Exam august 2020 Flashcards
Liquid chromatography (6p)
(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). Explain which parts of the van Deemter curve are affected by the particle size and how this is connected to the plate number N
HETP=A+B/u+Cu
HETP = a measure of the resolving power of the column [m]
A = Eddy-diffusion parameter, related to channeling through a non-ideal packing [m]
B = diffusion coefficient of the eluting particles in the longitudinal direction, resulting in dispersion [m2 s−1]
C = Resistance to mass transfer coefficient of the analyte between mobile and stationary phase [s]
u = speed [m s−1]
Where a is mostly related to particle size and the smaller the particle size, the more efficient the column
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.
C18 silica
Mobile phase : mixture of methanol and water
State two ways in which the separation factor a can be affected in reversed-phase
HPLC. Explain briefly
affecting polarity of stationary and mobile phase, column lenght
Make a schematic sketch of a capillary electrophoresis instrument so that it is clear
what parts the instrument is made up of.
A typical capillary electrophoresis system consists of a high-voltage power supply, a sample introduction system, a capillary tube, a detector and an output device.
During the course, we talked about two injection techniques used in capillary electrophoresis (CE) analysis. What are these injection techniques called and how do they work?
- Injection
Hydrodynamic injection, Hydrodynamic injection is accomplished by the application of a pressure difference between the two ends of a capillary.
Electrokinetic injection: useful for lower concentrations , injected amount of sample depends on sample conductivity. Injection by applying a voltage is called electrokinetic injection.
For capillary elecxtrophoresis What do you want to achieve by adding sodium dodecyl sulfate (SDS) to the carrier
electrolyte? (0.5p)
The addition of SDS to the carrier electrolyte can modify the EOF in two ways. First, SDS forms a negatively charged micelle that interacts with the positively charged capillary wall, creating a strong and stable double layer that reduces the surface charge and therefore decreases the EOF. Second, SDS can interact with the analytes, coating them with a negative charge and thus enhancing their separation by increasing the electrophoretic mobility differences.
For capillary electro phoresis What do you want to achieve by adding tetradecyltrimethylammonium bromide
(TTAB) to the electrolyte buffer?
The addition of TTAB to the carrier electrolyte can modify the EOF in two ways. First, TTAB forms positively charged micelles that interact with the negatively charged capillary wall, creating a strong and stable double layer that reduces the surface charge and therefore decreases the EOF. Second, TTAB can interact with the analytes, coating them with a positive charge and thus enhancing their separation by increasing the electrophoretic mobility differences.
Therefore, the addition of TTAB to the carrier electrolyte in capillary electrophoresis can lead to a reduction in the EOF and an improvement in separation efficiency and resolution, particularly for neutral and anionic analytes that do not interact well with SDS.
For electro phoresis Two properties of the analyte ions affect the electrophoretic mobility (μep). Explain
how these properties affect μep.
Charge: The charge of an analyte ion affects its μep because the electrophoretic force acting on an ion is directly proportional to its charge. An ion with a higher charge will experience a stronger electrophoretic force and therefore have a higher μep than an ion with a lower charge. For example, in an electric field, a negatively charged ion will migrate towards the anode while a positively charged ion will migrate towards the cathode.
Size/Shape: The size and shape of an analyte ion affect its μep because they determine the resistance of the ion to movement through the medium. The larger or more complex an ion’s size/shape, the greater the resistance to movement, and the slower its migration through the medium. Therefore, an ion with a smaller size/shape will experience less resistance to movement and have a higher μep than an ion with a larger size/shape.
The following figure shows a schematic diagram of a scanning double-beam spectrophotometer.
a. Name two light sources that are compatible with this instrument. b. Describe a monochromator that most likely is used here. How does it work? c. Describe a detector that most likely is used here. How does it work?
a. Two light sources that are commonly used with scanning double-beam spectrophotometers are tungsten filament lamps and deuterium lamps. Tungsten filament lamps emit a broad range of visible and near-infrared light, while deuterium lamps emit ultraviolet light in the range of 160-400 nm.
b. The most common monochromator used in scanning double-beam spectrophotometers is a diffraction grating monochromator. The diffraction grating monochromator works by using a diffraction grating to disperse polychromatic light into its component wavelengths, which are then passed through a narrow slit to select a specific wavelength. The light then passes through a second slit that further narrows the selected wavelength to a beam of monochromatic light, which is directed onto the sample.
c. The most common detector used in scanning double-beam spectrophotometers is a photomultiplier tube (PMT). A PMT works by converting light energy into an electrical signal. When light strikes the photocathode of the PMT, it ejects electrons through a series of dynodes, each of which amplifies the signal by a factor of about 10. The amplified signal is collected at the anode and is proportional to the amount of light that struck the photocathode. The PMT can detect very low levels of light and has a wide dynamic range, making it ideal for use in spectrophotometry.
. Beer’s law applies only when certain conditions are fulfilled. Which are these? (2p)
The absorbing species should be in a uniform and homogeneous solution.
The incident light must be monochromatic, i.e., of a single wavelength.
The absorbing species should not undergo any chemical or physical change during the absorption process.
The incident light should be collimated, i.e., the light rays should be parallel to each other.
The concentration of the absorbing species should not be too high, i.e., the solution should not be too concentrated, as this can cause deviations from Beer’s law due to non-linear absorption effects or scattering.
Draw a schematic diagram for a GC-quadrupole MS instrument. Explain the function of the various parts.
A gas chromatography (GC) quadrupole mass spectrometry (MS) instrument consists of several components that work together to separate, detect, and analyze the components of a complex mixture.
Injection system: The injection system is responsible for introducing a small volume of the sample to be analyzed into the GC column. The sample is usually injected using a syringe or an autosampler.
GC column: The GC column is a long, narrow tube packed with a stationary phase material, such as a polymer or a silica gel. The column separates the components of the sample based on their volatility and polarity. As the sample travels through the column, the components are separated by their interactions with the stationary phase.
Carrier gas: The carrier gas is used to transport the sample through the GC column. The most common carrier gases used in GC are helium, nitrogen, and hydrogen.
Mass spectrometer: The mass spectrometer is the heart of the GC-MS instrument. It consists of several key components, including:
Quadrupole mass filter: The quadrupole mass filter is responsible for filtering and selecting ions based on their mass-to-charge ratio (m/z). It consists of four parallel rods that create a stable electric field. The quadrupole selectively allows only ions of a specific m/z ratio to pass through and reach the detector.
Ionization source: The ionization source ionizes the separated components eluting from the GC column. Common ionization sources include electron impact (EI) and chemical ionization (CI) sources.
Electron multiplier detector: The electron multiplier detector is responsible for detecting the ions that pass through the quadrupole. The detector consists of a series of dynodes that amplify the signal generated by the ion impact.
Data system: The data system collects and processes the data generated by the mass spectrometer. It identifies and quantifies the components of the sample based on their mass spectra and retention times.
Overall, the GC-quadrupole MS instrument combines the separation power of GC with the specificity and sensitivity of MS to identify and quantify the components of complex mixtures.
b. Which ion source is compatible with LC?
Electrospray Ionisation Source
c. Briefly explain the term “SCAN” and its significance in MS
Typically the mass spectrometer is set to scan a specific mass range. This mass scan can be wide as in the full scan analysis or can be very narrow as in selected ion monitoring. A single mass scan can take anywhere from 10 ms to 1 s depending on the type of scan. Many scans are acquired during an LC/MS analysis.
d. What is selected reaction monitoring? Why is it also called MS/MS. Why does it
improve the signal-to-noise ratio for a particular analyte?
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.
c. What kind of molecules are preferably analyzed by ESI-MS? Which are preferably
analyzed by GC-MS?
