figure shows the typical configuration of a flame or plasma emission spectrometer.

• There are three primary types of high temperature plasmas:

ICP = inductively coupled plasma DCP = direct current plasma MIP = microwave induced plasma • ICP and DCP are commercially available. • Both types of plasmas sustain temperatures as high as

10,000K. ICP: • It consists of three concentric quartz tubes through which streams of argon flows. The diameter of the largest tube is 2.5cm. • Surrounding the top of the largest tube is a water-cooled induction coil that is powered by a radio-frequency generator, which radiates 0.5 to 20KW of power at 27.12MHz or 40.68MHz. This coil produces a fluctuating magnetic field (H). • Ionization of the flowing argon is initiated by a spark from a Tesla coil. The interaction of the resulting ions, and their associated electrons, with H makes the charges to flow in closed annular paths. • The resistance of ions and electrons towards the flow of charges causes ohmic heating of the plasma. • The tangential flow of argon cools the internal walls of the ICP. • Spectral observations are generally maed at a height of 15 to 20mm above the induction coil, where the temperature is 6,000-6,500K and the region is “optically transparent” (low background).

125

DCP • It consists of three electrodes configured in an inverted Y. A

graphite anode is located at each arm of the Y and a tungsten cathode at the inverted base.

• Argon flows from the two anode blocks toward the cathode. • The plasma is formed by bringing the cathode into momentary

contact with the anodes. • Ionization of the argon occurs an a current develops that

generates additional ions to sustain the current indefinitely. • The temperature at the arc core is between 5,000K and 8,000K.

ICP versus DCP: • DCP present lower background than ICP. • ICP is more sensitive than DCP; LOD in ICP are approximately one order of magnitude better. • DCP and ICP have similar reproducibility of measurements. • DCP requires less argon usage. • ICP is easier to align because the optical window of a DCP is relatively small. • Graphite electrodes must be replaced every few hours, whereas the ICP requires little maintenance.

126

Instrumentation

• The main two types of instruments for AES fit into two general categories: sequential or multi-channel spectrometers.

• Sequential instruments are designed to read one line per element at the time.

• Multi-channel instruments are designed to measure simultaneously the intensities of emission lines for a large number of elements (50 or 60 elements).

• Both instruments require wavelength selectors with high spectral resolution.

Sequential instrumentation: • It uses a slew-scanning monochromator. • Hg lamp is used for calibration. • Two PMT, one is used for the UV and

the other for the VIS. • A flipping mirror selects the exit slit and

the PMT.

127

Scanning Echelle instrumentation • It can be used either as a single channel or as a

“simultaneous multi-channel spectrometer”. • Scanning is accomplished by moving the PMT in both x and

y directions to scan an aperture plate located on the focal plane of the monochromator.

• The plate contains as many as 300 slits. The time it takes to move the PMT from one slit to another is approximately 1s.

• This arrangement can be converted to a multi-channel system by placing small PMT behind several exit slits.

Polychromators • The entrance slit, the exit slits, and the grating

surface are located along the circumference of a Rowland circle.

• The curvature of a Rowland circle corresponds to the focal plane of a monochromator.

• Each exit slit is factory configured to transmit lines for selected elements.

• The entrance slit can be moved tangentially to the Rowland circle to provide scanning.

128

Charge-Coupled Device Instrumentation • Typically incorporates two CCD, one for the

UV (165nm – 375nm) and one for the VIS (375nm – 782nm).

• The Schmidt cross-disperser separates the UV from the VIS radiation and the orders at each emission wavelength.

Elements Determined

Tl and Nb curves are not linear probably because of incorrect background subtraction. Self-absorption is another cause of non-linearity. It occurs at

high concentrations where the non-excited atoms absorb emitted radiation.

130

An Introduction to Ultraviolet-Visible Molecular Spectrometry (Chapter 13)

• Beer’s Law: A = -log T = -logP0 / P = e x b x C See Table 13-1 for terms. • In measuring absorbance or transmittance, one should

compensate for reflections and scatter occurring at the interface of the cuvette.

• Compensation is always done by running the blank.

131

Application of Beer’s Law to Mixtures

• Before applying these equations, you need to know the following:

• For compound 1: e1 at l1 and l2 • For compound 2: e2 at l1 and l2 • If their values are not know, you should obtain them

via calibration curves. • It is also common to obtain e values from single

standard solutions. • You should always remember: e is a constant

that depends on: Wavelength Temperature Solvent

Concentration, M A

bsor

banc

e

l1

l2

e1

e2

132

Limitations of Beer’s Law

• Three types of limitations cause deviations from linearity:

a) Real Limitations b) Instrumental Limitations c) Chemical Limitations • Real Limitations: Result from analyte-analyte interactions at

high analyte concentrations (usually C > 0.01M).

• The upper limit of the LDR depends on the compound. For the same compound, it also depends on the solvent and the temperature.

• You should always determine experimentally the upper limit of the LDR.

LDR

Abs

orba

nce

Concentration

• Solute-solute interactions are mainly dipole-dipole interactions • Electrostatic interactions can also occur among strong electrolytes

High analyte concentration causes average

distances among analyte molecules to decrease

133

• Chemical deviations: Arise when an analyte dissociates, associates, or reacts with a concomitant (including solvent) to

produce a product with different absorption spectrum than the analyte. • Typical example: Acid-Base Indicators (HIn). HIn <=> H+ + In- (Color 1, l1) (Color 2, l2) Spectrum looks like: => • There is a dissociation constant associated to the equilibrium above: Ka = [H+][In-] / [HIn] • HIn and In- concentrations depend on the pH of solution: [H+] = Ka x [HIn] / [In-] => log [H+] = logKa + log [HIn] / [In-] => - log [H+] = - logKa - log [HIn] / [In-] => pH = pKa + log [In-] / [HIn] • Assuming an indicator with pKa = 5 (Ka = 10-5): pH log[In-] / [HIn] [In-] / [HIn] [In-] = ? X [HIn] 1 - 4 10-4 0.0001 2 - 3 10-3 0.001 5 0 100 1 6 1 101 10 7 2 102 100

• At any given wavelength, the total absorbance intensity of the solution is the sum of the individual

intensities of HIn and In-: A Total, l A HIn, l + A In-, l

134

• According to Beer’s law, the absorbance intensities of HIn and In- are the following:

A HIn, l = e HIn, l . b . [HIn] A In-, l = e In-, l . b . [In-] • Substituting in the total absorbance equation: A Total, l = e HIn, l . b . [HIn] + e In-, l . b . [In-] => At any given wavelength, the total absorbance intensity of the

solution (A Total) depends on the concentrations of HIn and In-. => Because [HIn] and [In-] depend on pH, A Total depends on pH. • You should also remember that the indicator concentration is

given by: C Indicator = C = [HIn] + [In-] HIn <=> H+ + In- C – x x x where x = [In-] = [H+]. From Ka: Ka = x2 / C – x => x2 = Ka (C – x) => x2 + Ka.x – Ka.C = 0 => x = -Ka ± {Ka2 + 4KaC}1/2 / 2

This equation demonstrates that the concentration of indicator varies non-linearly with the ionized fraction of the acid. The same is true for [HIn] ( = C – x).

• A graph of intensity of absorption as a function of concentration

of indicator of an un-buffered solution provides a non-linear plot.

At 430nm: the absorbance is primarily due to the ionized In- form of the indicator and is proportional to the ionized fraction, which varies non-linearly with the total indicator

concentration. At 530nm: the absorbance is due principally to the un-dissociated acid HIn, which increases non-

linearly with the total concentration.

135

Instrumental deviations due to polychromatic light

• Beer’s law is only followed when measurements are made with mocnochromatic radiation.

• Wavelength selection of continuous sources radiation made with filters or monochromators provides a Gaussian wavelength profile with a central wavelength of maximum intensity.

• Consider a beam of radiation consisting of two wavelengths:

• This condition is best met at the maximum absorption wavelength of the absorber.

Only when the two molar

absorptivities are the same, this

equation simplifies to A = e.b.C and Beer’s law is followed.

136

Instrumental deviations in the presence of stray radiation

• Similar considerations are true for stray radiation.

• In the presence of stray light radiation (Ps), absorption is given by:

A’ = log [P0 + Ps] / [P + Ps] • Depending on its relative magnitude, stray

light radiation can cause significant deviations from linearity.

• At high stray levels and high concentrations, i.e. strong absorbance and low transmittance, the radiant power transmitted through the sample can become comparable to or lower than stray-light level.

Mismatched Cells • If the analyte and blank cells are optically different and/or have different path-lengths,

deviations from Beer’s law can occur. • You should always use optically equivalent cells!

137

Effects of instrumental noise on spectrophotometric analyses

• The relative standard deviation (sc/c) of a concentration (c) obtained via a transmittance measurement (T) is given by the equation:

sc/c = 0.434sT / T. logT where sT is the absolute standard deviation of the

transmittance measurement. • This equation shows that the uncertainty in a measurement

varies non-linearly with the magnitude of the transmittance. • Non-linearity as a function of relative standard deviation is

also true for absorbance measurements. • The sources of noise (uncertainties) in transmittance

(absorbance) measurements can be divided in three cases.

• K1, k2 and k3 are proportionality constants.

• Only for Case I sT is independent of T.

• If the limiting source for uncertainty in a measurement is instrumental noise, the best standard deviation within the calibration curve will then be obtained at the absorbance value where sc/c is minimum.

138

Types of Instruments

• Single channel systems • Multi-channel systems

Multi-channel systems present the advantage of real-time spectra but the upper concentration limit of their LDR is usually lower than single-beam systems.

141

Luminescence Spectroscopy • Excitation is very rapid (10 - 15s). • Vibrational relaxation is a non-radiational process.

It involves vibrational levels of the same electronic state. The excess of vibrational energy is released by the excited molecule in the form of thermal or vibrational motion to the solvent molecules. It takes between 10-11 and 10-10s.

• Internal conversion is a non-radiational process. It is the crossover between states of the same multiplicity. It occurs when the high vibrational levels of the lower electronic state overlap with the low vibrational levels of the high electronic state. It can occur between excited states (S2 and S1) or between excited and ground states (S1 and S0). Between excited states is rapid (10-12s).

• Fluorescence is a radiational transition between S1 and S0. It occurs from the ground vibrational level of S1 to various vibrational levels in S0. It requires 10-10 to 10-6 s to occur.

• External conversion is a nonradiative process in which excited states transfer their excess energy to other species, such as solvent or solute molecules.

• Intersystem crossing is a non-radiative process between electronic states of different multiplicities. It requires a change in electronic spin and, therefore, it has a much lower probability to occur than spin-allowed transitions. Because the time scale is similar to the one for fluorescence (10-8 - 10-7s), it competes with fluorescence for the deactivation of the S1 state

• Phosphorescence is a radiational deactivation process between electronic states of different multiplicity, i.e. T1 to S0. It usually takes between 10-4 to 10s to occur because the process is spin forbidden.

142

Intensity of fluorescence (or phosphorescence) emission as a function of fluorophor (or phosphor) concentration

• The power of fluorescence (or phosphorescence) radiation (F or P) is proportional to the radiant power of the excitation beam that is absorbed by the system:

F = K’(P0 – P) (1) • The transmittance of the sample is given by Beer’s law: P/P0 = 10-ebc (2) • Re-arranging Eq. 1: F = K’ P0 (1 – P / P0) • Substituting Eq. 2 above: F = K’ (1 –10-ebc) (3) • The exponential term in Eq. 3 can be expanded as a Maclaurin

series to: F = K’.P0.[2.303ebc – (2.303ebc)2 + (2.303ebc)3 - …] (4) 2! 3! • For diluted solutions, i.e. 2.303ebc < 0.05, all of the subsequent

terms in the brackets become negligible with respect to the first, so:

F = K’.P0.2.303ebc or F = 2.303.P0.K’.ebc (5) • A plot of fluorescence (or phosphorescence) intensity as a function

of concentration should be linear up to a certain concentration. • Three are the main reasons for lack of linearity at high

concentrations: a) 2.303ebc > 0.05 b) self-quenching c) self-absorption

P0 P

LDR

In

tens

ity

Concentration

Detector

F

143

Fluorescence quantum yield • The slope of the calibration curve is equal to

2.303.P0.K’.ebc. • K’ is also known as the fluorescence

quantum yield (fF). • The fluorescence quantum yield is the ratio

between the number of photons emitted as fluorescence and the number of photons absorbed:

fF = # of fluorescence photons # of absorbed photons • Fluorescence quantum yields may vary

between zero and unity: 0 < fF< 1 The higher the quantum yield, the stronger

the fluorescence emission. • In terms of rate constants, the fluorescence

quantum yield is expressed as follows: fF = kf kf + ki + kec + kic + kpd +kd

kf

ki

kec

kic

• Consider a dilute solution of a fluorescent species A whose concentration is [A] (in mol.L-1). A very short pulse of light at time 0 will bring a certain number of molecules A to the S1 excited state by absorption of photons: A + hν → A* • The excited molecules then return to S0, either radiatively or non-radiatively, or undergo intersystem crossing. As in classical kinetics, the rate of disappearance of excited molecules is expressed by the following differential equation: -d[A*] / dt = (kf + knr) [A*] knr = ki + kec + kic + kpd + kd are the rate constants of competing processes that ultimately reduce the intensity of fluorescence. k units = s-1.

144

Fluorescence lifetime

• Integration of equation: -d[A*] / dt = (kf + knr) [A*] yields the time evolution of the concentration of

excited molecules [A*]. • Let [A*]0 be the concentration of excited

molecules at time 0 resulting from the pulse light excitation. Integration leads to:

[A*] = [A*]0 exp(-t / t) where t is the lifetime of the excited state S1. • The fluorescence lifetime is then given by: t = 1 / kf + knr • Typical fluorescence lifetimes are in the ns range. • Typical phosphorescence lifetimes are in the ms

to s range. • The fluorescence (or phosphorescence lifetime)

is the time needed for the concentration of excited molecules to decrease to 1/e of its original value.

• The fluorescence lifetime correlates to the fluorescence quantum yield as follows:

ff = kf. t

145

Excitation and emission spectra

• Excitation spectra appear in the same wavelength region as absorption spectra.

• However, it is important to keep in mind that absorption spectra are not the same as excitation spectra.

• Fluorescence and phosphorescence spectra appear at longer wavelength regions than excitation spectra.

• Phosphorescence spectra appear at longer wavelength regions than fluorescence spectra.

• In some cases, it is possible to observe vibrational transitions in room-temperature fluorescence spectra.

There are several parameters (many wavelengths and two lifetimes) for compound identification.

146

Fluorescence and Structure • General rule: Most fluorescent compounds are

aromatic. An increase in the extent of the p-electron system (i.e. the degree of conjugation) leads to a shift of the absorption and fluorescence spectra to longer wavelengths and an increase in the fluorescence quantum yield.

• Example: Aromatic hydrocarbon Fluorescence Naphthalene ultraviolet Anthracene blue Naphthacene green Pentacene red

• The lowest-lying transitions of aromatic hydrocarbons are of the p→p* type, which are characterized by high molar absorption coefficients and relatively high quantum fluorescence quantum yields.

No fluorescence

Fluorescence

147

• The effect of substituents on the fluorescence characteristics of aromatic hydrocarbons varies with the type of substituent.

• Heavy atoms: the presence of heavy atoms (e.g. Br, I, etc.) results in fluorescence quenching (internal heavy atom effect) because of the increase probability of intersystem crossing (ISC).

• Electron- donating : -OH, -OR, -NH2, -NHR, -NR2

This type of substituent generally induces and increase in the molar absorption coefficient and a shift in both absorption and fluorescence spectra.

• Electron-withdrawing substituents: carbonyl and nitro-compounds Carbonyl groups: There is no

“general rule”. Their effect depends on the position of the substituent group in the aromatic ring.

Nitro groups: No detectable fluorescence.

Substituted Aromatic Hydrocarbons

148

pH of solution

Additional resonance forms lead to a more

stable first excited state

Rigidity

Rigidity usually enhances

fluorescence emission

149

Quenching • Quenching: nonradiative energy transfer from an

excited species to other molecules. • Quenching results in deactivation of S1 without

the emission of radiation causing a decrease in fluorescence intensity.

• Types of quenching: dynamic, static and others. • Dynamic quenching: or collisional quenching

requires contact between the excited species and the quenching agent.

• Dynamic quenching is a diffusion controlled process. As such, its rate depends on the temperature and viscosity of the sample.

• High temperatures and low viscosity promote dynamic quenching.

• For dynamic quenching with a single quencher, the Stern-Volmer expression is valid:

F0/F = 1 + Kq[Q] Where F0 and F are the fluorescence intensities

in the absence and the presence of quencher, respectively. [Q] is the concentration of quencher (mols.L-1) and Kq is the Stern-Volmer quenching constant.

• The Stern-Volmer constant is defined as: Kq = kq / kf + ki + kic where kq is the rate constant for the quenching

process (diffusion controlled). • Static Quenching: the quencher and the

fluorophor in the ground state form a complex. The complex is no fluorescent (dark complex).

• The Stern-Volmer equation is still valid but Kq in this case is the equilibrium constant for complex formation: A + Q <=> AQ.

• In static quenching, the fluorescence lifetime of the fluorophor is not affected. In dynamic quenching, the fluorescence lifetime of the fluorophor is affected. So, lifetime can be used to distinguish between dynamic and static quenching.

Fluorescence quenching of quinine

sulfate as a function of chloride concentration

Oxygen sensors O2 is paramagnetic, i.e.

its natural electronic configuration is the

triplet state. When O2 interacts with

the fluorophor, it promotes conversion and deactivation of excited fluorophor

molecules. Upon interaction, O2 goes into the singlet state (diamagnetic).

150

Instrumentation

Typical spectrofluorometer (or spectrofluuorimeter)

configuration

Recording excitation and

emission spectra

Recording synchronous fluorescence

spectra

151

Spectrofluorimeter capable to correct for source wavelength dependence

Spectrofluorimeter with the ability to record total luminescence

152

Measuring phosphorescence

• Using a spectrofluorimeter with a continuous excitation source: be careful with fluorescence and second order emission!

• Rotating-can phosphoroscope: “good-old” approach to discriminate against fluorescence.

A. True representation B. Approximate representation te = exposure time; td = shutter delay time; tt =

shutter transit time; tC = time for one cycle of excitation and observation; tE = te + tt; tD = td + tt

• Spectrofluorimeters with a pulsed source: employ a gated PMT for fluorescence discrimination.

Excitation source on => PMT off = fluorescence decays Excitation source off => PMT on – phosphorescence measurement

153

Introduction to Chromatographic Separations • Analysis of complex samples usually involves

previous separation prior to compound determination.

• Two main separation methods based on instrumentation are available:

Chromatography Electrophoresis • Chromatography is based on the interaction of

chemical species with a mobile phase (MP) and a stationary phase (SP).

• The MP and the SP are immiscible. • The sample is transported by the MP. The

interaction of species with the MP and the SP separates chemical species in zones or bands.

• The relative chemical affinity of chemical species with the MP and the SP dictates the time the species remain in the SP.

• Two general types of chromatographic techniques exist:

• Planar: flat SP, MP moves through capillary action or gravity

• Column: tube of SP, MP moves through gravity or pressure.

SP Detector MP + sample

154

Classification of Chromatographic Methods

• Chromatographic methods can be classified on the type of MP and SP and the kinds of equilibrium involved in the transfer of solutes between phases:

Column 1: Type of MP Column 2: Type of MP and SP Column 3: SP Column 4: Type of equilibrium

Concentration profiles of solute bands A and B at two different times in their migration down the

column.

155

Migration Rates of Solutes

• Two-component chromatogram illustrating two methods for improving separation:

• (a) Original chromatogram with overlapping peaks; (b) improvement brought about an increase in band separation; (c) improvement brought about by a decrease in the widths.

Distribution constant or partition ratio or partition coefficient: It describes the partition equilibrium of an analyte between the SP and the MP.

If K = Kc and SP = S and MP = M Kc = cS / CM = nS / VS nM / VM Where VS and VM are the volumes of the two phases and nS and nM are the moles of A in SP and MP, respectively.

156

Retention Time

• Retention time is a measured quantity. • From the figure: tM = time it takes a non-retained species

(MP) to travel through the column = dead or void time.

tR = retention time of analyte. The analyte has been retained because it spends a time tS in the SP. The retention time is then given by:

tR = tS + tM • The average migration rate (cm/s) of the

solute through the column is:

Where L is the length of the column. • The average linear velocity of the MP

molecules is:

157

Relationship Between Retention Time and Distribution Constant

The Rate of Solute Migration: The Retention Factor

Where VS and VM are the volumes of SP and MP in the column. Knowing that:

An equation can be derived to obtain KC of A (KA) as

a function of experimental parameters. =>

Retention factor = capacity factor = kA = k’A. However: k’A ≠ KC or k’A ≠ KA

=>

158

• The selectivity factor can be measured from the chromatogram.

• Selectivity factors are always greater than unity, so B should be always the compound with higher affinity by the SP.

• Selectivity factors are useful parameters to calculate the resolving power of a column.

Band Broadening and Column Efficiency

• The “shape” of an analyte zone eluting from a chromatographic column follows a Gaussian profile.

• Some molecules travel faster than the

average. The time it takes them to reach the detector is: tR – Dt.

• Some molecules travel slower than the

average molecule. The time it takes them to reach the detector is: tR + Dt

• So, the Gaussian provides an average retention time (most frequent time) and a time interval for the total elution of an analyte from the column.

• The magnitude of Dt depends on the width of the peak.

159

• Considering that: v_ = L / tR => L = v_ x or L ± DL = v_ x [tR ± Dt] • The equation above correlates chromatographic

peaks with Gaussian profiles to the length of the column.

• Both Dt and DL correspond to the standard deviation of a Gaussian peak:

Dt ≡ DL ≡ s • If s = Dt, s units are in minutes. If s DL, s units

are in cm. • The width of a peak is a measure of the

efficiency of a column. The narrower the peak, the more efficient is the column.

=> • The efficiencies of chromatographic columns

can be compared in terms of number of theoretical plates .

160

The Plate Theory • The plate theory supposes that the chromatographic

column contains a large number of separate layers, called theoretical plates.

• Separate equilibrations of the sample between the

stationary and mobile phase occur in these "plates". • The analyte moves down the column by transfer of

equilibrated mobile phase from one plate to the next. • It is important to remember that theoretical plates

do not really exist. They are a figment of the imagination that helps us to understand the processes at work in the column.

• As previously mentioned, theoretical plates also serve

as a figure of merit to measuring column efficiency, either by stating the number of theoretical plates in a column (N) or by stating the plate height (H); i.e. the Height Equivalent to a Theoretical Plate.

• The number of theoretical plates is given by: N = L / H • If the length of the column is L, then the HETP is: H = s2 / L

• Note: • For columns with the same

length (same L): The smaller the H, the narrower

the peak. The smaller the H, the larger the

number of plates. => column efficiency is favored

by small H and large N. • It is always possible to using a

longer column to improve separation efficiency.

161

Experimental Evaluation of H and N

• Calling s in time units (minutes or seconds) as t:

=> s / t = cm / s • Considering that: v_ = L / tR = cm /s • We can write: L / tR = s / t or t s s L / tR v_ • If the chromatographic peak is Gaussian,

approximately 96% of its area is included within ± 2s. This area corresponds to the area between the two tangents on the two sides of the chromatographic peak.

• The width of the peak at its base (W) is then equal to:

W = 4t in time units or W = 4s in length units.

• Substituting t = W / 4 in the equation above we obtain: s = LW / 4tR • Substituting s = W / 4 in the same equation we obtain: t = WtR / 4L

• Substituting s = LW / 4tR in the HEPT equation: H = LW2 / 16tR2 • Substitution of this equation in N gives: N = 16 (tR / W)2 • These two equations allow one to estimate H and N

from experimental parameters. • If one considers the peak of the width at the half

maximum (W1/2): N = 5.54 (tR / W1/2)2

In comparing columns, N and H should be obtained with the same compound!

162

Kinetic Variables Affecting Column Efficiency • The plate theory provides two figures of merit

(N and H) for comparing column efficiency but it does not explain band broadening.

• Table 26-2 provides the variables that affect band broadening in a chromatographic column and, therefore, affect column efficiency.

• The effect of these variables in column efficiency is best explained by the theory of band broadening. This theory is best represented by the van Deemter equation:

H = A + B / u+ CS . u + CM . U where H is in cm and u is the velocity of the

mobile phase in cm.s-1. The other terms are explained in Table 26-3.

• Table 26-3: f(k) and f’(k) are functions of k l and g are constants that depend on

the quality of the packing. B is the coefficient of longitudinal

diffusion. CS and CM are coefficients of mass

transfer in stationary and mobile phase, respectively.

• Before we try to understand the meaning of the van Deemter equation, a better understanding of chromatographic columns is needed.

163

Some Characteristics of Gas Chromatography (GC) Columns

• Two general types => Open Tubular Columns (OTC) or Capillary Columns => Packed Columns • OTC: => Wall Coated Open Tubular (WCOT) Columns => Support Coated Open Tubular (SCOT) Columns => The most common inner diameters for capillary tubes

are 0.32 and 0.25mm. • Packed Columns: => Glass tubes with 2 to 4mm inner diameter. => Packed with a uniform, finely divided packing material of

solid support, coated with a thin layer (005 to 1mm) of liquid stationary phase.

• Solid Support Material in OTC and Packed Columns

Diatomaceous Earth: “skeletons of species of single-celled plants that once inhabited ancient lakes and seas”.

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Some Characteristics of HPLC Columns

• Only packed columns are used in HPLC.

• Current packing consists of porous micro-particles with diameters ranging from 3 to 10mm.

• The particles are composed of silica, alumina, or an ion-exchange resin.

• Silica particles are the most common. • Thin organic films are chemically or

physically bonded to the silica particles.

• The chemical nature of the thin organic film determines the type of chromatography.

SP for normal-phase chromatography

SP for partition chromatography Typical micro-particle

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Another Look at the van Deemter Equation

• H = A + B/u + CSu + CMu • The multi-path term A or Eddy-Diffusion: => This term accounts for the multitude of pathways by which

a molecule (or ion) can find its way through a packed column.

• A = 2ldp where: l is a geometrical factor that depends on the shape

of the particle: 1 ≤ l ≤ 2. dp is the diameter of the particle. Using packing with spherical particles of small

diameters should reduce eddy -diffusion.

Injection Detector

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• The Longitudinal Diffusion Term B/u: • Longitudinal diffusion is the migration of solute from

the concentrated center of the band to the more diluted regions on either side of the analyte zone.

• B/u = 2gDM/u where: g is a constant that depends on the nature

of the packing and it varies from 0.6 ≤ g ≤ 0.8.

DM is the diffusion coefficient in the mobile phase. DM a T / m, where T is the temperature and m is the viscosity of the mobile phase.

• As u → infinite, B/u → zero. So, the contribution of longitudinal diffusion in the total plate height is only significant at low MP flow rates.

• Its contribution is potentially more significant in GC than HPLC because of the relatively high column temperatures and low MP viscosity (gas).

Initial band

Diffusion of the band with time

The initial part of the curve is predominantly due to the B/u term.

Because the term B/u in GC is larger than HPLC, the overall H in GC is about 10x the overall H in HPLC.

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• The Stationary-Phase Mass Transfer Term CSu: • CS = mass transfer coefficient in the SP. CS a df

2 / DS where df is the thickness of the SP film and DS is the

diffusion coefficient in the SP. => Thin-film SP and low viscosity SP (large DS) provide low

mass transfer coefficients in the SP and improve column efficiency.

• The Mobile-Phase Mass Transfer Term CMu:

• CM = mass transfer coefficient in the MP. CM a dp

2 / DM

where dp is the diameter of packing particles and DM is the diffusion coefficient in the MP.

• The contribution of mass transfer in the SP and MP on the overall late height depends on the flow velocity of the mobile phase. Both phenomena play a predominant role at high MP flow rates.

Liquid SP coated on

solid support

Cartoon with example of SP mass-transfer in a liquid SP: different degrees of penetration of analyte molecules in the liquid layer of SP

lead to band-broadening

Porous in silica

particle

Cartoon with example of MP mass-transfer: stagnant pools of MP retained in the porous of silica particles lead to band-

broadening.

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Summary of Methods for Reducing Band Broadening

• Packed Columns: • Most important parameter that affects band

broadening is the particle diameter. • If the SP is liquid, the thickness of the SP is

the most important parameter. • Capillary Columns: • No packing, so there is no Eddy-diffusion

term. Most important parameter that affects band broadening is the diameter of the capillary.

• Gaseous MP (GC): The rate of longitudinal diffusion can be

reduced by lowering the temperature and thus the diffusion coefficient.

• The effect of temperature is mainly noted at low flow rate velocities where the term B/u is significant.

• Temperature has little effect on HPLC.

Effect of particle diameter in GC

Effect of particle diameter in HPLC

Note: m = mm

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Optimization of Column Performance

• Optimization experiments are aimed at either reducing zone broadening or altering relative migration rates of components.

• The time it takes for chromatographic analysis is also an important parameter that should be optimized without compromising chromatographic resolution.

• Column Resolution: • It is a quantitative measure of the ability of

the column to separate two analytes. • It can be obtained from the chromatogram

with the equation:

• In terms of retention factors kA and kB for the two solutes, the selectivity factor and the number of theoretical plates of the column:

• From the last equation we can obtain the number of theoretical plates needed to achieve a given resolution:

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• For compounds with similar capacity factors, i.e. kA ≈ kB:

• The time it takes to achieve a separation can be predicted with the formula:

Where k = kA + kB / 2

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The General Elution Problem

• The general elution problem occurs in the separation of mixtures containing compounds with widely different distribution constants.

• The best solution to the general elution problem is to optimize eluting conditions for each compound during the chromatographic run.

• In HPLC, this is best accomplished by changing the composition of the mobile phase (Gradient Elution Chromatography).

• In GC, this is best accomplished by changing the temperature of the column during the chromatographic run.

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Qualitative and Quantitative Analysis in GC and HPLC

• Both are done with the help of standards. • Qualitative analysis, i.e. compound identification is done

via retention time. The retention time from the pure standard is compared to the retention time of the analyte in the sample.

Note: retention times are experimental parameters and as such are prone to standard deviation.

• Quantitative analysis is done via the calibration curve method (or external standard method) or the internal standard method.

• Calibration curve or external standard method: the procedure is the same as usual. The calibration curve can be built plotting the peak height or the peak area versus standard concentration.

The same volume of sample was injected in each case, but “Sample B ” has a much smaller peak. Since the tR at the apex of both peaks is 2.85 minutes, this indicates that they are both the same compound, (in this example, acrylamide (ID)). The “Area” under the peak (“ Peak Area Count ”) indicates the concentration of the compound. This area value is calculated by the Computer Data Station. Notice the area under the “Sample A” peak is much larger. In this example, “Sample A” has 10 times the area of “Sample B”. Therefore, “Sample A” has 10 times the concentration, (10 picograms) as much acrylamide as “Sample B”, (1 picogram). Note, there is another peak, (not identified), that comes out at 1.8 min. in both samples. Since the area counts for both “samples” are about the same, it has the same concentration in both samples.

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Internal Standards • An internal standard is a known amount of a

compound that is added to the unknown. • The signal from analyte is compared to the

signal from the standard to find out how much analyte is present.

• This method compensates for instrumental response that varies slightly from run to run and deteriorates reproducibility considerably.

• This is the case of mobile phase flow rate variations in chromatographic analysis.

• Internal standards are also desirable in cases where the possibility of loosing sample during analysis exists. This is the case of sample separation in the chromatographic column.

• The internal standard should be chosen according to the analyte. Their chemical behavior with regards to the SP and MP should be similar.

• How to use an internal standard?: a mixture with the same known amount of standard and analyte is prepared to measure the relative response of the detector for the two species.

• The factor (F) is obtained from the relative response of the detector.

• Once the relative response of the detector has been found, the analyte concentration is calculated according to the formula:

Area of analyte signal = F x Area of standard signal Concentration of analyte Concentration of standard

A known amount of standard is added to the unknown X. The relative response is measured to obtain the

detector’s response factor “F”.

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Example of Internal Standards

• In a preliminary experiment, a solution containing 0.0837M X and 0.066M S gave peak areas of AX = 423 and AS = 347. Note that areas are measured in arbitrary units by the instrument’s computer. To analyze the unknown, 10.0mL of 0.146M S were added to 10.0mL of unknown, and the mixture was diluted to 25.0mL in a volumetric flask. This mixture gave a chromatogram with peak areas AX = 553 and AS = 582. Find the concentration of X in the unknown.

• First use the standard mixture to find the response factor:

AX / [x] = F x {AS / [S]} Standard mixture: 423 / 0.0837 = F {347 / 0.0666} => F = 0.9700 • In the mixture of unknown plus standard, the

concentration of S is: [S] = (0.146M)(10.0mL / 25.0mL) = 0.0584M where: 10.0mL / 25.0mL is the dilution factor • Using the known response factor and S

concentration of the diluted sample in the equation above:

553 / [X] = 0.9700 (582 / 0.0584) =>[X] = 0.05721M

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Liquid Chromatography

Size exclusion or gel: polystyrene-divinylbenzene

Silica with various porous sizes

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Instrumentation

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Pumping Systems

• Pumping systems that allow to change the MP composition during the chromatographic run provide better separation of compounds with wide range of k’ factors.

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Detectors

UV-VIS absorption cell for HPLC

ADVANCED ANALYTICAL LAB TECH (Lecture) CHM 4130-0001 · 2016-08-19 · ADVANCED ANALYTICAL LAB TECH...

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Transcript of ADVANCED ANALYTICAL LAB TECH (Lecture) CHM 4130-0001 · 2016-08-19 · ADVANCED ANALYTICAL LAB TECH...