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Anal. Chem. 1993, 65, 1125-1129
AC RESEARCH
Solid-Phase Injector for Open Tubular Column Supercritical Fluid Chromatography Iina J, Koski, Edgar D. Lee, Ivan OstrovskyJ and Milton L. Lee' Department of Chemistry, Brigham Young University, h o u o , Utah 84602
The design and use of a solid-phase injector for open tubular column supercritical fluid chromatography (SFC) are reported. For sample introduction, a liquid sample was loaded on a platinum wire. After the solvent had evaporated, the wire was inserted into, and sealed in, the injector. The chromatographic run was started by introducing the supercritical mobile phase into the injector. Absolute and relative peak area reproducibilities, as well as retention time reproducibility, of the injection method were evaluated. The relative standard deviations (% RSDs) of absolute and relative peak areas were 2.0-4.5 and 0.4-1.870, respectively, for both 1- and ~ - M L injections. Larger deviations were observed for compounds that had high vapor pressures. The % RSDs of retention times from six subsequent injections were 0.04-0.06. INTRODUCTION A liquid solvent is generally used in chromatography to transfer a sample aliquot into the stream of mobile phase. If the liquid solvent interferes with the chromatographic process, it must be separated from the solutes a t the beginning of the analysis. In practice, the solvent can be vented through the analytical column1*2or through a waste side stream before the column. In the 1960s and early 1970.9,solventless injection for GC was reported by several groups.3-5 Renshaw and Biran3 used a 'solid-phase" injector to introduce samples into packedcolumn GC. The sample holder was a t one end of a stainless steel rod which was made in the shape of a spoon and capable of holding 0.1-0.2 mL of liquid. A soft iron cylinder a t the other end of the rod enabled the spoon to be manipulated by * To whom correspondence should be sent.
' On leave from the Chemical Institute of Comenius University, Bratislava, Czech and Slovak Federal Republic. (1) Davies, I. L.;Raynor, M. W.; Kithinji, J. P.; Bartle, K. D.; Williams, P. T.; Andrews, C.E. Anal. Chem. 1988,60, 683A. (2) Cortes, H. J.;Pfeiffer,C. D.; Jewett,C. L.;Richter, B. E. J.Microcol. Sep. 1989, I , 28. (3) Renshaw, A.; Biran, L. A. J . Chromatogr. 1962, 8, 343. (4) Ros, A. J. Gas Chrornatogr. 1965, 3, 252. ( 5 ) Van den Berg, P. M. J.; Cox, T. P. H. Chromatographia 1972,5, 301. 0003-2700/93/0365-1125$04.00/0
1
1
6
P - 8
4 Flgurr 1. Failing needie injector for GC in the inject and load posltions. A more accurate figure can be found in ref 9: (1) capillary restriction, (2) injectionport and septum, (3)carrier gas inlet, (4)anatytlcalcolumn, (5) glass needie, (6) magnet, (7) main body of the injector (made of glass), and (8) oven wail.
means of a magnet. After the sample was loaded into the spoon, the solvent was allowed to evaporate a t room temperature. The spoon was then introduced into the chromatograph without admitting air or interrupting the flow of mobile phase. The spoon came into contact with the hot packing material of the column, which caused the sample to be volatilized instantaneously into the stream of mobile phase. Several researchers have modified the injection method described by Renshaw and Biran. R0s4 loaded the sample onto a fine silver thread of 1-mm diameter which was tapered to a point of 0.2-mm thickness and twisted into a spiral of 12 mm X 1 mm. McComas and Goldfiensused a modified syringe to introduce dry samples into GC. A piece of wire was attached to the plunger of a syringe. The wire could be drawn into the needle during insertion of the needle into the injector. The forward end of the wire was ground to a square and twisted (6) McComas, D. B.; Coldfien, A. Anal. Chem. 1963,35, 263.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
Table I. Resolution Loss as a Function of Injection Volume in SFCB
injection vol (nL)
resolution loss ( % ) 1 3
5
Values used in eq 1: 2.5 m m.
X
50 pm i.d. column; k = 3.2; h = 1.04
1 Ll-
ART - 11
(1+ I t ) (1)
internal diameter (i.d.),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% losses in resolution ( M = 0.01, 0.03, 0.05, and 0.10, respectively) in 50-pm-i.d. columns are given in Table I. Because even a 200nL injection volume in a 2.5 m X 50 pm i.d. column causes 10% resolution loss in a chromatographic separation, it is necessary to split during injection, refocus the solutes after injection, or eliminate the solvent before the chromatographic separation in order to reduce the peak broadening. The solvent effect1 is not as easily utilized in SFC as it is in GC because supercritical fluids normally have some solvating power even a t low densities. Soluble solutes as well as the solvent are carried down the column while other solutes that are not dissolved a t low densities are retained in the beginning of the column. Nitrogen gas has been used to purge the solvent from a precolumn, but because the sample becomes dynamically coated on the walls of the precolumn, poor column efficiencies have been observed when large inner diameter precolumns were used.8 ( 7 ) Peaden, P. A.; Lee, M. L. J . Chromatogr. 1983, 259, 1. (8) Koski, I. J.; Markides, K. E.; Lee, M. L. J . Microcol. S e p . 1991,3, 521.
2
3
into a spiral to minimize contact of the wire with the inside surface of the needle. The sample was loaded onto the wire, the solvent was allowed to evaporate, and the wire was drawn into the needle, which was inserted into the injector. The twisted wire was held in the hot mobile-phase stream until the sample had vaporized.6 Van den Berg and Cox5 referred to a similar injection method as the "falling needle" method. The injection needle was a pointed glass rod that was attached to a short piece of glass-enclosed iron rod (Figure 1). The vertical position of the needle was controlled by an external magnet (6 in Figure 1). When the needle was in the raised position (load position), several drops of sample solution were placed on the probe tip (5). The needle was held in the load position until the solvent had evaporated in the carrier gas, which was vented to the atmosphere. The time for evaporation depended on the carrier gas flow rate, temperature, solvent, and volume of the sample. To inject the sample, the needle valve on the top of the injector was closed and the magnet was removed, which caused the needle to fall into the inject p ~ s i t i o n . ~ In supercritical fluid chromatography (SFC), the internal diameters of the open tubular columns that are used are much smaller than those of typical GC columns. Due to these small internal diameters, injection is more difficult. The liquid solvent can easily flood the column, modify the stationary and mobile phases, and affect the selectivity. Peaden and Lee7 discussed how resolving power in open tubular column SFC was affected by the injection volume. The sample volume (injector volume), V, was related to the fractional loss in resolution, M , according to eq 1, where d, is the column
V = O.866ad:(Lh)li2
1
65 116 152 223
10
X
1
11
10
9 4
a 7
Figure 2. Schematic diagram of the soiMphase Injector for SFC: (1) screw for opening and closing the valve, (2) hole for the on-column needle to Introduce the sample, (3)main body of the Injector, (4) Valco butt connector,(5) male nut to prevent the glass connector from moving, (6) analytical column, (7) deactivated glass butt connector, (8) Teflon ferrule,(9)mobilephase Inlet, (10)platinum wlre, and (1 1)Tefsel seal.
All aspects considered, sample introduction in SFC is more difficult to perform than in GC. Particularly for trace analysis, elimination of the sample solvent prior to chromatographic separation has become essential. In this study, the design and evaluation of a solid-phase injector for open tubular column supercritical fluid chromatography is reported.
EXPERIMENTAL SECTION Samples. Pure n-alkane and steroid standard compounds were obtained from Sigma (St. Louis, MO). Maltodextrins were obtained from corn syrup and they were premethylakd according to Ciucanu and Kerek.s A coal tar sample was obtained from the National Institute of Standards and Technology (SRM 1597, Gaitherburg, MD). The analyzed explosives and their possible detonation residues were obtained from Aldrich (Milwaukee,WI), the exceptionsbeing diphenylamine (obtained from Mallinckrodt, St. Louis, MO), pyrene (purchased from Matheson Coleman and Bell, Cincinnati, OH), and 1,3,5-trinitrobenzene, 2,4,6-trinitrotoluene, and RDX, which were supplied by the U S . Department of Defense. Apparatus. The supercritical fluid chromatographic system was a Lee Scientific Series 501 SFC/GC (Salt Lake City, UT). Carbon dioxide was used as the mobile phase (Scott Specialty Gases, Plumsteadville, PA), and solutes were detected with a flame ionization detector. A schematic diagram of the injector is shown in Figure 2. Several different analytical columns were used, and they are identified in the figure legends. The column (6in Figure 1)was connected to a deactivated glass butt connector ( 7 , Lee Scientific) which was sealed to the main body of the injector using a Teflon ferrule (8)and a zero dead volume connector (4, Valco Instruments, Houston, TX). To prevent the glass butt connector from moving from its proper position if the seal was not sufficiently tight, a male nut (5) was connected to the opposite end of the zero dead volume connector. Procedure. The screw (1)was rotated out to the sample load position, the fused-silica needle on an on-column syringe was inserted through a hole in the main body of the injector (2), and the sample was loaded as a droplet onto the platinum wire (30 gauge) of the injector. The solvent was then allowed to evaporate before introducing the wire into the inlet of the injector. Evaporation of the solvent could be easily observed through cutouts in the main body of the injector. The rate of solvent evaporation could be increased by introducing nitrogen or other inert gas through the mobile-phase inlet (9), which flowed out of the sample inlet and around the wire. After the solvent was (9) Ciucanu, I.; Kerek, F. Carbohydr. Res. 1981, 131, 209-217.
ANALYTICAL CMMISTRY. VOL. 85. NO. 9, MAY 1. 1993
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Table 11. Reproducibilities of Retention Times Using the Solid-Phase lniectar in SFC. retention time (minl
BV
5, Std Dev
14.65 0.06
18.21 0.05
Time lMinl
21.02 0.04
a Conditions: 100 'C; density program from 0.18 to 0.76 g mL-' at 0.02 g mL-l min-' after a 6-min constant-density period.
Table 111. Reproducibilities of 2-pL Injections Using the Solid-Phase Injector in SFC. eampd absolute area RSD (%) relative area RSD (%) n-Cia nG"
n-Cm n-Cz, n-CB n-CI4
75 324 98 840 120504 749032 723430 834 685
.1.0 4.3 3.9 3.0 2.7 3.5
9.6824 7.4132 6.0673 0.9657 1.MxM 0.8697
1.8 0.8 0.9 0.4 0.7
Flpum 3. ~ f f e cof t evapratkm nme on sduie recavery using the solid-phase Injector. Condmns: 2-pL lntsctlon volume; 2 m X 50 pm 1.d. column coated wlth methylpoiyslbxanestationary phsse (4 = 0.25 pm); carbon dloxlde at 120 'C dlchlwomethane lnjectbn solvent; solid bar. ratb of hexadecane to eicosene; shaded bar, ratb of
tehadecane to elmsane. n
6 7 7 7 6 7
3
Conditions: 100 'C: density program from 0.18 to 0.76 g mL-' at 0.02 E mL-I min-' after a 6-min constant-density wriod. Table IV. Reproducibilities of I-pL Injections Using the Solid.Phase Injector in SFC' comDd absolutearea RSD (7%) relative area RSD (%) n n-Cta n-Cm n-Crr
n-Cz4 n-Cm n-C:t,
41 306 55250 66714 421020 410482 473 615
4.5 2.0 2.7 2.7 3.5 2.8
9.763 7.434 6.173 0.9758
1.8 1.5 1.4 0.8
LOOM)
0.8658
0.7
4
5
5
5 5 4 5
Conditions: 100 "C; density program from 0.18 to 0.76 g mL-1 at 0.02 g mL-' min-I after a 6-min constant-density period. 12
removed, the wire was inserted into the injector inlet by rotating the screw (1) to the closed position. Then the mobile phase was introduced and the chromatographic run was started.
*
RESULTS AND DISCUSSION The solid-phase injector designed in this study has some similarities to the previously described solid-phase injection systems for G C the loading of the sample onto the injection wire, theeliminationofthesamplesolvent,andthemovement of the wire in and out of the injection zone. However, the system described here was constructed to hold pressures up to 415 atm. Hence, septa could not be used to separate the mobile phase from ambient conditions. The injector body was made of stainless steel, and Tefsel was used as O-rings to seal the injector. Teflon seals were evaluated, but they did not hold the required high pressures. In this injector, the solutes were dissolved by the supercritical mobile phase (not vaporized by the heat of the column as in GC). Therefore. thesystemhadtobeconstructedinsuchawayastominimize dead volumes that would contribute to band broadening. The reproducibilities of the injection system with respect toretention time and peak area were evaluated. The relative standard deviations ( % RSD) of retention times of n-Cw, n-Cwand n-Ca,were0.06,0.05,and0.0% ,respectively (Table 11). The % RSD of peak areas of n-alkanes (n-Cls. n-Cm, n-Cns n-Cz4,n-CZ9,n-Csr)were 2.7-4.3% for 2-pL injections ( n = 6-71 and 2.0-4.5% for 1-pL injections ( n = 4-5). The
0
10
20
30 Tirne(Min)
m e 4. Chromatogram of a mlxtue of expbslvw and pomlble detonatlon residues. Conditkns: 0.2-pL Injectbn volume; 8 m X 50 prn 1.d. column coated wlth p.pcyanoblphenyl polyslbxane statbnaty phase(b= 0.1 pm);carbondloxldeatl00'CCn,CI,injectlonaolvent; mobilephase densily programmed horn 0.2000 to 0.50000 mL-' at 0.0075 g mL-' mln-'. Peak identlflcations: (1) dlbenzofuran.(2) 2 . 6 dinmotduene. (3)dlphenylamlne. (4) 2,Cdlnlbotduene. (5)z-nlbonaphthatane. (6) 2.4.6hlnnroto1uene. (7) 2-nltrodlphenylamlne. (8) 1.3.5binmobsnrene. (9)pyrene. (10) 1.3.5-hlnllro-1.3.5-blaracyclohexane. (1 l)benz[e]anthracene.(12)t-nmopyrene,and(t3)benro[e]pyrone. absolutepeakareasforthe2-and1-plinjectionsaretabdated in Tables 111 and IV, respectively. When nonadodecane (nC.d was used as an internal standard, the relative standard deviations were 0.4-1.8% for 2-pL injections and 0.7-1.8% for I-pL injections. The deviations were larger for lower molecular weight compounds, which are also more volatile. Temperature control of the injector was needed to obtain reproducible results when the oven was held at high temperatures and highly volatile compounds were analyzed. To cool the injector, copper tubing which was connected to a circulating bath was coiled around the lower part of the
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ANALYTICAL CHEMISTRY, VOL. 65,NO. 9, MAY 1, 1993
I
115
100
nme(Mln)
415
Presrure(atm)
0
I 0.18
Time (Mln)
58
hnsb (e mL)
0.76
Figure 8. Chromatogram of a coal tar sample. Conditions: 1-pL injection volume; 10 m X 50 pm 1.d. column coated with 5% phenyl methylpolysiloxane stationary phase (4 = 0.25 pm); carbon dioxide at 100 O C ; dichloromethane injection solvent. It
115
415
pnsrUre(Cmn)
Flgure 5. Chromatogram of a permethylated maltodextrin sample. Conditions: (A) 7- and (B) 14-pL injection volume; 10 m X 50 p m i.d. column coated with 50 % cyanopropyl methylpotysiloxane stationary phase (4 = 0.25 pm); carbon dioxide at 120 O C ; dichloromethane
injection solvent.
injector. The cooling prevented the volatile compounds from diffusing into the column before the mobile phase was introduced. The effect of evaporation time on the recoveries of volatile solutes was evaluated. The injector was left in the sample loading position for different periods of time after the sample had been introduced onto the wire and the solvent had evaporated. The test solutes were tetradecane (n-CId), hexadecane (n-C16),and eicosane (n-Cao). Eicosane was used as a reference solute to measure the loss of tetradecane and hexadecane. A recovery of 100% was assumed when the injector was closed as soon as the liquid solvent had evaporated (observed visually). As much as 33% of the tetradecane was lost if the injector was left in the load position for an extra 30 s, and further losses were observed with longer time periods. The loss of hexadecane was not significantly different (at the 95% confidence level) from full recovery during the 2-min period that the injector was left in the load position, even though some loss was observed. Supportive data are given in Figure 3. Hence, the injector should be closed as soon as the solvent has evaporated in order to prevent losses of volatile compounds. Also, solvents that have high vapor pressures at ambient conditions should be used. If the sample contains only solutes with high vapor pressures, other injection methods are recommended. Column efficiencies were measured and compared to column efficiencies that can be obtained with split injection
I' 5
t
1
30
50
70
rime (Mln)
Flgure 7. Chromatogram of a steroid sample. Conditions: 0.2-pL injection volume; 6 m X 50pm i.d. column coated wlthp,pcyanoblphenyl polysiloxane stationary phase (4 = 0.1 pm): carbon dioxide at 90 O C ; CHZCIZ4- CH30Hinjection solvent; mobile-phase density programmed from 0.1600 to 0.7700 g mL-l at 0.0048 g mL-l min-'. Peak identifications: (1) Sa-androstane, (2) 56-androstan-3@ol,(3) 58androstan9a-01, (4) 5~-androstan-l7@-0l, (5)5a-androstan-38-01,(6) Ba-androstan-l7@oI,(7)Bfi-androstane-36,176-diol,(8)5@-androstane3a, 17@diol,(9) 5u-androstane-3a,176diol, (10) 5a-androstane3@,17@diol,(1 1) da-androstana3,17dione, (12) 4-androstane-3,17dione, and (13) 5P-androstane-3a,1 16,176-triol.
using the same column at the same operating conditions. Tetradecane and hexadecane were used as test solutes. The capacity factors ( k )in split injection for n-C14and n-C16 were 6.3 and 14.1, respectively. The oven was held at 110 "C and the mobile phase was held at 0.16 g mL-I. The average column efficiencies ( n = 8 ) were 4900 and 5000 plates m-1 and the relative standard deviations were 19.6and 27.6%,respectively.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
The column efficiencies obtained with the solid-phase injector were lower than with split injection, approximately lo00 plates m-l. Column efficiencies were improved when the mobilephase density was lowered below the critical pressure a t the beginning of the analytical run. Immediately after the carrier flow was switched on, the density of the mobile phase was increased to a density of 0.16 g mL-1 (110 "C). The average column efficiencies (n = 4-6) using this method were 2700 and 6100 plates m-1 for n-C14 and n-C16,respectively, and the relative standard deviations were 27.9 and 6.7 7%. When the mobile phase was introduced a t low mobile-phase pressures, it did not have the solvating power to immediately dissolve and introduce the sample into the column. Hence, solute focusing during density programming occurred and narrow peaks were obtained. A high-efficiency separation can be seen in Figure 4, which is a chromatogram of a mixture of explosives and their possible detonation residues. Using this injection system, large volumes can easily be injected, making trace analysis possible. By increasing the sample volume from 0.2 (the sample volume of the commonly used internal sample loop injectors) to 14 pL, the concentrations of solutes can be 70 times lower and still be detectable. To illustrate the injection of large sample volumes, a permethylated maltodextrin sample was analyzed (Figure 5). The molecular weights of the solutes varied from 454 to 3514 (degreeof polymerization was 2-17). Good separation of the two anomers (CY and 0) could be obtained with a 50% cyanopropyl methylpolysiloxane column. In Figure 5, different volumes of the sample (7 and 14 pL) were introduced while the mass of the sample was kept constant. The chromatograms are nearly identical, except for the loss of some of the more volatile components from the 14-pL injection. This is a result of a longer period of time required to evaporate the solvent from the injection wire. The volume
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of the solvent that can be introduced onto the wire a t one time is dependent on the sample solvent used. In this study, approximately 1pL (dichloromethane) could be introduced before it dropped from the wire due to gravity. Therefore, when larger sampling volumes were desired, subsequent l-bL volumes were injected and the solvent was allowed to evaporate before the next 1-pL sample was applied. If the sample contains solutes that are only slightly soluble in the mobile phase, the wire should be rinsed before the following chromatographic run to prevent cross contamination. Two additional samples, a coal tar sample (Figure 6) and a mixture of polar steroids (Figure 7), were analyzed to further demonstrate the applicability of this new injector. Some peak tailing of the steroids can be observed due to slower kinetics of desorption of these more polar compounds from the injection wire. An advantage of this injection system is that sample components that are not soluble in the supercritical mobile phase will not be introduced into the analytical column, but will remain on the injector wire. Samples, therefore, can be analyzed without extensive cleanup, as long as the injector wire is rinsed with a suitable solvent between analyses. In this manner, the injection system operates as a small-volume supercritical fluid extraction cell.
ACKNOWLEDGMENT We gratefully acknowledge financial support from Lee Scientific (Division of Dionex Corporation) and the Academy of Finland. RECEIVED for review June 29, 1992. Revised manuscript received November 10, 1992. Accepted December 30, 1992.
