the chamber temperature is raised to 75" to SO" C., and the free diethylamine is removed b y a n exhaust fan. The dry sheets are sprayed with a solution of 0.257, ninhydrin in 1-butanol and heated for 10 minutes at 75" to 80' C. t o develop the color. The amino acids appear as colored q m t s against a light blue background. The cliaracteristic colors of amino acids and background developed only when the chromatogram was treated with diethylamine before being sprayed with ninii\.drin. Table 1 shows color reactions given by amino acids and some related ninhydrin-positive compounds with dieth\-inmine-ninhydrin (DEAN) as conipm eri t o thee given by ninhydrin alone. Of the 21 amino acids listed, 12 show new cmlore x i t h D E A S as compared t o ninhydrin aiid the range of colors is greater. The list of related compounds is not intended t'o be exhnustiw, h u t is included to demonstrate that the usefulness of the D E A N method i* not limited t o amino acids. The DEAK system corresponds closely to ninhydrin in its sensitivity. Either is capable of excellent color developmwt with amino acids a t a leve! of 0.01 t o 0.05 +If. I n many cases, in work with concentrations at or near the minimum limit of detection, the DE.4N e!-stcni is superior to ninhydrin done, becaiise it often intensifies the color, or is more evident because of increased cont,rast wit,h t h e background. TTe' h a w found the D E A K technique t o be 1wferal)le in many respects t o the X-C?; polychromatic method of itloflat and Lytle. The colors given with individual amino acids are usually different with the two techniques, so thzt they may complement one another in the identification of individual amino acids. The D E A N syst'em is
Table 1.
Compound Amino acids Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Homoserine Histidine Hydroxyproline Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Threonine Tryptpphan Tyrosine Valine
Color Reactions Using Diethylamine-Ninhydrin Reagent (DEAN) on Paper Chromatograms
Color with Ninhydrin DEAN Purple Purple Purple
Purple Purple Green-blue
Lt. purple Purple
Brom-n Purple
Purple Purple Purple Brown
Red Dk. blue Brown Salmon
Purple Purple Purple Purple Purple Purple
Purple Purple Gray-blue Purple Purple Yellowbrown Yellow Dk. brown Blue Brown Yellowbrown Purple
Yellow Purple Purple Blue-gray Purple Purple
more useful than the N-CX method in differentiating between glutamic acid and serine; both proline and hydroxyproline are more evident with DEAX because of their contrast with the background. The sensitivity of the two methods with most amino acids is almost identical, but the N-CN technique yields a much stronger color with cystine. We believe that under most circumstances the D E A N technique is somewhat simpler and easier to employ than the N-CN method.
Compound Related compounds Alanylalanine p-Aminobenzoic acid Asparagine Azaserine Cadaverine Carnosine Diaminopimelic acid Digitonin Dipicolinic acid Glutamine Glycylglycine Histamine Pyridpxamine Spermine
Color with Ninhydrin DEAN
Purple
Dk. blue
Yellon-
Tan
Lt. brown Blue Purple Gray-brown Lt . purple
Yellow Brown Purple Tan Dk. purple
Lt. salmon Lt. purple
Lt. brown White
Purple Brown
Purple Yellow
Blue Rust,
Dk. brown Rust
Lt. purple
Dk. purple
LITERATURE CITED
(1) Bl:ck,
R. J., Durruni, E. L., Zweig, G., Manual of Paper Chromatography and Paper Electrophoresis," 2nd ed., Academic Press, New York, 1958. (2) Kolor, hl. G., Roberts, H. R., Arch. Biochem. Riophys. 70, 620 (1957). (3) Moffat, E. D., Lytle, R. I., ANAL. CHEM.31, 926 (1959). INVESTIGATION supported, in part, by research grant 3-2895 (C2) from the National Institute of Allergy and Infectious Diseases, Public Health Service, and by a grant from the Dr. Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago.
A Device for the Introduction of Submicrogram Quantities of Solids into a Gas Chromatograph Darrell B. McComas and Alan Goldfien, Departments of Obstetrics and Gynecology and Medicine, and the Cardiovascular Research Institute, University of California School of Medicine, San Francisco, Calif.
m'
H4YE C0iT;STRUCTED and tested in our laboratory a device with which we crln introduce submicrogram quantiticc of solids onto a gas chromatographic column through the conventional r u l k m diaphragm user! for the injecticin of liquids. Other means of introduciiig d i d s h:b\-e been reported ( 1 , 2 ) . Tlii. device, however, is well suited to the introduction of minute quantities. clops not require modification of the injrction system of the instrument, a i d does not interrupt the flow of carrier gas. The absence of a solvent has two distinct advantages in the quantitative analysis of steroid hormones isolated from biological material. First, at the very high sen-
TE
sitivities rrquired for the estimation of niillimicrogram quantities, the solvent peak may be so broad as to ohscure much of the early portion of the chromatogram. Figure lil shows the trace produced by the injection of 3 ~ 1 of. a solution of cholestane and progesterone in n-hexane. Figure 1B shows the same steroid mixture injected without the solvent using the device described in the text. When the solvent was present, the cholestane peak was obscured. The use of a longer column to resolve the cholestane and solvent peaks would result in an increase in analysis time and loss of sensitivity. The use of this device does not, of course, obviate the necessity for isolation of a com-
pound from nonvolatile impurities. For example, lipid material found in biological extracts can lead to base line disturbances even more serious than that produced by the solvent. The preliminary use of paper or thin layer chromatography, solvent partition, and freezing may be required. The second advantage in this method is that a sample can be transferred quantitatively to the solid injector whereas we have found no way to concentrate a solution to a few microliters and get all of i t into a syringe. The first device (Figure 2 A ) consists of a 24-gage hypodermic needle and a stainless steel wire approximately 0.011 inch in diameter and about 2 VOL. 35, NO. 2, FEBRUARY 1963
263
inches in length and a plastic syringe. I n the hub of the needle is a tightfitting silicone rubber diaphragm. The wire pierces this diaphragm and passes through the needle. The needle is fixed to the syringe (Seringue S.E.S.I. 2-cc. nylon springe), and the wire is attached to the plunger so that by moving the plunger the wire can be drawn back into the needle and pushed forward until it protrudes about 3/4 inch beyond the point. The forward end of the wire has been ground square for about inch and tivisted into a spiral. The purpose of the spiral is so that a minimum of surface of the wire can contact the inside of the needle when it is drawn inward. T o apply the sample, the wire is extended beyond the tip of the needle, a solution is applied to the spiral a few microliters a t a time, and the solvent is allowed to evaporate. When the desired amount of material has been applied to the spiral or quantitative transfer has been achieved, the wire is drawn back into the needle. The needle is then inserted into the injection port diaphragm, and the wire is pushed forward into the gas stream and held until the sample has volatilized. I n this design, the purpose of the syringe is merely t o facilitate manipulation of the needle and wire. An alternate design, whicli is simple t o construct, is to attach the wire to the plunger of a gas tight syringe and omit the plug in t h e h u b of the needle (Figure 2B). At the low inlet pressures we are using (less than 20 p.s.i.g.) we have found an ordinary 1- or 2-cc. disposable syringe [Tomac (R) 1- or 2-cc. syringe] to be satisfactory. We have used these devices for the injection of a variety of steroids and
-
A
8 STAINLESS STEEL W/RC SPIRAL TfP
P L UNGCR
RUddER TfP
2cc DISPOSA8LB SVR/NG€
I Figure 2. Devices for injecting solids (A) with silicone rubber diaphragm in hub of needle ( B ) using syringe with a gas tight plunger
fatty acid esters in amounts ranging from a few micrograms to a few hundredths of a microgram. Volatilization is apparently rapid and complete for these substances as judged by the retention time and character of the recorded peaks. The major problem
SOLYEAW AND CHOLCSTANE
- A I R PISAK
Table I. Reproducibility of Repeated Injections of 0.1 3 Fg. of Progesterone with Dry Injector 7.5 ;..i H1 p.s.i.g. 6 . 5 ti.5 Carrier p.s.i.g. 9 0 7 0 9 0 7 . 0 Area sq. cm. 7 98 7 39 8 77 G 67 8 T i 7 00 8 44 7 33 '7 85 7 78 7 32 7 26 8 21 7 85 8 24 6 80 '7 65 6 67 8 64 T 06 8 11 7 33 8 31 T 00 Mean Std dev. & 0 43 0 50 0 49 0 29
c CHOLESTANP
A
\ 1
0
2
4
6
8
A
10 MINUTES
2
4
6
8
f
O
0
Figure 1. Recordings showing difference between injecting steroid mixture with (A), and without ( B ) , solvent (inlet pressure 1 6 p.s.i.g. Nz) Mixture of cholestane and progesterone injected with 3pl. of n-hexane using Hamilton lO-p\. liquid (A) inlection syringe (6) Some amount of cholestane and progesterone without solvent using dry injector described in text
264
rn
ANALYTICAL
CHEMISTRY
encountered has been variation in performance of individual injectors with respect to reproducibility and it may be necessary to t'est several to find one which performs satisfactorily. The performance of a good injector under varying operating conditions is illustrated by the data in Table I. The gas chromatograph used in these esperiinents (Research Specialties Co.: Richmond, Calif.) was equipped with a hydrogen flame detector. The %foot stainless steel column (2-mm. i d . ) was packed with Gaschrom Z. 60- to 80mesh, containing 1% QF-1 and mainta,ined a t a temperature of 245' C. LITERATURE CiTED
(1) Bowman, R. L., Kitmien, -4., Suture 182, 1233(1958).
( 2 ) Renshaw, A,, Biran, L. A,, J . Chromatog. 8, 343 (1962). WORKwas supported in part by Grants A-4426 and H-6285 from the Sational Institutes of Health.
