CHR OM ATOGR APHY
Application of OPLC for the Purification of Crude Samples at the Milligram Scale D. Papillard, S. Laroche , M. Manach
A new H P L C a cces s or y a llows the high- per for m a nce pur ifica tion of cr ude s a m ples a t the m illigr a m s ca le. T his new device ca n be integr a ted into a ny H P L C s ys tem . Cr ude ex tr a cts conta ining tens of m illigr a m s ca n be injected without pr ior pr epa r a tion. T he colum n des ign ( Òfla t colum nÓ) a llows the s cientis t to vis ua l ins pection the colum n a nd ens ur e tha t a ll fr a ctions ha ve a ctua lly been eluted. Optimum Per for mance L aminar Chr omatogr aphy, or OPL C, is a liquid chr omatogr aphy technology enabling high per for mance separ ations of complex samples for both, analytical or pr epar ative pur poses [1, 2]. T he OPL C technique is based on the use of a flat 192mm long capillar y column of var ious widths and thick nesses (up to 192x192x0,5 mm). Var ious separ ation phases ar e available, including R P18, Nor mal Phase and Diol. T he use of 5 or 10 µm par ticle sizes for the
Channel 1 : DETECT 535-1 mV
F ur ther mor e, pur ity checks of all the collected fr actions can also be car r ied out in a single r un on the same H PL C accessor y. Aliquots of a few micr oliter s of each fr action ar e spotted next to each other on the dr y, flat capillar y column. T he column is then intr oduced into the OPL C Pur ification Unit and elution is done via the H PL C pump. H owever, this time the flow of eluent is stopped befor e it r eaches the end of the column (compounds ar e not eluted out of the column, but ar e k ept inside). T he column is r emoved fr om the Pur ification Unit and pur ity can be evaluated qualitatively using a UV lamp, or quantitatively by spectr odensitometr y. T he OPL C pur ification Unit is an H PL C accessor y which can easily be inser ted into any classical H PL C system. I t is a useful tool for milligr amscale pur ification of cr ude samples for any r esear ch labor ator y. T he combination of moder ate cost of the flat capillar y HT Sor b columns, the elimination (or simplification)
T his unique featur e fur ther impr oves the peak shape and consequently, the pur ity of isolated fr actions. T he OPL C Pur ification Unit has been used to pur ify var ious cr ude samples, such as plant extr acts, synthetized chemical molecules, cell br oth, and biological fluids [3, 4]. As a fir st example, we descr ibe her e the scaling-up of a separ ation per for med on a commer cial whiskey sample (F ig. 1a, b, c). Pr ior to injection, the sample was par tially evapor ated to r emove alcohol and other volatile molecules. T he exper iment was per for med on a HT Sor b r ever se-phase R P18 column (200 µm thickness). Cr ude biological samples wer e also fr actionated. F ig. 2 shows the separ ation of a cr ude ur ine sample (inj vol 800 µl) injected without any pr ior tr eatment or solvent extr action. T he capacity to wor k with lar ge volume injections facilitates the r ecover y of tr ace constituents and their subsequent identification by mass spectr ometr y or nuclear magnetic r esonance.
2000
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23.59:9
200
columns enables high-per for mance separ ations. After a separ ation, columns can be inspected and any constituents r emaining on the stationnar y phase can be detected, e. g. with a UV lamp. Visual inspection of the column is a power ful and unique possibility which allows the analyst to have a full under standing of how the sample constituents behave in a par ticular chr omatogr aphic method. And when the sample is not too dir ty, the column can be r einser ted and used sever al times. T he OPL C Pur ification Unit is the housing for the flat column and together they have been designed to tak e the place of a standar d H PL C column. T he Pur ification Unit can be connected to an exter nal pumping system, typically an H PL C pumping system that will deliver the eluent(s) at a pr ogr ammed and steady flow r ate. I n or der to eliminate wall effects dur ing separ ation, a special, patented F E W (F lowing E luent Wall) holder has been developed.
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39.09:19 41.00:20 42.04:21
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Fig. 1: Pure malt whiskey separation (50 µL, 500 µl, 1000 µl) Eluent A: Water + 0.1 % TFA; B: CH3CN + 0.1 % TFA, gradient from 1 % B to 50 % B in 45 mn; a) injection 50 µL; b) injection 500 µL; 2 G .I.T. Laboratory Journal 1/2003
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CHR OM ATOGR APHY
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UV (254.0nm) UV (230.0nm) UV (215.0nm)
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37.86:15 38.84:16 40.42:17 41.96:18 43.39:19
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of sample pr epar ation steps, and the assur ance of r ecover y of all constituents - while avoiding the r isk of clogging mor e expensive HPL C columns - decr eases the cost of access to pr epar ative-scale, high-per for mance chr omatogr aphy.
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Fig. 2: Sample: 800 µl urine, Column HTSorb RP 18 Eluent; A: Water + 0.1% TFA. B: CH3CN + 0.1% TFA, gradient from 1% B to 40% B in 36 mn
Literature
tonen, P. Sunila: J .L I Q.Chr om. & R el. Technol., 1425-1434 (2001)
[1] E . Tyihak , E . Mincsovics, T. J . Szè e k ely: é
J.
Chr omatogr.
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[4] B . Dalmadi K iss, I . K lebovich, E . Mincsovics, K . B alogh-nemes: Pr oc.Planar Chr omatogr. 2000,
(1979) 75 [2] S. Nyir edy: T r ends in Analytical
Domitille Papillard Sylvie Laroche Application Laboratory, Bionis SA
L illafü u r ed, H ungar y pp. 57-65 ü
Michel Manach R&D Department, Bionisis SA
Chemistr y, vol. 20, n°2, 2001 [3] A. Pelander, I . Ojanper à, J . Sis-
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