ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
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Packed Microcapillary Columns with Different Selectivities for Liquid Chromatography Yukio Hirata' and Milos Novotny" Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Takao Tsuda and Daido Ishii Department of Applied Chemistry, Faculty of Engineering, Nagoya University, Chikusa-ku, Nagoya-shi 464, Japan
Packed microcapillary columns for hlgh-performance LC are prepared by drawing alumina or silica particles inside the thick-walled glass tubing, and a subsequent in situ bonding of various silanes to their surfaces. The columns with different selectivities were prepared, including a polar phase, a reversed-phase column, and an Ion-exchanger packing. Chromatographic results are presented with standard solutes. Mlcrocolumns with different selectivities and very high efficiencies are now available.
In spite of being a relatively new separation technique (I-3), capillary liquid chromatography (LC) has lately been attracting much attention. Although much development is still needed in this area, its potential has been indicated. The main advantages of LC microcapillary columns as compared to those now in use in conventional HPLC are: (a) a potential for greater resolving power; (b) much lower consumption of expensive (and sometimes environmentally hazardous) solvents; and (c) a greater compatibility of such columns with certain detection and ancillary techniques. The latter aspect is particularly crucial in approaching some technological problems of t h e L C / M S combination. The described microcapillary columns are of two basic types: (a) packed capillaries ( I ) ; and (b) open microtubular columns (2, 3 ) . Whereas further development of both column types for L C is highly desirable, it appears t h a t packed microcapillaries may provide (at least for now) a needed compromise between t h e column efficiency and sample capacity. An almost simultaneous arrival of t h e miniaturized liquid chromatograph ( 4 , 5 ) with unconventional sampling and detection techniques has been a very desirable complementary factor t o t h e LC capillary column developments. T h e previously described ( I ) packed microcapillaries were prepared by drawing alumina particles inside the glass tubes. Alumina is a highly suitable adsorbent material for this column technology because it easily survives the melting temperature of glass. However, this adsorbent will provide only a limited selectivity and the need for additional microcolumn separation media is clearly indicated. This paper describes stationary phases of widely different selectivities for microcapillary column work. While retaining alumina as the starting material, we have utilized a n earlier observation of Knox and Pryde (6) t h a t silanes can bond and polymerize on t h e alumina surface in a fashion similar to their reactions with surface silanol groups of siliceous materials. Both acidic and basic alumina packings were evaluated and found acceptable for column preparation. Several silanes of different structures were investigated and t h e prepared selective microcapillary columns were evaluated in chromatographic terms. Alumina and various structural moieties attached t o its surface provide a wide range of selectivities as needed for 'On leave from the School of Materials Science, Toyohashi University of Technology, Toyohashi, Japan. 0003-2700/79/0351-1807$01.00/0
diverse separation problems. However, i t has been of some interest to find out to what extent a chromatographic function of typical siliceous materials will be diminished after the drawing process owing to the well-known loss of certain surface groups.
EXPERIMENTAL AND RESULTS Preparation of Columns. Microcapillary columns were prepared by the drawing procedure reported earlier ( I ) . The packing materials used were acidic alumina (30 bm LiChrosorb Alox T, from E. Merck Reagents, Darmstadt, West Germany), basic alumina (25-ym fraction, prepared by grinding, mechanical sieving, and sedimentation sizing from a commercial packing from M. Woelm, Eschwege, West Germany) and Spherosil XOB 075, (50 pm from Pechiney, Saint-Gobain, France, supplied from Gasukuro Kogyo, Tokyo, Japan). The microcolumns of various lengths were prepared, with internal diameters ranging between 60-80 bm. The microcolumns with bonded stationary phases were prepared by a procedure, reminiscent of the well-established dynamic coating procedure of capillary gas chromatography or the in situ column preparation technique described by Gilpin et al. (7). This procedure is necessary, since it is unlikely that any organic surface groups would remain intact at the temperatures of column drawing. The following silanes were employed in bonding experiments: octadecyltriethoxysilane, N-(@-aminoethy1)-yaminopropyltriethoxysilane, and P-cyanoethyltriethoxysilane (products of PCR Research Chemicals, Inc., Gainesville, Fla.). Hexane and toluene were spectroquality solvents dried over anhydrous CaS04. In this procedure, the drawn microcolumns were attached to an ordinary injector and a high-pressure pump, while being immersed in a constant-temperature bath maintained a t 50 "C. Hexane was first passed through for several hours at the flow-rate of approximately 10 pL/min. A 150-yL amount of a 2% silane solution in toluene was injected, and the same amount of reagent was introduced again after the first amount eluted. The columns were subsequently washed with hexane for 24 h and conditioned in the liquid chromatograph with the mobile phase until the base line stabilized. Liquid-Chromatographic System. The Varian Model 8500 high-pressure pump was used to generate the necessary pressures and flow rates through the microcolumns. The overall chromatographic system was modified from that previously described ( 2 ) . Namely, the standard Varian UV monitor, operating a t a fixed wavelength of 254 nm, was modified with a quartz microflow cell (inner volume of less than 0.1 yL) according to Tsuda et al. ( 3 ) . As demonstrated in their work as well as the present study, such a small detector still possesses adequate sensitivity. Just as in the previous study ( I ) , a majority of the described experiments were carried out using an injection splitter system. An alternative sampling technique using a "micro-feeder'' (3) is applicable, and its high-pressure version has been developed during this work. The technique permits a direct introduction of small samples and, according to our preliminary results (8), is responsible for no more band-broadening than the previously used splitting sampler ( I ) . Whenever desirable, the microcapillary columns were placed in a constant-temperature bath during the chromatographic runs. Column Performance Studies. Microcapillary columns of both the adsorption and bonded-phase types were evaluated for 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
_____ Table I. Relative Retention Data on Columns of Different Selectivitiesn relative retention time Solute
acidic alumina
basic alumina
octadecylsilane/ alumina
cyanosilane/ alumina
aminosilanei alumina
1.00
1.00
1.00
1.05 1.03
1.06 1.04 1.09 1.61
1.08
1.00 1.08
1.00 1.10
N,N-diethylaniline N-methylaniline diphenylamine quinoline indole a
1.16 1.40
1.04 1.14 1.55
1.13
1.26
1.20
1.08
1.61
3.09
Conditions: mobile phase, 1%isopropanoi in hexane; flow rate, 4 pL/min.___-___________-
-
'1) '
5
1 d
20
10
"---.-Ai-
al
YIMUTES
Separation of aromatic nitrogen-containing compounds on an alumina/cyanoethylsilane microcapillary column. Mobile phase, 1 % isopropanol in hexane; inlet pressure, 100 atm; linear velocity, 2 cm/s. Key: (1) N,N-diethylaniline,(2) N-methylaniline,(3) diphenylamine, (4) quinoline, (5) indole, amounts ranging from 40 to 70 ng
l
1
0
I
efficiency and selectivity. Just as in the previous study ( I ) , a high degree of reproducibility in retention and column performance was observed. Various silanes were found to bond easily to alumina surfaces. Columns with various selectivities as needed for different applications can become available. The in situ bonding technique is simple and reproducible. Several types of columns were chosen to demonstrate different modes of operation. Two types of polar phases, a reversed-phase system, and an ion-exchanger stationary phase, are shown in this report. Table I shows retention data of selected solutes on differently prepared columns. Here, several columns of identical length (15 m) and inner diameter (70 pm), prepared with different column chemistry and under the identical conditions of mobile-phase composition (1% isopropanol in hexane), were compared in terms of relative retention. The test mixture of aromatic nitrogencontaining compounds (N,N-diethylaniline, N-methylaniline, diphenylamine, quinoline, and indole) was used; retention of all tested compounds is related to N,N-diethylaniline. Chromatographic resolution of the standard compounds on a cyanoethylsilane-modified alumina microcolumn is shown in Figure 1. Although this chromatogram was recorded at a very high value of linear velocity (2 cm/s), the compound resolution is still good and symmetrical peaks are obtained. While differences in retention of most solutes (Table I) appear minor, a greater degree of selectivity is noticed with the polar bonded phases. An example of microcapillary reversed-phase chromatography is shown in Figure 2, where a standard mixture of polynuclear aromatic hydrocarbons is recorded with a 27-m microcapillary containing acidic alumina with bonded octadecylsilane. The mobile phase is acetonitrile/water (65:35) at the column lemperature of 50 "C. The numbers of theoretical plates vary from about 150000 for the early component, to 64000 for the last component (benzo[e]pyrene). The use of an aminosilane as a weak ion-exchanger was earlier recommended by Vivilecchia et al. (9). It is believed that the phases of this type will find only limited utilization because of capacity reasons. However, the previously described technology (10, 11) of silicone-based ion-exchangers can also be applied to microcapillary columns. Figure 3 demonstrates separation of three ionic substances on the aminosilane microcolumn with 0.01 M HC1 as the mobile phase. We have also found that several siliceous materials retained adequate chromatographic retention properties even after the drawing step. When relatively loa-melting glass tubes were used
W
60
50
Figure 1.
lo
I
1
L
MINUTES
Figure 2. Separation of model polycyclic aromatic hydrocarbons on an alumina/octadecylsilane microcapillary column. Mobile phase, acetonitrile/water (65:35);inlet pressure, 200 atm.; linear velocity, 0.5 cm/s. Key: (1) toluene, (2) naphthalene, (3) fluorene, (4) anthracene, (5) pyrene, (6) chrysene, (7) impurity, (8) benzo[ elpyrene, amounts ranging from 20 ng to 3 pg 1
1
12
3
, . L -
1
20
40
M,
UINUTES
Figure 3.
Separation of standard acidic compounds on an alumina/
N-(j3-aminoethyl)-y-aminopropylsilanemicrocapillary column. Mobile
phase, 0.01 M HCI; inlet pressure, 100 atm.; linear velocity, 0.5 cm/s. Key: (1) phenol, (2) o-nitrophenyl, (3) benzoic acid, amounts ranging from 130 to 520 ng
0
30 MINUTES
60
Separation of aromatic nitrogen-containing compounds on a Spherosil-packed microcapillary column. Mobile phase, 1.57YO acetonitrile in hexane; linear velocity, 1.3 cm/s. Key: (1) N,N-diethyhniline, (2) N-phenyCa-naphthylamine, (3) N-phenyl-p-naphthylamine, (4) aniline, (5) a-naphthylamine, (6) @-naphthylamine,amounts ranging from 10 to 30 ng Figure 4.
for the preparation of microcapillaries, good symmetrical peaks and column efficiencies were obtained for mixtures of aromatic amines and phthalic acid esters. Compound retention indicated that enough adsorptive sites remain on the surface even after the drawing process. Figure 4 shows the separation of amines obtained with a 16 m X 80 pm i.d., column drawn with Spherosil. Several
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
different silica materials were tested with approximately the same results. At this point, we prefer totally porous spherical packings. Superficially-porous materials can also be drawn into the microcapillaries, but the overall capacity of such columns is very small. Surprisingly,the crucial adsorptive sites are not destroyed even with using Pyrex (high-melting) glass as the initial tubing material. In fact, there was little difference in retention when compared with the low-melting soft glass. A further support to this fact is an easy surface modification (e.g., with an octadecylsilane) to form a chemically bonded phase.
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Although “selectivity” in liquid chromatography frequently accounts for a very complex (mixed) retention mechanism, differently prepared columns can exhibit significantly different retention. I t is widely known t h a t often a desirable combination of selectivity and column efficiency causes difficult separations to be accomplished. Unlike in capillary gas chromatography where the plate number does not increase with the column length in a linear fashion (12) and volatility limitations are rapidly imposed, longer microcapillary columns in liquid chroniatography are worth further explorations. Available inlet pressures and analysis times are the only restrictions. It is predicted that various reversed-phase systems will play a crucial role in future applications of microcapillary columns to complex and difficult mixtures. As shown in a following publication ( 8 ) , developments of suitable gradient elution techniques and direct sampling are also essential. Combination of microcapillary chromatographic columns with better detectors and novel ancillary tools are among the best hopes for the future utility of capillary LC.
DISCUSSION In agreement with the report of Knox and Pryde (6), we find it relatively easy to bond various silane compounds to the surface of alumina. We have already established in the previous work ( I ) that alumina packings pass unharmed through the hot zone of a glass drawing machine and retain their fundamental chromatographic properties. Compound retention itself may be affected by the degree of acidity of alumina surface, while the silane bonding process can be generally realized with either basic or acidic alumina. However, the residual activity of uncovered sites may occasionally cause undesirable tailing of certain “sensitive” solutes. If there is little freedom as to whether to choose acidic or basic material, some additional surface deactivation approaches must be developed. Since the efficiencies of columns prepared with different silanes are not particularly different and column-to-column retention reproducibility is also good, we believe that this column technology is superior to the conventional slurrypacking approaches. The results obtained with siliceous materials have been a positive surprise. Although future studies should easily establish whether silica and alumina-based materials are preferrable to each other in terms of selectivity, a role of the particle shape might also be important in microcapillary LC. Still better results are anticipated through optimization studies. Results obtained in this work extend the scope of utilization of microcapillary columns in terms of the phase selectivity.
LITERATURE CITED (1) Tsuda, T.; Novotny, M. Anal. Chern. 1978, 50, 271. (2) Tsuda, T.; Novotny. M. Anal. Chern. 1978, 50. 632. (3) Tsuda, T.; Hibi, K.; Nakanishi, T.; Takeuchi, T.; Ishii, D. J . Chromatogr. 1970, 158, 227. (4) Ishii, D.; Mochizuki, K.; Mochida, T. Abstracts of the 1977 Pittsburgh Conference on Analytical Chemistry and .Applied Spectroscopy, . . Clevebnd, Ohio, No. 385. (5) Ishii, D.; Asai, K.; Hibi, K.; Jonokuchi. T.; Nagaya, M. J . Chromatogr. 1977. 144. 157. (6) Knox: J. H.; Pryde, A. J . Chromatogr. 1975, 112, 171. (7) Gilpin, R. K.; Korpi, J. A.; Janicki, C. A. Anal. Cbent. 1974, 46, 1314. (8) Hirata, Y.; Novotny, M. J . Chromatogr., in press. (9) Vivilecchia, R. V.; Cotter, R. L.; Limpet?, R. J.; Thirnot, N. Z.; Little, J. N. J . Chromatogr. 174, 9 9 , 407. (IO) Asmus, P. A.; Low, C.-E.; Novotny, M. J . Chrornatogr. 1976, 119, 25. (11) Asmus, P. A.; Low, C.-E.; Novotny, M. J Chromtogr. 1976, 123, 109. (12) Grob, K.;Grob, G. J . Chromatogr. Sci. 1989, 7, 515.
RECEIVED for review March 8, 1979. Accepted June 18, 1979. This work was supported by Grant No. GM 24349 from the National Institute of General Medical Sciences, U S . Public Health Service.
Determination of Chlorinated Hydrocarbons in Water by Headspace Gas Chromatography Edward A. Dietz, Jr.’ and Kenneth F. Singley Hooker Chemical Company, Research Center, Grand Island Complex, M.P.O. Box 8, Niagara Falls, New York 14302
Concentrations of chloroform, carbon tetrachloride, trlchloroethylene, and tetrachloroethylene, ranging from 0.1 ppb to low ppm, are conveniently determined at ambient temperature using headspace gas chromatography in conjunction with electron capture detection. Effects of the analysis conditions, sample matrix, and sample history are evaluated in detail. The headspace method, which Is compared with the purge-trap procedure, Is shown to be accurate and exhibit a standard deviation of about 5 YO for routine analyses of drinking, natural, and industrial waters.
conjunction with Hall electrolytic conductivity or electron capture detectors. These analyses have employed four different sample handling techniques: direct aqueous injection ( I ) , solvent extraction (2, 31, gas stripping (4-8), and static headspace sampling (9, I O ) . Each method can be effective for analyses of selected halocarbons in water and each has advantages and disadvantages which relate to equipment needs, desired detection limits, sample matrix, and analysis speed. For our purposes a routine method was required that would determine chloroform (CHCl,), carbon tetrachloride (CC14),trichloroethylene (C2HCl,), and tetrachloroethylene (C2C1,) in drinking water, natural waters, and water from industrial operations; be applicable over a very large concentration range (0.1 ppb to 1 ppm); and not be adversely affected by less volatile chlorinated and nonchlorinated
-
Parts per billion concentrations of volatile halocarbons in water have been determined using gas chromatography in
e
0003-2700/79/0351-1809$01.00/0 1979 American Chemical Society
