3-Final Exam (Feb.2009)

Department of Pharmaceutical Chemistry Introduction to Instrumental Analysis Dr. Raimund Niess Dr. Raafat Aly Farghaly



PHCMt561, February 2009 Final Exam

Instructions: Read Carefully Carefully Before Before Proceeding. Proceeding. 1. The allowed time for this exam is 3 (three) hours. hours. 2. Write your solutions in the space provided. If you need more space, write on the back of the sheet containing the question and remark this near the question. 3. This exam booklet contains total 21 pages. 4. Calculators are permitted, yet it is not allowed to exchange calculators with others. 5. When you are told that time is up, stop working on the test. Good Luck!

Please, do not write anything on this page

Part I Question

I

II

III

IV

V

VI

Possible Points

12

10

42

34

15

12

Part II

Bonus

Total

10

5

135 (140)

Obtained Points

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Part I QUESTION I

Complete the Sentences (12 pts)

1. An example example of a universal detector for HPLC is the refractive index detector. 2. The thermal conductivity detector utilizes a heated filament in its detection mechanism. 3. In size-exclusion size-excl usion chromatography, chromatograp hy, larger molecules are eluted first. 4. A strong ion-exchanger ion-exchanger carries a permanent charge and can be used at any pH value. 5. "CH2COO" can be found on the surface of weak  cation exchangers. 6. A refractive index detector contains two triangular-shaped flow cells, a light source and a photodiode-array. 7. The fluorescence detector is more sensitive than the UV detector, but for most analytes derivatization is necessary if this detector is used. reciprocating ting pump is used. 8. A pulse-damper pulse-damper will be necessary when a pulsed flow or reciproca 9. In “splitless injection” of GC, 80% or most of the analyte will be delivered to the column. 10. The six-port valve of HPLC can be adjusted to load position and to inject position. 11. Superconducting magnet is the most sensitive and accurate magnet used in the modern FTNMR instruments. 12. As a condition of resonance in NMR spectroscopy, the Larmor frequency of the precessing nuclei should match the frequency f requency of the radio-wave.

QUESTION II

(10 pts)

True or False. Correct any False Statement 1. Ninhydrin, FeCl3 and molybdatophosphoric acid can among others be used for detection of  analytes in TLC. 2. Hexane is considered a strong eluent for reverse-phase chromatography. 3. In gas chromatography, the detector is maintained several degrees cooler than the column temperature. hotter or at higher temperature...... temperature ...... ………………………………………………………hotter ………………………………………………………

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4. The “hydrodynamic volume” is the volume of an analyte molecule that is moving in a liquid. 5. The stationary phase “aluminum oxide” can have acidic, basic, or neutral properties. 6. Gel Filtration and Gel Permeation Chromatography both are based on the size-exclusion principle. 7. If a magnetic field is applied on a sample containing protons, exactly 50% will be in -state and exactly 50% will be in -state. The number of nuclei in the lower -state is slightly more than those in the -state 8. Anisotropic behavior is observed for aromatic rings, as well as for double and triple bonds. 9. In pulsed NMR spectroscopy, we record the “Free Induction Decay” which is transformed into a spectrum by use of the Fourier Transform. 10. The unit of chemical shift  is “ppm”, this stands for “parts per million”.

QUESTION III

Give Short Answers (42 pts)

1. Briefly define the following terms: (2 pts)

a. Elution mobile phase washes analytes from the stationary phase

b. Rf  value in TLC: distance (start line  – spot) / distance (start line  – solvent front) OR the distance traveled by the solute divided by the distant traveled by solvent (mobile phase) 2. A chromatographic separation of a mixture of compounds was performed on a reversed phase column using Methanol : Water (80:20), as mobile phase. If mobile phase composition is changed in a second run to Methanol : Water (70:30), how would the retention times of solutes be affected? Justify. (2 pts) longer retention times. New mobile phase contains larger portion of water, is more polar than the previous one. On reversed stationary phase, this new mobile phase will have less eluent strength than the previous one, so analytes spend more time in the stationary phase

3. What does "isocratic elution" mean in HPLC? (1 pt) composition of mobile phase is kept constant during the run. Or the elution is carried out under constant conditions.

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4. Name two examples of stationary phases than can be used to separate enantiomers. (1 pt) -- cyclodextrine -- any stationary phase that is derivatized with a pure enantiomer of a chiral compound, e.g. with L-valin or derivatives or other optically pure amino acids 5. Sketch a HPLC-chromatogram that shows peak tailing. Give ONE reason for peak tailing. (2 pts) reason: irreversible or very strong binding of analyte to stationary phase; OR generally: if there is a “ negative ” deviation from the linear (ideal) partition coefficient

6. What are the advantages of supercritical fluid chromatography over GC and LC? (2 pts) -- non-volatile analytes can be investigated (unlike GC) -- more uniform flow of the mobile phase (better spreading) -- no interference of mobile phase with the detector (unlike HPLC). -- higher flow rates can be applied OR : Speed and resolution better compared to liquid chromatography (faster diffusion inside  the column cavity, thus faster equilibration .

7. What is the “molecular weight exclusion limit” in size exclusion chromatography? (1 pt) it is a property of a stationary phase indicating the largest molecular weight for which this stationary phase material can be used for separation Molecular weight above which no retention occurs

8. What is a “guard column” used for ? How does it work? (2 pts) a “guard column” is used to protect the column that is used for the chromatographic

separation, it contains the same stationary phase material and consequently will capture / trap anything that is present in the sample solution that might harm the column by too strong adsorption / irreversible binding / any decomposing reaction 9. Write the type of chromatography after the appropriate description. (2 pts)

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Analyte equilibrates between mobile phase and liquid-like stationary phase: partition chromatogr. Different-sized analytes penetrate voids in stationary phase to different extents: size-exclusion chr. Analyte equilibrates between mobile phase and surface of stationary phase: adsorption chrom. Ions are attracted by counter-ions covalently attached to stationary phase: ion-exchange chrom.

10.

Draw a cross-sectional sketch of  two different types of open-tubular columns; give their full names and common abbreviation. (3 pts)

2 of them:

11.

The Van Deemter equation describes the "height equivalent to a theoretical plate" in terms of mobile phase flow rate and three phenomenological constants: i- Give the names for and briefly describe the effects that are considered responsible for  A, B, and C . (3 pts)  A: multiple paths / Eddy diffusion: different molecules of the same analyte will

move on slightly different paths through the stationary phase  B: longitudinal diffusion: analyte molecules will migrate away from the

concentrated center of a band to the less concentrated zones C : equilibration time / mass transfer: the time required for equilibration between

stationary and mobile phase ii- Is it better to minimize or maximize HETP when performing chromatographic separations? Justify. (1 pt) minimize: the smaller the plate height, the more plates (i.e. the stages of perfect equilibration between stat. and mob. phase) will be present on a column, the better will the separation be.

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12.

When designing a binary (CH 3CN : H2O) mobile phase for gradient elution on using a C-18 stationary phase would you usually increase or decrease the proportion of water during elution? Why? (2 pts) Decrease. On reversed phase, the mobile phase that is less polar will have a greater eluent strength, so if we change the mobile phase to less polar one, we will elute the “late peaks” earlier and so avoid problems of too long retention (waste of time, solvent, lot

of diffusion) 13.

Consider this equation: V e = V 0 + KV i i. What does the equation mean? (1 pt) it is used to determine / calculate the “elution Volume” (V el) in size-exclusion

chromatography depending on the volumes we have on a column (void volume in the pores and external volume outside the stat. phase particles) ii. Why in this equation is the condition for K: 0 < K < 1 ? (1 pt) least required elution volume (analyte will not enter the pores): V el =- Vo, this means K = zero largest possible volume (analyte will completely enter the pores): V el = Vo + Vi , this means K =1 any other elution volume is expected to be in between the min. and the max.

14.

a. What does the table on the right show? (1 pt) relative affinities of anions to a certain anion exchange resin.

b. What are the properties of ions that determine their affinities to an ion-exchange phase? (1.5 pts)

Quaternary ammonium anion-exchange resin Anion F OH Cl Br NO3 I ClO4

Relative ---0.09 0.09 1.0 2.8 3.8 8.7 10.0

charge – solvatized radius  – polarizability

15.

Name three different pumps that are commonly used in HPLC, and give one advantage and one disadvantage of each. (4.5 pts) reciprocating pump: advs: small internal volume, independent of back-pressure  – disadv.: pulsed flow

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pneumatic pump: adv.: cheap, pulse-free  – disadv.: affected by back-pressure syringe pump: adv.: pulse-free

16.

disadv.: limited capacity

Briefly explain the following terms (sketches or drawings can be USEFUL): (5 pts) a. Shielding: (1 pt) The circulating -electrons is found to shield the protons from the external applied field. Thus protons actually sense lower field than expected. The extent of shielding of  each proton in a molecule depends on the electronic density (chemical environment) that surrounds it.  Beffective = Bapplied    Blocal b. Chemical shift (meaning not a rule): (1 pt) It measures how much NMR frequency of a proton in an organic compound is shifted downfield or to higher frequency compared with the protons in TMS. This shift in hertz should be related to the operating frequency of the instrument. c. Magnetic anisotropy of aromatic ring systems: (2 pts) It is a secondary magnetic field created by circulating -electrons that may, in one location, add to the external field or may be, in another location, subtracts from the external field. Consequently it appears as if it shields or deshields the aromatic protons. d. Spin-spin-splitting: (1 pt) The magnetic field created by spinning protons is found to affect the resonance position of protons on adjacent carbons. Consequently the peak of a specific proton will split depending on the number of adjacent hydrogens (n+1) rule.

17.

What does “Continuous Wave” measurement mean in NMR spectroscopy? (1 pt) In the continuous-wave experiment, the strength of the magnetic field is slowly scanned (changed from downfield to upfield) while the frequency of the source is held constant or vise versa.

18.

In NMR, describe a method to determine if an unknown solution contains an alcohol. (2 pts) We shake to the sample with deuterium oxide (D 2O) where the proton of the hydroxyl group is exchanged by deuterium (acidic proton). Since D is not an NMR active at the 1 conditions of  H-NMR, the peak of the hydroxyl proton will completely disappear or decreases in intensity indicating the presence of this kind of protons.

19.

What is the name of the depicted compound? What is it used for? (1 pt) Tetramethylsilane (TMS) is used as internal standard where the signal of protons in organic compound is compared to the signal of TMS protons.

CH3 H3C

Si

CH3

CH3

 \ 

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QUESTION IV  – Choose Answer table for MCQ

One  Best

Answer for Each Question

The answer outside this table will not be marked

Blacken the circle corresponding to the correct answer

(34 pts)

1. Which of the following statements is not correct? a) b) c) d)

Chromatography is a separation technique. The term “chromatography” comes from the separ ation of colored bands. In chromatography, we always need to have a 2-phase system. In chromatography, the 2 necessary phases of the separation system should be miscible to permit strong interaction. e) One of the 2 phases in chromatography must move through the system. 2. Which type of chromatography uses a liquid or liquid-like stationary phase? a) b) c) d) e)

adsorption chromatography. ion-exchange chromatography. partition chromatography. GSC None of the above.

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3. To which statement on organic polymers as stationary phases do you agree? a) b) c) d) e)

polymers of glucose can be used. polymers of styrene can be used. they can bear functional groups that are used for ion-exchange. (a) and (c). all of the above.

4. The partition coefficient in chromatography is defined as: a) The volume occupied by the stationary phase divided by the volume occupied by the mobile phase. b) The amount of stationary phase divided by the amount of mobile phase. c) The amount of analyte in the mobile phase divided by the amount in the stationary phase. d) The migration rate of B divided by the migration rate of A . e) The concentration of analyte in the stationary phase divided by the concentration in the mobile phase. 5. To improve a chromatographic separation, we should: a) b) c) d) e)

increase the number of theoretical plates on the column. increase the height of the theoretical plates on the column. increase both the number and the height of the theoretical plates on the column. decrease the column length and increase the diameter of the column. increases the flow rate of the mobile phase.

6. The most common mobile phases in GC are: a) b) c) d) e)

Ar, N2 and F2 N2O, C2H2, and O2 H2, Ar, and Ne He, Ne, and Ar H2, He, and N 2

7. If you wish to change your column to separate double the mass of your sample and still maintain the same resolution without changing the column length, you would need a) b) c) d) e)

a column with twice the radius of the original column. a volume flow rate twice the rate in the original column. triple the volume of solvent added to the solute before application on the column. a linear flow rate one-half the rate in the original column. a column with one-half the radius of the original column.

8. Which of the following will result in narrower peaks and better resolution for HPLC? a) b) c) d) e)

Increasing the thickness of the stationary phase. Using smaller particles for the stationary phase. Injecting the sample more slowly onto the column. Increasing the column length. (b) and (d).

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9. Which of the following detectors is commonly used in HPLC? a) b) c) d) e)

Electron-capture detector. Refractive index detector. Atomic absorption detector. Flame ionization detector. All of the above are commonly used in gas chromatography.

10. Consider the effect of  longitudinal diffusion on band-broadening in a packed liquid chromatographic column. How does this effect depend upon the flow rate of the mobile phase? a) b) c) d) e)

longitudinal diffusion will increase with an increase in the flow rate. longitudinal diffusion will decrease with an increase in the flow rate. longitudinal diffusion is independent of flow rate. longitudinal diffusion will first decrease, then increase as flow rate increases. there is insufficient information to answer the question.

11. In comparing open tubular columns to packed columns with a gaseous mobile phase, a) b) c) d) e)

a packed column is generally much longer than an open tubular column. both are used for preparative separation. the packed column does not give as good resolution as the open tubular column. if an open tubular column is used, it is possible to utilize a higher flow rate. (c) and (d)

12. Considering different injection techniques in GC, which series represents the correct increase of sample fraction that is delivered to the column? a) b) c) d) e)

splitless < split < on-column injection. split < splitless < on-column injection. on-column < split < splitless injection. splitless < on-column < split injection. None of the above.

13. What is the primary advantage of temperature programmed gas chromatography? a) b) c) d) e)

It allows separation of early eluted solutes. It facilitates the analysis of volatile compounds. It increases the number of theoretical plates for the column. It facilitates the use of the electron capture detector. None of the above.

14. A nuclear magnetic resonance signal cannot be observed with a)

2 1

 H 

b)

19 9

F 

14 c) 7  N 

d)

13 6

C 

e)

16 8

O

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15. Phosphorous-31 has a spin quantum number (I) of ½ and therefore exhibits two magnetic quantum states, m =  ½. Calculate the energy of photon that will be absorbed to cause a transition between these states in a 7.15 T external magnetic field. (gyromagnetic ratio for 31 8 -1 -1 P is 1.0841 x 10 T s , Planck constant = 6.62 x 10 34 Js) 8

a) 8.17 x 10 26 J -7 d) 2.34 x 10 J

b) 1.23 x 10 J e) None of the above

c) 123 J

16. Consider a magnetic nucleus with a total spin quantum number (I) of 7/2. The number of   possible orientations will this nucleus have in an externally applied magnetic field and the magnetic quantum numbers given for these orientations are: a) b) c) d) e)

2, (1/2, -1/2). 4, (3/2, 1/2, -1/2, -3/2). 6, (7/2, 5/2, 3/2, 1/2, -1/2, -3/2, -5/2, -7/2). 8, (7/2, 5/2, 3/2, 1/2, -1/2, -3/2, -5/2, -7/2). None of the above. 1

17. What is the significance of  H-NMR spectroscopy as an analytical method? a) b) c) d) e)

we can use it to find the molecular weight of an analyte. we can use it to find the exact elemental composition of an analyte. we can use it to find the chemical structure of an analyte. we can use it to find the solubility and pKa of an analyte. None of the above.

18. Why do we need to apply a magnetic field in NMR spectroscopy? a) b) c) d) e)

in order to align the sample probe with the radio-wave transmitter. in order to make use of the Fourier Transform operation. in order to create different energy levels for the nuclei under investigation. in order to improve the S/N ratio. (b) and (d).

19. Which term is NOT related to NMR? a) b) c) d) e)

Larmor precession. net magnetic moment. absorbance of radio-wave energy. bathochromic shift. Free Induction Decay.

20. How do we conduct Fourier Transform- NMR spectroscopy? a) b) c) d) e)

by applying short pulses of radio-waves to the sample and record the FID. by using an interferometer. by scanning the applied magnetic field while the radiofrequency is kept constant. by scanning the radiofrequency while the applied magnetic field is kept constant. (a) and (b).

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21. Which statement about the use of deuterium (D, a) b) c) d) e)

2 1H)

in NMR spectroscopy is TRUE?

it is not NMR-active. its oxide (D2O) can be used to replace exchangeable protons in an analyte molecule. 1 deuterium nuclei will not give a signal under the conditions of  H-NMR spectroscopy. (a) and (b). (b) and (c).

22. Choose the best statement about nuclear magnetic resonance frequency: a) b) c) d) e)

it is directly proportional to the magnetic field strength. a nucleus’ basic resonance frequency can be calculated from B 0 and . it will be the same for different isotopes of the same element. it will be the same for different elements that have the same spin quantum number. (a) and (b). 1

23. Choose the best statement concerning H-NMR spectra: a) b) c) d) e)

the number of signals reflects the number of carbon atoms present in the molecule. the scaling on the x-axis is MHz. signals may consist of one line only or of several lines. a molecule containing a total of 15 protons will give a spectrum with 15 signals. aromatic protons’ signals are found in the range of approx. 9-11 ppm. 1

24. Choose the best statement/s concerning the (n+1) rule in H-NMR spectroscopy: a) b) c) d) e)

add 1 to the number of all hydrogen nuclei on adjacent carbons. add 1 to the number of equivalent hydrogen nuclei on the adjacent carbon atoms. the rule is used to calculate the multiplicity of a signal. (a) and (c). (b) and (c).

25. How can you differentiate between a “doublet of doublets” (dd) and a “quartet” (q)? a) q will consist of 5 lines, while dd will consist of 9 lines. b) q will consist of 4 lines, while dd will consist of 6 lines. c) q will consist of 4 lines of equal intensities, while dd will consist of 4 li nes with different intensities. d) q will consist of 4 lines with different relative intensities, while dd will consist of  4 equal lines. 26. Which statement about proton NMR is not true? a) The proton NMR spectrum generally indicates the number of different types of protons. b) The spectrum gives some indication of the chemical environment  in which each type of  proton resides. c) The spectrum can be integrated to give the relative ratios of each type of proton. d) Spin-spin splitting in the spectrum gives information about protons immediately adjacent  to a given type of proton. e) All these statements are true.

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27. In nuclear magnetic resonance spectroscopy, protons in different molecular environments show resonances at different magnetic field strengths because a) b) c) d) e)

they have fast relaxation times. hydrogen has more than one isotope. of the variety of spin energy levels. of the closeness of rotational energy levels. of differences in shielding by local electrons.

28. The relative peak intensities of a proton multiplet arising from a proton adjacent to three neighboring protons is a) 1:1:1

b) 1:1:1:1

c) 1:3:3:1

d) 1:2:2:1

e) 1:2:1

29. Which of the following will show a single proton NMR signal at δ 7.1? a) (CH3)2CO

b) CF3COOH

d) CHCl3

e) CH3CH3

c) CCl3COCH3

30. How many different absorptions (sets of peaks) would there be in the 1 H-NMR of this molecule? a) b) c) d) e)

Three Four Five Six Twelve

31. What is the approximate multiplicity of the indicated protons in the 1 H-NMR? a) b) c) d) e)

Q (triplet), R (septet), S (singlet). Q (triplet), R (sixtet), S (triplet). Q (quartet), R (quintet), S (triplet). Q (triplet), R (multiplet), S (doublet). Q (quartet), R (multiplet), S (doublet). 1

32. The accompanying H NMR spectrum would correspond to which of the following molecules?

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a)

b)

d)

e)

c)

33. The pattern in the accompanying spectrum is immediately recognizable as what structural fragment? Assume that groups X and Y do not have protons that would affect the splitting patterns.

a)

b)

c)

d) CH3CH2X

e) X-CH2CH2-Y

34. The pattern in the accompanying spectrum is immediately recognizable as what structural fragment?

a)

b)

d)

e)

c)

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QUESTION V

Problems on Chromatography (15 pts)

1. The following data were obtained for separation of  Methanol and Ethanol on a GCchromatograph using a 50-cm long packed column at 200 ºC: Methanol: tR = 1.73 min w = 0.22 min Ethanol: tR = 1.90 min w = 0.24 min An injection of air (unretained) gave a peak at 0.30 minutes. i.

Draw the complete chromatogram considering all the given data. (4 pts)

ii. What equation/s do you need to calculate the height equivalent to theoretical plate (HETP) for Ethanol on this column under the given conditions? (1 pt) a)  HETP   A  d)  N  

 B u

 C * u

5.54  t R

w0.5

b)  HETP   H  

 L  N 

c)  N  

16  t R

2

w2

2

2

e) (b) and (c)

f) (b) and (d)

iii. Find the adjusted retention time for the compound with the smaller capacity factor. (1.5 pts) tR’(Methanol) = 1.73 –  0.3 min = 1.43 min iv. Calculate the carrier gas flow rate (cm/min) used to acquire the above data. (1 pt) vx = L / tM = 50 cm / 0.3 min = 166.6 cm / min v. Using the data given, calculate the capacity factor for Ethanol. (1.5 pts) k’(EtOH) = (tR(EtOH) –  tM) / tM = 5.33

2. An HPLC analysis was conducted for caffeine on “Super -Extra-Energy Formula 2.2 with Hyper-Drive Now!” sports drink. An amount of 10.1 ppm methanol was injected both into the sample and a standardized solution of 304 ppm caffeine. The data measured by a diodearray detector at each λ max for the absorptions for methanol and for caffeine are summarized in the table below.   sample: 304 ppm Caffeine standard:

AUC (methanol)

23141 28441

AUC (caffeine)

52777 77313

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i. What is the role of Methanol here? (1 pt) b) it is an internal standard c) it is the sample solvent e) none of the above

a) it is an external standard d) it is the mobile phase

ii. What equation/s do you need to find the concentration of caffeine in that sports drink? (1 pt) tR VR a)  AUC = 1.064 * h * w 0.5 b) R w av w av c) c analyte

AUC analyte

c standard

F

AUC standard

d) (a) and (b)

e) (b) and (c)

iii. Find the concentration [ppm] of caffeine in that sports drink? (4 pts) F = (AUCa  / ca) / (AUCst / cs) = (77313/304) / (28441/10.1) = 0.0903 ca = AUC / F * cst / AUCst = 52777 / 0.0903 * 10.1 / 23141 = 255 ppm

QUESTION VI

NMR Spectroscopy (12 pts)

1. For each of the following couples, state the expected most striking difference in their 1 H-NMR spectra: (4 pts) H3C

H3C

CH2

CH3 O

O

CH3

O CH2 O

vs.

In the range of 0-5, compound I shows three peaks that are all singlets while compound II exhibits in the same range three peaks, singlet, quartet and triplet. O OR R In the range of 0-5, compound II, shows triplet and quartet signals while compound I shows only singlet peaks. H3C

H3C CH3

vs.

O

CH3

Compound I shows all in all two signals that appear as singlets with integration (3:2), while compound II shows four signals (singlet (3H), singlet (3H), doublet (2H), and doublet (2H). O OR R Compound II is only one that shows doublet peaks in the range of 7-8 (aromatic protons).

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CH2Cl

CH2Cl CH2Cl

CH2Cl

CH3 CH2Cl

CH3 CH3

vs.

In the spectrum of compound I we have two signals integrated to 2:1 while in the spectrum of compound II the two signals integrate to 3:2.

BrCH2CH2CH2Br

vs.

BrCH2CH2CH2NO2

two signals

three signals 1

2. Predict the structures of the compounds that give the tabled H-NMR data: (4 pts) i. C9H10O: 1.2 ppm (t, 3H),

3.0 ppm (q, 2H),

7.4 – 8.0 ppm (m, 5H)

IHD = 5 ii. C8H7N: 3.7 ppm (singlet, 2H.),

7.2 ppm (multiplet, 5H)

IHD = 6

1

3. Plot the H-NMR spectra of the following compounds. (4 pts {2+1+1})  Don’t forget to indicate integration of the signals as well as the labels and scaling on the axes, where applicable.

CH3 O

O

NH2

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CH3 O

CH3

H2 C H2C H2C

CH2 C H2

CH2

Part II Choose 1.

Answer for Each Question (10 pts)

Which sequence reflects the eluotropic series for chromatographic separation with silica gel as stationary phase? a) b) c) d) e)

2.

One  Best

diethyl ether –– acetone – methanol – water. hexane - diethyl ether – acetone - methanol – water. diethyl ether – hexane – acetone – water – methanol. acetone – hexane – diethyl ether – methanol – water. diethyl ether – acetone – petroleum ether – water – methanol .

Which statement/s are correct? In Gas Chromatography, the retention ti me of an analyte is influenced by: a) b) c) d) e)

the temperature on the column. the partition coefficient between stationary and mobile phase. the carrier gas flow rate. (b) and (c). all of the above.

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3.

Arrange the depicted molecules according to increasing retention times upon isocratic HPLC separation on normal phase using hexane / 2-propanol (80 : 20) as mobile phase: OH

OH

OH

OH

OH

HO

(1) a) 1, 2, 3, 4 4.

(2) b) 2, 1, 4, 3

CH3

OH

(3) c) 2, 3, 4, 1

(4) d) 3, 2, 1, 4

e) 4, 1, 2, 3

What is meant by reversed phase chromatography? a) use of hydrophilic mobile phases and polar stationary phases b) separation of analytes on polar, not modified stationary phases c) thin-layer chromatographic separation of a mixture of analytes by second development 0 after 90 turn of the sheet d) separation of analytes on stationary phase that was modified with long-chained hydrocarbons e) gradient elution technique employing stepwise decrease of eluent strength

5.

Which variable/s do we use to determine the height equivalent to a theoretical plate? a) b) c) d) e)

6.

Which of the following detectors is/are common in Gas Chromatography? a) b) c) d) e)

7.

retention time half-height peak width column length (a) and (b) all of the above

Flame Ionization Detector Thermal Conductivity Detector Mass Selective Detector (Mass Spectrometer) (a) and (b) All of the above.

The performance of a chromatographic column with a certain length can best be described by the: a) b) c) d) e)

resolution signal to noise ratio retention time peak height number of plates

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8.

Which of the following schemes shows a flow cell (as used in HPLC) with an ideal ratio between internal volume and light path?

9.

A “spectrum” can be a plot of: a) b) c) d) e)

10.

frequency vs. wavelength wavelength vs. wavenumber transmittance vs. absorption absorbance vs. frequency transmission vs. absorbance

In nuclear magnetic resonance spectroscopy, a signal that appears at a great chemical shift can be characterized as: a) b) c) d) e)

“up-field” “deshielded” “diamagnetic” (a) and (b) all of the above

Bonus Question (5 pts) 1

The following H-NMR spectra of FOUR isomers with molecular formula C6H12O2 are shown below. Which isomer produces which spectrum?

Page 20

m m

m

m

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Formula Sheet - Chromatography  A    R  2

 

4

 D

 AUC = 1.064 * h * w 0.5   

t ' R 2

canalyte 

   

t  R 2

t ' R1



F 





C m

K 2

u

 AUC standard 

 C * u

 L  N 

w2

 N  

 N  

 R 

 B

16  t  R

 N  

K 1

t  M 

 K   2



 R 

V s

w0.5

wav



2

2

2

 A /  B  1.25

 

N 

      1  4   1  k 'av  

V  R wav

t  R '  t  R  t  M  u x   L

t m

d  p [  m]

 N      1   k '2  

t  R

t s

3500 * L [cm]

41.7(t R  / w0.1 )

 4  



V m

5.54  t R

cstandard 

t  R1

 HETP   H  

C s

k '1

 AUC analyte

 HETP   A 

K  

k '2

t ' R

k ' 

2

t  M 

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uV   u x * Amob V  M   t  M  * uV 

V  R  t  R * uV  V e  V 0  KV i V t  = V g + V i + V 0 I

100 n

(N

n)

log t' R (unk ) log t' R ( N)

log t' R (n) log t' R (n)

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