Anal. Chem. 1900, BO, 2301-2303
teins in the mass range above 10OOO daltons, stable a t least up to times of about a millisecond. Singly charged molecular ions were in all cases the base peak of the analyte sign&, no fragment ions were observed in the mass range above 1000 daltons. Additionally, multimers of the molecular ions and doubly charged molecular ions were detected improving the molecular ion detection and increasing the molecular weight determination accuracy. Also, a remarkable sensitivity is demonstrated. A detection limit in the subnanogram range for total sample mass needed for a s u m spectrum appears to be realistic. All these features, no doubt, can still be optimized as discussed above. Though the general applicability of UVLD still needs to be shown by the successful desorption of a larger variety of different compounds, matrix-UVLD promises to be able to extend the accessible range for mass spectrometry of nonvolatile bioorganic compounds considerablywith the added advantages of low sample consumption, ease of preparation and short measurement time. Registry No. Lysozyme, 9001-63-2; trypsin, 9002-07-7. LITERATURE CITED (1) MacFarlane, R. D.; Hill, J. C.; Jacobs, D. L.; Phelps. R. G. I n Mass Spectrometry In the Ana&sk of Large Molecules: Wiley: Chichester, 1986; pp 1-12.
2301
Sundquist, B.; Hedin. A.; Hakansson, P. I.; Kamensky, M.; Salehpour, M.; =we, G. Int. J . Mass Specfrom. Ion Rmsses 1985, 65, 69-89. Kamensky, I.; Craig, A. 0. Anal. Instrum. ( N . Y . ) 1987, 76, 71-91. Chait, B. T.; Field, F. H. Int. J . Mass Spectrom. Ion Ro~%ses1985, 65, 189-180. Tanaka. K.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Presented at the Second Japan-China Joint Symposium on Mass Spectrometry (abstract), Takarazuka Hotel, Osaka, Japan; Sept 15-18, 1987. Barber, M.; Green, B. N. Rapm Commun. Mass Spectrom. 1987, 7 , 80-85. Karas, M.; Bachmann, D.; Bahr, U.; Hlllenkamp, F. Int. J . Mess Spectrom. Ion processes 1987, 78, 53-68.
Michael Karas* Franz Hillenkamp Institute of Medical Physics University of M h s t e r Hiifferstrasse 68 D-4400 Munster Federal Republic of Germany RECEIVED for review May 16, 1988. Accepted July 5, 1988. This work was supported by the Deutsche Forschungsgemeinschaft under Grant No. Hi 285/2-5 and by a grant from the Ministerium fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen.
Enhancement of Uphill Transport by a Double Carrier Membrane System Sir: In spite of ita obvious potential usefulness, two major problems for analytical applications of uphill transport membrane phenomena are (1) lower efficiency of energy conversion and (2) slower rate of transport, compared to the eventa occurring a t biological cell membranes. It has often been noticed that thinner membranes are required to accelerate the transport rate in artificial systems. Besides extremely thin lipid bilayers for biological cell membranes, another fundamental difference between artificial organic membranes and biological cell membranes is the involvement of Na+,K+-ATPasefor the majority of active transport in the latter systems. The essential chemistry of Na+,K+-ATPase is the generation of Na+ gradient and ita maintenance using the energy provided by the hydrolysis of ATP (I, 2). For example, active transport of glucose is known to be driven by concentration gradient of Na+ ions together with a carrier protein for glucose. Accumulation of the cotransported Na+ ions in the intracellular compartment results in a drop of the driving force (concentration gradients of Na+ ions) for pumping up glucose. However, this is restored by a Na+,K+-ATPase enzyme that pumps Na+ ions from the intracellular compartment back to the extracellular one. By this action of the ATPase, high efficiency of active transport of glucose is maintained. Therefore, it seems very interesting to simulate this unique function of ATPase for enhancing efficiency of uphill transport of ions and molecules of interest by artificial membrane systems. In the present correspondence, we report a preliminary study on double carrier membrane systems that mimic, in principle, the function of ATPase, although chemical compounds involved are totally different. It will be shown that the transport of ions with a double carrier membrane system is facilitated more efficiently than the conventional symport system. 0003-2700/S8/0360-2301$01SO10
EXPERIMENTAL SECTION Reagents. Dicyclohexyl-18-crown4(99% content) and trin-octylamine (Pure Reagent grade) were obtained from Aldrich Co. (Milwaukee, WI) and Wako Co. (Tokyo, Japan), respectively. o-Nitrophenyl octyl ether (NPOE) was obtained from Dojin Chemical Laboratories (Kumamoto,Japan). Picric acid (Kanto Co., Tokyo, Japan) and lithium hydroxide (Wako Co.) were both of G.R. grade. A solution of lithium picrate was prepared by neutralizingpicric acid solution with lithium hydroxide solution. Buffer solutions of pH 3.8 were prepared with lithium acetate and hydrochloric acid solutions. Other reagents used were all of G.R. grade. Milli-Q (Millipore, Bedford, MA) water was used. Apparatus. The cell used for transport experiments is made of glass and has two compartments of different volumes (Figure 1): one for a feed solution (50 mL) and the other for receiving (1mL). A Seiko E&I metal furnace flameless atomic absorption spectrometer (AAS)SAS 727 (Tokyo, Japan) was used for determining K+ ions. A Shimadzu spectrophotometer UV-240 (Kyoto,Japan) was used for determination of picrate ions. A TOA glass electrode pH meter HM-60s (Tokyo, Japan) was used for pH measurements. Preparation of Liquid Membranes and Transport Experiments. A Sumitomo Denko (Osaka, Japan) Fluoropore membrane filter, type FP-010 (pore size, 0.10 pm; thickness, 80 pm; diameter, 47 mm), is cut into two equal pieces. One piece of the membrane filter is impregnated with 2.5 mM dicyclohexyl-18-crown-6 (DC18C6)4ichlorobenzene(DB) solution and the other with a 2.5 mM tri-n-octylamiie (T0A)-NPOE solution. A pair of the carrier-impregnated liquid membranes (DC18C6 and TOA membranes) thus prepared me placed side by side separately to cover two of each window at the exit of the feed compartment, which is then fastened tightly with the receiving part of the cell using a strong clip (Figure 1). The respective feed (50 mL) and receiving (1mL) solutions are provided into each compartment of the transport cell. The feed solution is stirred mechanically with a stirrer (the receiving solution was not stirred). For determination of K+ions transported, a very small volume (usually 0 1988 American Chemical Society
2302
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988 10
-
8-
I
* I
0
" I-
Flgure 1. Structure of the present uphill transport cell: (1) feed compartment; (2)receiving compartment; (3)pair of carrier-impregnated llquid membranes; (4) glass separator; (5) stirring bar; (6) port
6-
Y
u
4-
for sampling.
a)
K
KL'Pic~
~+
E
~
C
0
PIC-
PIC-
5
10
15
20
25
Time, h K+ ions as a function of time with (1) a double carrier membrane system and (2)a symport system. The membrane system is the same as that shown in Figure 2. Figure 3. Uphill transport of
TO1
TOAH' PIT
Flgure 2. Uphill transport systems for K+ ions: (a) double carrier membrane system, (b) conventknal sympott system: feed solution (50 mL), 1.0 X lo4 M KCI, 2 mM Li plcrate, and 0.01 M Liotl; receiving solution (1 mL), 0.01 M CH,COOLi-HCI buffer (pH 3.1).
10 fiL)of the receiving solution is sampled by a micropipet and transferred into a sample tube. The solution is diluted with 1 mL of water, shaken vigorously, and subjected to atomic absorption spectroscopy determination of K+ ions. For determining picrate ions, a 20-pL portion of the receiving solution is taken in a glass tube and diluted with 3 mL of water. The pH of the receiving solution was monitored with small pieces of papers cut from universal pH-test papers (Macherey-Nagel,Dueren, F.R.G.). Transport of K+ ions by a symport system was also performed to compare ita efficiency with that of the double carrier membrane. For the sake of comparison, the area of the DC18C6 membrane through which K+ ions are transported should be equal for both the double carrier and symport membrane systems. Therefore, for the eymport experimenta, a MA-free NPOE liquid membrane was mounted in place to the MA-incorporated NPOE membrane. Other solution conditions were the same for both transport systems. RESULTS AND DISCUSSION The function of Na+,K+-ATPasewas formally simulated by using a completely different chemical system as shown in Figure 2a. The unique feature of the present double carrier membrane system is that the system utilizes two carriers for enhanced uphill transport of K+ ions: The carrier DC18C6 is for uphill transport of K+ions from the feed solution to the receiving solution using picrate as a pumping ion, in other words, using a concentration gradient of pic-. The other
carrier, TOA, is used to pump out the cotransported pic- ions from the receiving solution back to the feed solution using H+ ions as pumping ions. This is a formal mimic of the multiple transport systems found in nature. Needless to say, the "double carrier" approach of the energy conversion of the present uphill transport is totally different from the enzymatic process of Na+,K+-ATPasein biological membrane systems: The present double carrier membrane involves only gradient pumping, while the Na+,K+-ATPaseis a reaction pumping system. However, the role of the carrier TOA for backpumping of pic- ions is, in principle, the same as that of ATPase in biological active transport in the sense that the concentration gradient of the pumping ions is maintained by the action of this second carrier. To make this mechanism actually work, the organic membranes containing the above two carriers, respectively, are physically separated from each other by a glass wall, as shown in Figure 1 , m that the forward and backward transport processes proceed parallel without interference caused by a mixing of both carriers. The time course of uphill transport of K+ ions by the present double carrier membrane system (Figure 2a) is shown in Figure 3. For comparison, K+ ion uphill transport by a simple conventional symport mode is also shown (Figure 2b). In both cases, the K+ ions are enriched into the small volume of receiving solution due to the wded feed/receiving volume effect ( 3 , 4 ) . However, a remarkable difference was found, as expected, between the two: The concentration of K+ ions in the receiving solution transported in the double carrier membrane system after a transport time of 23 h was 9 times higher than that in the simple symport system. Also, the concentration of pumping ions (pic-) in the receiving solution after some transport starting from the concentration zero was increased to a less extent in the double carrier membrane system than the simple symport system as shown in Table I, indicating that the cotransported pic- ions are actually recycled back to the feed solution in the double carrier membrane system. The expected function of the carrier TOA as a gradient pump for pic- ions is also confirmed by the fact that when the initialconcentrations of pic- ions in the receiving and feed solutions are made equal before transport (Figure 4), a decrease with time in the concentration of pic- in the receiving compartment was observed. The cyclic stoichiometry implied from the scheme in Figure 2 suggests that pic- on the
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
r
1
r
20
v
4
4
J
~~
25
20
OO
Time, h Flgure 4. Back pumping of pic- ions by carrier TOA from the receiving solution to the feed one as a function of time. Initial compositions are as follows: (1) the receiving solution (2 mL), 2 mM Li picrate, 0.01 M CH,COOLi-HCi buffer (pH 3.1): (2) feed solution (50 mL), 2 mM Li picrate, 0.01 M LiOH. Change in concentration of pic- in (1) the feed solution and (2) the receiving solutbn wlth the TOA-incorporated NPOE membrane system. Change in concentration of pic- in the receiving solution with (3) the NPOE membrane containing no TOA and (4) the DB membrane containing no TOA.
Table I. Concentration Change of Picrate Ions in the Receiving Solution
transport time, h 0 23
concentration of pic- ions,=M double carrier conventional membrane symport systemb systemb 0
