EXAMINATION IN COURSE KJ 2053 CHROMATOGRAPHY

NTNU Norwegian University of Science and Technology Faculty of Natural Sciences and Technology Department of chemistry Page 1 (of 13) Inquiries concerning the contents of the test to : Assoc. professor Rudolf Schmid, tel./phone: 735 96203 (evt. Mob/Cell: 91375546) Department of chemistry, 735 50870 Student nr. : EXAMINATION IN COURSE KJ 2053 CHROMATOGRAPHY - SATURDAY, MAY 24TH 2013, 09:00-13:00 (4 HOURS) Answers for questions 1-8 are to be written into this questionnaire, which is delivered to the examination officer at the end of the examination. If not enough space is available in the questionnaire for your answer(s) use the opposite side/back side, or separate answer sheets which are returned together with the questionnaire. Refer to separate sheets in the questionnaire for the questions where this is relevant. Permitted aids: code D B1-type calculator with empty memory (as specified in NTNU s list of calculators approved for use in examinations at NTNU) No other aids (i.e. printed or handwritten texts or notes) are allowed. This test consists of sixteen - 16 - pages : 1 cover page, 1 front page (p. 1), 14 pages with 8 questions (p. 2-15). Weighting factors for the questions are given in the heading of the question. A maximum of 102 points may be obtained. In order to achieve full score 100 pts. must be achieved. Sign... Rudolf Schmid Responsible examinator Examination text checked by: Sign. Lise Kvittingen Deadline for the results to be announced: Saturday, June 14th, 2014.

(of 15) Question 1 : (5+4+5= 14 p.) In chromatographic theory the terms/symbols k, α, t R, R S, N are often used : 1.a Give the full name of these terms/symbols and briefly explain what they (5p) are/ express: k = α = t R = R S = N = 1.b List the expressions you know that express the efficiency of a chromato graphic (4 p) column, and how you determine/calculate it from a chromatogram. Navn of expression Symbol description/equation

(of 15) 1.c (i) What are the lowest values for minimumsverdien for α og R S? (1 p) < α.. < R S (1 p) (ii) Which ones of the terms in 1.a (above) are dependent on column properties only, and are not influenced by the nature of the analyte? (1 p) (iii) Which ones of the terms in 1.a can be increased in order to increase R S? (1 p) (iv) Explain the relation between k, t R og t 0? (1 p) (v) Illustrate graphically below a situation where R S = 1,5 (include what the axes represent).

(of 15) Question 2 : (12+4= 16 p) 2.a Indicate whether the chromatographic techniques listed below are (i) partition or adsorption chromatography, respectively, and (ii) either GLC, GSC, LLC or LSC. (12 p) Chromatographic metod (i) (ii) Partition/adsorption GLC/GSC/LLC/LSC Paper Chromatography (PC) 1p 1p Thin-Layer Chromatography (TLC, silicagel) Size Exclusion Chromatography (SEC, Gel Permeation Chro., PSV-DVB col., THF) HPLC (silicagel 60, acetonitrile:water, 80:20) Gas Chro. (packed column, stationary phase: PDMS ('dimethyl siloxane')) Gas Chro. (PLOT-column, stationary phase: zeolite molecular sieve) Sold Phase Extraction (SPE octadecyl silica: 1.) 50% methanol, 2.) ethyl acetate) 1p 1p 1p 1p 1p 0,5p 1p 0,5p 1p 0,5p 1p 0,5p 2.b (i) What are the advantages of planar chromatography, e.g. TLC, when compared to column chromatography (HPLC)? (2 p) (2 p) (ii) Briefly, describe the principle and typical area of application of EITHER : (Bio )Affinity Chromatography (BAC), OR : Hydrophobic Interaction Chromatography (HIC), OR : Isoelectric Focusing (IEF).

(of 15) Question 3 : (5 p) A polystyrene oligomer mixture (poly-phenylethene, degree of polymerization n = 1 - ~8) should be analysed: the following experimental conditions are suggested used by a slightly unexperienced summer job student: Capillary column (10 m x 0,1mm) w. siloxane polymer film (95% methyl:5% phenyl, crosslinked); mobile phase: CO 2, pressure increasing from 3 bar to 20 bar, temperature increasing from 50 C to 250 C; flame ionisation detector (at atmospheric pressure, i.e. after the mobile phase-flow restrictor). (i) The intention is to do the analysis by Supercritical Fluid Chromatography, SFC: Express your opinion on this choice for this samples type (polystyrene oligomers) and compare it to possible alternatives. (1 p) (ii) Are the choice of fluid (CO 2, T c =31 C / p c = 73 bar), and the conditions of the fluid gradient, as suggested above, adequate for performing a successful SFC analysis? (1 p) (iii) Do you agree with the choice of detector (FID), and with its suggested position in the experimental set-up of the SFC system? (Briefly explain.) (1 p) (iv) A UV-detector is often used in SFC. Is it suitable for this sample type? Where in your system do you install a flow restrictor when using a UVdetector?

(of 15) Question 4 : (5+6+6=17 p) 4.a) (i) What is, and where do we find, stagnant mobile phase, smp? (Brief and (1p) general answer) (ii) Which (physical) parameters do influence the contribution to band broadening originating from the stagnant mobile phase? (iii) What can you say about stagnant mobile phase in : Technique is there smp? Yes / No Comments / explanations TLC (silicagel) SEC ('Gel Chromatography') RP-LC using core/shell, (pellicular, porous surface layer) particles Capillary GLC Ion exchange chromatography on xerogel (soft) particles Bonus 0,5 p. Zone electrophoresis Bonus 0,5 p

(of 15) 4.b (i) Draw a representative partial molecular structure of the surface of (untreated) silica. (ii) What is the most popular stationary phase (SP) for reversed-phase (RP-) HPLC? Give specific name, and draw a representative (partial) structure. (iii) Give a name, and draw a structure of the stationary phase we used in our lab. experiments in GLC (and which is the most popular SP type in GLC).

(of 15) 4.c (i) Describe briefly the intermolecular forces that act between species (molecules, atoms, ions) which are involved in chromatography. (ii) List the dominating intermolecular forces which are active in(-side) the following phases, between anayte, MP and/or SP), and rank them according to strength (from weakest to strongest): A in GSC MP: B in ion-exchange SP: C in RP(C 18 )-LC SP: D in NP-LSC (silica) SP: Ranking : < < < < (1p) (iii) EITHER: What is the role of water in mobile phases in RP-HPLC? OR : What are the three properties that are used to classify mobile phases for HPLC in Snyders Selectivity Triangle?

(of 15) Question 5 : (2+3=5p) 5.a EITHER: Describe the two alternative injection modes when using (manual) valve (2p.) injection in HPLC. OR : What is high-pressure mixing and what is low-pressure mixing in HPLC? 5.b) (i) Draw a representative (partial) molecular structure of a strong anion exchanger. (1p.) (1p.) (ii) Draw a representative (partial) molecular structure of a strong cation exchanger. (1p.) (iii) List 2 ways to increase eluent strength in ion-exchange chromatography on a cation-exchanger (without changing the ph significantly).

(of 15) Question 6 : (6+6= 12 p + 1 bonusp.) 6.a The gas chromatogram below shows the separation of a mixture of volatile (5p.) hydrocarbons on a speciality column.. Peak select. solvents bp.[ C] 1 n-hexane 69 2 n-heptane 98 3 n-octane 126 4. n-nonane 151 5 n-decane 174 6 benzene 78 7 toluene 111 8 ethylbenzene 136 (from Restek Catalogue 2011/12, p.654) 10 isopropylbenzene 153 (2p.) (i) Was this chromatogram obtained on a capillary column or on a packed column? Briefly explain. (1p.) (ii) Is the analysis done isothermally or by using a (linear) temperature program? Briefly explain. (1p.) (iii) Is the stationary phase in this analysis polar or non-polar? Briefly explain. (1p.) (iv) All 3 xylene (dimethylbenzene) isomers are present in the sample, however there are only 2 distinct non-identified peaks visible. Based on the chromatogram above, explain what happened - where the "missing peak" could be. (1p.) (v) Briefly describe, how you would proceed to safely identify the 3 xylenes in the chromatogram.

(of 15) 6.b Detection in the above chromatogram (6.a) is by FID. (i) Give the full name of the FID, and a brief description (may include an illustrative figure) of how the FID "works". (ii) Could use of GC/MS (coupled gas chromatography-mass spectrometry) with Electron Ionisation (EI) help with the identification of the "missing peak" in the chromatogram of Q. 6.a? Briefly explain. (1p Bonus) (iii) The "group separation" in the figure of Q. 6.a (volatile aliphatics before aromatics) is only possible with special columns. For analysis of the 2 groups separately on ordinary GC columns, a work-up method achieving "group separation" would have to be used before the GC analysis. Could SPE (Solid Phase Extraction) be applied here? Explain your conclusion. If the answer is yes, suggest a SPE separation ('SPE-chromatography') technique (a 'sorbent') you would expect to allow this separation. (iv) What are the 2 major advantages of Solid Phase Extraction (SPE) for sample preparation, when compared to classical liquid-liquid extraction? - -

(of 15) Question 7 : (2+5=7 p.) 7.a For 4 of the analytes eluted in the chromatogramt below, tebuthiron 1, diuron 5, propanil 6, diflubenzuron 10. the un-assigned molecular structures A D are given below: (from Restek-Catalogue 2011/12, p.712) (i) Assign the structures A - D to the names/peaks 1, 5, 6 and 10 based on the observed relative retention on the octadecyl-silica column in the chromatogram shown above. Argue for your choices based on chemical structure (the different functional groups present). Peak 1 = peak 5 = peak 6 = peak 10 =

(of 15) 7.b (i) What happens in the chromatogram between about 11 and 17 min, when the base line is sinking? (1p) (ii) Why is the analysis starting with an isocratic section of 8 minutes (at 40 % (1p) acetonitril in the buffer) before the gradient starts? (1p) (iii) Which one of the 3 peaks, 3, 6 and 10, will achieve the highest, correctly determined, plate number in this analysis? Explain your choice (no measurements or calculations should be required). (iv) Is it possible to calculate a, correctly determined, separation factor, α, for peaks 4 and 5 in the chromatogram of Q. 7.a? If the answer is affirmative, give an estimate for the value of α 4,5 (set up the equation with symbols, and with the measured or estimated numbers).

(of 15) Question 8 :. (25p) Answer yes/no, and add a (compulsory) brief explanation ( because ) : (1 point pr. correct and explained answer : without explanation a yes/no answer is considered guessing and is valued as follows : correct answer is + 0,2 points, wrong answer is -0,2 points (subtraction!). 1. Peak A elutes after 1.0min, which also is t 0 = t MF, and peak B at 3.0min. In this case the separation factor is = 2.0. 2. In Hydrophilic interaction chromatography (HILIC) the particle surface must be hydrophobic. 3. In Micellar Electrokinetic Chromatography (MEKC), the typical analytes are inorganic ions, separated according to ionic radius. 4. A strongly basic anion-exchanger is not basic/alkaline at all, actually. 5. Silica-based RP columns tolerate much better alkaline conditions than RP columns made from organic polymer particles. 6. Partition coefficients for crucial analytes in Size Exclusion Chromatography (SEC) should preferably be less than 1,0. 7. Gel permeation chromatography is Size Exclusion Chromatography, SEC. 8. Aluminium oxide (alumina) thinlayer chromatography (TLC) plates are typically used for reversedphase TLC analyses. 9. Eddy diffusion does not occur in simple capillary electrophoresis (CZE). 10. In ion chromatography normally uses the conductivity detector, a universal detector for ions. 11. In selectivity curves of SEC gel materials K av increases with increasing molecular mass of the analytes. 12. PLOT columns are capillary columns for use in GSC. Yes/No, because. continued on next page

(of 15) (Question 8 continued ) Answer yes/no, and add a (compulsory) brief explanation ( because ) : (1 point pr. correct and explained answer : without explanation a yes/no answer is considered guessing and is valued as follows : correct answer is + 0,2 points, wrong answer is -0,2 points (subtraction! ). 13. Enantiomeric compositions of chiral compounds can be determined by "achiral" chro., if the analyte can be derivatised with a pure chiral reagent. 14. In Capillary Electrophoresis (CE), ions will always move/migrate towards the electrode of opposite charge, dues to the electrostatic attraction. 15. In split injections on capillary GC columns the Solvent Effect is crucial for getting narrow peaks. 16. Carbowax is the name of a group of GLC SP's consisting of polyethylene glycols, considered polar SPs in GLC. 17. In RP-HPLC retention times increase when we use mobile phases with larger eluent strength. 18. The MAOT (Max. Allowable Operating. Temp) of GLC-SP s is the same as the SP s Flash Point (as it is based on fire safety considerations). 19. In an Amino Acid Analyser amino acids samples are applied at low ph onto a anion-exchange column. 20. The best separation power in TLC is at R F values of 0,2 - ~0,5. 21. The UV detector in HPLC is a masssensitive detector. 22. It is not possible to us e the External Standard calibration method when the analytes' identity is not known. 23 In detector-oriented derivatisations, the mixing and the reaction of analyte and reagents must be done pre-column. 24 The presently most common ionization technique in coupled LC/MS is Electrospray ionisation (ESI). 25 In separations by SEC of macromolecules it is best to keep the eluent velocity low. Yes/No, because.