Solvents for the Paper Chromatography of Amino Acids and Sugars without Spraying Hassan S. El Khadem, Zaki M. El-Shafei, and Mohammed M. A. Abdel Rahman, Department of Chemistry, Faculty of Science, University of Alexandria, Alexandria, Egypt, U.A.R.
amino Pacids and sugars were run with the spraying agents already dissolved in the 4PER
CHROMATOGRAMS
Of
solvent mixture. This eliminated the need for their drying and subsequent spraying and in the case of amino acids enabled the movement of the spots to be followed up during the running of chromatograms and avoided their elution from the paper when the solvent was left to drip. -4mino acid mixtures containing 1% glycine, alanine, valine, and threonine were successfully separated by this procedure using the solvent systems (1) shown in Table I after addition of about 0.1% ninhydrin. Soon after the solvent front reached the amino acids, the spots acquired a pink coloration but their contours were not sharp due to tailing. This, however, disappeared when the chromatograms were removed from the solvent and left to dry at room temperature for 3 to 6 hours. The spots were then sharper than those obtained in the usual manner by spraying the chromatograms, so that a smaller amount of amino acids could be detected. Amino acids are known t o react with ninhydrin at low p H (2). We have found however that the R, values of amino acids are not altered by the addition of ninhydrin to the solvent system zuggesting that such a reaction
Table I. Solvent Mixtures for Amino Acids Solvent mixtures Volumes, ml. Wt. of ninhydrin, mg. Ethanol-water 75 :25 100 n-Butanol-2,V acetic acid 20 :20 40 n-Butanol-acetic acid-water 40: 10: 10 60 Methanol-pyridine-water 80: 4:20 100 Isopropanol-phenol-water 7:TO:25 100 Vpper layer of: n-Butanol-acetic acid-water 40: 10:50 50 (in upper layer) n-Butanol-acetic acid-water 68:5:27 85 (in upper laver) n-Butanol-acetic acid-water 40: 10:60 50 (in upper laier) n-Butanol-acetic acid-water 25:6:25 25 (in upper layer) 20:20:5:40 n-Butanol-phenol-acetic acid-water 50 (in upper layer) Table 11.
Solvent Mixtures for Sugars
Solvent mixtures Upper layer of: n-Butanol-acetic acid-water n-Butanol-acetic acid-water
does not take place during the running of the chromatograms. It is probable that this method can be applied to other solvent systems provided they do not contain a compound that gives a color with ninhvdrin such as ammonia. Paper chromatograms of sugars were run with the solvent systems shown in Table I1 which contained benzidine or p-anisidine. I n this case, the spots appeared only after drying the chromatograms at 100’ C. for 10 minutes. Mix-
Volumes, ml. 40: 10: 50 40: 10350
Reagent Benzidine (100 mg.) p-Anisidine (60 mg. )
tures of glucose, galactose, arabinose, and rhamnose were separated satisfactorily by this method. LITERATURE CITED
(1) Bl;ck,
R. J., Durum, E. L., Zweig, G., Manual of Paper Chromatography and Paper Electrophoresis,” p. 148, Academic Press, New York, 1958. (2) Van Slyke, D. D., MacFayden, D. A., Hamilton, P. B., J. Bid. Chem. 150, 251 (1943).
Simplified Method for Determining Sensitivities in Activation Analysis B. T. Kenna, Sandia Corp., Albuquerque, N. M., and L. A. Kenna, U. S. Army Electronic Proving Ground, Ft. Hauchuca, Ariz. EVERAL RECEST P4PERS
have given
S excellent summaries of sensitivities
for activation analysis (3-5, 10). However, in the computations, specific flu^ levels and/or specific irradiation periods were assumed. Meinke (6-8) has extended thic nork by showing how the sensitivity for activation analysis can be varied and discrimination obtained using different times of irradiation. Although thwe data and compilations are important and serve a yaluable purpose, the assumption of specific fluxes and irradiation periods limits the immediate and general applicability of the data. The method for determining sensitivities in acth ation analysis described in this paper does not have these limitations. There are tn o minor drawbacks, however. nhich nil1 be discussed later. Although this method is not intended to be used evlwively in lieu of other techniques, the user nil1 find that it is a rapid niethod and that he can obtain 1766
ANALYTICAL CHEMISTRY
either accurate results or valid estimates with it. Other useful nomograms pertaining to radioactive buildup and decay are available (2, 11). However, these do not lend themselves readily to sensitivity calculations. Mesler (9) has described an excellent method whereby a rapid assessment of thermal neutron activation, in terms of the activity produced, can be obtained. For some esposure times, however, a correction factor must be applied. Equation 1 is the basis for sensitivity calculations in activation analysis
where W . is the weight of the sample nuclide, 2, in grams; A is the disintegrations per second of the product nuclide; M,is the atomic weight of 2; u is the cross section in square centi-
meters; f is the neutron flux in neutrons per sq. cm. per second; S is the saturation factor; and 8 is the fraction of the target iyotope in the element. S is defined by (1 - e - 0 . 6 9 3 T / twhere )~ T i s the irradiation time and t i s the half life of the radiation product in the same units of time as 5“. If one defines a variable, X, as X = T/t, the variation of S us. X is as shown in Figure 1. Note that a t X 2 6, S approaches the value of 1 very closely. Thus, although saturation is said to occur usually a t X = 10 ( T = lot), one may assume, for approximate calculations, that saturation occurs a t X 2 6. I n Equation 1, the true unknowns, which thus far have prevented a formulation of data of essentially general application, are u,f, and S (or X). While it would be difficult t o correlate these on a conventional X-Y or three dimensional plot, it is a relatively easy matter
'T 10 -
1.0
9-
0.9
8-
0. 8
1-
0.7
0
/
/
6-
0.6
/ /
/
5-
/
'
0.5
0 10
4.
.O. 4
0
3
0
0
0
-0.3 20
0
30
0
2
.0.2
, 1
I
I
2
3
60
I
4
5
6
70
X
80
Figure 1.
Variation of
S
with X
100
to prepare a nomograp'i correlating these three Yariables. To do this, Equation 1 is simplified by assigning the value of one disintegration per second to A . Then
2
1
-0.1 S
(of)
Figure 2.
Nomograph of SI (crf), and W N for activation analysis
x .10-2s)M. - (16.6 ____uf,f,ss
1 -
For the nomograph, TVN,S, and (uf)are used as the three axes and are plotted on logarithmic scales as siown in Figure 2. The S and (uf) axes ,ire one-cycle logarithmic scales, whercas the W N axis, n hicli is equidistant from the other axes, is a two-cycle logarithmic scale. When a value of W Nfor a nliclide is found on the nomograph, the sensitivity for this particiilar nuclide is given by Equation 3.
The value of n is the exponent of the product, (us). For e.tample, to determine the sensitivity for 6J3a136 ( 0 = 0.0781). K i t h a crosf! section of about 0.4 barn, assume that the available neutron source has a thermal flux of 5 X 10loneutrons per tiq. cm. per second and that the irradiation time is S = 0.8 ( - 2 . 3 half lives). Then (uf) = 2 X If a line is d r a m from S = 0.8 t o (cf) = 2. the Yalue of Wj7 may be wad as 10.03 on a nomograph (Figure 2 ) . Tl1crrfore.
= 0.17 pg.
(4)
A standard calculation with Equation 1 yields WBglal = 0.18 pg. Thus, the nomograph method is in good agreement with the standard method. A is not limited to 1 disintegration per second; rather it may be any value which the investigator feels necessary. Increasing A to 100 would alter Equarather than tion 3 to Apparently assuming A = 100, Studier et al. (18) estimated that in a flux of 3 X lola neutrons per sq. cm. per second is of the the maximum sensitivity for IlZ7 order of 0.1 nanogram (abbreviated ng.). From Figure 2, knowing that e is 1 .O and (bf) 2.01 X W p 7is calculated to be approximately 0.11 ng. This procedure also may be used to calculate sensitivities in activation analysis utilizing a 14-m.e.v. neutron flux. Assuming t h a t f = lo8, S = 1.0, and A = 100, the sensitivity for F19undergoing an (n, p ) reaction, as calculated
-
by Equation 1, is 23.4 mg., whereas the nomograph method yields 24.3 mg. One further value of the nomograph method is that it amplifies Meinke's work, which illustrates how sensitivities can be varied and discrimination obtained. It is evident that the nomograph method facilitates actual oalculations of T and any other variable which can influence the discrimination. As mentioned previously, there are two drawbacks to this method. Many of the papers which have given summaries on sensitivities have incorporated two important items which are absent in the nomograph procedure as it now stands-detector counting efficiencies and time intervals before counting to allow for chemical separation, to permit unwanted activities to die out, etc. These were deliberately omitted to make the nomograph method as generally applicable as possible. The detector counting efficiency is easily disposed of by introducing E , which represents counting efficiency, into the numerator of Equation 4 and incorporating its value into the value of W N . Then the values VOL 35, NO. 11. OCTOBER 1963
1767
oflWs It-ould be correspondingly different-Le., if e were 0.25, the values on tlie W s axis would be one fourth of t,hat shown in Figure 2. Thus, each investigator may use his own counting efficiency and prepare a nomograph rvhich is applicable to his work. The time interval is included by introducing into t'he numerator of Equation 4 the tcrni. exti, where t , is the time elapsed from irradiat,ion to detection ( I ) . The incorporation of this term into TT-.,- could cause difficulty in obtaining the normal axis for WTS and may have to remain an entity in Equation 3. This method is cumbersome in tliat calculaticns are necessary to olitsin quantitatiw results. However. this is
compensated for by the fact that, after a little experience, one can make excellent qualitative estimations without, written calculations. After obtaining TYhr, rapid approximate mental calculation.; with Equation 3 are possible. LITERATURE CITED
1 ~ 1Aalberts, ) J. H.,
Terheijke, J. H.,
d p p l . Physics Letters 1 , 19 (1962). ' , 2 ) Freiling, E. C . , ."\izccieonics 14, S o . S, G S (1956). ( 3 ) Gillespie, A. S., Hill, IT. IT., Sztcleonics 19, 170 (1Y61). ( 4 ) Jenkins, E. S . , Smalrbs, -1.A , , Quart. Rec. Chetti. Soc. London 10. 83 (19561. [S) Leddicotte, B. W.,Re?:nold~,9. _\., U. 8. Atomic Encry?- C omrn. Rep!. AECD-3489 (1953)
(6) Meinke, I\'. I!.) AXAL. CHEM. 31, 792 (1959). ( 7 ) lleinke. \V. IY.. Science 1 2 1 . 177 (1955). (8) Meinke, \V. \\. , Maddock, R . S., ASAL. CHEM.29, 1171 (1957). (9) Mrsler, R. I3 3"ucleonics 18, so.1 , ~
73 (1960). (10) Schindewolf, U., Angeii. Chr' l t l . 'io, 181 (1938). (11) Stelin, J. R., ('lancy, E. F., .I-cicl eonics 13. S o . 4. 27 (1955). (12) Studh-, x.' M.;Postmus, C., Jr., LIech, J., W'alters, R. R., Sloth: E. S . , Argonne Katiunnl Laboratory, ANL6577 (1962).
\VURK \viis done under the auspices i \ f the Unitcd St:itcss A\tomic Energy Coniniission. Contmts of this paper do not nccrsenrily r c f l w t tlic. position of t h r V.S. :\rrny.
liquid Sample Introduction System for a Mass Spectrometer Albert D. Pattillo and Howard A. Young, Mobil Oil Co.,Beaumont Refinery, Beaumont, Texas
work. Both instruments are equipped the past several year>. memwith heated inlet systems operated at of ASTAI Committee D-2, 135' C. (276' F,). 9 project was intiRD IV have cooperated in a program to ated to determine the best means of develop a method to analyze petroleum introducing a sample in the gasoline fractions in the gasoline boiling range for boiling range into the mass spectromhydrocarbon types. One of the principal drawbacks to such a method 1 1 ~ eter. ~ The first method tried consisted of been finding a suitable nipan- of wiiple using a microliter syringe to inject a introduction into the ma+ spectrometer sample through a silicone rubber plug vacuum. The device shown in Figure 1 into the ~ a c u u msystem. Further inhas been used routinely 3t 11013i1'3 vestigation revealed selective adsorpBeaumont Refinery Laboratory for tn-o tion, predominately with the aromatics. years and has eliminated the objection. -1more commonly used device was then presented by other methods. installed consiqting of a fritted disk Two CEC Model 21-103C Ala-s covered with a heated gallium-indiumSpectrometers were used to coiidwt this tin alloy. The sample way introduced through this n i t h a capillary "dipper." Reproduction was poor as a reiiilt of the elevated temperature of the alloy, some distillation occurring through the fritted disk. Since a device himilar to the one shown in Figure 1 had been used successfully in this laboratory on gas chromatographs, it was decided to build and inytall one on the mass spectrometer. URING
D bers
m
DESCRIPTION AND OPERATION
Figure 1. Cross section of liquid introduction system for mass spectrometer
@ @ @ @
'/B-inch drill rod Charging volume (approx. 0.0006 cc.). Drill hole in '/g-inch rod, solder appropriate size needle stock in hole, machine, and polish compression cap Teflon plug ('/a-inch thick) M.S. panel Connection to M.S. inlet system
1768
e
ANALYTICAL CHEMISTRY
The sample introduction -ystem IS mounted on the front panel of the control cabinet along with the conventional gas introduction system. The liquid introduction system consists of a piece of stainless steel (6, in Figure 1) drilled so that the drill rod, 1, can be moved through i t into the maSs spectrometer. The drill rod is polished n i t h crocuB cloth. The stainless steel, 6, is threaded, and a compression cap, 3, is screwed onto it. The compression cap contains a '4 inch thick Teflon plug, 4, drilled so that the drill rod fits into it snugly. The compression cap is pulled up tight enough to maintain
the mass spcctroiiieter vacuum. Tlic small hole, 2, in the drill rod ha3 :I capacity of approximately 1 11. To obtain a sinal1 hole, install a sevtioii of hypodermic needle. With the drill rod pulled out p i 1 tially, a sample is introduced into the small hole with a syringe or any other convenient means. The drill rod is then pushed in until the sample reaches the mass spectrometer vacuum. The sample is completely vaporized into the mass spectrometer. The mash spectrometer has been operated a t 204" C. (400' F.) n i t h a satisfactory sample introduction. -4 fine stream of laboratory air is directed on the drill rod while in the charging poiition to permit the reproducible introduction of samples boiling Rt 35" C. (9.5" F.). Any sample can be introduced that has a boiling point high enough to be volatilized a t the temperature of the inlet system maintained at a pressure of less than 5 X torr before sample introduction. KO difficulty has been observed with samples ha&g a 95% boiling point of 260" C. (500' F.). Many advantages result from the installation of 5uch a liquid introduction system. Since no distillation occurs, reproducibility of pressure measurements is better. Teflon is essentially inert to hydrocarbons, resulting in no selective absorption. There is little chance to lose high vacuum due to channeling or leakage such as occurs when using fritted disks and silicone rubber plugs. A relatively volatile sample can be introduced a t room temperature into the heated inlet system. One such device has been in constant use on a mass spectrometer without replacing any parts for tn-o years, while another one has been in service for a longer period of time on a gas chromatograph.
