Anal. Chem. 1988, 60, 1677-1680 (20) CRC Handbook of Chemistry and fhysics, 66th ed.; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1985; pp D167-169. (21) Reference 20, pp D161-163. (22) Terabe, S.; Otsuka, K.; Ando, T., submitted for publication in Anal. Chem (23) Reijenga, J. C.; Aben, G. V. A.; Verheggen, Th. P. E. M.; Everaerts, F. M. J . Chromatogr. 1883, 260, 241-254. (24) HjertBn, S. J . Chromatogr. 1885, 347, 191-198.
.
1677
Lukacs, K. D.; Jorgenson, J. W.
HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1885, 8 , 407-411. (26) Terabe, S.;Utsumi, H.; Otsuka, K.; Ando, T.; Inomata, T.; Kuze, S.; Hanaoka, Y. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Common. 1888, 9 , 666-670. (25)
RECEIVED for review January 5,1988. Accepted April 20,1988.
Chromatographic Behavior in Electrochromatography Takao Tsuda Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466, J a p a n
An equation for the height equlvalent of a theoretical plate In electrochromatography Is developed. The flow is the summatlon of pressurized flow and eiectrophoretlc flow. The terms of mass transfer In the moblie phase and the coupling term of eddy dlffuslon are reduced compared to those In conventional chromatography. To attain a constant retention time, about 20 mln must elapse after electrovottage Is applied along a column. This time lag might be equal to the period needed to alter the nature of the column supports due to the application of electrovoltage. I n eiectrochromatography, It is posslble to retaln a charged sdute In a column if tts moblltly Is toward the column head and equal or larger than the pressurlzed flow veloclty. Typical separations are demonstrated.
Electrochromatography, in which two functions (mobility and sorptive interaction) have been used a t the same time, may be a highly effective method (1-3). Otsuka and Listowsky (1)and O’Farrell (2) separated ferritin subunits by applying electric voltage along a column. Since applied voltages in these experiments were relatively low, such as 12 V/cm, the separation process took more than 10 h. In our former report (3) we demonstrated electrochromatography with a high electric voltage along a column (0.5 X 74 mm) and obtained an effective separation within several minutes. In the present study, we examine the chromatographic behavior of electrochromatography and demonstrate typical separation examples.
EXPERIMENTAL SECTION The apparatus used was nearly the same as in our earlier report (3) except as follows. The pumping flow was split in front of a injector in two ways, that is, an analytical column via the injector and a resistant column (4.6 X 150 mm, packed with silica with chemically bonded octadecylsilane groups (ODs), 7-pm particle diameter). To prevent the evolution of bubbles, 50-pm fused silica capillary tubing of 15cm length was connected to the line following the terminal after the UV detector (254-nm,UVD-2, Shimadzu, Kyoto, Japan). Analytical columns were tetrafluoroethylene tubings packed with ODs, having a particle diameter of 7 pm (Develosil-7,Nomura Kagaku, Seto, Aichi, Japan). Columns I (145 mm in length and 0.6 mm i.d.), I1 (100 X 0.5 mm), and I11 (130 X 0.5 mm) were used. A pump (LC-4A, Shimadzu) was used at a constant pressure mode. As we used microcolumns, most of the pumping flow was passed through the resistant column. Reagenk of guaranteed grade (Wako Pure Chemical, Ltd., Osaka, Japan) were used. Adenosine-5’-monophosphate(AMP) was purchased from Yamasa-Shoyu, Ltd., Tokyo. Retention times and plate heights, H, of solutes were generally measured after the 0003-2700/88/0360-1677$01.50/0
voltage had been on at least 20 min, except as otherwise noted.
THEORY OF BAND BROADENING In electrochromatography there are two new factors, electrophoretic mobility and electroosmotic flow, that do not exist in ordinary liquid chromatography. Therefore, the band broading of a solute in electrochromatography is different from that of ordinary chromatography. The apparent mean linear flow velocity of a solute, u(app), is u(app) = u(pres) + u(mob) + u(osm) (1) where u(pres), u(mob), and u(osm) are mean linear flow velocity due to pressurized flow, mean linear flow velocity due to mobility of a solute, and mean linear flow velocity due to electroosmotic flow. Generally, u(osm) in a slurry-packed capillary column is one-fifth to one-tenth of u(osm) in an open tubular capillary column ( 4 , 5 ) . Electroosmotic flow observed for a column packed with Partisil 10-ODS using 85/15 methanol/water was 0.16 mm s-l at 900 V cm-’ (4). If u(osm) in a slurry-packed capillary column is considerably slower than u(mob), eq 1 becomes u(app) = u(pres)
+ u(mob)
(2)
In general chromatography, four processes contribute to band broadening of component zones as they migrate through the column, namely, axial molecular diffusion, resistance to mass transfer in the stationary phase, resistance to mass transfer in the mobile phase, and the coupling term of eddy diffusion. The plate height is given by the following expression of Giddings (6):
H = B/u
+ C,u + C,u + (1/A + l/C,u)-l
(3)
where u is mean linear velocity and A , B , C,, and C, are coefficients for eddy diffusion, axial molecular diffusion, resistance to mass transfer in the stationary phase and resistance to mass transfer in the mobile phase, respectively. Only C,u in eq 3 is affected by the shape of the velocity profile in the column. Thus, the third and fourth terms in eq 3 are affected by it. In the process of flow in electrochromatography, the flow of u(pres) is operated with a Poiseuille flow profile, and we suppose that the flow pattern of u(mob) would be a plug flow profile. Therefore, the velocity inequality in the radial direction is only due to laminar flow, namely, u(pres). The flow pattern in electrochromatography is supposed to be mixed with Poiseuille and plug flow. The actual net flow profile assumed is given in Figure 1. At time to,a solute is introduced as a plug, F(to). After time t,, the flow profile becomes F(t,) due to laminar flow in the condition without applied voltage. As every point of F(tll) gains an additional flow of u(mob) with 0 1988 American Chemical Society
1678
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
zE Y
A2
0 8 1
/.
/ . I ’.
/
2
1
Ai
06
I
Figure 1. Flow profiles.
applied voltage, the actual net flow profile in electrochromatography is supposed to be F(t12)at time t,. The difference between t , and t o is unit time. The process of mass transfer in the mobile phase is a hybrid of lateral diffusion and velocity inequality of the flow pattern. The application of a random walk model for estimation of band broadening in general chromatography has been well developed by Giddings (6). With the random walk model, the contribution of mass transfer in the mobile phase, H3, is calculated in chromatography as follows. A molecule must diffuse a distance w,d, to reach one extreme velocity from another. The multiplying term w, acquires values from well below to well above unity in terms of different flow channels (6), and d, is the particle diameter. Giddings (6) considered five or six different w values for various eluent nonequilibrium conditions, not all of which are equally important. For simplification, we discuss them here in general. The average time, t,, needed to diffuse the distance w,dp is
0 4 /
02
0
1
2
3
v[pres)
4
5
[ m m / secl
Flgure 2. Relation of H and pressurized flow velocity. Solutes of A and B were AMP and uracil, respectively; applied voltage was 2 kV.
l
o
> 40
*
20
-3
I
t , = wU2dp2/2D,
(4)
where D, is the diffusion coefficient in the mobile phase. The length of step 1 is the distance gained or lost with respect to the mean (peak center) because of the temporary residence in one of the velocity extremes.
i = wp(pres)t,
+ u(mob))-’
(6)
On combining these expressions with a’ = 1% and H = o z / L (a is the standard deviation of zone in length units), we obtain
H3:
where C, is equal to (~,w~d,)~(2D,)-~. Therefore, the H in electrochromatography is given as follows:
H = B/u(app)
+ C,u(app) + H 3 + (1/A + l/H3)-l
I
3
0
0
(5)
where w is equal to Au(pres)/u(pres) and Au is the difference between the extreme and the mean. The number of random steps while a solute passes through the total pass length, namely, column length L , is expressed as follows:
n = Lt,(u(pres)
U
(8)
From the comparison between H values for general chromatography and electrochromatography, namely, eq 3 and 8, respectively, the contribution of u(mob) makes the value of each term smaller than in general chromatography. Therefore, H in electrochromatography has to be reduced, especially at lower and higher u(pres). The effect of the electric field for terms in H , eq 8, is the most effective for the third term, namely, mass transfer in the mobile phase.
RESULTS AND DISCUSSION Experimental results regarding the relationship of H and u(pres) are shown in Figure 2. Subscripts 1and 2 in Figure 2 indicate with and without applied voltage, respectively. Column I was used, and the effluent was 8 X M phosphate
Figure 3. Variation of retention time and peak h e i t due to the period of voltage application: peak height of methanol (0);retention times, AMP (A)and methanol (+); applied voltage, 2.5 kV.
buffer (pH 4.5). H values with applied voltage are smaller than those without applied voltage. In the case of AMP, H is reduced remarkably due to its large electrophoretic mobility ( 3 ) . These differences between H values with and without applied voltage might come mostly from eq 8. In electrochromatography, retention time and peak height of a solute depend on the time from the beginning of the voltage application. Variations of these values for methanol and AMP are shown in Figure 3. Column I1 and M phosphate buffer as effluent were used. As pure methanol was used as a solute, it gave a W detector response. Retention times and peak heights for solutes were measured before, during, and after the application of voltage (Figure 3). Retention times of methanol and AMP become constant 20 min after the application of electrovoltage, and when electrovoltage was released, the retention times become constant after 10 min. These time lags to attain a constant value of retention may be equal to the period that is necessary to alter the nature of column supports by electrovoltage. Without applied voltage, the methanol peak in Figure 3 is a low and broad one. But, with applied voltage, it becomes a high and sharp peak, and peak area is also twice as large when compared to that without any applied voltage. This phenomenon, also observed in the case of AMP, might be due to the alteration of the column support. More evidence for the alteration of the column support with applied voltage is shown in Figure 4. Applied voltage was
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
.-
30
-
20
-
10
-
1679
\
E ’ --
‘
0
20
IO Rt
6
30
(mini
s u 4 [r
Figure 4. Retained solute in column in the condition of applied voltage. N, was with applied voltage; N, and N, without. N, was obtained just after release of applied voltage at time zero. Solute was N-methylphenylpyridinium perchlorate (A) and its impurities (other peaks).
9.6 kV. Column I11 was used, and a mixture of 1.7 X M phosphate buffer (pH 7) and methanol (30/70) was used as effluent. N1,N2,and N, were a series of experiments. With applied voltage, the chromatogram of N1 shows three peaks. After several injections with applied voltage, the application of voltage was stopped at zero time of Nz,and a chromatog” of N2 was obtained without an injection. After a large peak was eluted out, the base line became straight, and the chromatogram of N3was obtained with an injection. N-methylphenylpyridinium perchlorate, peak A in Figure 4,was only eluted out when no voltage was applied. Under the condition of applied voltage, only minor components in the sample of N-methylphenylpy-ridinium perchlorate were eluted, as shown in N1 of Figure 4. During several injections with applied voltage, the main peak of N-methylphenylpyridiniumperchlorate had been retained in the column. Therefore, after the electrovoltage was released, it emerged as a large peak (peak A of Nz in Figure 4). A reverse phenomenon was also observed. Just after the application of electrovoltage to the column began, several peaks were eluted out without any injection. These peaks might belong to components in the sample solution or the eluent itself. From the above experimental results, we obtained the following conclusions. The time lag for attaining a constant retention time or peak height corresponds to the period for saturating or releasing adsorptive materials from the surface of the column support due to application or release of the electrovoltage. The surface of the support, when we apply voltage, might become more polar due to polar adsorptive materials of charged species and/or the environment of the electric field. Although these adsorptive materials are unknown, it is supposed that they are carried with the eluent into the column. If we apply electrovoltage along a column that has been just packed, several materials adsorbed on the surface of the supports will come out from the column support. Therefore, the application of electrovoltage is one method to clean the column so as to remove unfavorable charged substrates, which were tightly adsorbed during the preparation of the column support and the column packing procedure. The relation between retention time and pressurized flow for AMP is plotted in Figure 5. Experimental conditions were the same as in Figure 1. Since the solute moves because of both pressurized flow and its mobility under applied voltage, the retention times are reduced compared to those without applied voltage. As the ratio of u(mob) to u(app) for AMP becomes larger under slow pressurized flow, the difference between P1and P2becomes larger compared to that at faster pressurized flow. Typical chromatograms are shown in Figure 6. Column I11 and the mixture of phosphate buffer (pH 6.7)(109 M for A and M for B) and methanol (12/88) as effluent were used in Figure 6. In the chromatogram of phenylthiohydantoin (PTH) derivatives of asparagine, two unknown
3
-
2
-
018
1
, 0.2
, , , ,,, 0.3
0.5 0.7
/ ,
1
v[presl
,
,
,
2
3
4
[mm/secl
Flgure 5. Variation of retention times of AMP at different pressurized flows under a constant applied voltage. P, and P, were with and without applied voltage, respectively.
1
without applied voltage
A
2
~
with applied voltage
A
B
4
+ 2 min
Figure 6. Chromatograms of PTH derivatives of asparagine (A) and 2-naphthalenesulfonic acid (B) with and without applied vottage. Applied voltages for A and B were 7.4 and 11.8 kV, respectively.
peaks (1and 2 in Figure 6) are well separated with applied voltage, and in the chromatogram of 2-naphthalenesulfonic acid, peak 3 is retarded with applied voltage; its retention times become 3.5 times longer. However, peak 4 is not retarded with applied voltage, and thus may be a neutral compound. Electrochromatography is a good method to separate charged components from neutral compounds. This method
Anal. Chem. 1900, 60,1680-1683
1600
might be useful for the pretreating of a microsample by the process of electrical purification.
LITERATURE CITED (1) (2) (3) (4) (5)
Otsuka, S.; Listowsky, L. Anal. Biochem. 1980, 102, 419-422. O'Farrell, P. H. Science (Washington, DC.) 1985, 227, 1586-1589. Tsuda, T. Anal. Chem. 1987, 59, 521-523. Stevens, T. S.; Cortes, H. J. Anal. Chem. 1983, 55, 1365-1370. Tsuda, T.; Nomura, K.; Nakagawa. G. J . Chromatogr. 1982, 248, 241-247.
(6) Giddings, J. C. Dynamics of Chromatography; Dekker: New York, 1965.
RECEIVED for review December 16,1987. Accepted April 25, 1988. Part of this paper was presented at 8th International Symposium of Capillary Chromatography, Riva del Garda, Italy, May 19-21, 1987. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture (No. 62010037).
Determination of Reduced Sulfur Compounds in Aqueous Solutions Using Gas Chromatography Flame Photometric Detection Caroline Leck* Department of Meteorology, University of Stockholm, S-106 91 Stockholm, Sweden
Lars Erik Bagander Department of Geology, Section Microbial Geochemistry, University of Stockholm, S-106 91 Stockholm, Sweden
A method for simultaneous analysis of hydrogen wlfkle (H,S), methyl mercaptan (CH,SH), carbon disulfide (CS,), dimethyl sulfide (DMS, CH,SCH,), and dimethyl disulfide (DMDS, CH,SSCH,) In aqueous solutions is described. The reduced sulfur compounds are released from the aqueous sample (50-200 mL) by purging with N, and then trapped cryogenically in a U-shaped sample tube with llquld nitrogen. The sample tube was sealed with end caps and placed in a portable freezer. Under stable conditions In the laboratory, the sulfur compounds are released by controlled heating and Injected onto a packed column gas chromatograph with a flame photometric detector. The precision of the method for environmental samples was better than f5% for ail compounds except for H,S, for which the precision was f25%. The detection llmlts for H,S, CH,SH, CS,, DMS, and DMDS were 1, 0.6, 0.2, 0.2, and 0.4 ng-L-' S, respectively, in a 200-mL natural sample. Analyses of environmental samples have been successfully performed with the described method.
The estimated flux of 30-50 Tg of S/year of reduced sulfur compounds from the oceans ( 1 )contributes significantly to the atmospheric sulfur budget. In that flux, dimethyl sulfide (DMS) dominates (99%) over other reduced sulfur compounds (1). Due to analytical difficulties, the estimates of sea to air fluxes of carbon disulfide (CS,), methyl mercaptan (CH,SH), hydrogen sulfide (HzS) and dimethyl disulfide (DMDS) may have been systematically underestimated (2-4). These reduced sulfur compounds originate mainly from marine phytoplankton. So far, the mechanisms leading to the production of the reduced sulfur compounds in surface waters are not well understood. We have developed an analytical method where simultaneous detection of HzS, CH,SH, CS2, DMS, and DMDS can be performed in a single seawater sample. This is a great improvement compared to earlier methods (3-5) when interpreting measurements, especially when one wishes to understand the regulating mechanisms for the production
and fluxes of reduced sulfur from the oceans to the atmosphere. The analytical device consists of an easily handled field sampling system separated from the analytical system; the latter was kept under stable conditions in the laboratory. This two-step procedure allows sea surface sampling by helicopter where large areas can be covered within a short time span. A particular advantage with this sampling technique lies in the interpretion of the reduced sulfur emissions as a function of meteorological and biological parameters without considering induced variability due to long sampling times. The analytical method has been tested on more than 400 seawater samples, with total reduced sulfur concentrations ranging from
