The
II
A. Jackson school
Grammar
Blyth, N o r t h u m b e r l a n d England
A Simple High Frequency Transistor Recorder for Chromatography
High frequency apparatus has been used both for conductometric titration and for chromatographic recording, but the apparatus used for the latter purpose in general lacks stability. Suitable high frequency apparatus has been described by Stigmark and Johansson, among others (1-4). A simpler transistorized oscillator was found to give satisfactory results, especially on Sephadex columns with very small quaritities of material. Preliminary experiments having shown that a working frequency of 1-2 Mc/s gave the best results with the size of column used (30-50 cm long by 1.25 cm in diameter), the oscilator (Fig. 1) was constructed.
connected to the R F output from the oscillator; the lower was connected to the detector (Fig. 3). The rectified emf from the detector was balanced by an opposing emf obtained via a resistance network from a 9 v dry battery, and fed to a Sunvic High Speed Potentiometric Recorder (Model RSP 2) working on 1mv full
Aluminium she
R.F. to Detector
Glass W a o l k
V
Figure I. The oscillator. An O C 1 7 i tranrirtor should be used if a higher frequency than 1 OMc/s is required.
This is connected by coaxial cable to an electrode surrounding the column. Another electrode is connected, also by coaxial cable, to a simple germanium diode detector (Fig. 2). The electrodes, which may be clamped a t any suitable position, consist of thin aluminum sheet wrapped closely round the tube containing the column. The upper one, for a tube 1.25 cm in diameter, was 2 cm high and was separated by 0.1 cm from the lower, which was 1 cm high. The upper was ~ditery Tapped Every 1.5" R,
mn
I
t
EX 34
Figure 2. The detector. The four potentiorneterr ore 011 wire-wound. The recording meter war a Sunvis Hish Speed Potentiornettic Recorder IModei RSP21.
Figure 3.
The electrode system.
scale deflection.' It will be noted from the data (Figs. 4 and 5) that a sensitive recorder is necessary. The oscillator and detector were constructed in an aluminum case measuring 15 X 10 X 6 cm and were con~pletelyscreened from each other by an aluminum sheet. Separate batteries were used for oscillator and detector to avoid the possibility of R F leakage. The oscillator has only two controls, an on-off battery switch and a coupling capacitor C which is used a t the lowest setting which will give the desired sensitivity. The detector has a batterv switch and four ~otentionleter controls (R1-R4). kl is connected across the battery and enables a suitable voltage to be selected. Rz and Ra (zero controls) are used to apply a suitable fraction of this voltage to the recording meter, in opposition to the voltage derived from the rectifier circuit. Rz is the coarse adjustment, and Ra (driven by a 9: 1 reduction gear) is the fine adjustment. Ra is a meter shunt for reducing the sensitivity of the meter if this should be necessary. The screen of the coaxial cable and the oscillator and detector must all be connected to ground. Ideally, the Similar to a Leeds and Northrup Speedomax, type H, model S. Volume 42, Number 8, August 7 965
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Table 1.
,
10 ELUTION VOLUME
I
.
.
.
,
8
30 ml
20
Flgvre 4. Separation of the proteins of milk on Sephodex G-75. ...Elution rote of 1 8 mllhr; --Elvlon rate of 9 mllhr.
.
column and electrode system should also be protected by a grounded screen,as any movement within about 1m of the electrodes will cause a deflection of the meter. In practice, as long as the column is not approached during a separation, the screening may be omitted. The experimental columns were prepared in 50-ml burets. The constriction a t the base was plugged with glass wool, and then 4 rnl of glass beads (0.01 cm d i m eter) were placed on this and washed well with water. The instrument is particularly valuable for showing when the column is washed clean and when it has been fully regenerated after use. For instance, it was noticed that the washing of 4 ml of glass beads with distilled water took ll/z hours--the time taken for the meter to indicate a steady deflection once more. The electrodes were clamped around the glass bead section. Various materials were tried for the chromatographic OPTICAL AT
mv 0.60
-
0.50
.
n
Magnitude of Deflection Obtainable with Various Concentrations of Solutes
Solution in 0.2 ml
Concentration (mg/ml)
Weight of solute (mg)
Sodium chloride Lysozyrne chloride Albumin (bovine)
1.25 3.0 2.75
0.25 0.6 0.55
-Peak deflectionScale div.
mv
300 229 25
3.00 2.29 0.25
column itself, including alumina, Deacidite F F (hydroxide form), Zeobrb 225 (hydrogen form), and Sephadex (G-25 and G-75). Aqueous solutions of inorganic ions were easily detected, and good peaks were obtained with amino acids, proteins, and methyl-glucosides, especially on Sephadex. However when samples of l-methyl-6nitroindazole and 2-methyl-6-nitroindazole in benzene were passed through an alumina column washed with benzene, although they separated as two yellow bands, they were not detected by the recorder. Separations on Deacidite F F (hydroxide form) always showed a peak recording after the passage of one void-volume of eluant, due presumably to carbonation of the resin by atmosDheriC carbon dioxide when the s a m ~ l was e introduced. Separations on Sephadex were well demonstrated by the recorder, G-75 being more "conductive" to R F than G-25. ~he'chiefdifficulty arose when a salt was present in the mixture, as the method is particularly sensitive to salts and even a minute concentration causes the recorder t o move off scale (1 mv). If this happens the recorder may be reset manually by means of the zero control. An increase in the amount of sodium chloride passing between the electrodes of as little as lo-* g caused a deflection of 1scale division (0.01 mv). Some typical examples of peak deflections are given in Table 1. Even when the column was washed and eluted with a buffer solution, satisfactory peaks were obtained as fractionation proceeded. Owing to the DENSITY greater "background" conduction, a 750ms larger bias voltage had to be chosen. Here too the instrument was extremely useful in showing when the column was 0.50 2 5 0 washed clean, and when it had been regenerated.
1
ELUTION VOLUME 5
10
15 20 FRACTION NUMBER
25
Figure 5. Separation of the proteins of milk on Sephodex G-75. cording with optieol density meoruremenh
448 / journal of Chemical Education
30 Comparison of RF re-
Figure 6. A simple detector eircuit for routine work using a microommeter. All potenliometerr are wire-wound.
The separation of the proteins of milk was used to demonstrate the method, following the work of Pri3aux and Lontie (6). A column of Sephadex G-75 was prepared in a buret. The constriction was plugged with glass wool, and a 4-ml layer of glass heads (0.01 cm in diameter) was placed on this. The purpose of the heads is not only to support the column, but also to avoid further separation of the constituents after detection, and also to reduce the void-volume during and after detection. The electrode system was clamped round the buret over the bead section, and the column was washed with distilled water until the meter indicated a steady reading. The Sephadex column was prepared by a slightly modified Porath method (6, 7), and measured 45.5 X 1.25 cm in diameter (45.5 ml). The eluant was an acetate buffer (pH 4.63; 0.1 M) and the column was washed with this buffer until the meter reading was again steady. 100 ml of milk was slowly treated with 20 g ammonium sulfate and centrifuged a t O°C for 50 min a t 10,000 rpm. 65 ml of supernatant was treated with a further 16 g ammonium sulfate and centrifuged a t O°C for 30 min a t 10,000 rpm. The bulky precipitate was dissolved in 4 ml buffer, and dialyzed against the buffer. Some of the resulting solution was diluted 6-fold and some 10-fold with buffer, and 0.4 rnl of the dilute solution placed on the column which was eluted with buffer with a head of 10 cm. Fifty fractions of 1 ml were collected, and the total protein per fraction was determined by the method of Lowry (8,9). The rate of elution was 18 ml/hr for the first separation, and 9 ml/hr for the second. The higher rate of elution used in the first separation failed to give the desired resolution, although the broad outline of the elution curve showed that separation into three fractions was being achieved (Fig. 4). Better results were obtained with the slower elution rate, and in Figure 5 the recorder curve is compared with the results of the optical density determinations of the 1-ml fractions a t 750 mw after treatment by the Lowry method. In Figure 5 the volume scale for the optical analysis has been transposed by 2 ml to allow for the time interval between recording and collection of the fractions. Whitaker (10) has shown that under certain conditions a linear relation exists between the logarithm of the molecular weight of a protein and the ratio: void-volume of s Sephdex G-100 column elution volume of the protein
Squire (11) however points out that Whitaker's expression fails as the volume ratio approaches unity, and has shown that the relation between the elution volume and the molecular weight of a macro-molecule is of the form:
where Vp = volume available for the protein (elution volume) ; V , = void-volume; M = molecular weight of the protein; C = molecular weight of the smallest protein which cannot enter the gel; and g = a constant. For separations on Sephadex G-75 he therefore derives the expression: Using this relation, the highly speculative interpretation
Table 2.
Comparison of Results with Those of Prbux and Lontie
Elution volume (ml) 9.6-12.1 12. a 1 3 . 2 15.2-15.6 16.4-18.2 19.8 23.W25.0
Moleoular weight
Comparison with results of Pr&u & Lontie
74,00W43,000 1st peak; 3 components; M > 50,000. 37,00&34,000 2nd peak; 0-lactoglobulins; 34 35.000. 21,00&19,000 Shoulder; 0-lactoglobulins; cr lactalbumin; 2 other components; M > 17,000. 15,00&10,000 3rd peek; a-lactalhumin; 1 other component; M 1 17,000. Not recorded. 6000 Not recorded. 2,000 - 750
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of the above results shown in Table 2 may be obtained. The apparatus may be further simplified (Fig. 6) if a very cheap and highly portable detector is required for routine checking of rapid separations, or for checking that column washing is complete. In this version the oscillator is unchanged, but a 50 pa meter is used as indicator in the detector. The 50 kQ potentiometer is used as a shunt to control the sensitivity of the meter, and the 10 kQpotentiometer provides a bias voltage for setting the meter zero. Deflection of the meter provides a visual indication that one of the constituents of a mixture is passing between the electrodes. The more sophisticated model has proved successful in a variety of applications, its main advantages being its simplicity and cheapness and the fact that the sample being detected is not destroyed. Modifications not yet tested which preserve these advantages include the use of aluminum paint on the glass of the column under the electrodes in order to provide more intimate contact. It would also be desirable to provide a method of detection which would be applicable when elution in a salt gradient was being used. Possibly another pair of electrodes a t the top of the column could be used to provide an automatic bias voltage to the detector which would increase with the salt concentration, thereby preserving the sensitivity of the lower electrodes. The author is grateful to Professor J. Baddiley, Professor of Organic Chemistry in the University of Newcastle upon Tyne, England, for laboratory facilities and for the loan of apparatus, without which this investigation could not have been completed. Literature Cited 11) h- , o n a h ~ L.. ~ . AND J~HANs~DN, G.. Mikrochim. Acta., 131 ~(1963): ' (2) ISHII, KO, HAYASHI,SEOICBI,AND FUJIWARA, SHIZUO, Anal. Chem., 31, 1587 (1959). (3) JOAANSSON, G., KARRMAN, K. J., AND NORMAN, A,, Anal. Chem., 30, 1397 (1958). (4) F., AND BLAEDEL,W. J., Anal. Chem., 28, 2 . . BAUMANN, (1956). 15) P ~ i ~ nG.. x .AND LONTIE, G., Arch. Intern. Physiol. Bwchim. 69, im (1961). (6) P O R A J., ~ , Btochim. Biophys. Ada, 39, 193 (1960). (7) FWDIN,P., Dissertation, Uppsala, 1962. 0.H., ET AL.,J. Bwl. Chem., 193,265 (1951). (8) LOWRY, S. P.,AND KAPM, N. O., "Methods in Enzy(9) COTOWICK, mology," Academic Preas, New York, 1957, Vol. 3, p.
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(10) W H I G R J. , R., Anal. C h . ,35, 1950 (1963). (11) %WIRE,P. G., Biochim. Biophys. Ada, 107,471 (1964). Volume 42, Number 8, August 1965
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