Gas chromatography of radioactive substances

GAS

CHROMATOGRAPHY

OF

TECHNIQUES

RADIOACTIVE

AND

JEAN-PIERRE

SUBSTANCES

APPLICATIONS ADLOFF

Centrede Recherches Nucldaires. DQartement Strasbowg-Cronenbourg

I.

de Chimie Nuclbaire, (France)

INTRODUCTION

Gas chromatography, which has already been used with remarkable success in numerous fields, has become a very popular method in radioisotope work, such as in the separation and purification of labelled molecules, for research in radiation chemistry and hot atom chemistry. It has proved of outstanding value especially in this last field. HARBOTTLE~ commenting on recent progress in the chemistry of recoil atoms emphasises the important and ever increasing part played by gas chromatography: “This tremendously powerful technique, which permits rapid, clean separation and determination of a multitude of molecular species, and even of isotopic molecules such as HT and DT has permitted investigators to broaden the scope, while drastically shortening the time of chemical analysis. The use of this technique has in addition sharpened the awareness of numerous investigators to the presence of undesired side-reactions induced by ionizing radiation”. In this way EVANS AND WILLARD~ were able to separate more than twenty molecules labelled with s2Br after irradiating ti-propyl bromide, while LIBBY et aZ.3*4and CHIEN AND WILLARD~ in their first separations employing fractional distillation with added carriers, had only found about ten compoundse.

Z.TECHNIQUES OF GASCHROMATOGRAPHYWITHRADIOACTIVE

SUBSTANCES*

The essential difference between gas chromatography with radioactive substances and the normal techniques, lies in the use of a detector which registers the radioactivity of the substance at the point of emergence from the column. These radioactivity detectors present a much greater sensitivity than the most sensitive detector for inactive substances7, permitting the measurement of activities of some millimicrocuries (IO-~C) or ro-log of W, ro-13g of tritium, ro-15g of *2Br. The main radioactive tracers used in organic chemistry and biochemistry are 14C and tritium both emitters of soft p-radiations (14C:E = r58keV; 3H:E = IBkeV), hence most of the radioactivity detectors for use with chromatography columns that are described in the literature were constructed with these very soft radiations in mind. *Some of these techniques were recently I)OBBS~~ and JAMES AND PIPER’~.

described

in the Journal

of Chromatography.

See

20

J. P.

(a) Methods

ADLOFF

of discontinuous measurement of radioactivity

The simplest technique is that of the separate condensation of the substances at their emergence from the column, to be followed by measurement of their activities with apparatus suited to the activities of the various fractions. The separation is controlled by a standard detector, for example a thermal conductivity cell. HARRIS*employed this method for the identification of the various chemical species formed when bromoalkanes containing B2Br are bombarded with neutrons. The eluted compounds emerging from the chromatographic column are adsorbed on activated charcoal, in a tube cooled with liquid nitrogen, so that even the most volatile products are condensed. The tube is replaced at constant intervals (one per minute) and introduced into the hole of a NaI(T1) crystal. The activity-time curve so obtained shows a number of peaks, which are identified by chromatographing known bromo-compounds under the same conditions. KARMENAND TRITCH~have developed an efficient method of collection of the fractions, which permits the detection of compounds containing tritium and l*C: the stream of gases from the column is condensed in tubes containing crystals of anthracene impregnated with the stationary phase from the column, and the radioactivity of each fraction is measured with a scintillation counter. A commercial fraction collector+ has been designed on this principle. The vapours pass over a chromatographic detector and are then condensed and adsorbed on anthracene crystals impregnated with silicone oil. The tubes are changed according to the detector signals by manual control. Each substance (shown as a peak on the detector) is collected separately; it is also advisable to collect the gas between the peaks so as to detect any radioactive substances present in such minute amounts that they do not show on the chromatographic detector. DUTTONet aZ.l@used a similar method in the separation of tritium-labelled fatty acid esters; the gas stream issuing from the conductivity cell being directly introduced into a liquid scintillator. These discontinuous methods are, however, only used exceptionally, most workers preferring detectors which permit a continuous scanning of the radioactivity of the gas stream. (b) Continuous detection The coupling of a radioactivity detector with a chromatographic detector such as a catharometer permits the simultaneous recording of radioactive and inactive substances chromatographed. The advantages of this procedure are considerable: the catharometer indicates both inactive and radioactive substances present in macroquantities, while the radioactivity detector registers only the radioactive species. A radioactive substance present in macro-quantities is thus recorded by both detectors, By comparing the heights and areas of the peaks registered on the detectors (after suitable standardisation) the specific activity of the substance can be deter’ Packard Model 830 Tri-Carb Gas Fraction References

p. 24.

Collector.

GAS CHROMATOGRAPHYOF RADIOACTIVE SUBSTANCES mined.

In the presence

symmetrical, 1. Integral

of radioactive

impurities

thus giving an excellent

the peak widens and becomes

criterium

for radiochemical

un-

purityn’.

detectors

Detectors

of this type were described

by LOWE AND MOORER’ and in more detail by

POPJACK et a1.12. The carrier gas and the substances scintillating column.

21

solution

(diphenyloxazole

Owing to their solubility

in the scintillating tubes connected

solution

radioactivity

detector

range-changing

is proportional

device

the response

to the quantity

up to 30,000 impulses per second. The apparatus substances

prepared

and tritium

with ZOO/~.

by biosynthesis.

when a certain a constant

amount

of radioactive

(sensibility

a

stay

ratio IQ to 3)

of a gas-density

balance.

The response of the introduced,

of POPJACK is used for analysis r4C is counted

of

with 50 o/oefficiency

liquid should be periodically

substance

has been dissolved,

so as

sensitivity.

The method of KARMEN AND TRITCH~ (see p. 20) can be easily tinuous monitoring

substances

of the radioelement

JAMES AND PIPER’~ pointed out that the scintillation to maintain

through

is measured with photomultiplier

show the steps typical for integral detectors.

radioactive

replaced,

are bubbled

at their exit from the

and high boiling point the organic

unit which also registers

The curves obtained

or xylene)

and the radioactivity

with an automatic

and a registration

separated

in benzene

by retaining

modified for con-

the vapours in a column of anthracene

placed between

two photomultipliers. Another condensed to permit counter

integral

the 14C radiation

placed against

2. Detection

was developed

to traverse

it and be measured

and proportional

easy to pass the vapours

Geiger counter.

by BLYHOLDER~~. The

were recorded

after chromatography

and the design improved

principles:

(I) labelling

vation after chromatography matography copper

over the window of a

by using two thin end-window count-

them (ROGINSKY et a1.16).

substances

before

and (3) labelling

with radioactive the vapours

reagents.

issuing

may be carried out following

chromatography, the separated

He describes

a method

from the chromatographic

After isotopic

counter.

The sensitivity

exchange obtained

the labelled CO, is detected

(2) neutron

substances based

column

oxide, the CO, formed is then passed over a tube containing

at 200’. ductivity

with a thin end-window

counters

BEHRENDT~’ pointed out that radiochromatography

principle:

are thin

KOKES et al. r5 have already used this method since 1955, Some appli-

ers and passing the vapours between three

vapours

One of the cell walls is sufficiently

this wall.

with Geiger-Miiller

It is relatively cations

detector

in a cell cooled in liquid nitrogen.

after

actichro-

on this last

are burned

over

Na,14C03 heated

with a thin end-window

is claimed to be 10~ higher than with thermal

con-

cells.

However,

the efficiency

detected

with these methods.

Rejerences

p. .q.

of detection

of ‘“C is always low and tritium

cannot

be

J. P. ADLOFF

22

The efficiency

can be considerably

through the counter, the counter.

thus eliminating

increased

if the gas stream

WOLFGANG AND MACKAY~’have described

and more precisely a counter designed for the continuous of a gas issuing

from a chromatographic

As the counters

cannot

function

another gas, usually methane,

column

the

counters

was studied in detail and an expression

relation

to the volume

as ionisation (up to

detector

of the counter

chambers,

gas-flow counters of the radioactivity

(WOLFGANG AND ROWLAND~‘).

and the counter.

The performance

for the average

of

into the system of such

rate of counting

in

are perfectly

adapted

for the measurement

of

of IO -a to IO-SC. They are 2 to 25 times as sensitive

with a sensitivity

give a faster

Furthermore

200”).

and

and the rates of flow of the carrier gas and

was derived. These detectors

14C and tritium

recording

which gas is injected

between

methane

proportional

substances

with the carrier gas alone, the introduction

becomes necessary,

chromatographic

passes directly

the wall between the radioactive

response

and have a better

the results can be both differentially

temperature

and integrally

range

recorded

simultaneously. SILBERT AND TOMLINSON~used this counter IOO~/~; they confirmed

also that

only effect of the increasing Some disturb

substances

temperature

(nitro-

its performance,

for szBr with an efficiency

the performance

was still acceptable

being the lengthening

or halogen-compounds)

giving negative

of almost

at 2o0°,

of the counter plateau.

can poison

the

peaks due to the shifting

counter

introducing

a “poison” into the gas passing through the counter

(e.g. 4% of nitrobenzene

a constant

amount of vapours).

JAMES AND PIPER’~ use the same type of counter with one modification used as carrier gas and the gas stream is burned in the presence the column.

perchlorate, 5%

Water

sufficient inactive

vapour CO,

is then eliminated

: argon is

of copper oxide after

by passing

over magnesium

is added to the gas stream to bring it to a total of

CO, and then passed through

tritium

and

of the zero line.

ACHE et al.20 resolved this difficulty by deliberately

passing

the

the counter.

if calcium carbide is used instead

3. Applicatiorz

of scintillation

For y-emitters

sodium iodide crystals

The method

of magnesium

may also be used for

perchlorate.

counters activated

with thallium,

NaI (Tl) , are employed.

EVANS ANDW~LLARD~passed the gas stream through a glass tube placed in a hole in the crystal

and were able to detect

ro-13g of CHZa2Br and ro-15g of CH,*OBr (see also 21).

MOUSSEBOIS AND DUYCKAERTS~~ use the same arrangement

for organic

iodides

labelled with 1311. HERR et a1.23 passed the gas stream through a glass spiral of internal

volume of IO

ml, placed on a NaI(T1) crystal of 2.5 inches and thus detected with a reasonable cy organic bromides labelled witha2Br. Excellent ing the chromatographic

column

directly

With all these methods the sensitivity the radioactive discussed

gas remains

results may also be obtained

through

the tunnel of a NaI(T1) crysta124.

is proportional

inside the sensitive

efficienby pass-

to the length of time which

volume of the detector,

as has been

by HERR et aL2”.

Isotopes Refevences p. 24.

emitting

p-rays

can be detected

using plastic

scintillators.

STRANKS~~

GAS CHROMATOGRAPHY measured tillator.

the activity GRANDY

this was placed

of CO, in a cell which

AND KOCH~~ constructed

on a plastic

scintillator

multiplier.

FUNT AND HETHERINGTON

tube

out of plastic

made

4. Application

from

the column

in a suitable

chamber

of stainless

developed

of the ionisation of the

be kept about

from of the

procedure and

the column gas

is considerably

stream; reed

of low activities

to be detected

as well as in variations

temperatures

should

material

up to rgo’

current

xoll

Q.

This

to be measured.

the ionisation

chamber

interferes

an ionisation

chamber

of this chamber

the chamber

must

with themeas-

of the characteristics

constructed

chamber

sensitivity

of

the

of the

of the

cell. The ionisation

noise which

as the insulating

of the chamber.

chamber

which

func-

DOBBS~~ also described and gives

a good

a

response

0.5 PC of tritium. On the other

after

of

and 20 m,&

with

independent

a resistance

are examined,

in a background

can be used with

volume

with

diluted

an ionisation

of ensuring

heating The

AND

31. The gas

of the ionisation

conductivity

tions well up to 240°, using teflon which

is then the

diluted.

the

electrometer

nevertheless

advantage

the volume

however,

with a high boiling-point

and this can result

by WILZBACH

rate through

performance

of preventing

to the ratio between

millimicrocuries

MASON et aZ.32 have

with

on a photo-

of a capillary

cell and is then

has the double

whose

stream,

a vibrating

some

columns

conductivity

the same as that of the thermal with

If substances urement

the application

by CACACE AND INAM-UL-HA@O~

a thermal

chamber,

is proportional

velocity

be heated

described

scin-

the gas stream;

B” which in turn was placed

and passed at a pre-determined

carrier-gas

as the gas stream

allows

out of a plastic

cell for circulating

chromatographic

mainly

steel. This

velocity

still

over

mixer

stability

is measured

“Pilot

23

scintillator.

passes

nitrogen

and the

had one side made

a lucite

27 have

was first used with

and further

arrangement

SUBSTANCES

of ionisation chambers

This kind of detector RIES~@’

OF RADIOACTIVE

chromatography

is trapped

CACACE et a1.34 overcame

hand

in a quartz

and the CO,,

kept at o0 for maximum

N,

sensitivity

chamber.

This dilution

flow

ensuring

a constant

and

carrier-gas

flow. The sensitivity

can be measured Table

tube containing

and the carrier

ionisation

3. APPLICATIONS The gas chromatography

flow

rate

obtained

the various

dilution

with

oxide.

The water

through

the

is 0.5 m,&

the gases so formed

the catharometer nitrogen

of increasing

detection

which

pass through

is the

the speed of the gas

chamber,

independent

and the smallest

amount

of the which

methods.

OF THE GAS CHROMATOGRAPHY of radioactive

The radiochromatographic

in various

fields

p. 24.

and after

has the advantage

plications.

References

copper

gas pass over

by burning

is 2 m,&.

I summarises

to be exhaustive

this difficulty

of application,

substances technique

has found numerous has been

as shown in Table

but only to serve as examples

OF RADIOACTIVE

used

from

and varied

by numerous

2. The references

taken

SUBSTANCES ap-

authors

cited are not meant

the most recent

work.

J. P. ADLOFF

24

TABLE

1

DETZXCTION METHODS Efuiciency of

Obsrntations

dEtecttin of “C andtritlum

Detector

References

Geiger counter

14C:weak T : zero

Proportional counter with circulation

about IOO o/0The gas passes through the counter. Max. temp. about zoo’. Risks of “poisoning”. Widely used.

13, IS--20

Scintillator NaI(T1)

zero

Used for continuous analysis of y-emitters

2, 8, 22-24

Plastic scintillators

14C: 60 0/0 T:Io%

Liable to be attacked by certain organic vapours. Temp. range limited

25-27

Used as integral detectors

g-12

Organic and liquid lators Ionisation

chamber

scintil- l*C : 75 y0 T:zo%

Sample outside limited

counter;

temp.

range

about 100% Much used. Max. temp. dependsoninsulating substances used

TABLE APPLICATIONSOFTHE

14-17

28-33

2

GAS CHROMATOGRAPHYOFRADIOACTIVESUBSTANCES

Separation of the hydrogen isotopes: H,, HT, T, Separation of the radioactive noble gases Study of halogens (s”Br, ssBr, a*Cl, i2*1) produced by recoil reactions Study of tritium formed in the reaction6Li(n,a)T and 3He(n,p)T Study of i4C formed by the reaction 14N(n,p)14C WILZBACH'S gas exposure method for tritium labelling Analysis of products formed with accelerated 1% ions Separation and purification of labelled molecules Radiolysis of labelled compounds

(n,y)

37.38 35. 36 I, 2, 6, 8, 23 39-43 44.45 10, 28, 29, 46-50 51.52 53. 54 55

REFERENCES i G. HARBOTTLE, Conference on the Use of Radioisotopes in the Physical Sciences and in Industry, Copenhagen, 1960, RICC/zgo. 2 J. B. EVANS AND J. E. WILLARD, J. Am. Chem. Sot., 78 (1956) zgo8. 3 L. W. FRIEDMAN AND W. F. LIBBY, J. Chem. Plays., 17 (1949) 647. 4 M. S. Fox AND W. F. LIBBY, J. Chem. Phys., 20 (1952) 487. 5 J. C. W. CHIEN AND J. E. WILLARD,J. Am. Chem.Soc., 79 (1957) 4872. 6 M. D. SILBERT AND R. H. TOMLINSON, Can. J. Chem., 39 (1961) 706. 7 J. E. LOVELOCK, Anal. Chem., 33 (1961) 162. s W. E. HARRIS,C~~.J. Chem., 39 (1961) 121. 8 A. KARMEN AND H. R.TRITCH, Nature, 186 (1960) 150. is H.J. DUTTON,E.P.JONES,L.H.MASON AND R.F.NYSTROM, Chem.&Znd.(London),36(rg58) I 176. i1A.E. LOWE AND D. MOORE, Nature, 182 (1958) 133. la G. POPJACK, A. E. LOWE, D.MOORE, L. BROWN AND F. A. SMITH, J.LipidResearch, I (1959) 29. Is A. T. JAMES AND E. A. PIPER, J. Chromatog., 5 (1961) 265. l4 G. BLYHOLDER, Anal. Chem., 32 (1960) 572. 16 R. J. KOKES, H.TOBIN, JR. AND P. H. EMMET,~. Am.Chem.Soc., 77 (1955) 5860.

GAS CHROMATOGRAPHY

OF RADIOACTIVE

25

SUBSTANCES

I0 S. Z. ROGINSKY, M. I. IANOVSKY, G. M. ZHABROVA, 0. M. VINOGRADOVA, B. M. KADENATSI AND 2. A. MARKOVA, Doklady Akad. Nauk S.S.S.R., I21 (1958) 674. I7 S. T. BEHRENDT, 2. phys. Chem. (Frankfurt), 20 (1959) 367. I8 R. WOLFGANG AND C. F. MACKAY, Nucleonics, 16, No. IO (1958) 69. Is R. WOLFGANG AND F. S. ROWLAND, Anal. Ckem.. 30 (1958) 903. 20 H. J. ACHE, W. HERR AND A. THIEMANN, Symposium on the Chemical Eflects of Nuclear Transformations, Prague, 1960, CENT/56. 2I A. H. GHORDUS AND T. E. WILLARD, 1. Am. Chem. Sac., 79 (19.57) 4609. 22 C. MOUSSEBOIS AND 6. DUYCKAERTS, 7. Chromatog., I (1958)’ 200. 28W. HERR, F. SCHMIDT AND G. ST&KIN, 2. anal. Chem., 170 (1959) 301. *4 J. P. ADLOFF, unpublished results. 7~ D. R. STRANKS, T. Sci. Ins&., 33 (1956) I. ** G. L. GRANDY AND R. C. Koc
NOTE ADDED

IN PROOF

While this article

was in the press a review on the same subject

peared:

organics

“Labeled

in gas chromatography”,

Nucleonics,

by F.

CACACE

rg, No. 5

(1961)

ap45.