Radiological Detector for Gas Chromatography

A Radiological Detector for Gas Chromatography C. H. DEAL, J. W. OTVOS, V. N. SMITH, and P. S. ZUCCO Shell Development Co., Emeryville, Calif.

4 method for sensing changes in the composition of a gas stream has been developed, which exploits differences in beta ionization cross sections and is suitable for use as a detector in gas chromatography. It gives a detector instrument of very rapid response, high stability, high sensitivity, and broad applicability, which is insensitive to gas flow rate. The fundamental principles of the method are outlined, and the apparatus and its application to gas chromatography are described.

I

S THE use of gas chromatography for separation or analysis of mixtures of volatile substances ( 9 ) a sensing device is re-

quired that nil1 respond to the presence of any of the components in the carrier gas, usually nitrogen or helium. Various types of detectors have been reported, their principle of operation being based on chemical reactions ( d ) , thermal conductivity ( 7 ) , gas density ( S ) , and heat of combustion (9). In some applications the authors have successfully used an ionization chamber detector, which has the desirable features of high sensitivity, good stability and precision, rapid response, and insensitivity to changes in rate of gas flow.

where the z j ’ s are the mole fractions of the components and P and 7’ are the total pressure and the absolute temperature. In the absence of recombination reactions this rate of ion prodiiction is equal to the collected ion current. Effect of Pressure, Temperature, and Composition. The extent to which the conditions that lead to Equation 2 are satisfied in the instrument under consideration may be seen in Figures 1 to 3, where the total response in millivolts, before any current is balanced out, is plotted as ordinate. The data given in Figures 1 and 2 show that for a flow of pure nitrogen the ion current is proportional to total pressure and to the reciprocal of the absolute temperature. When heptane is introduced into the stream, the current rises, as shown in Figure 3. The cross sections of heptane and nitrogen may be estimated from the data of Otvos and Stevenson (6) as 45.1 and 7.7 relative to 1.0 for the hydrogen atom. From these figures it can be shown that the ion current should increase by 4.6% of its value between pure nitrogen and 1 mole 70 heptane in nitrogen, and should be linear with the mole fraction. Actually, Figure 3 shows an in-

PRIYCIPLES OF DESIGN AND OPER4TION

The principles of ionization chamber operation are well known and instruments for gas analysis based on ionization chamber techniques have been described ( 1 , 8). This discussion is thercfore restricted to principles that bear on the design and operation considerations discussed below. Production of Ions. If ions are formed by a source of pparticles, for example, in a gas at or near atmospheric pressure and if a potential difference is applied to a pair of electrodcs placed in the gas, then current will be carried by the ions. The current will increase with increasing voltage until a “saturation” current is reached. At saturation, all the ions that are being formed reach the electrodes before they recombine, whereas at lower voltages the drift rate of the ions is smaller and the rate of recombination becomes comparable with the rate of collection a t the electrodes. The voltage required for saturation depends on the electrode spacing, the pressure, and the nature of the gas. Hence, to eliminate the effect of voltage on ion current under a variety of conditions, it is desirable to work considerably above the saturation voltage. In the apparatus described below, 100 volts xas found to be adequate. The rate of production of ions depends on the strength of the ionizing source, the nature of the gas, and the concentration of gas molecules, which in turn depends on temperature and pressure. If the current is to be proportional to the concentration, the size of the chamber and the energy of the @-particlesmust be so related that only a small part of the p-particle energy is dissipated in the gas. Under these conditions the ionization produced per unit length of path is constant throughout the chamber and the rate of ion production is given by i = kcQ

740

780

760

800

820

840

860

NITROGEN PRESSURE, MM.Hg

Figure 1. Response of nitrogen-filled cell to changes in pressure at 50°C.

r

(1)

where c is the concentration of gas molecules per unit volumr and Q is the ionization cross section of the gas molecules (6). For a mixture of gases one can write

60

Figure 2.

80

100 120 140 T E M P E R A T U R E , ‘C.

160

Response of nitrogen-filled cell to changes in temperature

Atmospheric pressure, 50 ml. per minute nitrogen flow

1958

V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6

1959

400

0 5 0 " C. A IOO'C.

1

j

CENTRALELECTRODE ,,'COSDUCTOR TO CAR1 E

C 1COR I\SLZ4TOR

R A DIOACTIb E

C 4 S [\'LET CARTRIDGE

I

0 0

0.01

I

I

I

I

I

0.02

0.03

0.04

0.05

0.06

CEITR-ZL ELECTRODE

J

OLTER ELECTRODE

H E P T A N E I N NITROGEN, MOLE F R A C T I O N

Figure 3.

Response to heptane in nitrogen

TEFLOZ t\bCL4TOR

Atmospheric pressure, 50 nil. per minute nitrogen flow

itial increase of 75; per mole 5 heptane and a s l o ~ l ydecreasiiig slope xith increasing heptane concentration. A complete discussion of the reasons for this discrepancy is beyond the scope of the present paper, but the effect is believed to be similar to that found by Jesse and Sadauskis ( 4 ) , who explained such a nonlinear behavior by the formation of additional ions by collision of un-ionized excited molecules and normal molecules. Such an effect indicates that relative ionization cross sections should be used only as a qunlitative guide to the behavior of gas mixtures a t other than very low pressures. Sensitivity and Noise. The statistical fluctuations in the int,ensity of the 8-particle source produce a noise in the ionization current that sets a limit on the concentrat,ion of a serond gas that can be detected in nitrogen. This noise depends on the source strength and on the t,ime constant of the recorder. For example, in the instrument described here, a 10 me. smrce is used with a 1-second filter cirruit in the recorder. assuming a reasonable geometry, and allo\ving for absorption of some @-particles in the source holder, 1f-e can estimate that 2 x 108 ionizing particles traverse the gas in the period of one time ?onstant. The random fliictuation associated with this rate is d m ,which is a noise-to-signal ratio of 7 X 10-5. The noise lpvel shown in Figii~e11 is about 0.1 mv. out of 300, or 33 X 10-6.

I

ELECTRODE CONSECTOR

'

Figure 5 . Ionization chamber assembly

This is not bad agreement lor siic-h :I cr itlc calculation, coiisidei,irig that there are other sources of noi-e as well, and that the 0.1 mv. is a peak to peak value rather than an average value. The heptane results, together vith the noisc figure, indicate that a concentration of 0.01 mole 7 0 heptane ~ o i i l dproduce a signnlto-noise ratio of 2 to 1 and would thiia be detectable under the standard conditions of operation. Flow Sensitivity. An ionization chamber detector is inherently insensitive to changes in flow rate as long as the residence time of the gas in the chamber is long compared to the drift time of the ions in reaching the electrodes. The latter can be roughly estimated to be lowato lo-? second under the apl)lied electric field ( 5 ) . The residence times are always much longer, even a t the unrealistic rate of 9 liters per minute shown in Figure 12. The small increase in current with increasing flow rate is probably a pressure effect due to flow restriction in the (,sit tube from the chamber.

+IO0 v.-

Power Supply

I

1

Panel Meter

I

compensating Voltage Pole n t lorn e te r

R e s 1s tor ( I 0 lo o h m s )

1

DC I:

I IOSIZATION C H-4.441BER

,

Amplifier (Gain

1

L

1000)

-

Output

Attenuator (Ad J us ta bl e )

' I ~

- - -

I'OTENTIOMETER RECORDER

----A

ELECTRO\lETEK A\lI'LIFIER

Figure 4.

Schematic block diagram of radiological detector for gas chromatography

1960

ANALYTICAL CHEMISTRY GENERAL DESCRIPTION OF APPARATUS

The complete detecting apparatus consists of a special ionization chamber with self-contained radioactive source, an electrometer amplifier with built-in compensating voltage supply, a potentiometer-type strip chart recorder, and an electronic temperature controller for thermostating the ionization chamber. Diagrams and photographs of the over-all assembly and essential parts are shown in Figurea 4 through 9. The cost of the equipment, exclusive of the recorder, is approximately $11M). Of this total, approximately $326 is for the ionization chamber and source, 8525 IS far the elertrometer amplifier, and $250 is for the temperature controller. Ionization Chamber. The present design of the ionization chamber has evolved from experience with several designs over the past 3 years. The most important requirements that must be met by the design are: 1. Safety from radiation and electrical shock hazards

5 . Ease 6.

bf

cle'anine - bv. fluihihine - with solvent without dis~~~~

~~

~~~

assembly Simplicity to minimize cost and simplify maintenance

The ionization chamber shown in Figure 5 in CIOSS section, and in Figures G and 7 in photographic views, meets the foregoing requirements very Tvell.

Figure 7 . Components of ionization chamber

The body of the cell is a rectangular stainless steel block having over-all dimensions of Z1/3 X Z1/z X 4'/2 inches. A hole is made by a rod I/a inch in diameter, extending through the inch in diameter bored through the block provides space for the gasket and bottom cover plate. Three holes a t the lower end electrodes and is the aEtive reeion of the cell. The outer eler~~~. of the electrode permit gas flow through the annular space outtrode is a Etainless steel tube :/a inch in outside diameter with side the electrode and Dermit eomolete drainine of solvent after cleaning. The central electrode is a stainless steel hypodermic tube with 0.063-inch inside diameter and 0.005-inch thick wall to permit penetration of 0-particles into the active volume from the enclosed source. This electrode is supported by a conical end piece into which the radioactive source is screwed, The central electrode assembly is mounted on a Vycor (SF% silica glass manufactured by the Corning Glass Works.) disk insulator and sealing is provided by Teflon gaskets. The' Vycor or fused quartz insulator is required to maintain high insulation resistance at high temperatures. Glass is not satisfactory because its resistivity drops rapidly a t temperatures above 200" C. The electrical connection t o the central electrode is made through B coaxial conductor to a conventional connector a t the top of a 2'L2-incb extension tube. The ZL/1-inch long stainless steel extension tube provides thermal isolation of the connector and cable from the hot cell. The cell block is bored for insertion of four cartridge-type electrical heaters and a. nickel resistance thermometer element. The cell temperature is controlled by means of an electronic temperature controller (Resistotrol temperature controller manufactured by Hallikainen Instruments, Berkeley, Calif.) with the resistance thermometer element connected a8 one arm of the bridge input circuit. Temperature control is better than *0.1' C., which is more than adequate for the stability requirements. Flow line connections are made through tapped hales a t the side near the top of the block and on the bottom cover plate. These holes are located to minimize the escape of direct pradiation from the chamber when flow lines are disconnected. The total volume of the chamber is approximately 13 ml., less than 5 ml. of which is active volume. The design is such that, a larger bore and larger outer electrode may be used to accommodate f l o m from larger columns, if desired. ~

~

Figure 6.

Ionization chamber assembled

~

~~

The grounded shell construction used here has the advantage of shielding the high voltage electrode and simplifying connection of flow lines because they need not be insulated from ground. It also provides an inherent "guard ring" for the central collector electrode, preventing electrical leakage paths between the outer electrode and the collector electrode other than through the ionized gas. A word of caution is due here concerning the selection of the collector electrode. If the outer electrode is

V O L U M E 28, N O . 1 2 , D E C E M B E R 1 9 5 6

1961

shielding fastened to the cell walls. However, exposure to intense radiation that can produce serious burns is possible if the cell is disassembled. For this reason the source should he removed and placed in a shield block by qualified personnel, and the cell should be checked for internal contamination before the disassembly is hegun. Electrometer Amplifier and Recorder. The ion current ohtained 7r.ith nitrogen in the cell is of the order of 5 X 10-10 ampere and the minimum changes to he detected are approximately 0.25 X 10-12 ampere (*0.05% of total current). In order to accomplish this satisfactorily, the ion current due to nitrogen must he balanced out either by current from a compensating ion chamber filled with a flowing reference gas (8) or by a current obtained from a compensating voltage applied through a high resistance. Blthough the compensating ion chamber has the advantage of compensating for changes in temperature and pressure, the single chamber with compensating voltage has proved completely satisfactory and has been used in most applications because of its simplicity and lower cost.

Figure 8. Electrometer amplifier

used as the collector-i.e., connected to the amplifier input eirc u i t c t h e measured current includes ions produced in the annular space between the outer electrode and the cell body, The drift velocity is very low for these ions because of the low potential difference between the grounded cell body and the amplifier input. This results in excessive sensitivity to gas flow, sensitivity to surface conditions owing to surface potentials, and Bensitivity to polarity of the high voltage electrode, because the external current may either add to or subtract from the ion current in the active region. When the central electrode is used as the collector, these difficultiesdo not arire. Radioactive Source. For the radioactive source strontium00-yttrium-90 has been chosen because i t is a pure ,%emitter and has a relatively long half life (approximately 19 years). A 7-emitter is not desirahle for this type of instrument because of the lo^ ionization efficiency of r-rays and the greater shielding problem. Although a-emitters are desirable for their high specific ionization ( l ) , their poor penetrating power make8 it necessary to expose the active m a t e d to the g a stream, ~ which may permit contamination of the gas or of solvents used for cleaning the cell. The strontium-90 in equilibrium with its daughter product, yttrium-00, emits &particles with energies up to ahout 2,200,000 electron volts, which permits the u8e of two isolating stainless &eel tubes hetween the active material and the sample gas without undue loss of sensitivity. The source consists of 10 me. of the radioactive strontium in the form of a sliver of activated foil in a thin-walled (0.002 inch thick) stainless steel hypodermic tube having an outside diame~

~

~~~~~

~~

~

~

~

.

~

~

~

.

~

~

handling and stable mounting in the chamber. These special “needle sources” were designed and fabricated t o specifications by the U. S. Radium Gorp., 535 Pearl St., New York, N.Y. A special handling tool is used for inserting or retracting the source from the cell. A photograph of this tool with a “dummy” source attached is shown in Figure 9. The cylindrical geometry permitted by the needle Source greatly increases the efficiency of ionization of the gas and improves the uniformity of irradiation. All @-parbielespenetrating the u d l of the inner electrode must pass through the gas and produce ions. A high percentage of those reaching the outer Ndl are back-scattered and produce further ionization. The ion current with this 10 me. source is approximately equal to that obtained with a similar chamber using a 25-me. capsuletype source mounted a t one end. Radiation Hazard. The radiation hazard with this apparatus is negligible during normal operation. The heavy walls of the ionization chamber stop all the 6-particle8, so that the external radiation is only low intensity secondary x-ray (Bremsstrahlung). The radiation level a t the surface of the cell body is approximately 15 milliroentgens per hour (mr per hour), and this is further reduced to less than 3 mr per hour by ‘/,inch-thick lead

Figure

9.

Radioactive

source h a n d l i n g tool

version i f one used weviously with a n rtmaratus for measuring

0~~

~

~~

stabikzedby a constant voltage transformertnd gas ~dischargetype voltage regulator tubes. Panel-mounted coaxial connec-

ohms in the input circuit, and an additional lOi0-ohm resistor

1962

ANALYTICAL CHEMISTRY

connected to an adjustable compensating voltage supply is connected to the input grid. Xormally the lO10-ohm input resistor is used and the compensating voltage is adjusted by means of a panel switch and 10-turn dial to balance out the current due to nitrogen. The amplifier output is connected to a panel meter and recorder jack through a step attenuator. The output circuit is designed so that panel meter readings agree with the recorder when a 10-mv. recorder is used (this may be modified easily for use with recorders to 50-mv. sensitivity). The time constant normally used is approximately 1 second and is determined by a resistive, capacitive filter in the output attenuator circuit.

Amplifier noise and incidental noise are normally small compared to the statistical noise associated with the @-ionization source. The latter imposes the practical limit at which a change in gae composition can no longer be detected

OPERATIOX A N D XIAINTENANCE

To operate the apparatus the electrometer amplifier and temperature controller are turned on and the desired temperature is set on the temperature control dial. The cell may be operated at a temperature above the highest column temperature to be used, if desired, to avoid resetting of controls. By the time the cell has reached the operat,ing temperature, the electrometer amplifier will be sufficiently stabilized for zero adjustments to be made. First the amplifier zero adjustment is made with the input cable disconnected and t,he compensating voltage set a t zero. Then the input cable is reconnected and the Compensating voltage is adjusted to give zero output signal with nitrogen (or other carrier gas) flowing through the chamber. Subsequently, the am lifier zero and the setting of the compensating voltage should g e checked occasionally to correct for further warm-up drift or changes in pressure. Normally the amplifier zero drift is negligible and need be checked only once a day, and the compensating voltage setting needs to be reset only when the operating temperature or pressure is changed. Occasionally deposit,s Kill build up in the ionization chamber owing to condensation of high boiling materials, dirt in the system, etc. Cleaning can usually be accomplished by flushing the cell several times with a solvent such as acetone, followed by air drying. In cases vihere solvent, flushing is inadequate, it is necessary to remove the radioactive source and dismantle the cell for cleaning. Dismantling has rarely been found necessary with the present cell depigri.

200

I

0

I

I

I

I

I

5

10

15

20

25

I 30

I

NOISE C A . 0 . 1 MV.1

0

5

10

I 15

20

25

30

TIME, MINUTE8

Figure 11. Detection of separation of 1% n-hexane in n-octane Atmospheric nitrogen pressure, 40 ml. per minute flow, 150°C. cell temperature. Current due to nitrogen balanced out

In gas liquid partition chromatographic application the inherent flow insensitivity of the present detection system is particularly valuable, because an interfering surge in total flow of gas as a band is stripped from a column can frequently be detected with flow-sensitive detectors. The design of the present cell with a “through”-flow pattern and with a holding time of 0.05 to 0.10 minute at the 50 to 100 cc. per minute flows usually used ensures a rapid response with the cell providing an accurate measure of the average composition of the gas in the cell at any instant. Time delays in the electrical circuits and the recorder may be expected to give only slight distortions in the peaks plotted by the recorder. An illustrative chromatogram of a mixture of lY0 n-hexane in n-octane obtained with the unit operated in conjunction with a gas partition column 2 meters in length and 8 mm. in diameter containing SF-96 silicone fluid (General Electric Co.) suspended on crushed C-22 (Johns Manville Co.) is shown in Figure 10. The same chromatogram at higher recorder sensitivity is shown in Figure 11. With nitrogen at 50” C. and atmospheric pressure flowing in the cell, the ionization current is aboiit 6 X amperes. which

35

TIME, MINUTES

Figure 10.

Detection of separation of 1c/c n-hexane in n-octane

i\tmo.;plieric nitrogen pressure. 40 ml. per minute flow, 150°C. cell temperature. Current due t o nitrogen balanced out

OPERATIONAL CHARACTERISTICS

The signal from the detector cell is sensitive to the composition of the gas in the cell, the operating temperature of the cell, and the operating pressure in the cell, but is insensitive to the rate of flow of gas in the cell. In practical operation the cell temperature and pressure may he fairly easily controlled to the degree required to provide a stable base line trace on the recorder with regard to both short-term variations which might he confused with trace sample components and long-term drifts.

FLOW OF NITROGEN, LITERS/MIN.

Figure 12.

Response to changes in nitrogen Bow

-4tmospheric pressure, 25OC. cell temperature

V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 corresponds to a potential of 6 volts across the 10IO-ohm input resistor. At an amplifier attenuation of I / ~ Othis corresponds to 8 300-mv. output, as shown in Figures 1, 2, and 3. Under operating conditions this current is balanced out by means of the compensating voltage, and the recorder sensitivity is set a t 1 my. per inch, so that, for example, the response to heptane vapor is 560-mm. displacement (22 mv.) per mole per cent of heptane. The net response is substantially linear in the composition of the gas up to 0.02 or 0.03 mole fraction (Figure 3). Because there is a rough proportionality between ionization cross section and molecular weight, the net response of the unit to different vapors is about the same when the compositions are expressed on a lveight basis. The net response to changes in cell pressure and temperature is on the order of 0.4 mv. per mm. of mercury (3.3-mm. displacement per mm. of mercury) and 0.8 mv. per "C. (6.7-mm. displacement per "C.), respectively (Figures 1 and 2). An increase in nitrogen flow of 1 liter per minute produces a net response of only 0.2 mv. up to flow rates of at least 5 liters per minute (Figure 12). The statistical noise corresponds to a net response'of about 3 mm. (0.1 mv.) and is equivalent in magnitude to the net signal from a change in heptane concentration in the gas stream of about 0.005 mole yo.

1963 standard method is usually used for quantitative analyses. I n this case, for any pair of sample components, 1 and 2, a parameter which is to be expected to be independent of operating conditions and the relative concentrations of the sample components may be defined as

where X ' s are weight fractions of the components in the sample charged and the M ' s are their molecular weights. *is indicated earlier, the AQ's may be estimated from the cross sections of the constituent atoms ( 6 ) .

1. 30

p

1.20

M I

\

< m

r"

i

t

n

0

A

1.10

1.00

kf" N

J

n

v

I

I

I

I

I

10 20 30 40 50 60 I S O P R O P Y L ALCOHOL CONCENTRATION, %W

Y Figure 14.

__

I I

01 0

I 70

Effect of sample composition of binary sample on calibration parameter

sec-Butyl alcohol-isopropyl alcohol solutions, loooC.

_.

60

80

100

T E M P E R A T U R E , "C. c Y

400

" 600 800 O U T L E T P R E S S U R E , MM.Hg

Figure 13. Effects of column operating conditions on calibration parameters for equiweight solution of secbutyl alcohol (1) and propyl alcohol (2) Cell operated at column temperature

In practice, parameter K,,z is found to be substantially independent of sample size, gas flow rate, temperature, and pressure. The parameter for sec-butyl alcohol (2-butanol), 1, and isopropyl alcohol (Spropanol), 2, is plotted as a function of these variables in Figure 13. It is found also to be independent of the mechanical details of the detector cell over a wide range of designs. It is, however, somewhat dependent upon the relative concentrations of the pair of components and its magnitude can be estimated only roughly from atomic cross sections. A typical plot of K1,2 vs. Xz, where the concentrations have been calculated on a two-component basis ( X i XZ = I), is shown in Figure 14. For a large number of cases, plots of this sort have been shown to be linear within experimental errors of 1 to 2%, regardless of whether the data are taken from binary or multicomponent samples. Data which allow the comparison of calculated parameters and experimental parameters are given in Table I.

+

APPLICATION TO QUANTITATIVE ANALYSES

When the unit is applied in conjunction with a chromatographic column, it is to be expected that the area Ai, of each of the chromatographic peaks representing the separated components of a sample will be proportional t o the product of the total moles of the component, mi, and the difference betaeen its ionization cross section and that of the carrier gas, A Q ~ ,

A , = k'm,dQi where k' depends upon the flow rate, the temperature, and the pressure of the gas in the cell. (It is implied here that rate of flow of total gas effluent from the column is constant and that the concentrations of noncarrier gas components detected are sufficiently small that the net response is linear in the effluent gas concentration.) Usually, however, it is not convenient to reproduce gas flows from chromatogram to chromatogram quantitatively or to measure the flow quantitatively, and an internal

Table I.

Comparison of Experimental and Calculated K I , ~

2-Butanol

Acetone

Methyl ethyl ketone Diisopropyl ether 2-Butanol Methyl ethyl ketone

2-Propanol 2-Propanol 2-Propanol 2-Propanol Acetone Acetone

1.18 0.88 0.99 1.14 0.88 1.27

1.12 0.91 1.05 1.13 0.91 1.16

1964

ANALYTICAL CHEMISTRY ACKNOWLEDGMENT

The authors are indebted to D. J. Pompeo for suggestions and encouragement in the use of ionization chamber detectors, to S. T. Abrams, 1%'. B. Conner, Jr., and S. AT. Brassington for their part in the design and construction of the cells, and to J. F. iJ7aller and H. U. Cole for their part in applying the cells in practical gas partition chromatography, LITERATURE CITED

(1) Deisler, P. F., Jr., hlcHenny, K. W., Jr., Wilhelm, R. H., ANAL. CHEM.27, 1366 (1955).

(2) James, A . T., Martin, A. J. P., Bwchem. J . 50, 679 (1951). i3j James, A.T., Martin, 8 . J. P., Brit.Med. Bul., 10, 170 (1954). (4) Jesse, W.P., Sadauskis, J., Phys. Rev. 100, 1755 (1955). (5) Loeb, L. B.,"Kinetic Theory of Gases," p. 555, RlcGraw-Hill, New York, 1934. (6) Otvos, J. W., Stevenson, D. P., J . Am. Chsm. SOC.78,546 (1956). O" Phillips, C.S. G., Discusdons FaTaday SOC.7, 241 (1949). (8) Pompeo, D.J., Otvos, J. W., U.S. Patent 2,641,710(1953). (9) Scott, R. P.W., h'ature 176,793 (1955). (10) Smith, V. N.,Otvos, J. R., ANAL.CHEM.26, 359 (1954).

RECEI V E D for review April 26, 1956. dccepted J u n e 15, 1956. Division of Analytical Chemistry. Symposium on Vapor Phase Chromatography, 129th Meeting, ACS, Dallas, Tex., -4pril 19.50.

Quantitative Determination of Phenylalanine on Paper Chromatograms ARTHUR E. PASIEKA and JOSEPH F. MORGAN Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada

A quantitative method for the determination of phenylalanine in the presence of varying proportions of 19 other amino acids utilizes the characteristic blue color formed by phenylalanine when ninhydrin-developed paper chromatograms are treated with dilute sodium bicarbonate. The ninhydrin colors of the other amino acids can be removed by water washing without eluting the specific phenylalanine color, which is then extracted with 1-butanol and measured in a spectrophotometer at 600 m p . The intensity of the clearly defined phenylalanine spot can also be read directly on the chromatograms with a densitometer. The application of the method to complex biological media is reported.

D

URISG studies on the amino acid metabolism of animal tissues cultivated in vitro in synthetic media (8),paper chromatography indicated that an uptake of phenylalanine occurred during the cultivation period. In these studies, the synthetic medium employed was 31 150 (4, 6), which contains 60 ingredients including 20 amino acids dissolved in a modified Tyrode's solution ( 2 ) . The DL-phenylalanine content of this medium is 50 mg. per liter, representing 4.596 of the total amino acids present. The disappearance of phenylalanine from the medium suggested its importance for tissue culture nutrition and metabolism. Under certain conditions, phenylalanine forms a characteristic gray color when developed by the conventional ninhydrin procedure, and by this method microgram quantities can be detected (1, 3, 9). Under the conditions of neutral and acidic solvent systems used in the present study, however, the phenylalanine-ninhydrin color was found to be nonspecific and indistinguishable from that of various other amino acids. For this reason, an effort was made to develop a selective method for phenylalanine that could be applied in the presence of high concentrations of other amino acids following development by neutral and acidic solvents. Previous studies from this laboratory ( 7 ) have shown that a characteristic blue color, specific for phenylalanine, is produced when the ninhydrin-developed chromatograms are subsequently treated with dilute sodium bicarbonate. Development of this specific color is associated with a shift in the absorption maximum from 560 to 600 mp as well as increased color stability. The results reported in the present communication show that this characteristic blue color can be used as the basis for a quantitative method that permits the specific determination of phenylalanine on paper chromatograms in the presence of 19 other amino acids, even under conditions of poor separation and resolution.

/I

0.25 w

0.20

-

I

0 Figure 1.

IO 20 30 40 50 PHENYLALANINE CONCENTRATION, 7

Standard curve for specific phenylalanine determination

0 Graded concentrations of DL-phenylalanine dissolved in A1 150 with variable ratio of phenylalanine t o other amino acids present X Graded concentrations of DL-phenylalanine with constant ratio of phenylalanine to other amino acids PROCEDURE

Chromatography. Samples for analj-sis (5.0 ml.) are concentrated to dryness in vacuo over concentrated sulfuric acid and reconstituted in 0.2 ml. of deionized water. With the biological media studied, 10-pl. aliquots of the concentrates were found suitable for analysis. One-dimensional descending paper chromatograms are employed with Schleicher and Schuell S o . 597 or Whatman No. 1 paper. The solvent systems recommended for use are either 1-butanol-acetic acid-water or l-butanol-ethanolwater, prepared as described previously (6-8). The chromatograms are developed at room temperature by the descending technique for 18 hours, dried at 110' C. for 2 to 3 minutes, and redeveloped in the same solvent for an additional 18 hours in the same dimension. Separation and resolution aere considerably increased by this second development period. The chromatograms are then dried at 110' C. for 2 t o 3 minutes and sprayed with 0.4YC ninhydrin dissolved in either watersaturated 1-butanol or 95YG ethanol. The chroniatograms are dried at room temperature for 5 to 10 minutes, reheated to 110' C. for 3 minutes, and dipped into l.OYC sodium bicarbonate. The phenylalanine region on the chromatograms immediately appears as a deep blue spot, which is clearly visible even in the presence of the purple color of other amino acids. This blue color does not fade on prolonged standing, although the color intensity of the other amino acids on the chromatograms decreases steadily (7). Measurement of Concentration. SPECTROPHOTOLIETER. The blue phenylalanine area is then cut into several strips, which are placed in a test tube with 5.0 ml. of deionized water. The tube from each spot is shaken mechanically for 1 minute, the liquid decanted, and the washing procedure repeated. The tubes are