An Improved Technique for Handling Milligram Samples for Nuclear Magnetic Resonance Analysis

David W. Mastbrook and Edward P. Ragelis Division of Food Chemistry and Technology, Food and Drug Administration, Washington, D. C. 20204 (Received 10 March 1969; revision received 28 March 1969)

INDEX HEADINGS: NMR; Small samples, Spectroscopic technique.

We have been handling milligram samples for nu- clear magnetic resonance (NMR) spectroscopy in a capillary with a technique tha t was developed mainly with gas-liquid chromatography (GLC) collection in nfind; however, this technique can also be applied when the sample capillary is filled by a syringe or capillary action. The technique compares favorably with similar ones reported, ~-4 and combines the follow- ing advantages :

(1) There is greater flexibility as to the a m o u n t of sample collected by GLC.

(2) No transfer of the sample from the GLC collec- tion tube to the N S [ R tube is necessary.

(3) Filling and positioning of the collection tube in the probe is less cumbersome than with other microcells.

(4) All components are commercially available at low cost and can be assembled quickly.

The assembly is shown in Fig. 1 and differs from the one reported by Fla th el al., 1 in that , once the sample holder has been adapted, the capillary tube containing the collected (or added) sample can easily be inserted from the top. (Some of the newer holders do not need to be adapted.)

* ~ CAPILLARY TUBE

CONSTRICTION - ~ SLIGHTLY LARGER THAN CAPILLARY % ~ 1/8" TUBING

STAINLESS STEEL i (1-1/2" LONG)

SWAGE LOK NUTS ~-_ I - - 1 / 4 " TO I/8" ' TUBING REDUCER

- - - - - I/4" STAINLESS STEEL

EXHAUST ,~ FROM GLC .~

FIG. 2. Connection of apparatus to gas chromatograph.

microcell assembly ~ (Kontes Glass Co., Vineland, N. J .) ; precision N M R tubes, polished. Collection tubes as follows : Drummond "Microcaps," disposable, 100-#liter capillary pipettes (Kensington Scientific Co., Oakland, Calif.), and long-stemmed, 7R-in dis- posable pipettes ( N M R Specialties, New Kensington, Pa.). Cut the stainless-steel tubing with a s tandard tube cut ter and ream the constrictions left at the ends of the tubing to a diameter which will just accommo- date the capillary tube. This will permit efficient col- lection of the sample.

Assemble the apparatus and connect to the exit por t of a gas chromatograph, as shown in Fig. 2.

Sample Collection: High boiling fractions: Inser t the capillary as shown in Fig. 2 just before the effluent arrives at the exhaust port. I t may be necessary to conduct a test run to determine the proper position of

I. EXPERIMENTAL

GLC Collection Equipment and Assembly: Stainless- steel tubing, ~ X l in. and ~X1½ in.; Swagelok re- ducer, ~ to ~ in., stainless steel ; Swagelok ~-in. nut with ferrules, stainless steel; rod and holder from N M R

• NYLON FOR INSERTION OF SAMPLE

Hole drilled • through here, TEFLON SAMPLE HOL Fla.th et ol. I~,L,~SAMPLE HOLE.I.Smm, N o . S 3 " ~ . L . J I I

I - I o.i, lJd U CAPlLLARY~ ~.,~.~ J~_c m

MICROCELL PREPARED SAMPLE ASSEMBLY IN ADAPTED ASSEMBLY

FIG. 1. Microcell assembly for NMR analysis of milligram sampleR.

(a) ;

I i

PPM 4 3 B I 0

'" I! i

~1 I li

Vil, PM 4 :5 2 0 I -

(c)

PPM 4 :5 2 I 0

FIG, 3. These spectra were re- corded on a Varian A-60 spec- trometer: (a) Capillary tube containing 1 uliter of ethanol in CDC13 and TMS (present work). (b) Microcell (1) con- taining 1 tditer of ethanol in CDCl~ and TMS. This is the best spectrum obtained from a lot of five bulbs (Kontes Glass Co.). (c) Semimicro- tube (NMR Specialties) con- taining 1 ~uliter of ethanol in CDC13 and TMS.

376 Volume 23, Number 4, 1969

the capillary that will permit condensation of the sample in the middle of the capillary.

Low boiling fractions : Insert the capillary part-way in the assembly, and cool the middle portion of the capillary tube, e.g., with a piece of dry ice, just prior to sample elution in order to condense the sample in the desired region.

Samples in excess of 5 mg: Replace the capillary tube with a long-stemmed pipette, and follow the s~mle procedures outlined above for the collection of low and high boiling fractions.

N M R Analysis: Upon collection of the desired frac- tion, remove the capillary and seal the cold end uni- formly. By means of a microliter syringe, introduce 10-20 uliter of a solvent spiked with t etramethylsilane (TSIS), midway down into the capillary tube, while slowly withdrawing the syringe. Tap the capillary so that the solvent washes the sample down into the tube. A solution height between 1 and 3 cm is required to obtain suitable NS,IR spectra. Seal off the remaining portion of the tube so that the final length is 5-6 cm, t:~king care not to degrade the contents of the capillary tube in the process. Drill the Teflon holder all the way through with a No. 53 hit and insert the capillary (from the top if the upper seal is of a larger diameter than the capillary tube). Screw the nylon rod part-way into the holder and insert the assembly into a clean, polished, precision-wall NMR tube. Adjust as shown in Fig. 1, making sure that the bottom of the capillary tube touches the bottom of the NS'[R tube. This centers the tube for spinning.

I I . DISCUSSION

The present method provides an inexpensive and adaptable technique for recording NMR spectra of milligram quantities without time averaging and of smaller amounts with time averaging. Positioning of the assembly is easier than with bulbs ~ because of the increased length and the wobble problem of the free floating capillary 2,4 is virtually imnexistent because the tube is held at the top and bottom. More rf power and higher gain settings, e.g., rf>0.1 and spectrum amplitude>32, are required to record spectra because of the small filling factor. See Figs. 3(a), (b), and (c) for a comparison of spectra.

1. R. A. Flath, N. Henderson, R. E. Lundin, and R. Teranishi, Appl. Spectry. 21, 183 (1967).

2. C. S. Slaymaker, Appl. Spectry. 21, 42 (1967). 3. B. Milazzo, L. Petrakis, and P. M. Brown, Appl. Spectry.

22, 574 (1968). 4. L. R. Provost and R. V. Jardine, J. Chem. Ed. 45, 675 (1968).

Tape Recorder Speeds Photometry

W. H. Dennen and W. H. Blackburn Cabot Spectrographic Laboratory, Department of Geology, University of Kentucky (Received 28 January 1969)

INDEX HEADING: Tape Recorder; Microphotometer.

For anyone who must engage in the tedious mea- surements of many line deflections with a non-record- ing microphotometer, saving of time is worth con- sidering. Most photometers require several hand manipulations per reading--location of the line, cor- rection for parallelism, reading traverse, etc.--plus the recording of each item of data. We suggest that the recording step may be efficiently accomplished by speaking into a tape recorder rather than by writing the data down. The hand and eye are thus relieved of one assignment and the reading rate improved.

As used in this laboratory, a cassette-type recorder equipped with a foot-treadle switch provides the necessary operational flexibility. Line identification, step, deflections, and background values are simply spoken into the recorder as observed. Dead time is eliminated by use of the foot switch and, with a little practice, the pacing of readings can be adjusted to a comfortable speed for transformation of the data to relative intensities on playback.

The use of cassettes allows the readings from dif- ferent plates to be kept separate, easily stored, and readily available for playback and calculation. Cas- settes may, of course, be reused when they have served their purpose.

APPLI ED SPECTROSCOPY 377