Most of the questions from exam 220110 with ansers

Question 1

For liquid chromatography how do you calculate retention factor k?

Answer 1

Retention time is the time it takes for the solute to pass through the column and is usually measured in minutes. These values can then be used to calculate retention factor which uses the formula, K=(tr-tm)/tm

The time for an unretained solute to reach the detector from the point of injection is called the column dead time or the hold up time(tM). The solute retention time (tR) is the time difference between sample injection and the detector sensing the maximum of the retained peak.

Question 2

For liquid chromatography how do you calculate seperation factor a?

Answer 2

α = k2/k1

It is defined as the ratio of two retention factors. Two components can be separated only if they have different retention factors.

Question 3

For liquid chromatography how do you calculate resolution?

Answer 3

Rs=tr2-tr1/Wav

Question 4

For liquid chromatography how do you calculate linear vlocity?

Answer 4

Linear velocity of mobile phase (mm/s).

So the lenght of the column and the time of the first peak.

Question 5

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.

Answer 5

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)

Question 6

Why are focusing techniques needed when samples are injected splitless?

Answer 6

In splitless injection, the sample is over a long period of time (30-60s). A starting band that is as wide as the injection time (band broadening in time) is obtained. Therefore, focusing mechanisms are required. (1p).

Question 7

Explain briefly how “solvent effect” (also called “solvent trapping” and s”olvent focusing”) works. What rule of thumb applies?

Answer 7

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)

Question 8

Explain briefly how “cold trapping” (includes “thermal focusing” and “stationary phase focusing”) works. What rule of thumb applies?

Answer 8

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)

Question 9

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).

Answer 9

Stationary phase: Octadecyl silica (C18), Si-O-Si(CH3)2-C18. Mobile phase : mixture of methanol and water (40% MeOH, 60% water) (2p)

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
Amino has a medium polar surface, ideal for carbohydrates and nitrogen containing heterocycles and amines
Diol has a low polar surface, ideal for lipids
Alumina has a high polar surface, ideal for acid sensitive compounds

Question 9

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 will be the elution order of the analytes? Explain.

Answer 9

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)

Question 10

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. Would you expect tailing problems with these analytes? PLease explain

Answer 10

The analytes are carboxylic acids and hydrogen bonding interactions with free silanol groups of the stationary phase can be expected.

If any residual (free) silanol groups remain at the surface of silica gel after bonding treatment, they may affect the retention of solutes.

Question 11

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. How should you change the proportions of components of mobile phase if you would like the analytes to elute faster?

Answer 11

If the analytes should elute faster, their interaction with the stationary phase should decrease, which requires a stronger mobile phase. The mobile phase is made stronger by using a higher percentage of methanol, e.g. 50% MeOH. (1p)

Question 12

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?

Answer 12

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)

Question 13

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. How are the samples normally injecged in HPLC analysis? Explain briefly how the method works. What is the injection method called?

Answer 13

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

Question 14

True or false, When using a WCOT column, the separation is based on partition chromatography.

Answer 14

WCOT stands for Two type wall-coated open tubular and has two liquid phases. Partition chromatography is a type of chromatography in which separation is based on partition between the two liquids. Therefore it is , True

Question 15

True or false, A WCOT column is used in reversed-phase HPLC.

Answer 15

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.

WCOT is based on two liquid phases.

False

Question 16

True or false, In normal phase HPLC, a C18 column can be used.

Answer 16

False

Question 17

True or false, A PLOT column is a good choice if you want to analyze gases with GC.

Answer 17

True. Porous Layer Open Tubular (PLOT) GC Columns are the ideal Gas Chromatography (GC) columns for the analysis of light hydrocarbons and fixed gases. PLOT solid-gas chromatography columns are used for separations of small molecules.

Question 18

True or false, An HPLC column packed with small particles will have a larger plate number N than a column packed with larger particles.

Answer 18

True

Question 19

True or false, A GC column with thick a stationary phase film gives smaller retention factors k than a column with thinner film.

Answer 19

False

Question 20

A compound with molecular mass 292.16 g/mol was dissolved in a 5-mL volumetric flask. A 1.00-mL aliquot was withdrawn, placed in a 10-mL volumetric flask, and diluted to the mark. The absorbance at 340 nm was 0.427 in a 1.000-cm cuvet. The molar absorptivity at 340 nm is e340 = 6130 M-1cm-1. Beer’s law applies only when certain conditions are fulfilled. Which are these?

Answer 20

Beer’s law applies to monochromatic irradiation and it works well for dilute solutions (≤ 0.01 M). See Harris p. 437.

Question 21

A compound with molecular mass 292.16 g/mol was dissolved in a 5-mL volumetric flask. A 1.00-mL aliquot was withdrawn, placed in a 10-mL volumetric flask, and diluted to the mark. The absorbance at 340 nm was 0.427 in a 1.000-cm cuvet. The molar absorptivity at 340 nm is e340 = 6130 M-1cm-1. Which of UV/vis absorption or fluorescence when applied as analytical method is in general more sensitive? State your reason well!

Answer 21

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 22

A compound with molecular mass 292.16 g/mol was dissolved in a 5-mL volumetric flask. A 1.00-mL aliquot was withdrawn, placed in a 10-mL volumetric flask, and diluted to the mark. The absorbance at 340 nm was 0.427 in a 1.000-cm cuvet. The molar absorptivity at 340 nm is e340 = 6130 M-1cm-1. Propose a spectrophotometric method to analyze Se in blood. Explain why your method is suitable to quantify the analyte in the sample.

Answer 22

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.

Question 23

Briefly describe the following terms and their significance in MS. (6p)

a. Molecular ion

Answer 23

The molecular ion is the initial ion produced from a molecule (M) in an ion source of a mass spectrometer. Ion sources apply various ionization mechanisms, for example electron impact (EI), in which an electron hits the molecule and produces an odd electron molecular ion, M+˖.

Question 24

Briefly describe the following terms and their significance in MS. (6p)

b. TIC

Answer 24

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).

Question 25

Briefly describe the following terms and their significance in MS. (6p)

c. MALDI

Answer 25

c. MALDI is the abbreviation for “Matrix assisted laser desorption ionization” which is another example for ionization applied in ion sources. It is a soft ionization method often combined with a time of flight analyzer, MALDI-TOF. Analytes are mixed with a crystalline UV-absorbing compound (matrix) and then subjected to a laser beam whose energy results in the production of [M+H]+ or [M-H]-

Question 26

Briefly describe the following terms and their significance in MS. (6p)

d. Analyzer

Answer 26

“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

Question 27

Briefly describe the following terms and their significance in MS. (6p)

e. m/z

Answer 27

See answer to d. “m/z” stands for mass to charge ratio for ions produced in ion sources.

Question 28

Briefly describe the following terms and their significance in MS. (6p)

f. SIM

Answer 28

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.