2253
Anal. Chem. 1984, 56.2253-2256 Pyrene and
and 02,do catalyze the oxidation of pyrene by SbC13 These species were undoubtedly present in the reagent grade SbCl, used here. Enhancement cannot be solely ascribed to oxidation of pyrene by SbC13 prior to bombardment, because maximum secondary ion emission occurs at temperatures below the melting point of SbC13,i.e., below temperatures at which oxidation is known to occur. This suggests that other processes may be contributing to enhancement. Evaporation of SbC13,for example, would result in increased concentration of pyrene in the solution. However, the intensity of the SbC12' ion (m/z 195) remains relatively constant between 30 O C and 65 "C. This may indicate that evaporation of SbC13does not significantly affect secondary ion intensity over the duration of the experiment. Further experiments are under way to develop the capabilities of SbC13 as a SIMS matrix and to identify the nature of the enhancement effect. These include measurements similar to those reported here, with samples prepared and SIMS analysis done under completely anhydrous conditions using zone-refined metal halides.
SbCI, Secondary I o n
I n t e n s i t y versus T e m p e r a t u r e
Pyrene, m/z 2 0 2
0
ACKNOWLEDGMENT We thank Chris P. Leibman, Gleb Mamantov, and A. C. Buchanan I11 for helpful discussion. Registry No. SbC13, 10025-91-9; pyrene, 129-00-0.
LITERATURE CITED (1) Barber, M.; Bordoll, R. S.; Elliot, 0. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 54 645A. (2) Caprloll, R. M. Anal. Chem. 1983, 55, 2387-2391. (3) Day, R. J.; Unger, S. E.; Cooks, R. G. Anal. Chem. 1980, 52. 557A. (4) Grade, H.; Cooks, R. G. J. Am. Chem. SOC.1978, 100, 5615-5621. (5) Ross, M. M.; Coiton, R. J. Anal. Chem. 1983, 55, 150-153. (6) Ross, M. M.; Colton, R. J. Anal. Chem. 1983, 55, 1170-1171. (7) DePauw, E. Anal. Chem. 1983, 55, 2195-2196. (8) Buchanan, A. C., 111; Livingston, R.; Dworkin, A. S.; Smith, G. P. J . Phys. Chem. 1980, 84, 423-427. (9) Sorlie, M.; Smith, G. P.; Norvell, V. E.; Mamantov, G.; Klatt, L. N. J . Electrochem. SOC. 1981, 128, 333-338. (IO) Perkampus, H.-H.; Schonberger, E. 2.Nafurfofsch., 8 1978, 31, 475. (11) Todd, P. J.; Gllsh, G. L.; Christie, W. H. I n t . J . Mass Spectrom. Ion Phys ., in press. Russell, D. H.; Smith, D. H.; Warmack, R. J.; Bertram, L. K. I n t . J . Mass Spectrom. Ion Phys. 1980, 35, 381-391. Weast, R. C., Ed. "Handbook of Chemistry and Physics," 64th ed.; Chemical Rubber Company Press: Boca Raton, FL, 1983. Miner, C. S.;Dalton, N. N. "Glycerol"; Reinhold: New York, 1953; p 266. I
\ ,
20
I
30
40
50
60
70
80
90
Temper a t u re
Figure 3. IT/125evs. temperature, where pyrene radlcal cation.
I is the Intensky of
the
20% of that generated from the pyrene/SbC13 sample a t 21 "C. Upon heating, the intensity of the pyrene molecular secondary ion was observed to increase by a factor of 3.5; however, the intensity of molecular ions at any temperature never equaled that produced from the pyrene/SbC13 sample at room temperature. As before, at higher temperatures and longer time after introduction, signal decreased due to sample depletion. Furthermore, the probe tip appeared charred upon completion of the experiment, indicating significant sample damage. Attempts to generate pyrene molecular ions from a 4 mol % pyrene/glycerol mixture failed; we attribute this to component insolubility. SbC1, clearly enhances secondary emission of molecular pyrene ions. Proximity of SbC1, to pyrene molecules in solution may assist ionization by reducing the energy necessary to form the pyrene molecular ion, e.g., via formation of a charge-transfer complex. Such a mechanism is suggested by reports indicating that pure (i.e., zone refined) SbC13 cannot oxidize pyrene (8). However, minor impurities, such as SbCl,
Gary S. Groenewold Peter J. Todd* Michelle V. Buchanan Analytical Chemistry Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37831
RECEIVED for review April 2,1984. Accepted May 25, 1984. Research sponsored by the US. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC05840R-21400 with Martin Marietta Energy Systems, Inc., and by the US. Department of Energy Postgraduate Research Training Programs administered by Oak Ridge Associated Universities.
Reverse-Phase Polystyrene Column for Purification and Analysis of DNA Oligomers Sir: The rapid synthesis of DNA oligomers of defined sequence has been successively achieved by phosphotriester and phosphite triester methods on solid support (1,2).Now
the main effort in this area should be directed toward a rapid process for isolating the pure oligomer of desired sequence. There are, at present, two kinds of purification methods
0003-2700/84/0356-2253$01.50/00 1984 American Chemical Society
2254
ANALYTICAL CHEMISTRY, VOL. 56, NO. 12, OCTOBER 1984
Scheme Ia DMT- XXXXX...
...X - PS
HO-XXX......XHO-X....
Ps
..X
- PS
1 ....
( 1 ) oximnle
( 2 1 NH40H
DMT- BBBBB.
&OH
HO-BBB......B-OH HO-B......B-OH
rev e r 80 -p ha $8 HP LC
( 3 ) SO% A c O H
*
HO-BBBBB......B-OH HO-BBB
HO-B
.....B- OH
....,. B-OH
anion exchange HPLC
a X , protected base; B, base; DMT, 4,4'-dimethoxytrityl; Ps, polystyrene.
outlined in Scheme I. In anion exchange chromatography, one has to use columns like AAX (3),S A X (4), or Zorbax-NH, (5). These columns can resolve a series of DNA oligomers with different chain lengths due to the different number of diester charges. The desired sequence will have the maximum number of charges in comparison with the shorter fragments resulting from less than 100% coupling efficiency during the synthesis on the solid support. For longer oligomers, say with 15 or more bases, the desired sequence might coelute with shorter fragments from the anion exchange column. I t will be particularly problematic if monomers are used in the coupling reactions instead of the dimers. Another problem is that if the overall synthesis efficiency is low, the desired peak might be smeared out by a large peak corresponding to the second largest fragment. Other columns that have solved oligonucleotide purification problems using ion exchange chromatography are RPC-5 (6) and NACS (7). As in other methods involving ion exchange, these columns require high molarity salt as the eluting solution. High salt solutions might have long-term undesirable effects on the HPLC system itself. Reverse-phase chromatography uses salt concentrations that are about 100-fold less in concentration, and harmful ions (for the HPLC system) like C1- are never used. Also, purification of self-complementary oligomers might be a problem by anion exchange HPLC as a large number of possible molecules with complete or partial overlap by base pairing might exist. Such base pairing may be prevented, but a high temperature like 70 "C may be necessary. The high temperature, in combination with the excess salt used for elution, might be bad for the HPLC system. In DEAE cellulose chromatography, one can use 7 M urea to prevent base pairing between different DNA fragments. But it will be very difficult to use the viscous 7 M urea buffer in HPLC systems, if not impossible. The other method commonly used is reverse-phase chromatography such as C18 (B), alkylphenyl (9),etc., which can selectively isolate the desired DNA oligomer bearing a hydrophobic protecting group a t its 5' end. This group is usually the 4,4'-dimethoxytrityl group (DMT) or 4-monomethoxytrityl group (MMT) for sequences with guanosine at the 5' end. The shorter oligomers that result from inefficient coupling do not have this protecting group at the 5' end due to detritylation before the last coupling step. Due to the presence of the DMT or MMT a t the 5' end, the longest (and desired) oligomer has a strong hydrophobic interaction with the nonpolar bond phase coated on the silica gel. Thus the strong advantage of this method is that purification is possible even if the coupling yield is small, as long as it is nonzero. However, the conventional reverse-phase column has two problems. One is its low capacity because the nonpolar alkylsilyl group is only a small fraction, by weight, of the total packing material. The
DNA oligomer sticks to the column due to its interaction with the alkylsilyl groups, which are few in number; so, for good resolution, one cannot use more than a milligram or so of the DNA oligomer in a single run. Another problem is its low recovery, around 50% or less for each run, probably due to the trapping of DNA in the pore of the silica gel. If the trapping is reversible, the DNA oligomer can be released in a subsequent run, which means a serious loss of control. Both of these problems have been successfully overcome by using a polystyrene-copolymer (PRP-1) column which had been introduced as a reverse-phase packing medium (IO, 11).
EXPERIMENTAL SECTION A series of DNA oligomers, dCGCG (tetramer), dCGCGCG (hexamer),and dGCGCGCGC and dCGCGCGCG (both octamers) were synthesized by the solid phase triester method ( I ) . After assembly of the DNA oligomer was completed, the solid support (around 100 mg in weight after synthesis) was treated with 0.5 M tetramethylguanidine syn-p-nitrobenzaldoximate for 24 h at 37 OC. This was followed by 28% ammonia treatment for 8 h at 60 OC. The sample was dialyzed against a 30 mM solution of triethylamine in water. After dialysis the sample was condensed and ready for analysis and purification by the PRP-1 column made by Hamilton Co., Reno, NV. PRP-1, polystyrene column 150 mm X 1.4 mm i.d., was purchased from Hamilton Co. The sample, a mixture of the desired and shorter oligomers, was applied to the column by a 210 sample injector (Altex). The column was enclosed in a temperature controlled oven (Waters Associates). For gradient elution two buffer systems were used: A buffer (10 mM ethylenediamineacetate, pH 7.6) and B buffer (50% v/v acetonitrile and 10 mM ethylenediamineacetate). HPLC was carried out on a Model 112 solvent delivery system (Beckman Instruments) regulated by a Model 421 controller (Beckman Instruments) at a flow rate of 1.5 mL/min. Efluent was monitored at 254 nm for analytical scale and 297 nm for preparative scale, respectively, by variable wavelength spectroflow (Axxiom). The same column was used for both analytical and preparative scale purifications. DNA has a smaller value of the extinction coefficient (but nonzero) at 297 nm, so this wavelength was used to monitor the preparative scale runs. If 254 nm is used for preparative runs, the peak as detected by the detector becomes saturated at the beginning of the peak, and the shape of the peak is not as nice as detection with the 297-nm monitoring. Recovered, resolved oligomers can be concentrated simply by evaporating the acetonitrile and the water from the sample. Recovered oligomers were analyzed by polyacrylamide gel electrophoresis in the presence of 7 M urea. Only one band could be noticed in all cases, and the position of the band on the gel was consistent with the length of the DNA oligomer. RESULTS AND DISCUSSION The use of the PRP-1 column for the purification and analysis of oligodeoxyribonucleotides was studied. All HPLC operation was performed a t 60 "C, at which the formation of secondary structures of the oligomers can be prevented. The separation of the tetramer and the hexamer (both with 5'hydroxy groups) was performed with a linear gradient as shown in Figure 1. The retention times for the octamer dCGCGCGCG, the hexamer dCGCGCG, and the tetramer dCGCG were 7.3 min, 6.8 min, and 5.5 min respectively. Also, (PA), and (pT), had longer retention times on increasing n (12).
The retention time on the reverse-phase column seems to be directly proportional to the hydrophobicity of the solute, here the DNA oligomers. One can define the net hydrophobicity of an oligomer as a sum with two parts. The first part corresponds to the hydrophobicity due to the bases, and the second part due to the hydrophobicity of the polar groups (may be assigned a negative value), mainly due to the phosphodiester group. The second term is constant for all nucleotides in the oligomer, irrespective of the bases. But the first term is base specific. The net hydrophobicity can be
ANALYTICAL CHEMISTRY, VOL. 56,
NO. 12, OCTOBER 1984
2255
Peak
I n
2
I
'
2 6 10 ( M I N ) Retcntlon Time Figure 2. Large scale purification of DMT-CGCGCGCG on the PRP-1 column. A linear gradient from 20 to 50% of B buffer during 15 min was used and the efiuent was detected at 297 nm to avoid saturation in peak shapes. About 200 optical density units (254 nm) of the second peak was recovered. Injection volume was around 0.3 mL.
I \ 3--
Retention T i m e
(Mln.)
Flgure 1. HPLC elution profile for the series d(CGCG), d(CGCGCG), and d(CGCGCGCG) on the PRP-1 column. DNA oligomers were eluted with a linear gradient of B buffer from 0 to 20% in 10 min. Amounts Injected for each oligomer were between 0.3 and 0.5 optical density units at 254 nm. Volume of injection was around 0.3 mL.
-
E
h
2.4
r
l
1
regarded as a sum of the terms for all the constituent nucleotides n
H = C B ; + (n - 1)P
(1)
i=l
where B, is the hydrophobicity of base i and P is the effect of each phosphate. For an oligomer like poly-A or poly-T, the various B,'s are also identical, and H = nB + ( n - l)Pfor an oligomer of n bases of the same kind. The difference N(for n 1 oligomers) - H(for n oligomer) = (n + l ) B nP - nB - (n- l ) P = B P is the difference in the net hydrophobicity between such polymers of n + 1and n bases, all of the same kind. Thus, depending on the sign of B + P, the shorter or the longer homopolymer will be retained longer on the reverse-phase column. The proposal that the net hydrophobicity of the solute is a sum of the hydrophobicities of its subunits is probably reasonable. Correlations such as this have been used successfully with other types of solutes and stationary phases (13). The order of hydrophobicity for nucleosides a t neutral pH is given by the following order (IO): C < G < T < A. The difference in hydrophobicity between the pyrimidines can be explained by the presence of the methyl group in T a t the 5 position. The difference in hydrophobicity between purines may be attributed to the fact that G has more polar groups (carbonyl and amino) compared to A (only amino). As A and T have large B values, although P is negative, B P is still positive. Thus, for A and T, longer oligomers elute later from the reverse-phase column. It has been experimentally possible to resolve peaks for the series (PA), and (pT),, n = 2, 4, 6, 8, 10, by HPLC (12). Longer oligomers are eluted closer to each other than the shorter ones, because although the hydrophobicity difference is the same between successive polymers, the percentage of difference in the net hydrophobicity decreases as the length increases. The magnitude of B + P is smaller for C and G, but still positive. Thus, when we compare a series of oligomers dCGCG, dCGCGCG, and dCGCGCGCG, it is found that the tetramer elutes first, then the hexamer, and lastly the octamer (Figure 1). The feasibility of the polystyrene column for the
+
+
+
+
7 IO Retention l i m e ( M I N ) Flgure 3. Repurification of the octamer d(CGCGCGCG) after DMT removal on the PRP-1 column. The completely deblocked oligomer was reinjected on to the PRP-1 column and eluted by a linear gradient of B buffer from 0 to 20% in 10 min. Efluent was detected at 297 nm. All of peak I1 from the run In Figure 2 could be repurified by the present run. Injection volume was 0.3 mL.
purification of DNA oligomers in large quantities was also examined by using a mixture of DMT-CGCGCGCG and a series of shorter oligomers (i.e., dCGCGCG, dCGCG, dCG, and dG) without the DMT group. Figure 2 shows the separation of the desired oligomer with the DMT group (peak 11) from the shorter ones without the DMT group (peak I). Although peak I consists of oligomers of various lengths, they could not be resolved into individual peaks as the efluent contained a large amount of acetonitrile from the very start (gradient was from 20 to 50% B buffer). After detritylation, the fully deprotected oligomer was repurified by one more HPLC ri n (Figure 3). The recovery of DNA from the HPLC runs W:~S measured at least five times and found to be constantly over 95%. The net capacity of the column may be around 25 mg of DNA, but resolution may get worse with such high loading. It is advisable to limit the amount to 10 mg for a single run. We tested the column for quantitative recovery for even longer oligomer, one with 16 bases. The useful life of the column seems to be around 1year with moderate use, but in general might be dependent on the user's care. The capacity of the column is also dependent on the type of buffer used. EDAA seems to serve the purpose quite well.
2258
Anal. Chem. 1984, 56,2256-2257
Registry No. dCGCG, 58927-25-6; dCGCGCG, 58927-26-7; dGCGCGCGC, 80458-01-1; dCGCGCGCG, 89991-79-7;PRP-1, 9003-70-7.
LITERATURE CITED Ito, H.; Ike, Y.; Ikuta,
1755.
S.;Itakura,
K. Nucl. Acids Res. 1982, 10,
Matteucci, M. D.; Caruthers, M. H. J . Am. Chem. SOC.1981, 703,
3185. Miyoshi, K.; Miyake, T.; Hozumi, T.; Itakura, K. Nuci. Acids Res. 1980, 8, 5473. Gait, M. J.; Singh, M.; Sheppard, R. C. Nucl. Acids Res. 1980, 8,
1081. McLaughlin, L. W.; Romaniuk, E. Anal. 6iOCh8m. 1982, 724, 37. Wells, R. D.; Hardies, S.C.; Horn, G. T.; Klein, E.; Larson, J. E.; Nevendorf, S.K.;Panayotatos, N.; Patient, R. K.;Selslng, E. Methods Enzymol. 1980, 65, 327. Thompson, J. A,; Blakesley, R. W.; Doran, K.; Hough, C. J.; Wells, R. D. Methhods Enzymol. 1983, 100, 368. Ike, Y.; Ikuta, S.; Sato, M.; Haung, T.; Itakura, S. Nucl. Adds Res. 1983, 11, 477.
(9) Ikuta, S.,City of Hope Medical Center, Duarte, CA, unpublished work April 1981. (10) Lee, D. P.; Kindsvaster, J. H. Anal. Chem. 1980, 52, 2425. (11) Lee. D. P. J. Chromatoor. Sci. 1982..~20.. 203. (12) Haupt. W.; Plngoud, A. 2. Chromatop. 1983, 260, 419. (13) fchapla, A.; Colin, H.; Gulochon, G. Anal. Chem. 1884, 56, 621.
Satoshi Ikuta* Rajagopal Chattopadhyaya Richard E. Dickerson Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, California 90024
RECEIVED for review April 2, 1984. Accepted May 21, 1984. This work was carried out with the support of the University of California and National Science Foundation Grant PCM82-02775.
In Situ Cleaning and Activation of Solid Electrode Surfaces by Pulsed laser Light Sir: Whether used for analysis or for studying electrode processes, the surface of a solid electrode changes with time due to adsorption of species from solution or chemical changes to the surface itself. These changes often result in variations in sensitivity or reversibility, and in extreme cases lead to complete inhibition of charge transfer. Electroanalytical chemistry originated with the dropping mercury electrode (DME) because the DME surface was renewed every few seconds and a clean, reproducible surface was thereby assured. We report here an analogue to the DME for solid electrodes, where an intense laser pulse is used to clean and activate a platinum or glassy carbon surface directly in the solution of interest. The effects of intense pulsed laser radiation on electrodes include removal of polymeric or adsorbed films and large increases in electron transfer rate. While the DME has the important property of periodic renewal of the surface, its potential range is constrained to that of mercury, and its mechanical properties make it unsuitable for many applications. The original motivation for the development of carbon paste by Adams was the desire for a renewable solid electrode suitable for potentials positive of mercury oxidation (1). A variety of methods have been devised for pretreating solid electrodes, including polishing, chemical pretreatment, flaming, potential cycling, vacuum heat treatments, and ion etching (1-11). These procedures produce widely varying effects on charge transfer rate, mainly because they vary greatly in the resulting degree of surface cleanliness. There is no agreed upon standard procedure for preparing solid electrode surfaces, and results for similar procedures vary greatly from lab to lab. None of these processes is capable of removing adsorbed films in situ, and none is repeatable on a short time scale, like the DME. An ideal surface treatment for electrochemistry would be one which could be carried out directly in the solution of interest, would generate a clean, reproducible surface, and would be repeatable on a time scale of seconds or less. Laser radiation has been used to retard corrosion by pretreating a metal outside the solution to redistribute alloy components (12,13). Laser pulses have also been used in situ to initiate corrosion by removing oxide layers (14) and accelerate electroplating (15). In the present work, the second harmonic (532 nm) of a Nd:YAG laser was focused onto
platinum and glassy carbon electrodes, and single pulses of varying energy were used to treat the surface, in situ. Depending on power density, the effects of the laser vary from surface heating and desorption to drastic effects such as local melting and vaporization (16). Two experiments will be reported here: activation of glassy carbon toward ascorbic acid oxidation, and removal of passivating polymeric films caused by phenol oxidation. Ascorbic acid oxidizes reversibly on mercury on the polarographic time scale (In,and extrapolation of pH 2-7 data indicates a half wave potential of +0.250 V vs. SCE at pH 1.0. On Pt or glassy carbon electrodes, slow charge transfer shifts the apparent half wave potential to +0.5 to +0.6 V, as shown in Figure 1, curve a. Curve b in Figure 1 was taken after a single 2 mJ, 20 ns laser pulse was delivered to the center of the electrode, covering a few percent of the active surface. Curve c was taken after several hundred such pulses were applied over about 50% of the electrode surface. The peak at +0.305 V did not occur in the absence of ascorbic acid and was also observed if the ascorbic acid was introduced after the laser pulse. Its peak current was linear with the square root of the scan rate, and the peak appeared completely well behaved. One concludes that the peak at +0.3 V corresponds to ascorbic acid oxidation with fast charge transfer, occurring at electrode regions irradiated with the laser. The peak at +0.305 V decayed with a half-life of about 90 min but was easily restored by further laser treatment. A crude estimate of the increase in heterogeneous rate constant was made by comparing the reversible half wave potential on mercury with those observed on carbon, assuming (1- a)n, = 1 for simplicity. Such a calculation indicates that the laser pulse caused an increase in charge transfer rate constant by a factor of approximately 5000. Comparable large increases in charge transfer rate have been observed for the ascorbate/carbon system following surface modification (18) vacuum heat treatment (11) or potential steps to very positive potentials (7,19). However, the heat treatment and surface modification cannot be done in situ and the electrochemical procedure exposes the solution to potentials which may alter subsequent observations. In contrast, the laser pulse occurs in situ, lasts for only 20 ns, may be repeated 10 times/s, and is carried out at fixed potential.
0003-2700/84/0356-2256$0 1.50/0 0 1984 American Chemical Society
