Chemometrics exam - March 2021 (R. Losno) 1) Distributions. The three lists A, B and C are issued independently from three distributions.

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Chemometrics exam - March 2021 (R. Losno) Duration: 40 min - No document

Answers are accepted in English or in French. All of them have to be developed with arguments.

1) Distributions. The three lists A, B and C are issued independently from three distributions.

A B C

6.5 5.8 8.7

8.6 5.0 6.7

3.4 5.9 7.9

5.2 4.8 8.0

5.5 6.9 7.9

3.1 5.1 7.8

2.9 1.7 8.6

These three distributions are normal. Calculate their average and standard deviation.

Trace the centile of the distributions

Could two of these lists be issued from the same distribution?

Calculate the average and standard deviation expected for: A - B, C/B

2) Calibrations.

Measurements are done using an ICP-AES instrument for which the measured intensity I is plotted as function of the calibration standard concentrations:

The uncertainty on the slope (a) is 3% and 250 cps on the intercept (b). Five zero calibration standards are independently measured, giving the following values: 63, 104, 72, 104, 64.

a) Calculate the detection limit of the method expressed in µg/L

b) Calculate the concentration of a sample A with a signal intensity of 320 cps and of a sample B with a signal intensity of 9500 cps.

c) The uncertainty on sample B intensity is 4%.

Calculate the uncertainty associated to sample A and those associated to sample B

Student's table: t

1-α/2, n

0 5 10 15 20 25

0 2000 4000 6000 8000 10000 12000

f(x) = 504.7 x + 129.2

Concentration (µg/L)

Inetnsity (cps)

1 2 3 4 5 6 7 8 9 10

90% 6.3 2.9 2.4 2.1 2.0 1.9 1.9 1.9 1.8 1.8

95% 13 4.3 3.2 2.8 2.6 2.4 2.4 2.3 2.3 2.2

99% 64 9.9 5.8 4.6 4.0 3.7 3.5 3.4 3.2 3.2

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Chimie Analytique Quantitative

Partie ICPMS – Y. Sivry – 30min – Aucun document accepté (No document accepted)

Question 1 :

1) On souhaite mesurer avec un ICP-MS la concentration en fer (Fe) dans un échantillon d’eau de rivière. Quelle est la résolution nécessaire pour résoudre l’interférence produite sur le ⁵⁶Fe par :

40Ar¹⁶O⁺⁺ ?

40Ca¹⁶O⁺⁺ ?

28Si2++ ?

We aim at measuring with an ICP-MS the iron (Fe) concentration in a river water sample.

Which resolution is necessary to solve the interferences on ⁵⁶Fe isotope produced by:

40Ar¹⁶O⁺⁺ ?

40Ca¹⁶O⁺⁺ ?

28Si2++ ?

Information: m¹⁶O = 15.994915 amu ; m⁵⁶Fe = 55.934942 amu ; m⁴⁰Ar = 39.962384 amu ; m⁴⁰Ca = 39.962591 amu ; ²⁸Si = 27.976927 amu.

2) On souhaite à présent mesurer la concentration en sélénium (Se) dans le même échantillon d’eau de rivière. Si 1 ppb donne 10^{^6}cp.s^-1 quel que soit l’élément et que l’on mesure :

- 25000 cp.s^-1 sur la masse 76 - 15000 cp.s^-1 sur la masse 72

Quelle est la concentration en Se en ppt ?

We now aim at measuring selenium (Se) concentration in the same river water sample. If 1 ppb gives 10^6 cps whatever the element :

Measured on mass 76 = 250000 cp.s^-1 Measured on mass 72 = 150000 cp.s^-1 What is the Se concentration in ppt?

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Table 1:

Question 2 :

Reclasser, dans l'ordre de 1 à 10, les différentes étapes du "parcours" d'un analyte lors de sa détection par ICP-MS :

a) Déshydratation b) Détection

c) Injection dans plasma d) Ionisation

e) Filtration en masse f) Nébulisation

g) Cône échantillonneur h) Séparation ions/neutres i) Atomisation

j) Cône écrêteur

Re-organize, from 1 to 10, the different steps of the « journey » of an analyte while it goes through an ICP-MS :

a) Deshydration b) Detection

c) Injection into the plasma d) Ionisation

e) Mass filtration f) Production of a spray g) Sampler cone

h) Separation ions/neutrals i) Atomization

j) Skimmer cone

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Paris, Thursday, 30

^th

, March 2021

Master 1

Electron Paramagnetic Resonance exam

Exercice 1 :

In a recent paper Eisermann et al discuss the transition metal EPR spectrum in metallodrugs

(Insights into metalloproteins and metallodrugs from electron paramagnetic resonance spectroscopy Current opinion in Chemical Biology 2021, 61:114–122 )

1) Can you identify the transition metals observe in figure 1 and figure 2 ? Give a justification of your answer.

2) One of those two metals is missing in the scheme proposed in this subject. Which one ? One can get confused with

another metal present in the scheme, why, what are the difference ?

2) In figure 1, the spectrum is varying with an external parameter (pH, Temperature …). Can you explain the observation, and guess what could be the environment parameter varying ?

In formation : Nuclear spin of different transition metal : I(Cu)=3/2 ; I(Fe) =0, I(Mn) =5/2, I(V)=7/2, I(Ni)=0 ; I(Co)=7/2

Figure 2 Figure 1

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Exercice 2 :

In a publication from 2018, (Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept, Free Radical Research, Taylor & Francis, 2018, 52 (2), pp.171-179 ), S. Kuresepi et al studies radical in reconstructed epidermis using spin trapping. They identified derived radical from cumene hydroperoxyde et skin reconstruction.

In those experiment DEPMPO is used as a spin trap ; 1) Explain why we don’t observe any signal in EPR spectrum (a) ? What does it mean ?

2) Can you confirm the attribution for spectrum d and e, making a bar diagram.

3) Spectrum e is no very tall in the paper, can you give the postulated intensity ratio of the different lines.

4) Figure 2, can you propose a caption for this figure ?

Information :

aN stand for nitrogen nuclear spin-electronic spin coupling, N nuclear spin is equal to 1

aP stand for phosphorus nuclear spin-electronic spin coupling, P nuclear spin is equal to 1/2

aH stand for hydrogen nuclear spin-electronic spin coupling, H nuclear spin is equal to 1/2

Cu (copper) nuclear spin is equal to 3/2 Figure 1

Figure 2

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1

UE10 UE Analytical Chemistry Spectrométrie de masse (MS)

30 Mars 2021

Exercice 1

a) Quelles sont les principales parties qui composent un spectromètre de masse ? Décrire brièvement le rôle respectif de chacune.

b) Décrire le phénomène d’ionisation mis en jeu dans l’ionisation par impact électronique (EI).

Préciser le type d’ions formés et le nombre de charge observé.

Exercice 2

a) Le composé suivant est analysé par spectrométrie de masse à ionisation par impact électronique (EI). En utilisant le tableau en annexe 1, calculer le profil isotopique (masse nominale / masse exact / abondance relative) du composé :

CH

2

= CH – Br

Nb : pour un état de charge z = 1 et on admettra que l’abondance relative de l’isotope ²H est négligeable

b) Expliquer la nature du profil isotopique et comment il se transcrit sur le spectre de masse obtenu

c) Déterminer la résolution instrumentale minimale requise afin de pouvoir distinguer les ions moléculaires correspondant aux composés suivant :

C

4

H

7

OH and C

5

H

12

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2 Exercice 3

L’oxazépam (Seresta®) est une molecule de la catégorie des benzopiazépines, utilisées pour leur propriétés anxiolytique et sédatives. On souhaite développer une méthode pour l’analyse de l’oxazépam.

Après extraction, le composé pourrait être analysé par chromatographie liquide (ultra)-haute performance couplée à la spectrométrie de masse (UPLC-MS) ou par chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS).

Oxazepam (C15H11ClN2O2)

On souhaite évaluer les deux techniques analytiques pour identifier la plus performante. Pour cela une série d’échantillon est analysée respectivement avec les conditions suivantes :

Conditions 1: analyse UPLC-MS (Waters ACQUITY – API 2000 MS)

Phase stationnaire Waters BEH C18 stationary phase (50 × 2.1 mm, 1.7 µm) Phase mobile A: H2O (+ 0.1% acide formique)

Phase mobile B: Acétonitrile (+ 0.1% acide formique) Instrument MS : source ionisation électrospray (ESI)

analyseur de masse quadripôle (précision 50 ppm ; résolution 2000)

Conditions 2: analyse GC-MS (Agilent Technologies 7890 GC-MS)

Phase stationnaire Agilent HP-ULTRA 1 (length 15 m, 0.20 mm, 0.33 μm film thickness) Phase mobile : Helium (0.9 mL/min)

Instrument MS : source ionisation impact électronique (EI) analyseur de masse quadripôle

a) Pour les différentes conditions, donner le type d’ion généré correspondant à l’oxazépam et donner le m/z moyen mésuré

La méthode d’analyse UPLC-MS (condition 1) est sélectionnée car la limite de détection est plus faible :

b) Quel est le role de l’acide formique contenu dans la phase mobile ?

c) Lors de l’analyse UPLC-MS d’un échantillon biologique, l’ion m/z = 287,7320 est mesuré pour l’oxazépam. Calculer la précision sur la mesure de masse. Est-elle en accord avec les performances de l’instrument ?

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3 Exercice 4

Afin de déterminer la séquence d’acide aminés d’une protéine utilisée comme agent thérapeutique, un échantillon de cette protéine est digéré à l’aide de trypsine puis le mélange de peptides est analysé par UPLC-MS/MS.

Le spectre MS/MS ci-dessous est obtenu lors de la fragmentation d’un peptide composant la protéine. Pendant l’analyse, la dissociation par collision induite (CID) est utilisée pour produire la fragmentation.

L’ion précurseur est mesuré avec un m/z = 383.8775 avec un état de charge (3+)

Déterminer à l’aide du spectre MS/MS, la séquence d’acides aminés de ce peptide (utiliser le tableau en annexe 2).

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4 Annexe 1 :

Elément Symbole Masse nominale Masse exacte Abondance naturelle (%)

Masse moyenne

Hydrogène ¹H 1 1,0078 99,989

1,0079

²H 2 2,0141 0,115

Carbone ¹²C 12 12 98,93

12,0107

¹³C 13 13,0034 1,07

Oxygène ¹⁶O 16 15,9949 99,757

15,9994

17O 17 16,9991 0,038

¹⁸O 18 17,9992 0,205

Azote ¹⁴N 14 14,0031 99,632

14,0067

¹⁵N 15 15,0001 0,368

Brome ⁷⁹Br 79 78,9183 50,69 79,904

⁸¹Br 81 80,9162 49,31

Chlore ³⁵Cl 35 34,9688 75,78

35,453

37Cl 37 36,9659 24,22

Annexe 2 :

Acide aminé Symbole Masse

monoisotopic Masse moyenne

Alanine A 71.0371 71.0788

Arginine R 156.1011 156.1875

Asparagine N 114.0429 114.1038

Acide aspartique D 115.0269 115.0886

Cystéine C 103.0092 103.1388

Acide glutamique E 129.0426 129.1155

Glutamine Q 128.0586 128.1307

Glycine G 57.0215 57.0519

Histidine H 137.0589 137.1411

Isoleucine I 113.0841 113.1594

Leucine L 113.0841 113.1594

Lysine K 128.0950 128.1741

Méthionine M 131.0405 131.1926

Phénylalanine F 147.0684 147.1766

Proline P 97.0528 97.1167

Serine S 87.0320 87.0782

Thréonine T 101.0477 101.1051

Tryptophane W 186.0793 186.2132

Tyrosine Y 163.0633 163.1760

Valine V 99.0684 99.1326

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1

UE10 UE Analytical Chemistry Mass spectrometry exam

30 March 2021

Exercise 1

a) What are the principal sections composing a mass spectrometer? Describe briefly their respective role.

b) Describe the ionisation phenomenon involved in electron impact ionization (EI). Precise the type of ion formed and the number of charge observed.

Exercise 2

a) The following compound was analysed using electron impact ionization mass spectrometry. Using the table in annex 1, calculate the isotopic profile (nominal mass / exact mass / relative abundance) of the compound:

CH

2

= CH – Br

Nb: For the charge state z = 1 and one can admit that the relative abundance of the isotope

2H is negligible.

b) Explain the nature of the isotopic profile and how it will translate on the mass spectrum.

c) Determine the minimal instrument resolution required in order to distinguish the molecular ions corresponding to the following compounds (use average masses):

C

4

H

7

OH and C

5

H

12

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2 Exercise 3

Oxazepam (Seresta®) is a molecule belonging to the class of benzodiazepines, used for their anxiolytic and sedative properties. We want to develop a method for the analysis of oxazepam.

After extraction, the compound can be analysed using (ultra)-high pressure liquid chromatography coupled to mass spectrometry (UPLC-MS) or gas chromatography coupled to mass spectrometry (GC-MS).

Oxazepam (C15H11ClN2O2)

We want to assess the two analytical techniques in order to identify the most relevant.

Therefore, a series of samples was analysed respectively using the following conditions:

Conditions 1: UPLC-MS instrument (Waters ACQUITY – API 2000 MS) Waters BEH C18 stationary phase (50 × 2.1 mm, 1.7 µm)

Mobile phase A: H2O (+ 0.1% formic acid)

Mobile phase B: Acetonitrile (+ 0.1% formic acid) MS instrumentation: electrospray ionisation source

quadrupole mass analyser (accuracy 50 ppm ; resolution 2000)

Conditions 2: GC-MS instrument (Agilent Technologies 7890 GC-MS)

Agilent HP-ULTRA 1 stationary phase (length 15 m, 0.20 mm, 0.33 μm film thickness) Mobile phase: Helium (0.9 mL/min)

MS instrumentation: electron impact ionisation source quadrupole mass analyser

a) For the different conditions, define the type of ions generated corresponding to oxazepam and give the average m/z ratio measured.

The UPLC-MS method (conditions 1) was selected due to a lower limit of detection:

b) What is the role of formic acid contained in the mobile phase?

c) During the UPLC-MS analysis of a patient sample, the ion m/z = 287,7320 is measured allegedly for oxazepam. Calculate the mass accuracy. Is it in agreement with the acuracy of the instrument.

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3 Exercise 4

In order to establish the amino acid sequence of a therapeutic serum albumin, a sample of the protein was digested using trypsin enzyme and the analysis of the peptide mixture obtained was analysed using UPLC-MS/MS.

The MS/MS spectrum presented below was obtained from the fragmentation of one peptide composing the protein. During the experiments, collision induced dissociation (CID) was performed to generate the fragmentation.

The precursor ion was measured at m/z = 383.8775 exhibiting a charge state (3+)

Using the MS/MS spectrum, determine the amino acid sequence of this peptide (use annex 2 table).

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4 Annex 1:

Element Symbol Nominal mass Exact mass Natural abundance (%)

Average mass

Hydrogen ¹H 1 1,0078 99,989

1,0079

²H 2 2,0141 0,115

Carbon ¹²C 12 12 98,93

12,0107

¹³C 13 13,0034 1,07

Oxygen ¹⁶O 16 15,9949 99,757

15,9994

17O 17 16,9991 0,038

¹⁸O 18 17,9992 0,205

Nitrogen ¹⁴N 14 14,0031 99,632

14,0067

¹⁵N 15 15,0001 0,368

Bromide ⁷⁹Br 79 78,9183 50,69 79,904

⁸¹Br 81 80,9162 49,31

Chloride ³⁵Cl 35 34,9688 75,78

35,453

37Cl 37 36,9659 24,22

Annex 2:

Amino acid Symbol Monoisotopic

mass Average mass