could be employed if the sample is trapped at reduced pressure so that the saturation pressure of oxygen is never exceeded. Resolution by the dimethylsulfolane column is generally satisfactory for Cz to Cs hydrocarbons, as s h o m by Fredericks and Brooks ( 2 ) . However, under the conditions employed here, ethane and ethylene emerge too close together and too close to the “air” peak; thus, ethane appears as a small shoulder on either the air or the ethylene peak, and can only be approximated \Then relatively large amounts of ethylene are present. Nitrous oxide, which is generally present to some extent in exhaust gases and atmosphere, emerges with propane. Other possible coemerging compounds are indicated in Table 11. In later ndrk slightly better peak resolution was achieved with a longer (50-foot) column with less (207,) dimethylsulfolane on the firebrick. Also, a partial separation of nitrous oxide and propane was obtained by allowing the trapping column t o warm up slowly during the elution step. In all of the cruise exhaust gas analyses there was a low trailing peak for nitrogen dioxide, not evaluated, which reached its highest point just ahead of butadiene. Therefore, the butadiene peak, and any later ones, are superimposed on the tail of the nitrogen dioxide peak. Tests made with nitrogen dioxide alone indicated that this compound is not completely removed
by the Ascarite absorber. Nitric oxide emerges with the air peak and does not interfere. I n the procedure described, the trapping and flushing steps take 0.5 t o 1.5 hours, the analysis step 1 to 2 hours, and the peak area measurements and calculations 0.5 to 0.75 hour. The capacity of a single unit is, therefore, two to three analyses per 8-hour day, The time per analysis could be reduced by increasing the rate of charging the sample to the trapping column; however, this possibility was not investigated. An automatic integrator for measuring peak areas would save operator time. The possibility of reactions in the sampling vessel prior to analysis was not studied, but is a problem which will have to be considered in applying the method. Alqo, the technique used here for sampling exhaust gas should be modified to prevent possible losses by condensation in the container. Complete vaporization might be ensured by warming the sample container or by dilution with an inert gas. Adsorption of various hydrocarbons in the Ascarite tube probably needs further study. However, the good results obtained with the standard C2 to Cs hydrocarbon mixture indicate that this is not a serious problem, a t least not with the C5 and lighter components. Any hydrocarbons which might be retained in the Ascarite during charging could probably be swept into
the trapping column with helium. This could be done conveniently by merely connecting the Ascarite tube ahead of the trapping column during the helium flushing operation. The technique appears to have promise for determination of other components in air and exhaust gases. Different columns or combinations of columns would enable methane and hydrocarbons above CS to be determined, and perhaps certain oxygenated hydrocarbons. However, some of the latter, notably the aldehydes, may be removed by the Ascarite absorber. If only total hydrocarbons are desired, rapid estimation should be possible by using the short trapping column and heating it sufficiently for complete elution. ACKNOWLEDGMENT
The authors are indebted t o E. D. Peters, who initiated the study and helped plan the scheme of analysis. Special acknowledgment is also given to Sigurd Groennings for valuable counsel and encouragement. LITERATURE CITED (1) Dimbat, M., Porter, P. E., Stross, F. H., ANAL.CHEM.28, 290 (1956). (2) Fredericks, E. M., Brooks, F. R., Zbid., 28, 297 (1956).
(3) Pat,ton, H. W., Touhy, G. P., Zbid.,
28, 1685 (1956). RECEIVEDfor review September 26, 1957. Accepted January 27, 1958.
Automatic Equipment for Determination of Amino Acids Separated on Columns of Ion Exchange Resins D. H. SIMMONDS‘ Biochemistry Unit, Wool Textile Research laboratories, Commonwealth Scientific and Industrial Research Organization, Melbourne, Australia
b Development of automatic equipment for determination of amino acids was undertaken to reduce the manual labor and systematic variability of these analyses, and to increase the output. Analytical variations have been reduced b y a factor of 3 over the manual method. No manual work is involved beyond loading the columns, changing buffers, and finally calculating results from the graphical presentation. Because it can work 7 days a week, the machine approximately doubles the output of results obtained by the manual method. Besides its use in determination of amino acids, it may b e applied directly to estimation of any groups of substances separable b y column chromatography, provided a
common colorimetric, absorptiometric, or turbidimetric assay method is available. With minor modifications it could b e used for the routine assay of large numbers of individual samples.
S
Stein, and Moore (11) have recently described their equipment for the automatic determination of amino acids in protein hydrolyzates. I n these laboratories, progress has also been made toTvard automation of the excellent but somewhat tedious colorimetric assays involved in the manual ion exchange procedures developed by Moore and Stein (4-‘7). This paper describes the equipment developed and report5 some results obtained Tvith it. PACKMAX,
The following sequence of manipulations is concerned in manual estimation of amino acids by the ion exchange chromatographic procedures of Moore and Stein (5, 6).
-4. Collection of fractions from chromatographic column effluent. B. Adjustment of each fraction to pH 5.0, if the reagent of Moore and Stein ( 4 ) is used. If their other reagent (7) is used, this step is omitted. C. *4ddition of ninhydrin reagent. D. Heating t o develop color. E. Dilution with ethyl alcohol-water (1t o 1,v./v.). F. Spectrophotometric estimation of diluted color. 1 Present address, Waite Agricultural Research Institute, Adelaide, South rlustralia.
VOL. 30, NO. 6, JUNE 1958
0
1043
RECTIFIER
PLATINUM
CHROMATOGRAPI-IIL
IN
Figure 2. turntable
Circuit for operation of fractionating device and fraction collector RECTIFIER
Figure 3. Circuit for operation of equipment
-... ING VE5SE.L
$L)
40C PV
To TUBE
--1
Lt
c -
AIR IN
1 Hd#
SOLENOID COIL H 1,F'LASTLC SLEEVE
,
f IC
,
OVERFLOWC TUBE
, CUVETTEN)
I I
SOURCE
PROTECTIVE ALUMINIUM COVER SOLENOID COIL I
25 1
t
$ 0 D TUBING
t
To
WASTE
Figure 1. Automatic amino acid analysis equipment
G. Recording and graphical plotting of results. H. Calculation of results.
In the apparatus described by Spackman, Stein, and Moore (fl), steps A, C, D, F, and G are carried out continuously by pumping the eluting buffer solution into the top of the ion exchange column and the reagent solution into the effluent. Although this is the method of choice, pumps capable of accurately metering such a small rate of flow (8 to 12 nil. per hour) were not available to the authors, and an approach, based on a mechanization of the procedures normally carried out by hand, was therefore used. The resulting equipment is believed to be somewhat cheaper to construct than that described by Spackman, Stein, and Xoore ( 2 1 ) and it traces the results as a singlepoint graph, which may be slightly easier to read and interpret than the threepoint record produced by the equipment of Spackman, Stein, and Xoore. Furthermore it lends itself to inexpensive replication, so that by utilizing a single 1044
ANALYTICAL CHEMISTRY
photometer and a multipoint recorder a nuniber of colunins can be run simultaneously and independently. A machine is currently being huilt in which the effluent froni eight ion euchange columns n ill be sirnultaneoudy estimated and recorded on {in eight-point recorder. The equipment has t n o disadvantages compared with that described by Spackman, Stein, and Moore (11). Because each effluent fraction is collwted and treated separately, the iiiaxiniuni speed a t which the equipment can he run is just under four 2-nil. fractions. per hour. This means that, with the equipment described in the present paper, determination of acidic and neutral amino acids by the rapid procedure referred to by Spackman, Stein, and Moore (If) n-ould take 3 days, and of the basic amino acids by the same procedure, 24 hours ( 3 ) . Proline cannot be estimated with this equipment, unless the filter in the spectrophotometer is changed manually a t the correct time. Hon-ei er, because of its greater accuracy and reproducibil-
ity, the authors prefer to estimate proline on a separate column of Dowex 50X8 (8,Y), uhing the reagent described by Chniard (f), and hence find this no inconvenience. If necessary, the equipment can he set up to determine proline by this method by using the appropriate reagent and diluent solutions and altering the filter on the spectrophotometer. DESCRIPTION
OF
APPARATUS
The component parts of the equipment are arranged vertically above one another, as illustrated diagranimatically in Figure 1. Collection of Fractions from Chromatographic Column Effluent. The magnetic balance (10) or a n y suitable fraction dispenser may be used.
A cheaper, more compact and convenient device, s h o w in Figure 1, ii, consists of a glass tube constricted a t its lower end and sealed by a metal-inglass valre. The valve, V , consists of part of a ll/p-inch nail sealed into a short length of glass tubing, the bottom
'
and tiniing sequence can be manually operated a t will.
I
HEATING ACKET
F VLSSLL (ill)
LLVLL DLVICE.
P FOR
,ZML
PARAFFIN OIL
OR (OML
HYPODERMIC
:i: AIR OR NITROGCN INLET
REAGENT RESLKVOIR
Figure 4. Automatic diluent dispensers
reagent
and
of which is ground to fit the constricted end of the outer tubing. Three or four lumps of glass attached to the top of the valve ensure its rise and fall centrally in the outer glass tube. Tube B has a platinum electrode, C, sealed in a t a level below that corr(qonding to the volume to be collected. d second adjustable platinum electrode, D , is introduced vertically through the top of the tube. Effluent from the chromatography column drains into the glass tube through side arm E . The valve is lifted by means of the solenoid coil, F , which slides over the outside of the glass tube and is held in the correct position by a rubber sleeve. The dimensions of this device may be scaled u p or down according to the size of fraction to be collected. For 2-nd fractions the dimensions shown in Figure 1, ii, have been found optimum. For 230-volt operation of solenoid coil F , 14,000 turns of enameled copper wire 0.0045 inch in diameter, on a Bakelite former 1.25 inches high by 1.25 inches in diameter, is satisfactory. For 110volt operation, 8000 turni of 0.0068inch tlianieter wire on a formcr 1 inch high ant1 1 inch in diaiiietei has been used. This measuring device can be used in conjunction with a neight- or motor-operated fraction collwtor turntable, if preparative scale separation, or manual estimation of a column effluent is desired. I n this case lifting of the valve and operation of the turntable are achieved through the circuit shown in Figure 2 . Effluent from the chromatographic column, nhich need be only .lightly more conductive than distilled n ater, fills the glass tube, B , through the side
DIAZLTLR *
Figure 5. Thermosiphon circulator for heating bottom jacket of heating vessel
arm, E , until electrolytic contact is made between electrodes C and D . Relay A then closes for approximately 5 seconds, allowing current to flow for this period of time through coil F and thus lifting valve I-. Relay B closes simultaneously. allowing condenser Cp to charge slomly up to the striking roltage of the neon tube, S E 96. At this voltage the neon strikes and closes relay C long enough to operate the fraction collector turntable. The delay between vah-e lifting and table operation is arbitrarily chosen. K i t h the component values shonii in Figure 2 , it is about 15 to 20 seconds. K h e n this fractionating device is used with the automatic amino acid analyzing equipment, the operational requirements are sonieu hat different. Figure 3 shows the complete circuit of the machine, with the exception of the thermosiphon water heater, the recorder, and the compressed air pump, m-hich are separately connected to the mains voltage and require no additional s~ itching. I n operation, the platinum electrodes,
C and D (Figure 1. ii) are bridged by effluent. Relay d (Figure 3) closes for about 5 seconds until condenser C, discharges. The contacts on this relay energize the solenoid, F, of the fraction dispenser, the solenoid of the reagent dispenser, and the coil of relay B. Contacts on the latter start timing motor 1 and this relay remains energized until the two-position srritch (KO.1, Figure 3) changes over, holding the motor on and releasing relay B. From this point on the timing and energizing of the equipment are taken over by timing rnotors 1 and 2 , n hich, by cam operated switche., control transfer of solution from one section of the equipment to another. addition of diluent. and reading and recording of absorbances. Preqs button switches (not shown in Figure 3) are also nired in, so that the reagent and diluent dispenscrs, fraction diylen-er. valres on the heating, diluting, and measuring vessels,
Adjustment of Fractions to pH 5.0. T h e reagent used is described by Moore and Stein ( 7 ) ; preliminary adjustment of the effluent fractions to pH 5.0 is not necessary. Addition of Ninhydrin Reagent. A conimercially available automatic ieagent dispenser could be used for nieasuring the aliquots of reagent and diluent required, if it could be adapted for single-stroke delivery. As such equipment was not readily available to the author, t v o were built to the design shonn in Figure 4. Each dispenser consists of a borosilicate glass hypodermic syringe (Z, 5 . or IO-nil., depending on the size of the aliquot to be delivered), sealed to a length of capillary tubing carrying a valve and side arm as shown in Figure 4. A second valve is located in the capillary tubing of the side arm, which is sloped upward to facilitate its closing. A ground joint is sealed to the capillary tube just below the side arm, so that the assembly can be placed in a suitable reservoir. Construction of the valves and stop mechanism is siniilar to that described by Human ( 2 ) . I n this form, the dispenser can he operated manually. If automatic operation is required, a short length of iron or mild steel iod to 5 / 8 inch in diameter is cemented to the top of the syringe plunger. A solenoid valve replacement coil of suitable dimensions is then mounted in such a way as t o lift the plunger and iron slug until they come in contact with the volume adjusting stop. The weight of the iron slug is sufficient to ensure that the syringe plunger returns to its seated position !\-hen the power is cut off from the solenoid coil. A 2-ml. hypodermic syringe is suitable for dispensing 1 ml. of the reagent, and a 10-ml. syringe for dispensing 5 ml. of the ethyl alcoholwater diluent. Both reagent and diluerit are added through glass capillary and polyethylene lines to the heating vessel shonii in Figure 1, iii. Heating to Develop Color. The glass vessel in n-hich t h e mixing v i t h reagent and heating are carried out is shoirn in Figure 1, iii.
It consists of a central tube j/g inch in outside diameter, to n hich two outer jackets are sealed. The bottom jachet extends below the constricted outlet, \vhicli is closed by a solenoid-operated valve. Boiling water is circulated t h o u g h the bottom jacket conveniently by means of the thermosiphon circulating device shown in Figure 5. The latter is fitted with a 250-watt inimersion element, the heat output of which is controlled by a Simmerstat snitch The outlet of the upper jacket of the heating vessel (Figure 1, iii), through which cold water is circulated. is connected to the constant-level n a t e r supply of the thermosiphon circulator. I n thi- way a constant flow of cold VOL. 30, NO. 6 , JUNE 1958
1045
water for both purposes is ensured. Precautions must be taken, so that the cold water supply does not slowly turn itself off. The authors have used a small header tank and ball-cock arrangement located a few feet above the equipment, so that low pressure water, controlled by a gas cock, is drawn at a constant rate through the water jacket and constant-level device. Between the cooling and heating jackets of the heating vessel (Figure 1, iii) are located the reagent inlet, diluent inlet, and overflow pipe. The first two are connected to the appropriate dispensers, the last, through rubber tubing, to a suitable drain. The central valve reaches to a point above the cooling jacket and has an iron slug sealed into the top. The solenoid coil which operates it slips over the glass tubing extension above the cooling jacket. The lower extension of the heating vessel is connected by plastic tubing to the diluting vessel below, so that no liquid can be lost by spilling or splashing. Heating and development of color proceed for a timed 13.5 to 14 minutes before the valve of the heating vessel is lifted and the contents are allowed to fall into the diluting vessel (Figure I , iv) below it. 4 t the same time the diluent dispenser is operated; this washes out the heating vessel. The valve then closes and the vessel is ready to receive the next effluent fraction. Dilution. The vessel in n-hich dilution and mixing are carried out (Figure 1, iv) is a glass tube 5/* inch in outside diameter, closed at its lower end by a metal-in-glass valve similar t o those described. It is surrounded by a 230-volt solenoid coil, H,similar to those used to operate,the valves of the heating vessel (solenoid G) above it and the reading cuvette (solenoid I ) below. These solenoids are wound on bobbins 2 inches high and 2 inches in diameter with a clearance hole b/g inch in diameter through the center. Cemented to their upper surface is an aluminum deflector plate to prevent liquid from soaking into the coil. As a n additional precaution the coils should be baked out and coated with an epoxy or similar resin, or vacuumimpregnated with paraffin wax. The upper part of the diluting vessel has an overflow tube on one side and a n air inlet on the other reaching to the bottom. It is joined to the heating vessel above by a plastic sleeve. The air inlet must be arranged so that it does not foul the operation of the outlet valve, but gives a fine stream of air bubbles which thoroughly aerate and mix the solutions. The air is conveniently pumped in by a small aquarium aerating pump. The bottom of the diluting T-essrl is connected by a plastic sleeve to the measuring cuvette. The actual quantity of diluent added depends upon the maximum absorbance expected; 5 ml. is a convenient volume. Spectrophotometric Estimation. After a timed 1 minute in t h e diluting vessel, t h e valve of this container is opened by t h e timing mechanism and the contents are allowed to fall into the
1046 *
ANALYTICAL CHEMISTRY
HIGH 5 T A B I L l T Y C A R B O N RLSlSTER
82 K
POTENTIOMETER
1oooJ-l W.W. TRMLMITTING PRECISION
POTWTlOMfTER
P~TLNTIOMETLR SWITCH
1.5" BRTTERY
TO COURTESY SlaRlST A N 0 WEISS. LTD
10MV
%'EEDOMAX
RECORDER
Figure 6. Arrangement of components in direct-reading Ultraphotometer
Figure 7. Circuit for connection of recorder to scale drum potentiometer
A.
High gain amplifier C1, Cz. Glass cells for standard and test solution D. Diophragm, automatically controlled b y scale drum F. Colored filter 1. Incandescent lomp (mains operated) M. Drive motor for scale drum and diaphragm D OM. Oscillating mirror (light modulator) PhA. Photoelectric cell for absorption measurements PhT. Photoelectric cell for nephelometric turbidity measurements R. Reflector S. Selector switch f o r measuring absorption or turbidity Sc. Scale drum for direct reading of p e r cent transparency o r turbidity
measuring cuvette. The dimensions and design of this vessel are shown in Figure l,-v. It is constructed in three parts: The bottom. of glass tubing. , - inch in outside diameter, is constricted a t its lower end and is fitted with a metal-in-glass valve which is operated by a 230-volt solenoid coil, I . The top of this section is flanged and ground to fit the center section. The latter is cut from 1 by 1 cm. square-bore glass tubing which has two opposite sides ground flat so that the faces are parallel and 1.2 cm. apart. The top section is fitted with an overflow tube and is flanged and ground to fit the center section. It is connected to the bottom of the diluting vessel by means of a plastic sleeve. The three sections are cemented together with Araldite or similar cement and the cuvette is set u p in the spectrophotometer so that the light beam passes centrally through the center section without being reflected from the sides in any way. The capacity of the cuvette should be such that the diluted solution reaches a level well above that a t which the light beam passes. K i t h the dimensions given in Figure 1, v, the final volume of 8 ml. reaches to within inch of the junction of the center and top sections. After the diluted solution has been held in the reading cuvette for 5 minutes for reading and recording, the timing mechanism opens the valve and discharges the contents to waste. I ,
PHoToarETm. Probably any simple colorimeter capable of being attached to a recording potentiometer could be used, if it had sufficient operational stability and gave a linear relationship between concentration and absorbance over the
range 0 to 1.0. However drift-free operation is exceedingly difficult to obtain 11-ith single-beam instruments, and where a single-point recorder is used there is no possibility of checking and recording drift if and when it occurs. For this reason the authors have used a self-compensating photometer (SigristPhotometer, Type UP2LD, manufactured by Sigrist and Jl'eiss, Ltd., Falkenstrasse 23, Zurich 1/8, Switzerland). The optical and compensating arrangements in this instrument are illustrated in Figure 6. The only modifications required have been removal of the turbidity photocell housing below the cuvette compartment and the direct coupling of a 1000-ohm wire-wound potentiometer to the main shaft of the scale drum. For ease of access the instrument was removed from its case and mounted in the framework, so that the amplifier could be separately enclosed and protected from spilt chemicals. The glass bottom of the cuvette compartment was removed and the cuvette vessel described was set u p in the light beam. A sealed cuvette containing distilled water was placed in the compensating beam and the compartment was enclosed above and below by lighttight metal shields. Recording a n d Graphical Plotting
of Results. The recorder should give a linear response over t h e range being studied, and should be stable and self-standardizing. A Leeds & Northrup Speedoniax Type G, Ilodel S, 60,000 single-point recorder has been found satisfactory for this purpose. It is connected to the scale drum potentiometer through the circuit shon-n in Figure 7 . The chart speed of the recorder is 4 inches per hour. At this speed, switching the recorder chart drive motor on for 2 minutes during each reading gives a horizontal trace about 1/8 inch long, n hich is con\-enicnt for subsequent reading. d complete acidic and neutral amino acid determination can thus be fitted into 15 inches of chart length, and a basic amino acid analysis into 5 inches. TIIIISG JIECHBNISM. -4s the coni-
Figure 8. Timing control mechanism Figure 9 . Complete equipment
I .DO1
.002
ffi *.AMINO NITROGEN
403
.m
L
.m
Figure 10. Relation of color to concentration for leucine, lysine, and aspartic acid plete sequence of events is controlled by this unit, care has gone into its design and construction t o ensure its complete reliability. Quick make and break action on the switched contacts is cssential, and has been achieved by a double cam system for each pair of contacts. These cams and their associated switch gear are arranged in pairs along two central shafts, each driven separately by a small synchronous clock motor. Motor 1 (Figure 3), rotating at 4 revolutions per hour, operates the switches controlling solenoid G of the heating vessel (Figure 1, iii), the diluent dispenser, solenoid H of the diluting vessel (Figure 1, iv), and the starting of the second motor. hlotor 2 (Figure 3), rotating at 6 revolutions per hour, operates the cams switching on the photometer motor, the recorder chart motor, and solenoid I of the reading cuvette (Figure 1, v). I n addition, each motor ha,s a self-locking switch (switches 1 and 6, Figure 3) arranged so that once started it completes one revolution before switching itself off. Esch motor then remains off until restarted by the closing of relay contacts B, (Figure 3) in the case of motor 1, or switch 4 (Figure 3) in the case of motor 2. Figure 8 shows a general view of the timing control mechanism, and Tahlc
I summarizes the switching sequence during one complete cycle of operations. Figure 9 shows the complete equipment. It occupies a floor space 18 inches by 4 feet and, excluding the chromatography column, stands ahout 4 feet high. Operation and Performance of Equipment and Calculation of Results. A single wall switch controls the equipment, except for the constant-temperature bath circulating water through t h e column jacket. rlfter allowing ahout hour for the thermosiphon heater and photometer amplifier t o reach operating temperature, the equipment is ready for use.
Table I. Switching Sequence for Timing Control Mechanism (Figures 1 and 3) Time, Min. Switching Event 0 Electrolytic contact made between electrodes C and D. Relay A closes. onerates solenoid F of iractibn dispenser, and reagent dispenser. Relay B closes and starts motor 1. 0 . 5 Snitch 1 closes to keep matar 1 running. Relay B opens. 13.5 Switch 2 operates solenoid G to lift valve of heating vessel. 13.5 Switch 3 operates diluent dispenm. 14.25 Switch 4 closes to start motor 2. ' 14.5 h i t c h 2 opens to drop valve of heating vessel. 14.5 Switch.6 closes to keep motor-2 running. 14.5 Switch 5 closes to energiae solenoid H and lift valve oi diluting "eSSd.
14.75 Switch 4 opens. 14.75 Snitch 5 opens t o drop valve of
diluting vessel. 15.00 Switch 1 opens t o stop motor 1. Section of timing control mechanism driven by motor 1 is now rpady to receive next effluent fraction from chrometography column. 20.00 Switch 7 starts Ultraphotometer motor. 20.35 Switch 7 opens to stop Ultraphctometer motor. 20.5 Switch 8 starts recorder. 22.5 Switch 8 opens to stop recorder. 2 3 . 0 Switch 9 energizes solenoid I to open valve of cuvette. 23.5 Switch 9 opens to drop valve of cuvdte. STANDARDIZATION OF EQUIPMENT. 24.25 Switch G opens to stop motor 2. The equipment is standardized by passing through it a series of standard leucine solutions. These solutions &re prepared by dissolving a known weight (20 to 25 mg.) of pure anhydrous 3) of the fraction dispenser. After 15 minutes the next aliquot is dispensed leucine in 1 liter of pH 5.0 citrate buffer into the top of the heating vessel while ( 5 ) , and diluting aliquots of this soluthe previous aliquot is read and retion 1 to 3, 1 to 1, and 3 to 1 with p H corded. 5.0 buffer, which also serves as a reagent blank. Two milliliters of each of these It is advisahlc to pipet out triplicates solutions is pipetted in turn directly at each strength-if., a total of 15 into the top of the heating vesscl. The readings for the initial Standardization machine is started manually by pressing of the equipment. Subsequent standthe starter button, thus short-circuiting terminals C and D (Figures 1, ii, and ardization, which should be carried VOL. 30, NO. 6, JUNE 1958
1047
out n i t h each fresh batch of reagent, need involve using only the blank and the half and full strength leucine solutions to ensure that the relationship remains constant. Similar experiments have also been carried out with standard solutions of lysine, histidine, arginine, alanine, aspartic acid, glutamic acid, glycine, isoleucine. leucine, methionine, phenylalanine, serine, threonine, tyrosine, and valine; Figure 10 shows the plot of amino acid concentration against color, obtained with this equipment, These and other experiments have shown that the photometer employed gives a n approximately linear relationship between absorbance and concentration over the range 0 to 1.3. CALCULATION OF RESULTS. Percentage transmittance figures are read directly from the recorder trace both for the standard and the unknon n peaks. These are converted into absorbance units by direct reference to a table constructed from the formula D1cm = 2 logloT where T is percentage transmittance (Table 11). From the mean absorbance readings for the full strength leucine standard and the blank, the amount of color due to the leucine can be obtained. JJ7hen this is divided by the amount of leucine nitrogen in the solution, a regression coefficient of color on nitrogen concentration is obtained. The absorbance readings for each peak are summed, the base line contribution (assessed from the base line before and after the peak) is subtracted, and the resulting figure is corrected for the color yield of the amino acid concerned to convert the color to “leucine equivalents.” Division of this figure by the leucine standard regression coefficient gives the amount of a-amino nitrogen in the peak. As can be seen from Figure 11. the base line shows little variation, and assessment of the blank value usually offers no difficulty. \There differences
Table II. Conver,sion of Per Cent %‘oT
1 2 3 4 5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
48 49 50
-
2 4 i 8 9 3 5 6 2I000 1.959 1.921 1,886 1,854 1 824 1 796 1.770 1,745 1.721 1.699 1,678 1.658 1.639 1.620 1 602 1 585 1.569 1.553 1.538 1.523 1.509 1.495 1.481 1.469 1 456 1 444 1.432 i 4io 1.40~ 1.397 1.387 1.377 1,367 1.356 1 347 1 337 1,328 1.319 1.310 1.301 1.292 1.284 1,276 1.268 1 260 1 252 1.244 1 237 1 229 1.222 1.215 1.208 1.201 1.194 1.187 1.180 1.174 1 167 1 161 1,155 1,149 1.143 1.137 1.131 1.125 1.119 1.114 1 108 1 103 1.097 1,092 1.086 1.081 1.076 1.071 1065 1.060 1 056 1 051 1.046 1.041 1.036 1.031 1.027 1.022 1.018 1.013 1 009 1 004 1.000 0.996 0,991 0,987 0.983 0.979 0.975 0,971 0 967 0 963 0,959 0.955 0.951 0,947 0.943 0.939 0.935 0.932 0 928 0 924 0.921 0.917 0.914 0.910 0,907 0.903 0.899 0.896 0 893 0 889 0.886 0,883 0,879 0,876 0 . 873 0.870 0.866 0,863 0 860 0 857 0.854 0.851 0.848 0.848 0.842 0.839 0 836 0,833 0 830 0 827 0,824 0.821 0.818 0.815 0.812 0.810 0 807 0.804 0 801 0 799 0 , 796 0.793 0 . 790 0.788 0.785 0 782 0 780 0 . 777 0.775 0.772 0 . 770 0.767 0.764 0.762 0,759 0 757 0 754 0,752 0.750 0.747 0.745 0.742 0.740 0.737 0 . 735 0 733 0 730 0,728 0.726 0 723 0.721 0.719 0.717 0.714 0.712 0 710 0 708 0,705 0.703 0.701 0,699 0,697 0.695 0.692 0,690 0 688 0 686 0.684 0.682 0.680 0.678 0.676 0.674 0.672 0,670 0 668 0 666 0.664 0.662 0.660 0.658 0.656 0,654 0.662 0,650 0 648 0 616 0,644 0.642 0.640 0.639 0,637 0.635 0.633 0.631 0 629 0 627 0.625 0.623 0.622 0,620 0.618 0.616 0.614 0.613 0 611 0 609 0,607 0.605 0.604 0,602 0.600 0,599 0,597 0.595 0 593 0 592 0,590 0.588 0 587 0,585 0.583 0.582 0.580 0,578 0 577 0 575 0.573 0.572 0.570 0.569 0.567 0.565 0,564 0.562 0 561 0 559 0,557 n ; 5 . ~ o 554 0 539 0.553 0.551 0.550 0,548 0.547 0 545 0 544 0.542 0 5,l 0.538 0,536 0,535 0.533 0.532 0 530 0 529 0,527 0 526 0 525 0.523 0.521 0.520 0.519 0,517 0.516 0.514 0.513 0 511 0 510 0.509 0.507 0,506 0.504 0.503 0.502 0.500 0.499 0 498 0 496 0.495 0.493 0.492 0.491 0.489 0 488 0 487 0.485 0 484 0 483 0.481 0,480 0.4i9 0,478 0,476 0 475 0 474 0.472 0 471 0 470 0.469 0.467 0.466 0,465 0.463 0.462 0.461 0.460 0 458 0 457 0.456 0.455 0.453 0.452 0.451 0 450 0 449 0.447 0 446 0 445 0,444 0.442 0.441 0.440 0.439 0.438 0.436 0,435 0 431 0 133 0.432 0.431 0.429 0.428 0.427 0.426 0.425 0.424 0 422 0 421 0.420 0.419 0.418 0.417 0.416 0.414 0.413 0.412 0 411 0 410 0.409 0.408 0,407 0.406 0.404 0.403 0.402 0.401 0 400 0 399 0,398 0.397 0.396 0.395 0.394 0.392 0.391 0.390 0 389 0 388 0.387 0.386 0.385 0.384 0.383 0.382 0.381 0.380 0.379 0.378 0.377 0.376 0.375 0.374 0.373 0.372 0.371 0.3iO 0.369 0.368 0,367 0.366 0.365 0.364 0,363 0.362 0.361 0,360 0.359 0,358 0.357 0.356 0.355 0.354 0.353 0.352 0.351 0.350 0.349 0.348 0,347 0.346 0.345 0.344 0.343 0.342 0.341 0.340 0.339 0.338 0.337 0.336 0.335 0.334 0.333 0.332 0.332 0.331 0.330 0.329 0.328 0,327 0,326 0,325 0.324 0.323 0.322 0.321 0.321 0.320 0.319 0.318 0.317 0.316 0.315 0.314 0.313 0,312 0.312 0.311 0.310 0.309 0.308 0.307 0.306 0.305 0 304 0.304 0.303 0.302 0,301 0.300 0.299 0.298 0.298 0.297 0.296 0.295 0.294 0.293 0
1
j
1
I
Figure 11.
Traces
150-LEU LEU
A. B.
1048
TYR
PHE
70-hour hydrolyzate, insulin, acidic a n d neutral amino acids eluted from 150-cm. column of Dowex 50 (passing 200 mesh) Synthetic mixture of lysine, histidine, ammonia, and arginine eluted from 15-cm. column of Amberlite IR 120 (passing 200 mesh) ANALYTICAL CHEMISTRY
.
Transmittance to Absorbance
%T 51 52 53 54 55
56 57 58
59 GO
61 62 (3i 64 G5
66 67
68 69 70 71
72 73 74 -I
0
76 77 78 79 80
81 82 83 84 85 86
87
88 89
90 ‘11
02 93
94 95
96 97 98 99
100
0
0.292 0,284 0,276 0,268 0.260 0.252 0.244 0.237 0.229 0.222 0.215 0.208 0.201 0.194 0.187 0.180 0.174 0.167 0.161 0,155
0.149 0.143 0.137 0.131 0.125 0,119 0.114 0.108 0.103 0,097 0.092 0.086 0.081 O.Oi6 0.071
0,065 0.060 0,055 0.051 0.046 0,041 0,036 0.032 0.027 0.022 0,018 0.013 0,009 0.004 0.000
1 0.292 0.283 0.275 0.267 0.269 0.251 0.243 0.236 0.228 0.221 0.214 0.207 0.200 0.193 0.186 0.180 0.173 0.167 0.160 0.154 0.148 0.142 0.136 0.130 0.121 0.119 0.113 0.107 0.102 0.096 0.091 0.086 0.080 0.075 0,070 0.065 0.060 0.055 0,050 0.045 0,040 0.036 0.031 0.026 0.022 0,017 0.013 0.008 0.004
2 0.291 0.282 0,274 0.266 0.258 0.250 0,213 0.235 0.228 0.220 0.213 0.206 0.199 0.192 0.186 0.179 0.173 0.166 0.160 0.154 0,147 0.141 0.135 0,130 0.124 0.118 0.112 0.10; 0.101 0.0‘36 0,090 0,085 0.080
0,075 0 .070
0,064 0,059 0,054 0,050 0,045 0,040
0.035 0.031 0.026 0.021 0.017 0.012 0.008
0.004
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 290 281 273 265 257 249 242 234 227 220 212 205 199 192
4
0.289 0.281 0.272 0,264 0.256 0.249 0.241 0.234 0.226 0.219 0.212 0.205 0.198
0,191
185 0.184
178 172 166 159 153 147 141 135 129 123 117 112 106 101 095
0.178 0.171 0.165 0.150 0,152
0.146 0.140 0,134 0.128 0.123 0.117 0.111 0.106 0,100
0.095 090 0.089
5 0.288 0.280 0,272 0.264 0.266 0,248 0.240 0,233 0.225 0.218 0.211 0.204 0,197 0,190 0.184 0.177 0.171 0.164 0.158 0.152 0.146 0.140 0.134 0.128 0,122 0.116 0.111 0.105 0.100 0,094 0.089
085 0.084 0 , 0 8 4 079 0,079 0,078 074 0 , 0 7 4 0,073 069 0.069 0.068 064 0.063 0.063 059 0.058 0.058 054 0.053 0.053 049 0.049 0.048 044 0.044 0.043 040 0.039 0.039 035 0.034 0.034 030 0.030 0.029 026 0.025 0.025 021 0.020 0.020 016 0.016 0,016 012 0.011 0.011 007 0.007 0.007 003 0.003 0.003
6 0,287 0,279 0.271 0.263 0.255 0,247 0.240 0.232 0.225 0.217 0.210 0.203 0,196 0,190 0.183 0.176 0.170 0.164 0.157 0.151 0,145 0.139 0.133 0.127 0.121 0.116 0.110 0.105 0.099
0.094
0,088
0.083
0.078
0,073 0.068 0.062 0,057
0.053 0.048
r
I
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
286 278 270 262 254 246 239 231 224 217 210 203 196 189 182 176 169 163 157 151 1.14 138 132 127 121 115 110 104 098 093
8 0,286 0,277 0.269 0.261 0,253 0.246 0,238 0.231 0.223 0.216 0.209 0 202 0 195 0.188
0.182 0,175 0,169 0,162 0.156 0.150 0.144 0.138 0.132 0.126 0.120 0.115 0.109 0.104 0.098
0,093
9 0.285 0,276 0.268 0,260 0.253 0.248 0.237 0.230 0,223 0,215 0,208 0.201 0.194 0.188 0.181 0.175 0.168 0.162 0.155 0.149 0.143 0.137 0.131 0.125 0.120 0.114 0.108 0.103 0.097 0.092
ACKNOWLEDGMENT
It is a pleasure to acknonledge the assistance rendered hy K. I. Wood, n h o designed and constructed the relay circuits controlling the fraction collector (Figure 2 ) and the timing mechanism (Figure 3 ) and improved the performance of the ultraphotonieter. Thanks are also clue to W. Sutherland, n-ho constructed the timing mechanisiii, and to R . J. Ronlands and Monica McShane for expert technical assistance and man\- ubeful wggestions. The author thanhi d . lIoore, Rockefeller Institute. Ken York, for supplying details of the rapid ion e\cliaiige procedure.
088 0.087 0,087
082 077 072 067 062 057 052 047
0.082 0.081 0 ,077 0.076 0.072 0.071 0.066 0.066 0.061 0.061 0,056 0.056 0.052 0.051 0,047 0.046 0.042 0,041 0.037 0,037 0.032 0.032 0,028 0.027 0.023 0.023 0.019 0.018 0.014 0.014
042 0.038 038 033 0,033 028 0.029 024 0.024 019 0.020 0.015 015 0.011 0 010 0.010 0,009 0.006 0 006 0,005 0.005 0 002 0 001 0,001 0.000 0.043
occur before and after a peak, the mean of sonie sis readings on each side is taken. Calibration of the equipment n ith synthetic mixtures of amino acids has giyen recoveries of 100 rt 3% (range) in the majority of cases. The use of this equipment for the analysis of protein hydrolysates and a coniparison of these results with those obtained manually ivill be reported in a subsequent paper.
LITERATURE CITED
(1) Chinard, F. P., J . B i d . Cheui. 199,
91 (1952). (2) Human, J. 1’. E Chern. cP: Ind. (London) 1955, 108. (3) Moore, S.,unpublished data. 141 ~, hfoore. S..Stein. TT’. H.. J . Bioi. Ch&. 176, 367 (1948). ’ (5) Zbid., 192, 663, (1951). (6) Zbid., 211, 893 (1954). (7) Zbid., p. 907. ( 8 ) Simmonds, D. H., unpublished data. ( 9 ) Himnionds. I). H.. Stell. I. G.. P/.oc. ~
Conf. Ausfrall‘a C l , 75 (195i). (10) Simmonds, D. H., Wood, K. I., . ~ \ . I L . CHEJI.26, 1860 (1954). ( l l i Spackman. D. H., Stein, IT. H., -lloore, ’S.!Federation Proc. 1 5 ; 358 (195G). RECLITEDfor revie!\* JUI? 8, 1957. Acrepted Januarv 13, 1958.
Partition Separation of Carotenoids by S iIica-Met hano I Columns ALBERT E. PURCELL
U. S. Fruit and
Vegefable Products laborafory, U. S. Department of Agricuhure, Weslaco, Tex.
b A method for partition chromatography of carotenoids is described. Petroleum solutions of carotenoids are passed through silica gel columns saturated with methanol. Three fractions can b e obtained. The first fraction, which contains carotene hydrocarbons, is removed from the column with petroleum. The second fraction
containing the monohydroxy carotenoids is removed with petroleum-ethyl ether, and the third fraction containing polyhydroxy carotenoids is removed with methanol.
I
K STUDPINQ the caroteiioid pigments
of plant tissue, it is difficult to iso-
late aiici identify minor pigments. In usual chromatographic procedures they are often lost on columns sufficiently large to separate the major pigments. The separation of carotenoids by adsorption chromatography is simplified if they are first separated into classes according to their solubility in various solvents. Partition separation betn.een VOL. 30, NO. 6, JUNE 1958
1049
