482 , NOTES

A tramistorised solvent front detector

Tllis circuit was designed to assist in the paper chromatography of unstable derivatives of vitamin B,, and folic acid, which have to be chromatographed at low temperatures, under an inert ‘gas, in the dark. At very low concentrations of folate and vitamin B,, derivatives, the problem is that of locating the solvent front on ascending or descending chromatograms in the dark, or by the light of a very low intensity safelight. It is also useful to locate the solvent front at a prearranged distance from the origin so that the area of chromatography paper covered by the solvent will fit a bioautograph tray without h.aving to cut the chromatogram to At, The detector described here was produced to trigger an alarm system when the end of a prede- termined solvent path-length had been reached.

The detector locates the leading edge of electrically conductive solutions by arranging the transistor (TRL in Fig. I) to conduct when the solvent resistance appears between points A and B (Fig. I). Relay RLI then closes and fires an alarm or other

SW1 On loft

Rl

Fig. I. Circuit diagram of the detector, RI represents interelectrode solvent rcsistancc. Latching circuit shown in broken lines (- - -).

circuit. The two detector electrodes (one connected to A, and one to 13) can be applied to sense either across the width, thickness, or length of the paper (Fig. 2). Suitable electrodes may be made from small paper-clips soldered to thin flexible polyvinyl- chloride-insulated flex, such as is supplied with hearing-aid earpieces. The spring tension in the clips and their connecting leads, and their combined weight should be insufficient to distort the wet chromatography paper. If using papers with low wet- strength, the paper and electrodes may need to be supported in an insulated frame which may be removed,when the paper is dry. Whatman No. I, No. 20 and ion- exchange papers have been used successfully without additional supports. Some ion- exchange papers have low wet-strength and need very careful handling; acid pre- treatment of these papers may improve their wet-strength characteristics. The tran- sistor and alarm circuits are placed outside the chromatography tank, the electrode leads being connected to insulated gas-tight conductors passing through the tank lid or wall.

Construction agad circa& details The circuit has proved to be adequately sensitive for use with electrically con-

J. Ckromatog;, 24 (Ig66) 482-484

NOTES 483

1Okfl

21cm ‘*

__..____4_

55kQ 1.5c mI

L35 cm

tinned P;,F$+r-

Pie;. 2. Some altcrnativc electrocle nrrangcmcnts. Interclcctrocle rcsislancos shown for Whatman No. 20 paper and 50 mM N&l at + x0.

ductive solvents. Some solvent resistance values are shown in Fig. 2. When using a Mullard 0C76 transistor for TRl a solvent resistance of 1.25 M32 or lower caused the relay RLl to close, the base voltage being -75 mV. The sensitivity could be increased by the use of higher gain transistors or possibly a Darlington pair. Substituting a selected low-gain Mullard OC71 for TR1 the relay closed: with a solvent resistance of 330 kS2 or lower. In this case a base voltage of --ISO mV was required to trigger the relay. The circuit values in Fig. I are those found suitable for a Mullard 0C76. The emitter current is limited to 400 PA by R4 at maximum base drive. With a solvent resistance of IOO kQ a base current of 30 /AA was recorded with the circuit in Fig. I. R2 is inclucled to limit base current when low solvent resistances are present. The current, voltage and resistance values were measured on the I mA, 2.5 V d.c., and RXIOO ranges of a Taylor Multimeter type A (IOO l&?/Vcl.c.). Using silicon or germanium transistors, temperature stability was founcl to be satisfactory in the range 0’ to +37”.

An alarm bell is preferably driven from a separate dry battery. If both the transistor and the alarm bell are driven from the same battery the circuit should be checked for the presence of high-voltage transients. These may occur when the bell operates, possibly leading to irreversible damage to the transistor with subsequent unreliability of the device. The battery terminal voltage should also be checked with the alarm operating to ensure that the transistor is supplied with at least 3.8 V. A crude test for the absence of high-voltage transients is to connect a neon-bulb across the bell; even a feeble glow should not be seen in the neon-bulb with the bell operating. Such transients may usually be suppressed by the use of simple resistance-capacitance filters, although i% this fails a thyrector diode may have to be connected across the bell or buzzer to ensure that the transistor is not damaged, The relay RLl should have a d.c. coil resistance of about 350 $2, the contacts (X in Fig. I) being suitably rated for the current and voltage load of the alarm circuit(s). Electrical latching of the relay in the ‘on’ position when the transistor conducts may be provided by the use of extra contacts (Y in Fig. I) and a reset switch (SW2), base drive being provided

J. Chvowxztog., 24 (IgGG) 482-484

484 NOTE5

via Rg. Using a uniselector or multi-way switch and electric clock, with electrodes placed at measured intervals down the paper, solvent run times may be recorded. This has been of considerable use when working at o”.

The author gratefully acknowledges the assistance of G. T. WARNER, and the receipt of a grant from the Medical Research Council.

De~artnaent of Biochemistry, Unit of

M&o.Xology, OTfoJbrd (Great Bktain) NIGEL HARDING

Received March 24th, IgGG

1. C?crol?a&O~., 24 (IgGG) 4.82-484

Some technical improvements in the paper chromatography of sugars

A method of sample desalting and a sensitive staining reagent

Pyridine extraction procedures for desalting sugar samples are knownllz. Hot pyridine, however, is known to cause chemical transformations in some sugars3. In the method to be described in this paper, pyridine comes into contact with sugars at room temperature and for a short period.

Aromatic amine-acid mixtures are well known asspecific staining reagents for sugars*, but most of them lack sensitivity”. The p-aminobenzoic acid-phosphoric acid mixture to be described is more sensitive than most of the commonly recommended reagents of this type, It has the further advantage of being stable at room temperature for a long time.

The desalting $roccdure z ml of pyridine (B.D.H., A.R.) is added to a 0.5 ml sample (urine) to precipitate

the salt@. After mixing and allowing to stand for IO min., the tube is centrifuged at 2000 r.p.m. for 5 min. Supernatant is spotted over the area CDFGE of a strip of Whatman No. I filter paper, chromatographic grade (Fig. I), pyridine being evapo- rated off with a blast of cold air. Two washings, each of 0.5 ml pyridine, are similarly spotted. Next, the strip is immersed in ether in a jar, for a period of IO min, after which the strip is taken out and kept under a fan for another IO min. This completely removes pyridine from the filter paper strip.

The sugars are washed in 0.3-0.5 ml distilled water by a descending technique, running time I h at 70”. The elution is conducted in an incubator 40 x 40 x 60 cm adjusted to the requisite temperature, Rectangular staining jars 8.5 x 6.5 x 4.0 cm, supported on wooden blocks, are used as solvent troughs. A glass rod which fits tightly between the two opposite walls of the staining,jar is used to support the paper strip along one wall. The strip is rolled down over a thin glass rod.placed on the upper

.I. Clcromatog., 24 (1966) 484-486