Instrumental Analysis Final Exam

partition chromatography

-liquid stationary phase bonded to solid surface (SiO2 capillary), liquid or gas mobile phase. Flowing gas is the mobile phase in gas-chromatography

In many (or most) real analytical problems, analyze must be separated from a complex mixture before it can be identified and quanitified

-extraction -gas chromatography - liquid chromatography -others -electrophoresis -molecular exclusion - ion chromatography -affinity chromatography -thin-layer chromatography -paper chromatography

Kirchhoff's three laws of spectroscopy provide a basis for spectroscopic instrumental methods of chemical analysis. Briefly state, in your own words, each of the three laws.

1. A hot solid object produces light with a continuous spectrum. 2. A hot tenuous gas produces light with spectral lines at discrete wavelengths which depend on the energy levels of the atoms in the gas. 3. A hot solid object surrounded by a cool tenuous gas produces light with an almost continuous spectrum which has gaps at discrete wavelengths depending on the energy levels of the atoms in the gas.

Consider a general Jablonski Diagram as typically used to explain excitation and de-excitation in luminescence (fluorescence and phosphorescence) spectroscopy. In your opinion, and making use of the Jablonski Diagram, what can influence the magnitude of the Stokes shifts observed in either fluorescence or phosphorescence?

1. Fluorescence is short term excitation whereas phorescensce is long term excitation. Since Fluorescence is shifted greater from polar solvents, solvents and fluophore impact fluorescent stoke shifts. Phosphoresces is based on intersystem crossing. Solvent relaxation can result in dramatic effects on stoke shifts. Non radiative energy occurs through the transfer of small amounts of energy through molecular/atomic collisions. Energy is slowly transfered away and is moved from an excited state to a ground state in internal conversion.

Considering the NMR experiment one last time (here anyway), state how you think the sensitivity and limit of detection in the method might be dependent on the strength of the external magnetic field, and why.

1. Less analyte will be needed since increasing external magnetic field will improve sensitivity. When a nuclei is placed in an external magnetic field a bulk magnetation on the z-axis is formed. The nuclei's spins are coherent. The NMR signal can be detected as long as the spins are coherent. With a strong external magnetic field the spins are more likely to be coherent. The stronger the magnetic field the higher the sensitivity. As the magnetic filed increases so does the signal reducing unwanted noise which then reduces false negative and postiives. Less noise= more clarity, increasing the limit of detection. The strength of the external magentiv field impacts the sensitivity and limit of detection. When increasing the magnetic field the enrgy needed to execute the parallel spins to anti spins increases. Then smaller increases in temperature would be able to achieve smaller amounts of electrons from ground state. The majority of electrons or all will be within the ground state due to the strong magnetic n]=field and low temperature. Variable amount of energy can be inputted within the system. The energy input can be reduced and diference in the amount of electrons is decreased. The sensitivity of the NMR is increased. Lower energy can be used to excite electrons then increasing limit of detection. Using the idea of the Boltzmann equation the signal to noise ratio is dependent on the energy difference and the temperature. Which proves what was previously stated.

A version of the Larmor equation is shown below. Given the terms included in the equation, which relates the resonant frequency associated with a nuclear spin transition to the magnetogyric ratio and strength of an external magnetic field, how do you explain the apparent "chemical shift" that perturbed the physicists who found that the measured Larmor frequencies for, say, 1H or 13C nuclei depended on the identity of the molecule being used to make the measurement?

1. Shielding experience by a given nucleus is directly related to the electron density surrounding it. In the absorbance of other factors its expected shielding would decrease with increasing electronegativity of adjacent groups. Electrons are attracted by a magnetic field which affects differences seen in frequencies. Resonance frequency for a particular nucleus depends on the compound it was present in chemical shift.

In Raman spectroscopy, the lasers typically used for excitation emit monochromatic, coherent radiation at 785 nanometers or 532 nanometers, whereas the vibrations excited in most organic molecules require excitation wavelengths between 3 micrometers and 25 micrometers. First, explain why the near IR or visible wavelength photons are used (in instead of infrared wavelength) for excitation in Raman spectroscopy. Second, explain why it is that a monochromatic light source can be used to generate a vibrational spectrum by Raman scattering.

1. Smaller wavelengths mean higher radiation. Visible wavelength has higher energy than IR wavelength this energy is needed to excite electrons from ground state passed the vibrational state to the virtual state. When energy is released and the electron is relaxed it is then Ramen spectroscopy energy. Energy released in an altered state. If relaxed down to vibrational state it is a stokes line and if relaxed to ground state it is a anti stokes line. Monochromatic line is needed because the energy needs to be constant to excite electrons to the current excitation state to get a Raman spectroscopy and minimized the Raman scattering.

Stokes shifts are important to Raman spectroscopy and fluorescence spectroscopy. Why is the term used in the two methods, when Raman spectroscopy and fluorescence spectroscopy are supposed to be probing transitions of such different energy (vibrational versus electronic)?

1. Stoke shifts are used for both since it is not specific to different energies. It describes the differences in the emission of energy and absorbance. It describes the difference between stoke and anti-stoke lunes in Raman. Both of compare the absorbance and emission of energy difficulty. Whereas in fluoresce it describes the difference between energy at ground and excited state. The energy that in outputted and inputted.

Consider, in general, the relative intensities of the Stokes and Anti-Stokes Raman bands as they might be for a given molecule at atmospheric pressure and room temperature. First, predict how decreasing and increasing (in independent experiments) the temperature of the sample by 50 K might alter the relative intensities of the two types of bands. Second, state whether the change in temperature influences the magnitude of the Stokes shifts.

1. With an increase in temperature the intensity of the antistokes lines would increase while the stoke lines decrease. At higher temperature the antistokes would be more probable since there are more molecules in the excited state according to the Boltzmann equation. Heat increases frequency so the molecules move faster proving they would be in the excited state. Faster molecules would increase the frequency. Decreasing the temperature decreases the number of molecules in the excited state and more are in the ground state. Increasing the difference in energy between each state which changes the relative intensities. A decrease in temperature would be a greater intensity as it is more probable the molecules are at a lower vibrational state which allows for more efficient stoke lines. Under the idea that temperature is inversely related to frequency. Frequency is then inversely related to magnitude. Temperature would be proportional to magnitude.

Dynamic range

A measure of the concentration range from the limit of quantification, to the point at which the calibration curve departs from linearity

Bias

A report of the systematic (determinate) error of an analytical method

ion exchange chromatography

Anions or cations are covalently attached to the stationary solid phase, usually a resin. Solute ions of the opposite charge are attracted to the stationary phase. The mobile phase is a liquid.

Consider the equation of motion shown below, which is relevant to the trajectory of charged species in an applied magnetic field. In the equation, r is the radius of curvature, B is the strength of the applied magnetic field, and Vacceleration is the potential used to accelerate an ion to a given kinetic energy. What approaches might you take to measure the m/z ratio of a given ion based on the equation.

Based on the equation would be measuring the ion radius when applied to a magnetic field after acceleration. Through maintaining a constant for the other variable the radius can be measured and used as a determinant form of quantitate measurement. The mass impacts the radius. A larger mass will have a larger radius post acceleration due to the greater momentum which is more difficult to change direction. A simpler method would be the use of an instrument with a known r&b only changing the potential. Both use the potential as the dependent variable to solve for m/z.

(b) Based on your calculations, can you, with 95% confidence, say that the concentration of lead is below a mandated level of 10.000 ppb? (3 points)

Because the 95% confidence interval for the data set INCLUDES the 10.000 mandated value, you CANNOT say that the concentration is below the mandated level.

Both magnetic sector and Fourier-transform ion cyclotron resonance mass spectrometers use an external magnetic field for separation of ions based on m/z. How are the two methods similar and how do they differ? For the magnetic sector instrument, what do you think influences the resolving power (the ability to distinguish an ion of one m/z from another) of the approach?

Both use an external magnetic field to separate the ions based on their mass to charge ratio. FTIC uses associated cyclotron frequency of the ions within a fixed magnetic field to measure m/sz magnetic field. Magnetic sector's resolving power stems from the width of image slit. Increasing reaction by narrow slides fewer ions.

The rare earth element gadolinium is often used as a contrasting agent for magnetic resonance imaging. The concentration of gadolinium was measured in the discharge from a wastewater treatment plant near a large medical facility. The following eight measurements were obtained: 1.231 ppm, 1.233 ppm, 1.227 ppm, 1.225 ppm, 1.791 ppm, 1.227 ppm, 1.238 ppm, 1.229 ppm If the tabulated G values for 8 measurements is 2.032, use the Grubbs test to determine if one of these values is an outlier and can be rejected with 95% confidence.

By visual inspection of the data set, the value of 1.791 ppm seems suspect. The mean value (𝑥̅) and standard deviation (𝑠) for the data set are 1.300 and 0.198, respectively. The calculated G value is: 𝐺𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 =|𝑞𝑢𝑒𝑠𝑡𝑖𝑜𝑛𝑎𝑙 𝑣𝑎𝑙𝑢𝑒− 𝑥̅|𝑠 = |1.791−1.300|0.198 =2.474 The calculated G value is greater than the tabulated value of 2.032 for 8 measurements. Therefore, the value can be discarded.

Which of the following combinations are a reasonable approach to minimize thermal noise in an experimental measurement?

Decrease the bandwidth, decrease the electrical resistance, and decrease the operating temperature of the transducer.

Quasi-equilibrium theory (QET) allows physical chemists to rationalize the appearance of electron impact mass spectra ( for example, the relative abundances of molecular ion and fragment ion peaks) because it relates reaction rates to ion internal energy. Thinking about ion mean free paths and the operation of mass spectrometers in general, can you come up with plausible reasons why the relative intensities of the peaks in EI mass spectra may also depend on the specific type of mass spectrometer used in an analysis?

Different path lengths and fragmentation stem from different instruments. Having a shorter or longer wavelength would change the relative intensities of fragments.A slower path length will probably have a lower inesitiy of mass fragments compared to an instrument with a longer pathlength.

Consider electron impact ionization , in general, for a simple organic molecule. To what extent do you think thermochemistry and kinetics influence the relative intensities of the radical cation (molecular ion) and fragment ions in the mass spectrum?

How fast a reaction will occur is described as kinetics. If a favorable reaction has a slow reaction rate it may not fragment prior to hitting the detector which results in a lower peak then thermodynamically expected. How favorable a reaction is or how likely it is to occur is described by thermochemistry specific radical fragmentation reactions may have a higher transition state then another forming a lower peak. Other reactions may have a lower peak. Other reactions may have lower free energy making a higher relative abundance and therefore favorable.

Which type of ionization would you expect to produce the most fragmentation? If one or the other produces less fragmentation, state why.

I would expect chemical ionization to produce less fragmentation because impacts less energy to an analyze molecule.

Signal averaging is one approach to improving the signal to noise ratio in an instrumental analysis. In your own words, briefly explain why signal averaging can also significantly improve the limit of detection of an instrumental method.

In general, the limit of detection/detection limit is that point when the mean value for a set of measurements is 3x the standard deviation from a series of measurements with a blank. For an instrumental method of analysis, the criterion is that the level of signal must be 3x the level of the noise. Therefore, the greater the signal to noise ratio (because of signal averaging), the lower the limit of detection possible.

Sensitivity and selectivity have been two performance characteristics discussed many times this semester. What about a mass spectrum is selective, in terms of identifying a molecule. And what factors do you think would influence and/or determine the sensitivity for mass spectrometry.

M/z is an identifying factor since each molecule has its own way of fragmenting. With many ways to fragment a molecule. The ability of a compound to provide a gas phase ion and solvent use impact sensitivity. The correct use of a solvent allows for a molecule to be volatilized easier. If it is easier for a molecule to produce a gas phase ion then less molecules need to be inserted into the system to test it.

molecular exclusion chromatography

Separation by molecular size and permeation of porous gel. Mobile phase can be liquid or gas.

affinity chromatography

Separation carried out using specific interaction between solute and an immobilized molecule (eq. protein and antibody to the protein)

Which combination best describes the difference between signal, baseline and noise.

Signal is the output from an instrument that can be attributed to the presence of an analyte, baseline is the output of the instrument when measuring a blank, and noise is random fluctuations in output when either the blank or a sample containing analyte are being measured.

You have been introduced to three different instrumental methods that use the Fourier transform to produce a frequency-domain spectrum from a time-domain signal. What are the three, and what which are most similar? What benefits are gained by using the FT-MS approach?

The 3 methods are Ft-Ms, FT-IR, and FT-NMR. NMR and IR are most similar since radiation (radio and infrared) is being measured and there is not a direct physical interaction like MS. FT-MS's benefit is collision as it allows for mass of the ion to be read as well as forming a ion prior to measurement. Allowing fragmentation of the analyte and results give information on the structure.

Specificity

The ability of a method to differentiate the analyte from the rest of the sample

The change in velocity of propagation of light when it travels through a medium (as opposed to vacuum) is important to the concepts of refraction and refractive index, and to the use of refraction for the dispersion of light. Briefly explain why the velocity of propagation depends on the medium through which light travels.

The frequency of a beam of radiation is determined by the source and remains the same. The velocity of the radiation depends on the composition of the medium through which it travels. In vacuum, the velocity of radiation is independent of wavelength and travels at 2.99792 x 108 m/s. In any medium containing matter, propagation of radiation is slowed by the interaction between the electromagnetic field (of the radiation) and the bound electrons in the matter. Because the frequency of the radiation is constant, the wavelength must decrease as radiation passes from vacuum to another medium, and then again when passing from matter/medium to vacuum.

Which of the following is NOT an advantage to using an interferometer is a spectroscopy experiment?

The interferometer is ideal for use with a line source of radiation

Consider the method of least squares for the generation of a calibration curve from a set of data points. Which of the following statements best describes how the calibration line is fit to the data points, and the assumptions made in doing so?

The line is generated by minimizing the sum of the squares of the residuals for the data points; the assumptions are that there is a linear relationship between the y and x values, and that any error is in the measured values and not in the x-axis values.

The results of 6 measurements of lead ion concentration (in parts per billion, ppb) in ground water are shown below. The student's t value for 5 degrees of freedom is 2.571. 10.012, 9.964, 10.020, 9.823, 10.016, 9.875 (a) Calculate the mean and the 95% confidence interval for these measurements.

The mean value for the set of measurements is 9.952, and the standard deviation is 0.084. Therefore, the confidence interval can be calculated using: 𝜇=𝑥̅± 𝑡𝑠√𝑛=9.952±2.571(0.084)√6 =9.952±0.088 9.865 < 𝜇 < 10.040

The detection limit can be defined as the smallest amount (quantity) of analyte that is "statistically different" from a measurement performed using a blank. To satisfy the "statistically different" part of the definition is generally satisfied using the equation 𝑆𝑚 =𝑆̅𝑏𝑙 +3𝑠𝑏𝑙 - the mean value plus three times the standard deviation associated with measurements collected with the blank. In your own words, explain why this is an acceptable way to determine the limit of detection for an analytical method.

The mean value plus three times the standard deviation is acceptable to determine the limit of detection as it allows for a 45% confidence that there is a statistical difference in analyze in a non-ideal distribution. Moreover false positives and negatives are approximately 10% which supports the ability to determine limit of detection. Moreover, since quantification is not required this lower value of 3 is acceptable for detection. This is caused by the level effectively reducing random errors and signal noise which allows for greater precision in determining limit of detection. Moreover, within a normal distribution this confidence interval becomes even higher at a 49% confidence. Therefore, this method is successful at following the definition of limit of detection which analyzes smallest amount of analyze according to a confidence interval.

Which of the following statements most accurately describes the importance of a population inversion to the efficient operation of a laser?

The population inversion creates a higher probability for stimulated emission of radiation

The Boltzmann relation/equation allows one to predict the relative population of higher and lower energy states for a system for a given transition energy and temperature. Using the equation, predict that will happen to the ratio Ni/No (where Ni is the population of an excited state and No is the population of the ground state) if the temperature of the system is decreased.

The ratio will decrease because the excited state population (Ni) decreases

Sensitivity

The slope of the calibration curve, which shows the change in signal or measured value per unit change in concentratio

Limit of Detection

The smallest quantity of analyte that can be distinguished from the blank

The fluorescence lifetime for a given molecule is an important part of the fluorescence spectroscopy experiment. How would you define what the fluorescence lifetime is, and what physical processes do you think influence the length of a fluorescence lifetime?

The time between when the fluorophore enters its excited state until a photon is emitted and returns to ground state. Temperature and lifetime are inversely related Polarity and fluorescence are other external influences. Internal factors such as the structure of the fluorophore influence lifetime.

Consider the respective equations of motion, in general, for a magnetic sector and time-of-flight mass analyzer. How are the magnetic sector and time-of-flight analyzers similar, and how do they differ, in their mode of operation?

They both involve the distance of the IR and the difference magnetic sector deal with the radius of the IR trajectory and field and time of light analyzer observes the distance of the electrons/ions travel. Both conserve in the distance of travel which helps to distinguish the mass of the ions which allows the distinguish of heavier and light ions. The magnetic sector uses a magnetic field to separate the ions by mass. The flight analyzer uses time flight to help separate the ions by mass. Both separate the ions based on mass, but use different sources to do the separation.

Describe chemical ionization

Through the use of a gas phase acid-base reaction with a gas like ammonia as the reagent solvent. After the analyze and reagent gas mix in the chamber a stream of electrons enter. The gas is more likely to be impacted since it is more prevalent in the mixture.

Describe how ions are created via electron ionization

When a (70ev) beam of electrons hits the sample molecules in the gas phase

By now you are all familiar with the Beer-Lambert law (more commonly known as Beer's Law). In general, the law links the attenuation of radiation to properties of the medium through which it passes. In quantitative chemical analysis, there are three parameters that influence absorbance. State what the three parameters are, and more importantly, specifically why each should influence the measured absorbance for a given sample.

a. Extinction coefficient: i. Directly proportional ii. The more favorable the transition from ground to excited the higher this number increases iii. Describes analyte's ability to absorb the particular radiation wavelength b. Concentration i. Higher concentration leads to higher absorbance ii. Amount of light absorbed is dependent on the number of molecule interactions iii. Higher concentration means more radiation was absorbed c. Pathlength i. Longer path means more molecules within the path are receiving the beam of radiation ii. Directly proportional to absorption

As you have heard a couple of times in class, NMR will be the only spectroscopy discussed for which the frequency of the radiation used to excite a transition is dependent both on attributes of the "chromophore" and the experimental apparatus (namely, the strength of the external magnetic field). Briefly explain why this is the case for NMR spectroscopy - why is the transition energy (measured using the Larmor frequency) dependent on the strength of the external magnetic field?

a. Higher magnetic field means higher resistance which means more energy needed to go from parallel to anti parallel. b. Transition energy is dependent on the strength of the external magnetic field. Since the NMR flips the parallel spin to anti parallel and measures the energy used to flip back to the unquantized state. The flipping is measured by the transition energy. The magnetic field acts as a resister against the flipping. The resistance is what the transition energy needs to overcome. Meaning the stronger the magnetic field a higher resistance state is needed. Which causes a higher transitional state. Making the transitional energy dependent on the strength of the external magnetic field.

adsorption chromatography

solid stationary phase, liquid or gas mobile phase