2150 ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978

AIDS FOR ANALYTICAL CHEMISTS

Modified Photography Tank for Continuous Development of Thin-Layer Chromatographic Plates

Reinhold Vieth, * ' Donald Fraser, and Glenville Jones

Departments of Physiology and Paediatrics, University of Toronto, and the Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1x8

During our studies of enzymic hydroxylation of tritiated 25-hydroxyvitamin D3 (25-OH-DJ it became necessary to find a simple, efficient means of separating the substrate and its principal products, 24,25-dihydroxyvitamin D3 (24,25-(OH),D3) and 1,25-dihydroxyvitamin D3 (1,25-(0H),D3). Although conventional thin-layer chromatography (TLC) provided sufficient resolution of 25-OH-D3 from its products, we found tha t the technique did not provide adequate resolution of 24,25-(OH)?D3 from 1,25-(OH)&,. In order t o increase separation of these two compounds we considered modifi- cations to TLC tha t are known to improve the resolution obtained with the conventional method.

It is generally accepted that resolution can be increased in adsorption chromatography by decreasing solvent polarity and increasing solvent flow (1-3). Multiple-development TLC in which the plate is developed in a given solvent, dried, and developed in the same direction through one or more addi- tional cycles is one approach to increasing solvent flow in TLC (4, 5 ) . Alternatively, continuous-development TLC, which involves the evaporation of the solvent from the top of the plate, can be used to resolve slowly-moving components if buildup of fast-moving components a t the top of the plate is not a n important consideration (6). Several gadgets for continuous-development TLC have been described (7-10) but all require the chamber to be kept open, resulting in poor chamber saturation.

We have made slight modifications to a n inexpensive readily-available photography tank which make it well suited for continuous development of TLC plates.

EXPERIMENTAL Apparatus. The tank used for continuous development TLC

is a commercial polyethylene chamber for fixing slide tran- sparencies (Dippit No. 646; Polaroid Corporation, Cambridge, Mass.). These chambers are available from photographic sup- pliers. To prepare a tank, we discard the fixative solution in it, rinse it thoroughly with water and detergent, and cut away the dividing bar between the lower and upper chambers with a knife. The tank is filled with ethyl acetate and left to stand overnight to remove residual fixative. Inward warp of the walls of the tank during this treatment can be corrected by inserting an object into the upper section of the chamber for a few hours (we inserted an open-ended half-inch wrench longitudinally and twisted it 90').

Materials. Sheets of silica gel on aluminum foil, 20 cm x 20 cm and 0.25 mm thick (catalog no. 55381, were purchased from EM Laboratories, Elmsford, N.Y. 25-Hydroxy[ 26(27)-methyl- 3H]vitamin D3 (25-OH-D3) was purchased from Amersham Searle Ltd. (Taronto, Ont.). 1,25-Dihydro~y[26(27)-methyI-~H]vitamin D3(1,25-(OH)2D3) and 24,25-dihydro~y[26(27(-methyl-~H]vitarnin D3 24,25-(OH),D3) were produced biosynthetically, using chick-kidney mitochondria (11, 12). Identity and purity of the compounds were established on two high-pressure liquid chro-

Address correspondence to this author at the Hospital for Sick Children, Room 5128B, 555 University Avenue, Toronto, Ontario, Canada M5G 1x8.

matography systems by co-chromatography with authentic metabolites (13, 24) .

Procedure for Continuous-Development TLC. The eluent (15 mL of benzene:ethyl acetate, 1:l) is pipetted into the tank, which is then closed, shaken, and set aside for at least 30 min to allow saturation with solvent vapor.

The silica gel is cut into strips 2 cm X 16 cm and 1 WL of a saturated ethanolic methyl red solution is applied at the origin, 1.5 cm from the bottom edge. Samples (containing [3H]-25-OH-D3, [3H]-1,25-(OH)2D3, and [3H]-24,25-(OH)2D3, in our experiments) are applied on top of the methyl red and dried under a stream of nitrogen.

Two TLC strips are inserted into each tank and the metal clamp atop the device is closed so that the flexible lips of the tank form a tight seal around them (Figure 1). Solvent passes up the strips and through the seal; it evaporates along a line (the evaporation front) approximately 1 mm above the seal and 12.5 cm from the origin. The methyl red indicator, which separates into two closely-running spots during chromatography, is clearly visible through the semitransparent walls of the tank and shows the amount of development; as soon as the more intense, faster moving red spot reaches the evaporation front, the tank is opened and the strips are removed.

The strips are cut transversely at 1-cm intervals and each slice is placed in a 20-mL scintillation vial. To recover the vitamin D, metabolites we eluted each slice with 5 mL Aquasol ( 2 5 ) . We determined radioactivity with the foil remaining in the vials using a Beckman LS-355 liquid scintillation counter. Counting efficiency for tritium was 41 % .

Comparison with Other Chromatographic Systems. R, values for vitamin D3 metabolites and methyl red were determined by conventional TLC in glass tanks using 10 different solvent systems to a 17-cm front. Solvent strength (cos102) was calculated by Snyder's formula (2).

In another experiment, identical aliquots of a mixture of tritiated 25-OH-D3, 24,25-(OH)*D3, 1,25-(OH)*D3, and methyl red were subjected to either continuous-development TLC or mul- tiple-development TLC in the photography tank using benz- ene:ethyl acetate, 1:l as solvent. Three cycles of multiple-de- velopment TLC were carried out in the 2 h necessary for a single continuous development.

RESULTS Results of conventional chromatography of the three vi-

tamin D, metabolites and methyl red using 10 solvent systems are shown in Table I. Several solvent systems resulted in equal amounts of separation of the radioactive peaks of [1H]-1,25-(OH)2D3, and [3H]-24,25-(OH)2D3, but the maximum distance between these peaks was only 1 cm when chroma- tograms were developed to a 17-cm front.

The amounts of vitamin D metabolites produced in the reaction we were studying were too small for UV detection. We therefore employed [3H]-25-OH-D3 as substrate and quantitated the products by measuring the radioactivity in slices of the chromatograms by scintillation counting. It was not feasible to cut the chromatogram into slices of less than 1.0-cm length. I t is apparent from Table I that the resolving

0003-2700/78/0350-2150$01 OO/O C 1978 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978 2151

Table I. R f Values of Three Vitamin D , Metabolites and Methyl Red with Various Solvent Systems in Conventional Chromatography

solvent system

methanol, 100% ethyl acetate:methanol, 97:3 hexane:isopropanol, 17: 3 ethyl acetate, 100% ch1oroform:ethyl acetate, 1 : 1 benzene:ethyl acetate, 1:l benzene:ethyl acetate 2: 1 hexane:ethyl acetate, 4: 1 chloroform, 100% benzene. 100%

f SiO,

0.75 0.61 0.51 0.38 0.33 0.33 0.31 0.26 0.26 0.25

R f values - R f 25-OH-D,/ 24,25- 1,25- methyl R f 1,25-

25-OH-D, (OH),D, (OH),D, red (OHLD, 0.74 0.65 0.65 0.75 1.2 0.55 0.45 0.35 0.36 1.6 0.33 0.28 0.18 0.10 1.8 0.50 0.30 0.24 0.33 2.1 0.36 0.10 0.29 3.6 0.36 0.19 0.10 0.29 3.6 0.29 0.08 0.22 3.6 0.30 0.15 0.1 2.0 0.25 < 0.1 > 2.5 0 0

Figure 1. Modified Polaroid Dippit, showing development of two foil-backed TLC strips 2 cm X 16 cm. The black spots represent the darker, faster component of methyl red indicator. The chamber will accommodate a single TLC sheet up to 5 c m in width

power of conventional TLC was inadequate to ensure un- ambiguous quantitation of ['H]-1.25-(OH).,D3 and ['HI- 24,25-(OH),D3; a method with better resolution was required. From Table I, it is evident that as toSio2 of the solvent mixture decreases, the relative separation of the two metabolites (expressed as R, 25-OH-D,: R, 1,25-(OH),D3) tends to increase. Benzene: ethyl acetate, 1:1, was selected in our method for continuous-development TLC because, using this solvent, relative separation of the metabolites was good and the time of development short.

Figure 2A illustrates a typical chromatogram obtained with our continuous-development TLC method using benzene:ethyl acetate, 1:l. The separation between 1 , 2 ~ 5 - ( o H ) ~ D ~ and 24,25-(OH),D3 was, typically, 4 cm. 25-OH-D3 was compressed at the evaporation front and was discrete from 24,25-(OH),D3. With this amount of separation of sample components, slicing the chromatogram a t 1-cm intervals permitted unambiguous quantitation of each metabolite.

Comparison of continuous- and multiple-development TLC showed very similar patterns for the radioactive peaks (Figure 2, A and B). With multiple-development TLC, the methyl red spots appeared sharper and 24,25-(OH)2D3 and 25-OH-D, were closer together because there was a gradual compression of sample spots and a diminution in their separation in the upper third of the chromatogram.

DISCUSSION Our method of continuous-development TLC using the

modified photography tank has several advantages over other TLC approaches. It is inexpensive and simple, and the apparatus is readily available. It offers improved resolution of slowly-moving components of the sample by taking ad- vantage of the separation selectivity (3) afforded by weaker solvents than those used for conventional TLC. Another

b ::I 200

2 0 r/)

\

1600

1400

600

400

200

B

0 R I

G I

r?

1 d

n

F R 0 N T I - 13

I

r DISTANCE OF CUT FROM ORIGIN (cm)

Figure 2. Radioactivity recovered from successive 1-cm slices of TLC strips developed in the photography tank using benzene:ethyl acetate, 1 : 1 , (A) Chromatogram obtained by the continuous-development technique. (B) Chromatogram obtained by mutliple development through 3 cycles. (a) [3H]-1,25-(OH),D,, (b) [3H]-24,25-(OH),D,, (c) [3H]- 25-OH-D3 The spots represent the positions and outlines of the two components of methyl red

advantage of the photography tank is that efficient chamber saturation is ensured by its thin shape and, unlike previous continuous TLC methods, by its vapor-tight seal. Contin- uous-development TLC eliminates the evaporation cycles of multiple-development TLC which expose samples to the oxidative effects of air which could be detrimental in chro- matography of labile compounds such as vitamin D. The time required for development is conveniently short and not significantly different from that required to achieve a com- parable separation of metabolites by multiple-development TLC. Furthermore, continuous-development T L C is a one-step procedure.

2152 ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978

In our application, we allowed 25-OH-D3 t o be deposited at the front because there were no other fast-moving ra- dioactive components in our sample. Our procedure permitted optimal use of the available length of the T L C plate for separation of the more polar metabolites. Other applications may require additional chromatography t o distinguish fast-moving sample components.

In TLC, the rate of development and R, values may vary from one run t o the next. If development is controlled by co-chromatographing a visible marker so that it reaches a predetermined point on the chromatogram, continuous-de- velopment TLC is reproducible. Ideally, the marker should have a n R, value similar t o that of the compounds to be isolated. Methyl red was suitable for our application; however, a much slower-moving dye (e.g., neutral red) would be preferable if longer development were necessary. The "CRC Handbook of Chromatography" gives the R, values of many dyes in several solvent systems (16) .

In conventional TLC, the primary approach to increasing resolution is limited to selection of an appropriate solvent system. T h e technique described in this paper provides an additional means with which to improve resolution-the amount of solvent flow.

LITERATURE CITED D. L. Saundeys, Anal. Chem., 46, 470-473 (1974). L. R. Snyder, Principles of Adsorption Chromatography. The Separation of Nonionic Organic Compounds", Dekker, New York, 1968, p 208. L. R. Snyder and J. J. Kirkland, "Introduction to Modern Liquid chromatography", Wiley-Interscience, New York, N.Y., 1974, p 68. J. A . Thoma, Anal. Chem., 35, 214-224 (1963). J. A. Thoma. J . Chromatogr., 12, 441-452 (1963). J. M. Bobbit et al., "Introduction to Chromatography", Reinhold-Von Nostrand, New York, N.Y., 1968. M. Brenner and A. Neiderwieser, Experientia, 17, 237-238 (1961). N. Zollner and G. Wolfram, Klin. Wochenschr., 40, 1098-1101 (1962). E. V. Truter, J . Chromatogr., 14, 57-61 (1964). L. M. Libbey and E. A. Day, J . Chromatogr., 14, 273-275 (1964). R. W. Gray, J. L. Omdahl, J. G. Ghazarian, et al., J . Biol. Chem., 247, 7528-7532 (1972). J. C . Knutson and H. F. DeLuca, Biochemistry, 13, 1543-1548 (1974). G. Jones and H. F. DeLuca, J . Lipid Res., 16, 448-453 (1975). A. M. Rosenthal, G. Jones, S. W. Kooh, and D. Fraser, "Endocrinology of Calcium Metabolism", D. H. Copp and R. V. Talmage, Ed., Excerpta Medica, Amsterdam-Oxford, 1978, p 371. D. Kritchevesky and S. Malhotra, J . Chromatogr., 52, 498-499 (1970). G. Zweig and J. Sherma, "CRC Handbook of Chromatography", Vol 1, CRC Press, Cleveland, Ohio 1972.

RECEIVED for review December 8, 1977. Accepted June 15, 1978. This work was supported by a grant (MA 5139) from the Medical Research Council of Canada.

Gas Chromatographic Injector Attachment for the Direct Insertion and Removal of a Porous Polymer Sorption Trap

Harney Peterson

Adolph Coors Company, Golden, Colorado 8040 1

Gary A. Eiceman, Larry R. Field,"' and Robert E. Sievers

Department of Chemistry, University of Colorado, Boulder, Colorado 80309

In recent years, there has been a growing interest in the analysis of trace organic compounds in the environment (1-3). Three of the most widely used methods to t rap and con- centrate organic compounds from an aqueous matrix have been: direct sorption of organics from water onto charcoal, followed by solvent extraction and concentration ( 4 , 5 ) ; direct liquid-liquid extraction, followed by solvent evaporation (6, 7); and, inert gas stripping of volatile organics onto polymeric resins, followed by thermal desorption (8-10). It is our belief that the third method, despite its limitations, affords the best approach to analysis of trace volatile organics in the envi- ronment. With this method, long pre-concentration times, extensive sample handling, and the use of large quantities of ultrapure solvents can be avoided.

One major objection to this method has been the complex and expensive hardware required to thermally desorb a sample from a porous polymer t rap onto a gas chromatographic column. Some researchers using this technique use expensive heated switching valves, heated desorption ovens, and heated sample transfer lines (11 , 12). Two previous attempts to circumvent this hardware problem have been: first, removal of the septum and septum cap, direct insertion of the t rap into the GC injection port, and replacement of the septum and cap; and, second, insertion of either sealed glass tubes or metal caps containing the polymer followed by destruction of the seal inside the injection port (13, 14) .

These techniques have disadvantages. Problems en- countered in the first approach include backflushing of volatile

Present address, Department of Chemistry, University of Washington, Seattle, Wash. 98195.

compounds before the septum is resealed and limited de- sorption efficiency of trapped compounds due to poor carrier gas flow through the t rap and injection port. .4 severe dis- advantage to the second method is that it is necessary to clean the polymer from the injector port following each analysis. I t was our intention to devise a method in which most of the external hardware could be eliminated. In addition, we wished t o design and construct a device which would overcome the disadvantages listed for the previously used techniques.

EXPERIMENTAL Design and Construction. The two independent devices we

have designed each offer different advantages to potential users. Injector attachment A (see Figure 1) requires little machining and virtually no modifications to the gas chromatograph (GC), while fabrication of injector attachment B (see Figure 2) demands considerable machining and some GC injector port alteration.

Design A was developed for a Hewlett-Packard model HP- 5730A gas chromatograph in which the injection port internal diameter is -5 mm. The advantages of this design are its simplicity and minimal change of the instrument. Since each GC manufacturer has a different injection port design, the description of the injector attachment is general, and exact dimensions are not specified.

The injector body (design A) is made from a 15.2-cm piece of 6 mm o.d. x 4 mm i.d. Pyrex tubing with composite Swagelok fittings attached at each end (see Figure 1). On the end that attaches to the injection port, one half of a Swagelok inch-'/,-inch union is silver soldered to a l / inch Swagelok nut, the threads of which match those on the HGi730 injection port. Teflon ferrules are used throughout this design to provide air-tight seals. On the other end, a reducing Swagelok union I / , -

inch-1/8-inch allows a 0.3-cm o.d. brass rod to slide through a

0003-2700/78/0350-2152$01 OO/O 1978 American Chemical Society