All about the instruments Flashcards
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,
(ii) a spectrophotometer used for molecular absorption.
Both uses monochromators often gratings to create monocrhomatic light.
For AAS you can either use a hollow cathode lamp, nothing at all or a line source. The sample is then in a liquid and placed over a flamr which will cause the absobtion, emission or fkourescense signal depending on what system you use. A monochromator is then used to sepearete this nto a monocrhomatic light which a detector then reads.
For molecular adsobtion a lamp will act as a light source and then a scanning monochromator will break the light into one wavelength at a time and a rotating beam chopper will let different wavelengths of light hit the sample one at a time. There is a sample compartmeant with a slit so only one wave length can come in at a time. A detector will then detect the transmittance and a computer will display the results
Describe a detector that is used in a conventional fluorescence spectrophotometer.
In fluorescence spectrophotometry, a photomultiplier tube (PMT) is often used as the detector. This is because PMTs are highly sensitive to light and can detect low levels of fluorescence emitted from a sample.
a. Draw a schematic diagram for a GC-quadrupole MS instrument. Explain the function of
the various parts.
Gas chromatograph: The gas chromatograph separates the components of a complex mixture based on their physical and chemical properties. The sample is vaporized and injected onto a chromatographic column where the separation occurs. The column is typically packed with a stationary phase material that interacts differently with each component, causing them to elute at different times.
Injector: The injector introduces the sample into the gas chromatograph. It vaporizes the sample and injects it onto the column at a specific temperature and pressure.
Oven: The oven is a temperature-controlled compartment that houses the chromatographic column. The temperature of the oven is programmed to increase at a specific rate, allowing the components to elute from the column in a specific order.
Quadrupole mass spectrometer: The quadrupole mass spectrometer is a highly selective and sensitive detector that identifies and quantifies the separated components. It consists of four parallel metal rods that act as a filter to separate and detect ions based on their mass-to-charge ratio (m/z).
Electron ionization source: The electron ionization source generates ions from the separated components that enter the mass spectrometer for detection. This is accomplished by bombarding the separated components with high-energy electrons, which cause them to ionize and fragment.
Detector: The detector measures the number of ions that reach the detector and converts this information into an electrical signal, which is then processed and displayed as a mass spectrum.
b. Which ion source is compatible with LC?
The electrospray ionization (ESI) source is the most commonly used ionization source in LC-MS (liquid chromatography-mass spectrometry) due to its compatibility with the liquid phase of LC. ESI allows for the ionization of a wide range of polar and nonpolar molecules in LC, including small molecules, peptides, and proteins, as well as more complex biomolecules like nucleic acids and lipids.
d. What is selected ion monitoring? Why does it improve the signal-to-noise ratio for a
particular analyte?
d. Selected reaction monitoring is an acquisition mode that needs two analyzers
(Q1 and Q3) and a collision cell (Q2) inbetween them. The fact that two
analyzers are needed makes it a MS/MS method. In Q1 only a particular ion
from an analyte is chosen to go through. In Q2 this ion is fragemented and Q3
only lets one fragment ion from Q2 go to the detector. Since time is spent only
on two ions, these ions can be collected to a much higher degree with resulting
sensitivity.
d. Explain shortly how ionization gets achieved in ESI.
Electrospray ionization (ESI) is a technique used in mass spectrometry to ionize and detect large, polar, and/or non-volatile molecules. In ESI, a high voltage is applied to a liquid sample, causing it to form a fine spray of charged droplets. The charged droplets are then evaporated in a heated capillary, producing gas-phase ions that can be analyzed by mass spectrometry.
Table 1: Selected specifications&descriptions for Toyota UV-1800 PC model UV–VIS
Spectrophotometer (Kyoto, Japan)
Source lamp Hollow cathode
Beam type Quadrupole
Bandwidth 1.5 nm
Min wavelength (nm) 190
Max wavelength (nm) 1100
Detector Silicon photodiode
Description UV/Visible Scanning Spectrophotometer; 115 VAC
a. While reading the specifications you realize that there are obvious mistakes in the table.
Which are these and need to be revised by Toyota? Propose adequate alternative
specifications!
a. Lamptype is wrong. Hollow cathode is used for AAS. For this instrument a Xenon
lamp would work or a combination of tungsten and deuterium lamps. Beam type is
wrong, too. Quadrupole is an analyzer used in MS instruments. Here it has to be
“single” or “double beam”.
Table 1: Selected specifications&descriptions for Toyota UV-1800 PC model UV–VIS
Spectrophotometer (Kyoto, Japan)
Source lamp Hollow cathode
Beam type Quadrupole
Bandwidth 1.5 nm
Min wavelength (nm) 190
Max wavelength (nm) 1100
Detector Silicon photodiode
Description UV/Visible Scanning Spectrophotometer; 115 VAC
b. Suppose your sample contains two compounds of which one absorbs strongly at 284
and the other at 285 nm. Will you be able to quantify these with the instrument? State
your reason well.
b. No. Monochromator bandwidth of the instrument is 1.5 nm, so the two wavelengths
cannot be separated by the instrument.
Table 1: Selected specifications&descriptions for Toyota UV-1800 PC model UV–VIS
Spectrophotometer (Kyoto, Japan)
Source lamp Hollow cathode
Beam type Quadrupole
Bandwidth 1.5 nm
Min wavelength (nm) 190
Max wavelength (nm) 1100
Detector Silicon photodiode
Description UV/Visible Scanning Spectrophotometer; 115 VAC
The beam type is wrong what type of beam type can be used and how does it work?
Single beam is a light source that goes directly through the monocrhomator and then hits the sample. Double beam used mirrors to reflect the light. A grater can be used for a double beam spectrometer which rotates and breaks the light into one wave lenght at a time.
c. Briefly explain the term “SCAN” and its significance in MS. (1p)
c. SCAN is an acquisition mode in MS. The analyzer allows by changing
potential in rods all ions within a mass range to reach detector one by one.
Heavier ions pass first followed by lighter ions. Once all ions within the mass
range have gone through the process starts again with. This is called scanning
over a mass range and one cycle is called SCAN. Different to SIM, where the
instrument is set to measure only ions of selected m/z.
d. What kind of molecules are preferably analyzed by ESI-MS? Which are preferably
analyzed by GC-MS?
d. With ESI-MS polar (water soluble) samples of considerable molecular mass are
analyzed. For example biomolecules like proteins, peptides and carbohydrates. With
GC-MS unpolar volatile organic compounds of low molecular mass are analyzed.
Make a schematic sketch of a capillary electrophoresis instrument so that it is clear what parts of the instrument is madeup of
There is two beakers with background electrolytes, in the middle there is a power source and in between these beakers are a fused silica capillary. There is an anion and a cation and a detector.
Describe two injection techniques for capillary electrophoresis and how they work
Hydrodynamic injection and electro kinetic injection.
The steps involved in hydrodynamic injection are as follows:
The capillary is first filled with the electrolyte buffer and placed in the CE instrument.
The sample is loaded into a sample vial or well.
A syringe filled with buffer is used to apply pressure to the sample vial or well, forcing the sample to enter the capillary.
The pressure is maintained for a brief period to ensure the sample is injected into the capillary.
Once the sample is injected, the pressure is released, and the sample is separated by electrophoresis.
Hydrodynamic injection is a relatively simple and rapid method for sample introduction in CE, and it is particularly useful when dealing with small sample volumes. However, it can result in broad peaks due to the injection volume and variable injection times.
The steps involved in electrokinetic injection are as follows:
The capillary is filled with the electrolyte buffer, and the sample is loaded into a sample vial or well.
A high voltage is applied across the capillary to create an electric field.
The end of the capillary containing the sample is placed in the sample vial or well.
The voltage is switched to a lower value, and the electric field drives the sample into the capillary.
Once the sample is injected, the voltage is returned to its initial value, and electrophoresis begins.
Electrokinetic injection is a simple and rapid method for sample introduction in CE that allows for precise and reproducible injections. It also reduces sample loss and broadening that can occur with hydrodynamic injection. However, it may require higher sample concentrations and can lead to sample overload if not carefully controlled.
What do you achieve by adding sodium dodecyl sulfate to the electron carrier electrtolyte?
Makes it so neutral compounds can be separated, since it will form charged micelles and interact with neutral analytes giving them electrophoretic mobilities.
What do you achieve by adding tetraadecyltrimethylammonium bromide (TTAB) to the electorlyte buffer?
Increase the separation resolution: TTAB is a surfactant that forms micelles in the buffer solution. These micelles can interact with analyte molecules and cause them to have different electrophoretic mobilities, resulting in better separation resolution.
Suppress electroosmotic flow: Electroosmotic flow (EOF) is the movement of buffer solution due to the presence of a charged surface in the capillary. This flow can interfere with the separation of analyte molecules. TTAB can adsorb onto the capillary surface and suppress EOF, allowing for more accurate and reproducible separations.
