580
Anal. Chem. 1983,
55,580-583
ACKNOWLEDGMENT
(8) Kesler, S. E.; Jones, L. M.; Walker, R . L. €con. Geol. 1975, 70, 5 15-526. (9) Kietz, L. A,; Puchuckl, C. F.; Land, G. A. Anal. Chem. 1982, 3 4 , 709-7 10. (10) Ridley, R. G.; Daly, N. R.; Dean, M. H. Nucl. Instrum. Methods 1985, 3 4 , 163-164.
We thank L. Landau for performing the spark source mass spectrometric analysis, H. Simmons for performing some of the early isotope dilution analyses, and H. C. Smith for preparing the samples. Registry No. l’ISLu,14391-25-4;l’16Lu,14452-47-2.
David H. Smith* R. L. Walker C. A. Pritchard J. A. Carter
LITERATURE CITED (1) Hertel, G. R. J . Chem. Phys. 1988, 4 8 , 2053-2058. (2) Smith, D. H.; Christie, W. H.: McKown, M. S.: Walker, R. L.; Hertel, G. R. Int. J . Mass Spectrom. Ion Phys. 1972, 10, 343-351. (3) Todd, P. J.; McKown, H. S.; Smith, D. H. I n t . J . Mass Spectrom. Ion Phys ., in press. (4) Walker, R. L.; Eby, R. E.; Pritchard, C. A,; Carter, J. A. Anal. Lett. 1974, 7, 563-574. ( 5 ) Smith, D. H.; Walker, R. L.; Carter, J. A. J . Inst. Nucl. Mterials Management 1980, 8(4), 66-71. (6) Smith, D. H.; Christie, W. H.; Eby, R. E. Int. J . Mass Spectrom. Ion Phys. 1980, 36, 301. (7) Christie, W. H.; Cameron, A. E. Rev. Scl. Instrum. 1988, 3 7 , 336-337.
Analytical Chemistry Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37830
RECEIVED for review July 29, 1982. Accepted December 7, 1982. Research sponsored by the U S . Department of Energy, Office of Safeguards and Security, and Office of Basic Energy Sciences under Contract W-7405-eng-26 with the Union Carbide Corporation.
AIDS FOR ANALYTICAL CHEMISTS Nanoliter Injection System for Microcolumn Liquid Chromatography V. L. McGuffln and Milos Novotny” Department of Chemlstry, Indiana Unlverslty, Bloomlngton, Indiana 47405
In high-performance liquid chromatography (HPLC), the separation efficiency may be critically influenced by extracolumn contributions to band broadening, especially those which originate from the injection system. Precise, low-volume sample introduction is particularly important in high-speed liquid chromatography ( I ) and in microcolumn HPLC (24% where flow rates are typically on the order of microliters per minute and total peak volume may frequently be less than 1 pL. If the peak variance due t o the injection technique becomes significant in comparison with the variance due t o the column processes, themselves, then the column performance will be adversely affected. The maximum permissible injection volume (V,,,), which will produce a fractional (e2) increase in the volumetric variance (u:) of a nonretained peak, is given by the following equation (6): Vmm2 =
( T ~ K ~ ~ E ~ L ) ~ = Pa: N
for a chromatographic column of radius I“, length L , total porosity tT, and plate number N . The constant is characteristic of the injection profile and is equal to 12 for an ideal plug injection. Maximum injection volumes were calculated assuming an ideal injection profile for conventional, microbore, packed capillary, and open tubular columns. If the injection profile is not ideal, then permissible injection volumes h a y be considerably less than those indicated in Table I. The required injection volumes for conventional and microbore packed columns clearly appear to be within the capabilities of state-of-the-art technology. However, the stringent requirements of capillary columns preclude the use of conventional syringe or valve injection techniques. Several approaches to the injection problem in microcolumn HPLC have been reported, including various methods of split injection (4, 7, 8) and internal-loop valve injection (3, 9). Low-volume injection has also been achieved by filling a short length of stainless-steel or fused-silica capillary tubing with 0003-2700/83/0355-0580$0 1.50/0
sample and subsequently connecting this sampling tube to the microcolumn with shrinkable P T F E tubing (10). Direct sample introduction has recently been described in which the inlet of the microcolumn is briefly heated and sample is drawn into the column by capillary action (11). These injection techniques require a great deal of manual skill; they are generally cumbersome and imprecise and deliver only a limited range of injection volumes. A novel injection system has been constructed in our laboratory which is compatible with both packed and open tubular microcolumns. This system is capable of delivering injection volumes which range from 1 nL to 1 pL or more. Furthermore, the system is constructed of readily available materials and can be easily automated to improve precision and simplify operation for routine applications.
EXPERIMENTAL SECTION A high-pressure syringe pump (Model 8500, Varian Instrument Division, Palo Alto, CA) was utilized in this investigation, which allowed operation in both the constant-pressureand constant-flow modes. A schematic diagram of the injection system is shown in Figure 1. This device was based on a high-pressure six-port injection valve (Model AH-CV-6-UHPa-NG0,Valco Instruments Co., Inc., Houston, TX), equipped with a 10-pL sample loop. Two stainless-steel union tees (Swagelok SS-100-3,Crawford Fitting Co., Solon, OH) were connected in series at the valve outlet by using narrow-bore stainless-steel tubing. The branch of the first tee was connected to a restricting capillary and, subsequently, to a high-pressure shutoff valve (“SPLIT”). The second branch tee was connected with wide-bore stainless-steel tubing directly to a shutoff valve (“PURGE”). The HPLC microcolumn was inserted through both tees, and extended approximately 1 cm into the connecting tubing, so that turbulence and mixing at the column inlet were minimized. Two modes of operation were investigated with this sampling system: split injection and “heart-cut” injection techniques. Split injection was achieved with the syringe pump in the constant@ 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
581
Table I. Maximum Injection Volumes for Conventional and Microcolumns in High-Performance Liquid Chromatography column type conventional micro bore packed capillary open tubular Capillary
d,mm 4.6 1
L, m 0.25 1
0.1 0 0.07 0.03 0.01
10 10 10 5
N” 25 000 100 000 100 000 140 000 420 000 625 000
vmax
b
8-17 pL 0.7-1.6 pL 86-190 nL 36-80 nL 4-8 nL 0.2-0.4 nL
ab
OV
5-25 pLa 0.045-0.22 pLa 620-3100 nLz 110-530 nL2 1-6 nL2 0.002-0.12 nLa
-
-
a Optimum velocity and plate height are assumed; 5 pm particle size for conventional and microbore columns (eT 0.85), 30 pm particle size for packed capillaries ( e ~ 1). Range of volumes that will contribute 1-5% to the volumetric peak variance. WASTE
INLET
’ -~
I
i
HICROCOLUMN C HPLC
HPLC PUMP
DETECTOR
Figure 1. Schematic diagram of the injection system.
pressure mode. The split valve remained open and the purge valve was closed during the entire injection procedure. The sample (1-10 pL) was loaded by syringe into the injection valve, and the valve was actuated pneumatically. The injection volume was split between the microcolumn and the restricting capillary according to their respective inner diameters and lengths. In general, the restricting capillary was BO to 500 times more permeable than the analytical column to provide the necessary injection volumes (200-20 nL). Alternatively, a high-pressure metering valve may be used in place of the restricting capillary to allow variable splitting ratios. The split injection method has been widely utilized in microcolumn lliquid chromatography (4, 7,8) and will not be discussed further in this paper. In the “heart-cut”injection technique, the syringe pump was utilized in the constant-flow mode. The purge valve was initially open, so that no flow entered the analytical column. The sample (10 pL) was injected in ithe usual manner, and flowed through the connecting tube until the center of the peak was located at the column inlet. At this point, the purge valve was closed for a predetermined period ( A t ) ,during which time the sample entered the microcolumn. After the appropriate injection time had elapsed, the purge valve was opened again, and the remainder of the initial sample plug was washed away (Figure 2). By utilization of a large initial sample volume relative to that of the “heart-cut” fraction, the exact timing of the initial (Ti)and final (Tf) delay periods was not critical. These time periods were determined empirically by connecting the injector directly to a detector with a short length of fused-silica capillary tubing. Then by systematic variation of the sampling intervals, the optimum values for Ti and Tp were readily ascertained. These time intervals were dependent upon the volume of the sample loop and cunnecting tubing and also varied with the flow rate during injection. It must be emphasized that the injection flow rate is entirely independent of the chromatographic flow rate, which may be either pressure- or flow-controlled with this chromatographic system. The performance of this novel injection device was evaluated with both packed capillary and open tubular microcolumns of various dimensions. The results presented here were obtained with an open tubular fused-silica column of 30 pm nominal inner diameter and 13.0 m length. The column variance was estimated from the Taylor equation (12) to be 214 nL2. Hexane was used
Figure 2.
INJECTIOh T I M E (At) T --+ e Tf Schematiic representation of the “heart-cut” injection
technique.
as the mobile phase at a linear velocity of 1.3 cm/s, and neat toluene served as a nonretained model solute (D, = 3.7 X loT5 cmz/s at 20 “C). A variable-wavelength UV detector (Model Uvidec 100-11,Jasco, Inc., Tokyo, Japan) was modified for oncolumn detection though the fused-silica capillary column. ‘The illuminated volume was approximately 3 nL, and the calculated detector variance was 0.7 nL2. The volumetric variance of each chromatographic peak was calculated by computer from the second statistical moment (13-15). The variance attributable to the injector was then calculated by subtracting the estimated column and detector contributions from the total peak variance. The apparent volume of each injection was determined by comparing the peak area, calculated from the zeroth statistical moment, with that generated by the total sample loop volume (10 pL).
RESULTS AND DISCUSSION The “heart-cut“ injection method has several distinct advantages over the techniques currently utilized in microcolumn HPLC. This device is compatible with narrow-bore paclked columns (8),packed capillaries (4),and open tubular colurnns (5) of different dimensions without modification of the existing hardware. The sample volumes required for these varbous microcolumn types may be conveniently and accurately controlled by varying the injection time ( A t ) and volumetric flow rate, as shown in Figure 3. The range of desired injection volumes is roughly controlled by choosing an appropriate flow rate; for example, 1-5 mL/h might be selected for open tubular microcolumns, 5-10 mL/h for packed capillaries, and 15 mL/h or more for narrow-bore packed columns. Within each of these ranges, the desired volume can then be more accurately adjusted by varying the elapsed time of the “heart-cut” fraction. Neither injection time nor flow rate is related to injection volume in a straightforward manner, as evidenced by ithe notable curvature shown in Figure 3. These nonlinear calibration curves are most likely attributable to compressibility of the mobile phase and other compliances within the system. With both time aind flow rate as injection variables, a wide range of sample volumes can be conveniently delivered. Injection volumes between 2.0 pL and 9 nL are represented graphically in Figure 4. Furthermore, volumes on the order of 1 nL and less have been estimated from their respective peak areas. The variance of such small injection volumes was difficult to calculate accurately, since it was largely dominated by the column dispersion, and is therefore not included in
582
~
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
1500
i
rp
4
~ ~ " " ~ 50 " " 1 I "0 8 " " " I' 6 01 INJECTION TIME
208
"
'
j
250
i
Flgure 3. Effect of Injectlon time (At) on chrometographlc peak varlance, as a functlon of the flow rate during Injectlon: column, open tubular fused-slllca microcolumn, 30 pm Ld., 13.0m length; mobile phase, hexane at 13 cm/s; solute, toluene (k = 0);Injector, (0)15 mL/h, (0)10 mL/h, (A)6 mL/h, (fr)1 mL/h lnjectlon flow rate. Stralght line represents the column contrlbutlon to the total system variance, calculated from the Taylor equatlon (72).
IO'
1001
o,.
'
I 1 ~ 1
a
I
102
'
103
'
I
104
'
I
lo6
'
I
108
'
'01
I
'
0 1 8 3 4
VOLUME (yL)
Figure 1. Experlmental peak proflle obtalned wlth the "heart-cut" lnjectlon technlque, Chromatographic condklons are described in Flgure 3. Injectlon volume was 1,l pL (20 mL/h, 150 8).
l " " " ' " " " l r ~
'ozv
--m-33-,
J
INJECTION VOLUME SQUARED
Flgure 4. Reiatlonshlp between Injector varlance and the square of lnjectlon volume. Chromatographic conditions are descrlbed in Flgure 3.
Figure 4. Nevertheless, these injection volumes are significantly smaller than any previously reported and approach the theoretical requirements of open tubular capillary columns (16).
Both injection volume and variance are highly reproducible functions of the injection variables. With manual control of the injection sequence timing, the volume was 2.4 f 0.15 nL for replicate 10-s injections a t 15 mL/h. The absolute precision was reduced with an increase in either injection time or flow rate, as might be predicted from the slope of the calibration curves shown in Figure 3, assuming a constant relative error in the timing sequence. More importantly, however, the relative precision was improved with increasing injection time and flow rate. As indicated in eq 1,the variance of the "heart-cut'' injection should be linearly related to the square of the injection volume. This relationship, illustrated in Figure 4, was linear over a t least 6 decades. The constant P, which is characteristic of the injection profile, was determined from the y intercept of this logarithmic graph to be approximately 4. Although this value is small in comparison with the theoretical value for an ideal plug injection, it has been commonly observed for other HPLC injection systems (17,18). The shape of the "hearbcut" fraction is determined primarily by the speed of the switching valve and the subsequent rate of pressure elevation or relaxation. Because these processes are relatively fast, the leading and tailing edges of the injected sample plug are sharp and symmetrical transitions, as illustrated in Figure 5 .
Although the "heart-cut'' injection system has numerous advantages for application in microcolumn HPLC,there are several potential limitations which should also be considered. The initial sample volume required for "heart-cut'' injection is somewhat greater than the direct sampling methods but is comparable to that required for split or valve injection, If the available sample volume is limited, the direct sampling methods (10, 11) will be preferred. For a specified injection time and flow rate, the volume delivered by the "heart-cut" injector varies with the permeability of the analytical microcolumn. This dependence indicates that the sampling volume is determined by the pressure drop acrose the column and not directly by the volumetric flow rate during injection. There are several practical consequences of this pressure dependence. First, for columns of low permeability, such as packed microcolumns, longer injection times and/or faster flow rates will be necessary to deliver the required volume. If the permeability of the packed column changes substantially over a period of time, then injection volumes will concurrently vary. This problem, also exhibited by split injection and "temporary" valve injection methods (19),may be overcome by employing an internal standard. Second, this injection technique may not be suitable for highly permeable columns, since the injection volume cannot be precisely controlled. For open tubular columns of inner diameter greater than 50 bm (KO > 7.5 X mm2), the split injection method will be preferred.
CONCLUSIONS The further development and application of microcolumn HPLC is justified on both practical and theoretical grounds. Recent results clearly favor the use of very small columns and correspondingly low flow rates (8,11,20). While the reduction of extracolumn band dispersion in the detection system is a t least of equal importance (21-23), there has been a conspicuous lack of reliable sampling technology in this area. A novel injection system has been constructed in our laboratory which is compatible with both packed and open tubular microcolumns. A sample plug is introduced with a conventional valve injector, and a "heart-cut'' fraction of the sample is diverted onto the microcolumn using sequentially timed valves. If the injection time and flow rate are varied, injection volumes from 1 nL to 1 KLor more can be delivered. The versatility and precision of this technique originate be-
AnaL Chem. 1983, 55, 583-584
cause time, rather than three-dimensional space, is used to control the injected sample volume. As an injection variable, time is far easier to control accurately than distance, particularly with the aid of a computer. The timing sequence may be readily automated by using either pneumatically or electrically actuated valves. Thus, the "heart-cut" injection technique described herein may be an important step toward the routine analytical application of microcolumn HPLC. ACKNOWLEDGMENT The fused-silica capillary tubing utilized in this investigation was obtained through the courtesy of Kenneth Mahler and Ernest Dawes of Scientific Glass Engineering, Inc. LITERATURE C I T E D (1) DiCesare, J. L.; Dona, M. W.: Atwood, J. G. J. Chromatogr. 1981, 277, 369-386. Novotny, M. Anal. Chern. 1981, 53, 1294A-1301A. Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 769, 51-72. Tsuda, T.; Novotny, M. Anal. Chem. 1978, 50, 271-275. Ishii, D.; Takeuchi, T. J. Chromatogr. Scl. 1980, 78, 462-472. Martin, M.; Eon, C.; Guiochon, 0 . J. Chromatogr. 1975, 708, 229-24 1. Tsuda, T.; Nakagawa, G. J. Chromatogr. 1980, 799, 249-258. Yang, F. J. J. Chromatogr. 1982, 236, 265-277. Takeuchi, T.; Ishii, D. HRC CC, J. Hlgh Resoluf.Chromatogr. Chromafogr. Commun. 1981, 4 , 469-470.
583
Hirata, Y.; Novotny, M. J. Chromatogr. 1979, 786,521-5213, Tsuda, T.; Tsuboi, K.; Nakagawa, G. J. Chromatogr. 1981, 274, 283-290. Taylor, G. R o c . R . SOC.London, Ser. A . 1953, 219A, 186. Sternberg, J. C. "Advances In Chromatography"; Giddings, J. C., Keller, R. A., Eds.; Marcel Dekker: New York, 1966; Vol. 2; pp 205-270. Kirkiand, J. J.; Yau, W. W.; Stokiosa, H. J.; Dilks, C. H. J. Chromafogr. Scl. 1977, 75,303-316. Chesler, S. N.; Cram, S.P. Anal. Chem. 1971, 43, 1922-1933. Knox. J. H.; Giibetit, M. T. J. Chromatogr. 1979, 786, 405-418. Karger, B. L.; Martin, M.; Guiochon, G. Anal. Chem. 1974. .46, 1640- 1647. Coq, B.; Cretier, G.;Rocca, J. L.; Porthauit, M. J. Chromatogr. fici. 1981, 19, 1-12. Harvey, M. S.;Steams, S. D. "Liquid Chromatography in Environmental Analysis"; Lawrence, J. F., Ed.; Humana Press: Cliffon, NJ, 1982; Chapter 10. McGuffin. V. L.;Novotny, M. J. Chromatogr., In press. Henion, J. D. J. Chromafogr. Sci. 1981, 79, 57-64. McGuffin, V. L.; Novotny, M. J. Chromatogr. 1981, 278, 179-187. Hirata, Y.; Lin, P. T.;Novotny, M.; Wightman, R. M. J. Chromatog./ Homed. Appl. 19130, 787, 287-294.
RECEIVED for review July 21, 1982. Accepted November 114, 1982. This research was supported by the Department of Health and Human Services, Grant No. GM 24349. V.L.IM. was the recipient of a full-year Graduate Fellowship from the American Chemical Society, Division of Analytical Chemistry, which was sponsored by the Upjohn Co.
Determination of Anionic Surfactants in Presence of Cationic: Surfactants by Two-Phase Tit ration Masahfro Tsubouchi" Laboratory of Chemistry, Kochi Medical School, Oko, Nankf~ku,Kochi 78 1-5 1, Japan
Yuroku Yamamoto Deparfment of Chemistry, Faculty of Science, Hiroshima University, Hiroshima 730, Japan
Ionic surfactants are widely used in both industrial and medical applications. Anionic surfactants (AS) are precipitated by the addition of cationic surfactants (CS),but the fine turbidity is scarcely deposited in sewage or drainage samples, cases where the concentration is low. It is necessary t o determine total AS in the presence of the fine turbidity in water. Two-phase titration is one of the most frequently used methods for the determination of AS. However, it is generally difficult to determine total AS in the presence of CS because CS is used as a titrant (1,2).This paper presents a titrimetric method for the determination of total AS or CS in mixtures. EXPERIMENTAL SECTION Apparatus. A 25-mL buret was used. Materials. Solutions of cationic surfactants were prepared by dissolving zephiramine (tetradecyldimethylbenzylammonum chloride), benzethonium (benzyldimethyl[2-[2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxyJethylJammoniumchloride), and R = CBH1,-C18H97). They benzalkonium (C6H5CH2N(CH3)2RCl, were standardized according to the official titrimetric method (3) using Methyl Orange as an indicator and used after accurate dilution. Solutions of anionic surfactants were prepared by dissolving sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, and Aerosol OT (sodium bis(2-ethylhexy1)sulfosuccinate). They were standardized according to the official titrimetric method ( 4 ) using Methylene Blue as an indicator and used after accurate dilution. A 0.02 M solution of tetraphenylboron sodium salt wm checked according to the official gravimetric method (5)#andused after accurate dilution. Victoria Blue E3 (color was dissolved in ethanol to make a index: 44045,C33H32N3C1) 0.01% solution; it was used as an indicator. A pH 9.0 buffer solution WHS prepared by mixing 0.3 M sodium dihydrogen phosphate solution and 0.05 M sodium borate solution. All
reagents used were from Wako Pure Chemical Ind. (Tokyo, Japan). Procedure A. To 10 mL of a AS and CS mixture, each at (1-12) X 10" M concentration (AS > CS) in a 300-mL Erlenmeyer flask, were added 5 mL of buffer solution (pH 9.0), 1-2 drops of the indicator, and 3 mL of 1,2-dichloroethane. The mixture was titrated with a standard CS solution ( 5 X M or 1 X lo4 14) with vigorous shaking after each addition, until a color change from blue (Arna = 615 nm) to red,,A,( = 505 nm) took place in the organic phase. 1 mL of 1 X M titrant = 1 mL of the difference (AS - CS)(l x hl) Procedure B. To 10 mL of the same sample solution, as in procedure A, were added 5 mL of 6 N sodium hydroxide solution, 1-2 drops of the indicator, and 3 mL of 1,2-dichloroethane. The mixture was titrated with a standard solution of tetraphenylborate or 1 X (5 X M') in the same way as in procedure A, until a color change from red to blue took place in the organic phase. 1 mL of 1 X M titrant = 1 mL of 1 X M CS independent of AS present The totalconcentration calculated from the two titres in procedwe A and B corresponds to the total amount of AS. R E S U L T S AND DISCUSSION With a 5 X W5M[titrant, a blank titration is necessary because the color change at the end point is not sharp. The aqueous phase remains colorless throughout the titration in both prrocedures, due to the insolubility of the Victoria Blue B in alkaline water. 'The titre was constant between pH 8.5 and 9.5 in procedure A. In procedure B, the best color change and most constant titre were obtained a t a concentration of
0003-2700/63/0355-0583$01.50/00 1983 American Chemical Society
