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Anal. Chem. 1992, 64, 1669-1675
Microliter Sample Introduction for Open Tubular Column Supercritical Fluid Chromatography Using a Packed Capillary for Solute Focusing Iina J. Koski,?Karin E. Markides,t Bruce E. Richter,$and Milton L. Lee' Department of Chemistry, Brigham Young University, Provo, Utah 84602-1022
Llquld samples were Introduced Into an open tubular column wpercrltlcal fluld chromatograph(SFC) udng a short packed caplllary column as a solute focurlng devlce after a typlcal sample loop valve Injector. The packlng materlals were held lndde the caplllary by porous ceramlc frlts. Excess solvent was vented Into the atmospherethroughthe packedcaplllary. Slnce the exit end of the packed caplllary column was at atmosplwrk pressure, a denslty gradlent was established along the length of the column and solutes preclpltated at the polnt where the dendty dropped below the carbon dloxlde solvatingdensity for these compounds. The solutes were then backflushed from the precolumn Into the analytlcal column wlth wpercrltlcal COS. Thls method made lt pomlble to Inject rdatlvdy large volumes (1-3 pL) Into a 50-pm4.d. opentubular wpercrltlcal fluld chromatography column wlthout floodlng the column wlth the solvent. The reproduclbllltyof thls InJectlon methodwas compared wlth other lnjectlon methods usedwlth SFC; the relatlve standard devlatlon (five measurements) of peak areas of n-hexadecane was 5.5 %.
INTRODUCTION In trace analysis, large injection volumes are preferred because preconcentration of the sample is not necessary. If a 200-nL sample volume, which is the most commonly injected sample volume in supercritical fluid chromatography (SFC), is introduced into an open tubular column with split injection (split ratio 1:100) the concentration of the solutes must be 50 ppm for the solutes to be detected (0.1-ngFID detection limit). If the sample volume can be increased to 1or 3 pL, the required concentration becomes only 100 or 33 ppb, respectively. Peaden et al.1 discussedhow resolving power in open tubular column SFC is affected by the injection volume. The sample volume (injector volume), V, was related to the fractional loss in resolution, AR,according to the equation
V = 0.866~d,2(Lh)'/~
L1-
- 111'2(1+ k)
where d, is the column diameter,L is the length of the column, h is the theoretical plate height, and k is the capacity ratio. Examples of permissible volumes causing 1% , 3 % , 5 % ,and 10% loss in resolution in 50-pm4.d. columns are shown in Table I. The liquid sample solvent is usually a stronger solvent for the sample than the low-density supercritical fluid. Hence, solutes are carried down the column with the solvent until
* To whom correspondence should be sent.
f Current address: Harvard School of Public Health, Department of Nutrition, 665 Huntington Ave., Boston, MA 02115. t Department of Analytical Chemistry, University of Uppsala, 75121 Uppsala, Sweden. I LeeScientificDivision,DionexCorporation,SaltLakeCity,UT84123. (1)Peaden, P. A,; Lee, M. L. J. Chromatogr. 1983,259,l-16.
0003-2700/92/0364-1669$03.00/0
Table I. Resolution Loss as a Function of Injection Volume. resolution loss ( % ) ~~~~
injector volume (nL) ~
1 3 Conditions: 2.5-m
resolution loss ( % )
injector volume (nL)
5
152 223
~
65 116 X
10
50-gm4.d. column, k = 3.2, h = 1.04 X lo4
m.
the dissolvingpower of the solvent decreases due to the mixing of the solvent and the mobile phase. Peak broadening occurs in a similar fashion as in liquid chromatography (LC) when the sample is injected in a solvent that is greater in solvent strength than the mobile phase.2 Because even a 200-nL injection volume causes 10% resolution loss in a chromatographic separation using a 50-pm-i.d. open tubular column, it is necessary either to refocus the solutes or to eliminate the solvent and, hence, to reduce peak broadening. Hirata et al.2,3described a splitless injection method in which up to 1p L of sample was injected. The sample solution was diluted with supercritical carbon dioxide in a mixing chamber. Dilution made it possible to focus the solutes at the beginning of the column. Peak tailing was reduced by purging the mixture chamber to atmosphere after a specific period of time. Sample volumes were usually 0.5 pL, but when two mixing chambers were connected in series, injection of 1pL of some solvents became possible. The difficulty of injection increased with increasing polarity of the solvent.3 When the mixing chamber was connected with a solvent vent injection technique, the injection volumeswere increased even up to 100 pL.4 Previously we have injected l-bL samples into open tubular columns using a solvent vent injection method.5 The sample was introduced into 15-25-pm-i.d. precolumns by a 1-pL volume internal sample loop injector. The solvent was vented with nitrogen. The chromatographic run was started by introducing supercritical carbon dioxide into the precolumn which was connected to a 50-pm4.d. analytical column. Because the solvent was eliminated prior to the chromatographic run, column efficiency was not reduced due to the liquid solvent. As the precolumn internaldiameter decreased, column efficiency increased. Column efficiencieswere similar to those obtained with split injection. When large volumes were injected into these small i.d. precolumns, part of the sample was lost during the solvent vent. When large i.d. (100 pm) precolumns were used, the length of the precolumn was usually increased if the sample was lost during the solvent vent. The length of these small i.d. precolumns cannot be (2)Hirata, Y.; Tanaka, M.; Inomata, K. J.Chromatogr. Sci. 1989,27, 395-398. (3) Hirata, Y.; Inomata, K. J. Microcol. Sep. 1989,1,242-248. (4)Hirata, Y.;Kadota, Y.;Kondo, T. J. Microcol. Sep. 1991,3,17-25. (5)Koski, I. J.; Markides, K. E.; Lee, M. L. J.Microcol. Sep. 1991,3, 521-529. 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 15, AUGUST 1, 1992
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increasedwithout limits because small internal diameter precolumns act as restrictors in front of the column making the chromatography difficult. The use of different internal diameter open tubular precolumns in solvent vent injection has also been reported by other research groups;6J generally it can be stated that as the injection volume increases, the column efficiency decreases. Munder et al.8used a packed LC guard column as a solute trap for supercritical fluid extraction-chromatography (SFESFC). The restrictor and the analytical column were connected to one end of the guard column while the vent/ backflush line was connected to the other. In our study, it was evaluated whether or not an adsorbent could be used for solute focusing when the sample is introduced in a liquid phase.
EXPERIMENTAL SECTION PackingMaterialDeactivation. All of the packing materials used in this study were deactivated and coated accordingto Payne et al.9 The deactivation and coating method was based on a dehydrocondensation reaction between a polymeric silicon hydride reagent and the silanolgroups on the surfaceof the particles. The method is described here only briefly. Purified and acidtreated particles were washed with LC-grade water, weighed, and transferred into a reaction vessel. In the reaction vessel the particles were heat-dried under Argon gas. Then deactivation reagent (50% n-octylpolymethylhydrosiloxaneor 25 % n-octadecylpolymethylhydrosiloxane) in n-hexane was added to the particles. Argon gas which was introduced into the vesselthrough the sintered-glassbottom kept the particles moving in the coating solution. After the solvent had evaporated, the whole system was heated to bond the deactivation and coating reagent onto the particles. Prior to the packing of the capillariesthe particles were washed with solvents. The particles were from Vydac (The Separations Group, Hesperia, CA), Deltabond (Keystone Scientific, Bellefonte, PA), or Nucleosil (Macherey-Nagel, Diiren, Germany). The capillaries were packed with 5-pm of n-octylpolysiloxane-bonded silica particles, 30-pm of n-octylpolysiloxane-bondedglass particles, or 50-pmof n-octadecylpolysiloxanebonded glass particles. Packed Column Preparation. Short (10-cm)lengths of 200pm-i.d. fused silica tubing were packed with various packing materials. Two different methods for retaining the particles in the column were used: one using zero dead volume connectors (Valco Instruments, Houston, TX) and microscreens (V16-in. diameter X 0.2-pm mesh, Mectron Industries, City of Industry, CA)8 and one using a porous ceramic support.l0 A schematic diagram of the zero dead volume connector and the packed capillary is shown in Figure 1. The porous bed supports were made from potassium silicate solution (Kasil #1,The PQ Corp., Valley Forge, PA) with a weight ratio of SiOdK20 of 2.5, which was mixed with formamide (Matheson, Cincinnati, OH) in a weight ratio of lO/l-5/1 at room temperatureaccording to Cortes et al.l0 The ceramic material was deposited inside the fused silicatubing by inserting the end of the tubing into the premixed solution. The solution was drawn into the tubing by capillary action. The material was polymerized by heating with a hightemperature blow drier for about 5 min. After the ceramic support was polymerized, the columns were packed using supercriticalcarbon dioxide. The packingreservoir (Figure 2) was filled with excess,bulk, dry packing material. The reservoir was sealed and connected to the SFC pump. Then low-density carbon dioxide (0.2 g mL-l) was introduced into the reservoir at room temperature. Carbon dioxide pushed the (6) Farbrot-Bushke, A.; Berg, B. E.; Gyllenhaal, 0.;Greibrokk, T. J. High Resolut. Chromatogr./Chromatogr. Commun. 1988,ll, 16-20. (7)Johansen, H.;Doehl, J.; Greibrokk, T. J . Chromatogr. Sci. 1989, 27,378. (8)Munder, A.; Christensen,R. G.; Wise, S. A. J. Microcol. Sep. 1991, 3,127-140. (9)Payne, K.M.; Tarbet, B. J.; Bradshaw, J. S.;Markides, K. E.; Lee, M. L. Anal. Chem. 1990,62,1379-1384. (10)Cortes, H. J.; Pfeiffer, C. F.; Richter, B. E.; Stevens, T. S. J.High Resolut. Chronatogr.lChromatogr.Commun. 1987,10,446448.
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