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ANALYTICAL CHEMISTRY, VOL 50, NO 6, MAY 1978
LITERATURE CITED (1) S D James and L E DeVries, J
Electrochem soc , 123, 322 (1976)
(2) L E DeVries and E Gubner. this issue
RECEIVED November 18, 1977. Accepted January 20, 1978.
T h i s work was supported by t h e Independent Research Program a n d t h e Molten Salt Battery a n d Lithium-Chlorine Ratter5 Programs of t h e Naval Surface Weapons Center, F'hite Oak, Md.
Effluent Detector for Chromatographic Columns Using Volatile Solvents Norman S. Radin" and Dan del Vecchio Mental Health Research Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48 109
Preparative liquid chromatography is ordinarily operated in conjunction with a fraction collector a n d t h e individual fractions are subsequently analyzed by some separate procedures. Recent developments in monitoring systems have in many applications m a d e it possible t o follow t h e elution a n d assist t h e chemist in understanding t h e effected separation. All of t h e monitoring devices described or commercially available a r e of t h e continuous type, producing a record on a strip chart corresponding t o some physical property. Most monitoring devices are limited in their applicability, depending on a single physical property or chemical reaction, a n d interference by t h e elution solvent can be a serious obstacle. W e thought it would be useful t o have a device which would be independent of the solvent composition, provided t h e solvent was reasonably volatile, which could be used with a variety of detection techniques. T h e principle utilized was the evaporation of portions of the column effluent in t h e form of discrete spots on a long strip of paper, corresponding t o individual fractions in t h e fraction collector.
EXPERIMENTAL Materials and Design. The physical arrangement of the device components is shown in Figure 1. The column effluent goes through a stream sampler, A, operated by compressed air. When the sampler is activated, an aliquot of the effluent (1.6 pL) is transferred t o the paper strip just below. The transfer is produced by a stream of air (0.2 mL/min) coming from a peristaltic pump. We use a Brinkmann Ismatec Mini-Micro 2/6 pump, fitted with a tube 0.015-in. i.d. The paper strip is held taut against the delivery tube of the valve by a weight attached to the front end of the strip, a "bulldog" paper clamp. After delivery of the desired number of 1.6-pL drops, the paper strip is advanced 11 mm by simultaneous activation of the air solenoid valve, B, and solenoids C and D. Solenoid D normally holds the paper strip against stopping block E and prevents slippage; when D is activated, it frees the strip. When solenoid E is activated, it pushes the notching block, F, down against the paper strip, producing two bosses (notch-like depressions), one on each edge of the strip. The bosses mark the location of each dried spot and act to minimize contact of the dried material with other surfaces that might contaminate or adsorb the material in the spot. Valve B causes the plunger in the pneumatic linear actuator, G, t,o move forward. Because time is required for the air to build up pressure in the cylinder, the plunger does not begin to move until solenoids C and D have completed their movements. Solenoid C is mounted in frame H so it moves-together with the paper strip-until the frame reaches stopping block E. The bottom plate in the frame has two small holes to accommodate the two projections at the bottom of notching block F. The latter is fastened firmly t o the solenoid plunger with two screws, which serve as set screws and attachment points for two return springs. A fraction of a second after frame H hits block E; power to the three solenoids is removed and springs in the solenoids and air cylinder return them to the rest positions (as drawn). The paper strip cannot move further because it is held by solenoid D. The distance between spot centers is determined by the distance 0003-2700/78/0350-0824$01 . O O / O
between the left edge of block E and the right edge of frame H. This gives ample separation between spots, which are about 5 mm in diameter. The timing control for the device also operates the fraction collector. It is set to control the collection time for each fraction and the number of drops to be dried on each position on the paper strip. One must estimate how much liquid to apply to each spot. This is done by estimating the weight of the material of interest that is expected, and the volume of liquid in which it is hoped elution will occur. For example, one might use a solvent pump delivering 2 mL/min and collect 5-min (10-mL) fractions. If 90 mg are expected to elute in 30 mL, the average concentration will be 3 pg/pL. If one wishes to deposit, on the average, 24 pg in each spot, one must set the timer to apply 8 p L , or five 1.6-pL drops during each 5-min collection interval. To make the five drops more representative of the collected fraction, we have set the timer to deliver the first drop after one half of the first subinterval; this is 0.5 min in the above example. The remaining drops in the run are deposited after full subintervals (1 min). This arrangement also ensures sufficient time to move the last drop from the sampling valve to the paper strip prior to movement of the strip. A stream of house air is used to dry each drop promptly after transfer to the paper. In the case of materials that are very soluble in the column solvent, there is migration to the edges of the drop, with formation of a ring of dried material. This helps to make the dried material more visible with the detecting reagent; if a more uniform spot is desired (for photometric determination), the air stream can be heated. To prevent loss of material and contamination ofthe fraction collector while the collector is moving, the column effluent is passed through a valve held just above the collection vessels (Figure 2). When the timer actuates the fraction collector and paper advance device, it also produces a brief latching pulse to close the valve. Effluent then accumulates in the reservoir above the valve. When the fraction collector movement is complete, a second latching pulse opens the valve. We use two different tips on the bottom of the valve: a narrow one (E) for small columns and a wider one (D) for higher flow rates. T h e latter is not suitable for slow columns because deposition of dry material can occur at the tip; the former is not suitable for fast columns because the reservoir will then overflow. Not shown in Figure 2 is a protecting glass cylinder held around the drip tip of D by means of an O-ring. This reduces evaporation of solvent and subsequent deposition of material on the tip. The timer used to control the spotter, fraction collector. and collection valve is a solid state device that was built in our shop. A suitable timer could be made with a variable speed cam-type microswitch controller: supplemented by a separate pair of short interval cam timers for the paper advance solenoids and the collection valve. We are presently adapting a microprocessor unit to fulfill the same functions while producing a solvent gradient. Procedure. For counting radioactive spots, we simply cut the strip with a scissors and deposit the sections in scintillation vials. This technique was used t o evaluate the uniformity of spot formation and look for trailing. A radioactive solution mas allowed to flow through the stream sampling valve and eight groups of spots were collected, consisting of 3, 6, and 1 2 drops/spot. c' 1978 American Cherrical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978
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Figure 1. Paper strip effluent spotter. (A) sample injection valve (Model CSV-2) with pneumatic actuator and spring return, Laboratory Data Control, Riviera Beach, Fla., nominally 2-pL capacity; (B) 3-way air solenoid valve with muffler for exhaust air; (C) push type solenoid, intermittent duty, 110-oz push at ' / a in., 3/4-in.stroke (Gaurdian Electric No. 16P); (D) a smaller solenoid, two return springs attached to E not shown; (E) stopping block, Nylon, screwed to support plate; (F) notching block, Nylon; (G) air cylinder, linear actuator, '/*-in. bore, 1-in. stroke, spring return, single ended, spring in front; (H) brass advancing frame, holding solenoid C; (I) '/4-in. copper tubing for air stream to dry aliquots; (J) guide wheels for paper strip; (K) small pressure regulator, delivering 25 psi air; (L) box of Whatman No. 1 chromatography paper reel, ' I 2 in. X 100 yards, with slot cut in side for visual check on amount of paper left; (M) metal frame to hold reel. The entire assembly is mounted on a vertical plate of 9-mm thick plastic sheet, 12 X 12 in., with the paper and transport components on one side and the solenoids on the other. Not drawn to scale
Counting the three series produced mean activities of 1032, l034, and 1031 cpm/drop. The relative standard deviations were 1.4, 0.9, and 1.1%,respectively. These values include the variability due to counting statistics but are primarily a reflection of variation in completeness of transfer from the valve to the paper. After collecting the last radioactive spot, we rinsed out the reservoir and tubing above the sampling valve with clean solvent and collected four more spots, comprised of three drops each. The first spot contained 268 cpm, equivalent to 0.4 pL of the initial solution, and the next three spots contained 68,25, and 12 cpm. Evidently there is a slight amount of trailing inside the valve and its delivery tube, but this should be insignificant in ordinary chromatographic use where compounds elute as peaks in several fractions rather than in one fraction as in our test. We investigated several techniques for visualizing the dry spots on the strip. Good sensitivity was obtained with dinitrophenylhydrazine for carbonyl compounds, ninhydrin for amines, the Dittmer-Lester reagent ( I ) for phospholipids, and 1% iodine in methanol for unsaturated lipids and amides. The phospholipid reagent, a strong acid, prevented storage of the paper. With iodine. less than 1 wg of fat could he seen. Solid nonpolar lipids, like cholesterol, were easily seen by merely wetting the paper with water (this could be done without removing the paper from the apparatus). Other sprays for detecting lipids on thin-layer silica gel plates were not very sensitive; alkaline hromothymol blue (2) seemed the best beside iodine. In initial tests, a dark solution of Sudan black B in ethanol was passed through the stream sampling valve and the resultant paper spots were examined visually. Too high an air flow through the valve caused spattering of the dye, while too low a flow rate caused incomplete transfer to the paper during the period of time that the valve was in the dispensing position. Attempts at producing a slow air flow with restriction valves, tubing, and tees immersed in liquid were unsatisfactory.
Figure 2. Fraction collector valve. (A) Teflon microvalve, Angar Scientific Corp., East Hanover, N.J., 2-way latching with 1/4-28 thread ports and inserts for Cheminert fittings, No. 340013; (B) Cheminert male Luer adapters, such as Altex No. 200-19; (C) reservoir, made from a 13 X 100 mm test tube sealed to a female Luer joint, Kontes Glass No. K-663500-426; a side tube is sealed near the top for a waste drain in case of valve malfunction; (D) delivery tip, made by sawing a glass female Luer joint at an angle; (E) delivery tip, made from a Cheminert tube end fitting: the tip is cut at a 45' angle
DISCUSSION Our device could be modified for use with ultraviolet absorbing compounds by attaching a small light source and sensor close t o the stream sampling valve. This would allow one to monitor such samples even if the elution solvent absorbs in the ultraviolet region. Probably the application of a drop of mineral oil would improve t h e optics of such a system. Similarly one could monitor fluorescent substances without t h e complication of quenching by t h e column solvent. Another possible refinement would be t h e attachment of a reagent dispenser and photometric quantitation device t o operate directly on t h e paper strip. More simply, one could cut each spot off t h e strip with a scissors and drop it into a test tube. Many reagents for quantitative analysis are usable even in t h e presence of a small piece of paper. Where one wishes t o examine each fraction by paper or thin-layer chromatography, one can c u t u p t h e strip a n d transfer t h e spots of interest by stitching t h e spotted paper t o the chromatographic paper sheet or by eluting the spot onto t h e point of origin on t h e plate or sheet (3). Examination of t h e intact paper strip by a n appropriate detection technique is useful when one wishes t o know which fractions are worth further examination; t h e amount needed for a second chromatogram can often be estimated from t h e intensity of t h e spots t h a t were applied to t h e paper. An alternate design for t h e paper movement mechanism might be easier to build. It is possible t o purchase clutch controlled low speed motors, which can be turned on for short, specific periods of time, such as 1 s. T h e motor could drive a rubber surfaced wheel a n d thus advance t h e paper strip a specific distance. T h u s , solenoids R and D, air cylinder G, and regulator K could be omitted. T h e notching device C + F could be placed under valve A. A similar paper spotter has been described by S m i t h ( 4 ) for use with aqueous solutions, in which the effluent stream was sampled by a peristaltic pump. T h e paper strip was then sewn along t h e bottom of a large sheet of chromatographic
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978
paper and t h e individual spots were chromatographed for additional resolution. Another device has been described ( 5 ) for sampling t h e effluent from a small analytical high performance column, using a continuously moving thin-layer plate coated with silica gel. T h i s produced streaks corresponding t o eluted components. Because of t h e limited length of t h e plate (20 cm), only short separations could be monitored.
ACKNOWLEDGMENT W e are indebted to William Strauch and James L. Mullison for building t h e device.
LITERATURE CITED (1) J. C. Dittmer and R. L. Lester, J . LipidRes., 5. 126 (1964). (2) H. Jatzkewitz and E. Mehl, 2 . Physioi. Chern., 320, 251 (1960). (3) D. G. Irvine and M. E. Anderson, J . Chromatogr., 20, 541 (1965). (4) D. B. Smith, Can. J . Biochem., 43, 521 (1965). (5) P. R. Boshoff, 6.J. Hopkins, and V. Pretorius, J . Chrornatogr.,126, 35 (1976).
RECEIVED November 14, 1977. Accepted January 23, 1978. Supported by t h e U S . Public Health Service, National Institute for Neurological and communicative Disorders and Stroke, grant N S 03192.
Modification of the Arsenic Speciation Technique Using Hydride Generation E. A. Crecelius Battelle, Pacific Northwest Laboratories, Marine Research Laboratory, Route 5, Box 1000, Sequim, Washington 98382
An analytical method for the determination of nanogram levels of arsenite (As3'), arsenate (As"), methylarsonic acid (MAA) and dimethylarsinic acid (DMAA) in solution was developed by Braman and co-workers several years ago ( I , 2). Improvements in the method were recently published (3). We have been using, basically, the published technique (3) for several years for t h e analysis of arsenic species in geothermal waters, wine, tissue homogenates, and urine (4-6). However. we have made several modifications t h a t are reported here. which improve the method. T h e modifications described allow t h e technique to be applied to more diverse environmental samples and still maintain a standard error of approximately *lo%. T h e original system (3) consisted of a reaction chamber where t h e arsines are generated, a cold-trap which concentrates t h e arsines, a C 0 2 trap, and an atomic emission detector. Our modifications include adding three additional traps t o the system. These are: a water vapor trap, a second COz trap, a n d a H 2 0 trap. A major problem has been water vapor freezing out in the arsine trap, thus causing changes in the gas flow rate through the system. By adding a water vapor trap between the sample reaction chamber and t h e arsine trap, this problem was eliminated (Figure 1). T h e water vapor t r a p is constructed
of 8-mm glass tubing as a U-tube with 10-cm long arms and, in operation, it is immersed in a n ice-water/salt bath. Although the boiling point of dimethylarsine is 36 "C, trapping of dimethylarsine in the water vapor trap was not a problem under t h e conditions employed. Samples that contain relatively high concentrations of C 0 2 or H2S, such as wine and geothermal waters, may require special handling t o avoid interferences from these gases. A t low concentrations of COPand HzS, t h e NaOH-packed C 0 2 trap used by Braman et al. (3)was adequate. Two approaches have been used to eliminate interferences from high concentrations of these two gases. Either the sample is acidified a n d degassed in t h e reaction chamber for 5 min a t 500 mL/min-' t o remove the H2S and COP,or a H2St r a p and an additional C 0 2 trap are added t o t h e system. If t h e sample is acidified and degassed, extreme care must be taken t o remove all traces of sodium borohydride t h a t may remain in t h e reaction chamber from previous samples. T h e sample could be degassed in a separate chamber before addition t o t h e reaction chamber to avoid this potential problem. If H2S and C 0 2 are not removed by degassing t h e sample, their removal can be accomplished in the volatilization process by placing in the gas stream a lead acetate trap and an additional CO2 TRAP SODIUM HYDROXIDE PACKED 6rnm PYREX
CO? TRAP ., \ He C A R R I E R ,
GA 8
Figure 1. Sample reaction chamber, traps, and detector arrangement 0003-2700/78/0350-0826$01.00/0
C' 1978 American Chemical Society
D C POWER
