Failed questions of 2022-01-10 exam Flashcards
For liquid chromatography how do you calculate resolution?
Rs=(tr2-tr1)/0.5x(Wav 1+Wav 2)
Wav stands for average peak widht
For liquid chromatography how do you calculate linear vlocity?
Linear velocity of mobile phase (mm/s).
So the column lenght in mm divided by the time for the first peakx60 seconds
a. Explain in words and a simple sketch how a splitless injection works. The explanation must include the terms (i) total flow (ii) septum and septum flow, (iii) column flow and (iii) split flow, split vent and splitless time. (2p)
The total will be the column flow + split flow + septum purge, so adding them together gives the total.
The septum is used to seal the injection port and it is an interface for injecting the sample.
A continuous septum purge - a flow of 3–4 mL/min carrier gas along the septum - will take away the majority of the septum bleeding products that arrives from the high temperatures.
The split time is usually 30-60 seconds which is the time where the split vent an other opening in the GC chromatagorphay machine where a lot of the gas can leave to increase the flow rate.
The sample is injected through the septum in is vaporized in the hot injector (0.5p). There is a low septum flow, which prevents that organic compounds leaking from the septum enter the column (0.5p). The split vent is closed when the injection is made (0.5p) and the aim is that 95-99% of the sample/analytes enter the column. The split vent is opened after 30-60 s (splitless time) and the remaining analytes leave the inlet via the split vent (0.5p). (tot 2p)
c. Explain briefly how “solvent effect” (also called “solvent trapping” and solvent focusing”) works. What rule of thumb applies? (1p)
Solvent effect: The starting temperature of the oven is set 20-40 ºC below the boiling point of the solvent (rule of thumb). This causes the solvent to condense at the beginning of the column. The condensed solvent traps volatile analytes that are focused (need not describe the process of how the flooded zone decreases etc). (1p)
In chemistry, the word “volatile” refers to a substance that vaporizes readily. This will give better results because it traps the analyte and prevents band broadening.
d. Explain briefly how “cold trapping” (includes “thermal focusing” and “stationary phase focusing”) works. What rule of thumb applies? (1p)
Cold trapping: The starting temperature of the oven is set 150 ºC below the boiling point of the analyte (rule of thumb). Thermal focusing and stationary phase focusing take place simultaneously. Thermal focusing means that the temperature is so low that the analyte itself condenses. When the temperature is lowered, the equilibrium constant K will also change; a larger proportion of the analyte enters the stationary phase when the temperature is lowered, which focuses the analytes. (1p)
You want to develop an isocratic reversed-phase HPLC method to separate 4-methylbenzoic acid, 4-ethylbenzoic acid, 4-propylbenzoic acid and 4-butylbenzoic acid.
a. Propose a suitable stationary phase and a suitable mobile phase (give percentage of mobile phase components). (2p)
Stationary phase: Octadecyl silica (C18), Si-O-Si(CH3)2-C18. Mobile phase : mixture of methanol and water (40% MeOH, 60% water) (2p)
In reversed-phase chromatography, which is the more common form of HPLC, the stationary phase is nonpolar and the mobile phase is polar.
Silica has a high polar surface, it is the most popular stationary phase, ideal for conventional applications
C18 has a non-polar surface, ideal for peptides and proteins when using reversed chromatography for analyzing non polar compounds.
Reverse-phase HPLC involves binding an organic molecule to a stationary phase, often silica derivatized with alkyl chains, in a relatively polar environment (the mobile phase), which could contain water, and then eluting the organic molecule using a gradient of a less polar organic solvent.
Methanol is polar and commonly used, 40, 60 is usually the standard ratio
You want to develop an isocratic reversed-phase HPLC method to separate 4-methylbenzoic acid, 4-ethylbenzoic acid, 4-propylbenzoic acid and 4-butylbenzoic acid.
c. Would you expect tailing problems with these analytes? Please explain. (1p)
The longer the alkyl chain the stronger will the hydrophobic interaction with the stationary phase be. Hence, the elution order will be 4-methylbenzoic acid, 4-ethylbenzoic acid, 4-propylbenzoic acid and 4-butylbenzoic acid. (1p)
This is reverse phase so they will be interacting with the non polar stationary phase creating hydrophobic bonds.
f. How is the sample normally injected in HPLC analysis? Explain briefly how the method works. What is this injection method called? (1p)
Loop injection. A loop (e.g. 20 µl) is filled using a syringe. Once filled, a valve is switched such that the mobile phase pass through the loop, thereby bringing the sample to the column
You want to develop an isocratic reversed-phase HPLC method to separate 4-methylbenzoic acid, 4-ethylbenzoic acid, 4-propylbenzoic acid and 4-butylbenzoic acid. What are typical column dimensions of an analutical HPLC column (lenght, column i.d, and particle diameter?) What are the particles made of?
L=10cm, i.d. 2.1 mm, dp=3.5 µm. Particles are typically made of porous silica to which stationaru phase is bonded. (1p)
Beer’s law applies only when certain conditions are fulfilled. Which are these?
The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance.
In physics, monochromatic light is electromagnetic radiation of a single wavelength.
Beer’s law applies to monochromatic irradiation.
The medium must be homogenous.
The medium must not scatter the radiation.
It must not influence the molecules or atoms but act independently.
Which of UV/vis absorption or fluorescence when applied as analytical method is in general more sensitive? State your reason well!
Fluorescence (F) methods a much more sensitive than UV/vis absorption methods.
In other words, fluorescence is measured over a dark background, while absorbance is measured over a bright background. It is relatively easy to detect low levels of light, but difficult to identify small differences in intensity. Therefore, fluorescence is more sensitive than UV-Vis absorption spectroscopy.
Propose a spectrophotometric method to analyze Se in blood. Explain why your method is suitable to quantify the analyte in the sample.
Se is an element and not a molecule, so atomic spectrophotometry has to be applied, like AAS or AES. In analogy to molecular spectrometry, atomic absorption and emission values are proportional to the concentration of the analyte, thus enabling quantification.
Atomic spectroscopy is the study of the electromagnetic radiation absorbed and emitted by atoms. Since unique elements have characteristic (signature) spectra, atomic spectroscopy, specifically the electromagnetic spectrum or mass spectrum, is applied for determination of elemental compositions.
Briefly describe the following terms and their significance in MS. (6p)
b. TIC
TIC is the abbreviation for “Total ion chromatogram” or “Total ion current” and is the initial result/graph after a MS experiment. It represents the total number of ions (y-axis) detected over time (x-axis).
Briefly describe the following terms and their significance in MS. (6p)
d. Analyzer
“Analyzer” in the context of mass spectrometry is the device in the instrument that filters ions produced in the ion source with regard to their mass to charge ratio, m/z. Each value of m/z reaches the detector one at a time
Briefly describe the following terms and their significance in MS. (6p)
f. SIM
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.
