Exam august 2021

Question 1

Enter the van Deemter equation and explain the designations (förklara ingående
beteckningar). State what type of phenomenon is behind each of the three parts of the
equation.

Answer 1

The van Deemter equation is a mathematical expression used to describe the efficiency of a chromatography column as a function of flow rate. It takes into account the effects of three main phenomena on column efficiency: diffusion, mass transfer, and pressure drop.

The van Deemter equation can be written as:

H = A + B/u + Cu

where:

H is the height equivalent to a theoretical plate (HETP), which is a measure of the efficiency of a chromatography column

u is the linear velocity of the mobile phase (i.e., the flow rate)

A is the term related to axial diffusion, which is the diffusion of solute molecules along the direction of flow

B is the term related to mass transfer, which is the transfer of solute molecules between the mobile phase and the stationary phase

C is the term related to pressure drop, which is the reduction in pressure along the length of the column due to frictional resistance to flow.

Each of the three parts of the van Deemter equation corresponds to a different physical phenomenon in chromatography:

Axial diffusion: This term is related to the molecular diffusion of solute molecules along the direction of flow. As the linear velocity of the mobile phase increases, the rate of axial diffusion decreases, leading to improved column efficiency.

Mass transfer: This term is related to the transfer of solute molecules between the mobile phase and the stationary phase. As the linear velocity of the mobile phase increases, the rate of mass transfer decreases, leading to reduced column efficiency.

Pressure drop: This term is related to the reduction in pressure along the length of the column due to frictional resistance to flow. As the linear velocity of the mobile phase increases, the pressure drop increases, leading to reduced column efficiency.

The van Deemter equation provides a quantitative framework for understanding the effects of these three phenomena on column efficiency in chromatography, and it is widely used to optimize the performance of chromatography columns.

Question 2

a. You develop a GC method and focus on getting resolution (Rs) between the peaks
(analytes). In what two ways can you influence the separation factor α? Explain how the
separation factor is affected in each of the two cases.

Answer 2

The lower the boiling point, the lower will be the temperature of vapour formation and shorter will be the retention time of the eluting peak. The greater the difference between the boiling points of the constituents the better will be the resolution between the separated peaks.

Capacity Factor (temperature)
The first one, capacity factor. Capacity factor means you have to let the analytes interact with the stationary phase. No interaction, no separation. So the capacity factor is all about the temperature. You need to lower the temperature in your GC enough to make sure the analytes interact with the column.

Selectivity (stationary phase)stationary phase that works for most people. That would be a DB-5, 5% phenyl 95% methyl.

Efficiency (column dimensions)
The final term is efficiency. Efficiency is a measure of the skinniest of the peak. Long Column – To get more efficiency, which will lead to more resolution, you could use a longer column. Double the column length and you’ll double your efficiency. Resolution goes up by the square root of two. So double the column length, you get 40% more resolution guaranteed. Double column length, get 40% improvement in resolution. It takes longer. It takes twice as long. Small Diameter – The second thing we do is use a smaller diameter. If we decrease the diameter of the column we increase the efficiency. Peaks are skinnier. Peaks are taller. Better separation.

Thin Film – Along with that we decrease the film thickness. By doing that we also make the peak skinnier.

Question 3

b. Explain what chemical tailing is in chromatography. What causes the tailing to occur
and what type of compounds (analytes) suffers from this phenomenon?

Answer 3

Taling : the peaks are tailing when analytes are interacting with free silanol groups in the stationary phase. Polar compounds that can interact via hydrogen bonding are often tailing (alcohols, carboxylic acids, amines)

Question 4

c. Which two properties make analytes unsuitable for analysis by GC

Answer 4

Too high boling points (>approx 400 ºC), thermal instability

Question 5

d. What is meant with isothermal analysis? What is meant with temperature-programmed
analysis?

Answer 5

Isothermal analysis: constant oven temperature during entire analysis. Temperature programming: Temperature starts at low temperature and is increased during the analysis.

Question 6

a. A two-component mixture is analyzed with two columns packed with particles of
different diameters (the particle diameters 4.0 µm and 1.7 µm). Pair the columns with
the chromatograms (A and B) in the figure below. Explain which parts of the van
Deemter curve are affected by the particle size and how this is connected to the plate
number N.

Answer 6

The particle size of the stationary phase in a chromatographic column affects both Hmin and Hm, which are two important parameters in the van Deemter curve.

Hmin is primarily affected by the particle size, as smaller particles lead to a larger surface area and increased diffusion of the solute in the mobile phase. This results in a lower Hmin, meaning that a larger number of plates can be packed into a given column length.

Hm is affected by both particle size and the diffusion of the solute in the mobile phase. Smaller particles lead to a higher Hm, as they provide a higher mass transfer rate between the stationary and mobile phases. This results in a more efficient separation and a higher number of theoretical plates.

The plate number N, which is defined as the height of the column divided by Hm, is directly related to the efficiency of a chromatographic column. A higher N indicates a higher separation efficiency and a greater number of theoretical plates in the column.

In conclusion, the particle size of the stationary phase has a significant impact on the efficiency of a chromatographic column and is reflected in the van Deemter curve through the parameters Hmin and Hm. A smaller particle size can result in a higher N and a more efficient separation.

Question 7

Draw the chemical structure of a stationary phase that is commonly applied in revered-
phase HPLC. Give example of a mobile phase that could work for isocratic analysis
using the suggested stationary phase.

Answer 7

C18 silica Mobile phase: suitable mixture of two organic solvent (e.g. toluene, hexane, chloroform).

Question 8

State two ways in which the separation factor α can be affected in reversed-phase
HPLC. Explain briefly.
what solvent/stationary phase and how non-polar the analyte is.

Answer 8

The separation factor α in reversed-phase high-performance liquid chromatography (RP-HPLC) can be affected by various factors, including the solvent/stationary phase and the non-polarity of the analyte. Here are two ways in which α can be influenced:

Solvent/Stationary Phase Composition: The choice of solvent/stationary phase can have a significant impact on the separation factor α. In RP-HPLC, the stationary phase is typically a non-polar or partially non-polar material, such as C18 or C8, while the mobile phase is a polar solvent, such as acetonitrile or methanol. The composition of the solvent/stationary phase can affect the separation factor α by altering the strength of the interactions between the analyte and the stationary phase. By changing the composition of the solvent/stationary phase, it is possible to adjust the separation factor α and optimize the separation.

Non-polarity of the Analyte: The non-polarity of the analyte can also affect the separation factor α. In RP-HPLC, analytes with a higher degree of non-polarity will have a stronger affinity for the stationary phase and will elute later in the chromatogram. On the other hand, more polar analytes will elute earlier in the chromatogram and have a lower separation factor α. By adjusting the non-polarity of the analyte, it is possible to alter the separation factor α and optimize the separation.

In summary, the separation factor α in RP-HPLC is influenced by various factors, including the composition of the solvent/stationary phase and the non-polarity of the analyte. By understanding how these factors affect α, it is possible to optimize the separation and improve the overall efficiency of the RP-HPLC system.

Question 9

True or false, HPLC : In reversed-phase HPLC, it is common to use pure silica as the stationary
phase.

Answer 9

false

Question 10

t or f, GC : The retention factor increases if the film thickness is decreased.

Answer 10

false

Question 11

If you double the column length, you will also double the plate number N.

Answer 11

true

Question 12

I you double the column length, you will also double the resolution Rs.

Answer 12

It increases but does not double, false

Question 13

HPLC : the plate height decreases with decreased particle size

Answer 13

true

Question 14

GC : The peak size of the analyte will increase when changing the split ratio from 20:1
to 50:1.

Answer 14

false

Question 15

c. Measuring absorbance the values should be within a limited range, generally between
0.3 and 2. Why is that? Give a short explanation.

Answer 15

Because if adsbotion is too high too little light reaches the detector and if adsobtion is too low there is little difference between the sample and the reference for accurate measurmeant

Question 16

d. Using UV/vis absorption and fluorescence for quantitative analyses, which of the two
methods is generally more sensitive? State your reason well!

Answer 16

Fluorescence (F) methods a much more sensitive than UV/vis absorption methods. One way to explain that is that when measuring F you get a signal from a “black” background whereas in absorption you want to measure a small difference in irradiation.

Question 17

e. Describe a detector that is used in a conventional fluorescence spectrophotometer. (1p)

Answer 17

In fluorescence spectroscopy it is common to use Photo Multiplying Tubes (PMT) as detectors due to the high sensitivity and fast response of these detectors.

Question 18

Selenium plays an important role in the health of your immune system. This antioxidant helps
lower oxidative stress in your body, which reduces inflammation and enhances immunity.
However, both elemental selenium and (especially) selenium salts are toxic in higher doses,
causing selenosis.
You have been asked to analyze a biological sample for the content of Se using
spectrophotometry. Which methods are suitable for doing this?
Describe in detail two experimental procedures of which one (only) refers to atomic
spectrophotometry.

Answer 18

There are several spectrophotometric methods that can be used to analyze a biological sample for the content of selenium (Se). Some of the most commonly used methods include:

Atomic absorption spectrophotometry (AAS): This method is based on the absorption of light by atoms of the element being analyzed. In the case of selenium, the sample is atomized and the absorption of light by the atomic Se is measured.

Inductively coupled plasma-atomic emission spectrophotometry (ICP-AES): This method involves the excitation of the sample in an inductively coupled plasma, which causes the emission of light at specific wavelengths that are characteristic of the element being analyzed. In the case of selenium, the light emitted by the Se atoms is measured and used to determine the concentration of Se in the sample.

Inductively coupled plasma-mass spectrometry (ICP-MS): This method involves the introduction of the sample into an inductively coupled plasma, where it is ionized and the ions are sorted by mass. The concentration of Se in the sample can be determined by measuring the abundance of Se ions of a specific mass.

These are just a few of the many spectrophotometric methods that can be used to analyze a biological sample for the content of selenium. The choice of method will depend on factors such as the sample matrix, the required sensitivity, and the equipment available.

Question 19

Draw a schematic diagram for a GC-quadrupole MS instrument. Explain the function of
the various parts.

Answer 19

The quadrupole mass analyzer consists of four parallel conducting rods of hyperbolic cross section spaced about a central axis along which ions are conducted. Depending on their mass and charge they will interact differently with the rod, large molecules or very charged molecules will interact more and smaller less charged will interact less.

Question 20

b. Which ion source is compatible with LC?

Answer 20

Electrospray Ionisation Source

Question 21

d. What is selected ion monitoring? Why does it improve the signal-to-noise ratio for a
particular analyte?

Answer 21

SIM is the abbreviation for “Selected ion monitoring” and is a mass spectrometry acquisition mode in which only a few selected ions are transmitted/detected by the instrument, as opposed to all ions in the full spectrum range, SCAN. This mode of operation typically results in significantly increased sensitivity.

Question 22

c. Which compounds are preferably analyzed with ESI-MS? Which are better suited for
GC-MS? State your reason well!

Answer 22

Soft ionization is a useful technique when considering biological molecules of large molecular mass, such as the aformetioned, because this process does not fragment the macromolecules into smaller charged particles, rather it turns the macromolecule being ionized into small droplets.

EI which is a hard method is also the method that is most commonly used for GC-MS.

Question 23

Explain shortly how ionization gets achieved in ESI.

Answer 23

ESI, Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol.

Question 24

om du är bög c. Make a sketch of a van Deemter curve so that the basic appearance of the curve is
clear. Enter the quantities on the shoulders, their designation and appropriate units
(Storheter, storheternas beteckningar och enheter). What can you use the van Deemter
curve for?

Answer 24

The van Deemter curve can be used to determine the efficiency of a chromatographic column. It shows how the efficiency of a column changes with different plate heights, which is a measure of how well the components of a sample are separated. The higher the plate height, the better the separation, but at some point, increasing the plate height will not result in any further improvement in separation. This point is known as the optimum plate height and is indicated by Hopt on the van Deemter curve.

The quantities on the shoulders are:
- The minimum plate height (Hmin), measured in meters (m)
- The optimum plate height (Hopt), measured in meters (m)
- The maximum plate height (Hmax), measured in meters (m)

Question 25

Draw figures showing typical (i) atomic and (ii) molecular absorption spectra. Explain
shortly why the spectra look different.

Answer 25

Atomic and molecular spectra look different because they arise from different physical processes and involve different types of transitions between energy levels.

Atomic spectra are produced when electrons in an atom are excited from one energy level to another. This can occur through various mechanisms, such as absorbing light or collision with another particle. The energy difference between the two levels is determined by the quantization of energy in the atom, and it results in a unique set of spectral lines for each element. Atomic spectra are characterized by well-defined and discrete lines, which correspond to specific transitions between energy levels.

On the other hand, molecular spectra arise from transitions between energy levels in a molecule. Unlike atomic spectra, molecular spectra are much more complex due to the larger number of degrees of freedom in a molecule, such as rotations, vibrations, and electronic transitions. This results in a broader range of spectral lines, including rotational, vibrational, and electronic transitions. Molecular spectra also exhibit absorption and emission bands, which are characteristic of the molecular vibrations.

In conclusion, the differences between atomic and molecular spectra are due to the different physical processes involved in producing the spectra, as well as the different types of transitions between energy levels in atoms and molecules. Atomic spectra are characterized by well-defined and discrete lines, while molecular spectra are much more complex and exhibit a range of spectral lines and bands.

Question 26

b. For measuring atomic and molecular absorption the light needs to be sufficiently
monochromatic. Explain shortly how this is achieved in
(i) a conventional AAS-instrument,

Answer 26

In a conventional atomic absorption spectrometer (AAS), monochromatic light is achieved through the use of a hollow cathode lamp. The hollow cathode lamp contains the atomic species of interest in a low-pressure gas, and when an electrical current is passed through the gas, it excites the atoms and causes them to emit light at specific wavelengths that are characteristic of the element.

The emitted light is then passed through a monochromator, which is a device that selects a single, narrow wavelength band of light and passes it on to the sample. The monochromator is typically a diffraction grating or a monochromatic filter, and it works by reflecting or transmitting light of a specific wavelength, while blocking light of other wavelengths.

In this way, the monochromator ensures that only light of the specific wavelength that is characteristic of the element being measured is passed on to the sample, allowing for precise measurement of atomic absorption.

In molecular absorption spectroscopy, monochromatic light is typically achieved through the use of a monochromator or a tunable laser, which can produce light of a specific wavelength, while rejecting light of other wavelengths. The monochromatic light is then directed at the sample, and the amount of light absorbed is measured to determine the concentration of the molecular species of interest.

Question 27

b. For measuring atomic and molecular absorption the light needs to be sufficiently
monochromatic. Explain shortly how this is achieved in

(ii) a spectrophotometer used for molecular absorption.

Answer 27

In a spectrophotometer used for molecular absorption, monochromatic light is achieved through the use of a monochromator. A monochromator is a device that selects a single, narrow wavelength band of light and passes it on to the sample. The monochromator typically works by reflecting or transmitting light of a specific wavelength, while blocking light of other wavelengths.

One common type of monochromator used in molecular absorption spectrophotometry is a grating monochromator. In a grating monochromator, light from a broad-spectrum source is passed through a diffraction grating, which splits the light into its component wavelengths. The grating can be rotated to select a specific wavelength band, which is then directed at the sample.

Another type of monochromator is a filter monochromator, which uses one or more filters to select a specific wavelength band. The filters can be switched to select different wavelengths, allowing for measurement of absorption at multiple wavelengths.

In molecular absorption spectrophotometry, the monochromatic light is directed at the sample, and the amount of light absorbed is measured to determine the concentration of the molecular species of interest. The monochromatic light ensures that only a single wavelength is absorbed by the sample, allowing for precise measurement of molecular absorption.