River King, which contains 3.30% sulfur. The G P C elution curve indicates that this coal tar contains a high percentage of aromatic and tar acid compounds and a relatively low percentage of aliphatic hydrocarbons. Coal tar from a subbituminous coal, Big Horn, is little different from coal tar from Spring Canyon coal. However, this low rank coal (17.2 O,), when carbonized, did produce more water and somewhat more acids than did the Spring Canyon coal. The three low temperature coal tars studied show very little weight per cent in fractions numbered less than 50. This is O a n indication that aliphatic hydrocarbons of length C ~ and higher are not produced in the low temperature carbonization process a t 550 “C. However, the GPC curve from a pyrolysis coal tar (FMC), also shown in Figure 9, does show a significant weight per cent in fractions numbered less than 50. This curve indicates a predominantly aliphatic content rather than aromatic or tar acid composition. This is due to the fact that this material was pyrolyzed at a temperature of 870 “C. Shale oil distillates, petroleum crude oil, and benzene extracts from a tar sand sample, Figure 10, show high percentages of aliphatic hydrocarbons. The increased proportion of high carbon number paraffins can be seen in the higher weight per cent in fractions numbered less than 50. Using GPC, it is possible to separate coal tar products into aliphatic and aromatic fractions by molecular size and shape. Tar acids are separated into a third fraction by chemical affinity with the polar Sephadex column support material. This is in strong contrast to previous observations made with
styrene-divinylbenzene (Styragel) column support gels with tetrahydrofuran as the solvent (7). For instance, phenol elutes at about the same fraction as n-octane with Styragel but follows benzene with Sephadex. This fact indicates that a judicious choice of column support gel will yield a strictly size (molar volume) separation or a separation by size tempered by a n affinity of some components for the support material. The separation of coal tar samples into aliphatic, aromatic, and tar acid fractions is as precise and accurate as the standard chemical methods which are used for coal tar analysis (8, 12, 13). The GPC procedure using Sephadex as a column support material is especially interesting because it is capable of making separations of high boiling, high molecular weight coal tar and related products at room temperature. The problems of thermal cracking and polymerization when distillation is used for fractionation are minimized. High boiling pitches and tars that cannot be analyzed with GC, mass spectrometry, the ASTM FIA, or the USBM chemical extraction methods can be fractionated with this procedure.
RECEIVED for review February 12, 1969. Accepted April 21, 1969. Research sponsored by the U. S. Office of Coal Research and the University of Utah. (13) “Standard Methods for Testing Tar and its Products,” 5th ed., W. Haffer & Sons, Ltd., Cambridge, England, 1962.
An Automatic Sample Loader for Column Chromatography A. R. Thomson and J. W. Eveleigh’ Wantage Research Laboratory (A.E.R.E.), Wantage, Berks, England The apparatus to be described enables a number of samples to be loaded automatically at preselected times, on a single chromatographic column. The samples to be analyzed--e.g., protein hydrolysatesare first adsorbed on short tubes filled with a suitable adsorbent. These tubes are introduced in turn between the chromatographic column and the eluant feed. The device is generally applicable to analytical systems based on column separation. In conjunction with a suitable valve system, cycles of sample loading, analysis, and column regeneration can be carried out completely automatically.
RECENTADVANCES in the technology of ion exchange chromatography, particularly the use of small particle size spherical resins (13-22 p) and increased flow rates ( I ) , have led t o greatly increased sensitivity and to dramatic reductions in the time required for analysis-e.g., for amino acids from 24 hours to 2 hours (2). Also, automatic data handling systems described recently, greatly reduce the time required for evaluating analyses and give increased precision (3-6). The full potential of such automated equipment, can only be realized, however, if they are in continuous use for 24 hours a day, 7 days a week. This can only be achieved by the use of multicolumn assemblies which are very expensive, or by automatic sample loading. This paper describes a device 1 Present address, Molecular Anatomy Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
which loads samples automatically, in turn, on a single separation column (7, 8). It requires only minor alterations to existing equipment such as the amino acid analyzer on which it has been tested. Its performance has been assessed by comparison with the conventional manual loading procedure. EXPERIMENTAL
Sample Loader. The loader, which in the present design can accommodate twelve samples, is shown in detail in Figure 1. Samples for analysis are adsorbed on a suitable material-e.g., ion-exchange resin-contained in small tubes. (1) P. B. Hamilton in “Advances in Chromatography” Vol. 11, J. C. Giddings and R. A. Keller, Eds., Marcel Dekker Inc., New York. N. Y., 1966, pp 3-62. (2) R. W. Hubbard, Biochem. Biophys. Res. Commuti., 19, 679 ( 1965).
(3) A. Yonda, D. L. Filmer, H. Pate, N. Alonzo, and C. H. W. Hirs, Atral. Biochem., 10, 53 (1965). (4) M. I. Krichevsky, J. Schwarz, and M. Mage, ibid., 12, 94
(1965).
(5) R. A. Evans, A. J. Thomas, R. F. E. Axford, J. A. de S. Siriwardene, and A. J. Robins, Proceedings, 5th International Symposium on Automation in Analytical Chemistry, London,
October 1966. (6) A. R. Thomson, D. G. Gibbons, J. W. Eveleigh, and B. J. Miles, Biochem. J., 109, 8P (1968). (7) J. W. Eveleigh and A. R. Thomson, ibid., 99,49P (1966). (8) A. R. Thomson, 5th Colloquium on Amino Acid Analysis, Technicon Monograph 2 , 116 (1967). VOL. 41, NO. 8,JULY 1969
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Figure 2.
Figure 1. Sample loader These tubes are introduced, automatically and in turn, into the fluid line between the column feed and the analytical column, and each sample is loaded and analyzed in turn, Both the sample adsorbent and the main column are regenerated between each analysis. The solutions to be analyzed are pipetted onto adsorbent contained in Teflon (DuPont) tubes (3.8 X 1 cm) fitted with porous disks (Figure 1). Excess fluid is removed by suction and an appropriate solution is then used to ensure quantitative transfer of the samples. The tubes fit snugly into holes in the perspex block (Figure 1) and are fitted with 0 rings which are flanged at the top to ensure their retention in the block. The perspex sample block slides into the main body of the loader (Figure l), while the base of the block rests on two guide rails which are bolted to the holder. The bolts are fitted with rubber bushes t o provide some resilience and to assist in forming pressure tight seals. Pressure tight connections are made at each end of the sample tube. The upper connector, which is mounted in a stirrup (Figure l), has a ‘/16”-diameter connection from its edge to the center of its lower face. It has a small deformable plastic disk at the center of its upper face and a n 0 ring set in its lower face. It is connected to the column pump by 1/16”-i.d. stainless steel tubing and high pressure Teflon (DuPont) tubing. A spring, incorporated in the stirrup mounting, lifts the connector clear of the perspex block when the latter is being moved to the next position. The lower connector is a block of stainless steel with a 1/16’’-diameterhole through its center and an 0 ring set in its upper surface. This connector is attached t o a 1/16”-i.d. stainless-steel tube, the other end of which connects with the chromatographic column. The Geneva wheel system and cam (Figure 1) clamp the sample tube and con-
Table I. Conditions for Elution All buffers are 0.5N with respect to citrate, 0.2N with respect to Na+, and contain thiodiglycol (0.5% v/v), and Brij 35 (0.3% v/v); pH 9.6 buffer is prepared as indicated in the text
PH
Time (min)
3.0 10 % ethanol 3,50 4.10 9.6 0 . 2 N NaOH 3 .O (equilibration)
0-80 80-160 161-241 24 1 4 0 0 401-420 421-480
+
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ANALYTICAL CHEMISTRY
Circuit diagram of control unit
Relays R1 and R2 - G.P.O. Type K3000,50V, 2000 Q Time delay relay (T.D.R.) - A.E.I. type TDF Motor (of loader) Drayton type RQR 1rpm Pump (column buffer pump) - Milton Roy Mini-Pump CH 1-B-36P 0-153 mlihour nectors together forming a pressure tight seal. Each sample is brought on line in turn by means of the motor and cam assembly. All parts of the loader which are likely to come in contact with column solutions are constructed of stainless steel, and Teflon (Du Pont) is preferred to nylon for the sample tubes because nylon tends to swell in aqueous solutions. Eluant Selector Valve. A multichannel valve (9) is used t o program the repeating cycle of eluant changes to the chromatographic column. It also enables regeneration of the column and flushing of the analytical system to be carried out automatically. Control Unit. The loader and valve are controlled by microswitches operating cia relays, through the circuit in Figure 2. Time pulses are generated by a simple programmer consisting of a disk driven by a synchronous motor. Removable pins a t the periphery of the disk actuate the microswitches. Because the position of the cam relative to the pegs on the Geneva wheel is adjustable, the timing can be arranged so that: (i) the column pump is switched off and the pressure falls to atmospheric, (ii) after a delay of about 1 minute (caused by the time-delay relay, Figure 2 ) the loader motor is switched on, and movement of the cam releases mechanical pressure on the top connector, (iii) the sample block is moved to the next position by the pegs on the Geneva wheel, (iv) continued rotation of the motor brings the cam firmly down on the block and sample tube, ensuring that pressure tight seals are formed, and (v) one of the pegs actuates the microswitch so that the loader motor is switched off, the electrical circuit is reset, the column pump is switched on, and the analysis commences. Analytical System. The automatic loader was tested using a single column Technicon Amino Acid Analyzer. The chromatographic column (75 X 0.636 cm) was filled with Zeo-Karb 225, 8 % cross-linked (nominal), particle size 16 + 2 p [separated by the hydraulic method of Hamilton (IO)], and was thermostated at 60 “C. It is essential to keep the analytical column topped up with resin because any dead space at the top of the column may lead to loss of resolution. The sample tubes were filled with ion exchange resin (Chromobeads A) equilibrated with p H 2.2 sodium citrate buffer. Samples for analysis were adjusted to p H 2.2 or dissolved in 0.1NHC1 beforeloading. The analytical system was standard. Nitrogen was deoxygenated by passage through a solution of 1 vanadyl sulfate in 5 % H2S04 over amalgamated zinc. (9) J. W. Eveleigh and A. R. Thomson, Biochem. J . 99,49P (1966). (10) P. B. Hamilton and R. A. Anderson, ANAL.CHEM., 31, 1504 (1959).
lil
i
l
t
P
I$
1
*:
Figure 3. Chromatogram of standard amino acid mixture containing 0.1 pmole of each amino acid (Tryp Amm = ammonia). The 440-mp record for proline is also shown
1031-.-C O W L CLOCK
u I
,
:
C - - . _
I
__._.._
-FLUID CWES -----ELECTRICAL CONNECTIONS
L-i
VALVE
Figure 4. Block diagram of amino acid analysis system incorporating the automatic sample loader (not to scale)
The amino acids were eluted by step-wise changes of buffer (Table I) to eliminate possible errors due to diffusion of buffers in the Autograds. A typical chromatogram is shown in Figure 3. All buffers were 0.05N with respect to citrate and0.2Nwith respect to Na+. The pH 9.6 buffer was prepared from the stock concentrated Na citrate as follows: An appropriate volume was adjusted to pH 7.0 with 6 N HCI, the pH was then raised to 9.6 by the addition of saturated Na2C03 solution. Buffer flow rate was 0.7 ml/min, operating pressure 200-300 psi and the total time required for analysis was 8 hours. A block diagram of the complete apparatus is shown in Figure 4. RESULTS
Reproducibility. To test whether the automatic sample loader significantly affected the reproducibility of the analyzer, the results of analyses after manual and automatic loading of samples were compared. The results are shown in Table 11. There are distinct differences in the C,values, but the sample loader does not introduce serious systematic errors in the determinations, and recoveries are the same with both methods. There are no trends with successive analyses, even for methionine, caused by the drying out of the sample resin which sometimes occurred. Indeed in one experiment, there was no detectable loss of methionine three days after loading on the sample resin, (Table 111).
=
tryptophane
Table 11. Reproducibility of Analysis, Manual and Automatic Methods Compared For conditions see text. Column loading was 0.25 fimole of each amino acid, (the number of analyses is indicated in parentheses) ClJa
Amino acid Asp Thr Ser Glu Pro Gb Ala Val Met
Manual (4) Automaticm 3.27 3.48 2.54 6.05 3.78 3.74 5.08 4.63 8.75 4.39 7.22 4.36 4.32 4.55 3.95 4.19 5.33 6.54 4.22 2.46 Is0 5.87 5.67 Leu 5.40 5.97 TYr Phe 7.13 3.59 1.94 3.56 LYS 4.15 4.25 His 4.93 7.26 '4% 100 X standard deviation C, coefficient of variation = Mean Table 111. Stability of Amino Acids after Loading Aliquots of the standard were loaded on sample resin at the same time, but were analyzed consecutively. Duration of each analysis was 12 hours Area as of mean area Asp Glu Met TYr Analysis No. 1 104 102 102 96 2 104 98 99 102 3 98 100 98 101 4 99 99 100 103 5 98 100 100 94 6 97 97 100 96
Resolution. Because the sample resin is connected via a tube, admittedly of narrow bore, to the analytical column, some widening of the amino acid peaks might be anticipated. There are differences in peak widths but these amount at a maximum to about 10% except for the early peaks in the chromatogram which are sharper. The broadening is in part at least, due to, the small dead space left at the top of the column during the reproducibility tests to allow manual VOL. 41, NO. 8,JULY 1969
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loading of samples while the column length was kept constant. That this had no significant effect on reproducibility can be seen from Table 11. Reliability. During two years of continuous use, the loader has been operated regularly at pressures of up to 700 psi. No major mechanical breakdowns have occurred, and no significant modifications in the design have been necessary. DISCUSSION
The sample loader described necessitates only minor alterations to existing equipment, and the stepwise elution system used obviates the use of Autograds, an obvious advantage for rapid analyses. Loading of the samples to be analyzed on the sample resin takes only a few minutes and, with care, several samples can be loaded simultaneously. The time required for loading therefore is approximately 30 minutes a day. Because all other functions such as regeneration and reequilibration of the column, buffer changes, and sample loading are automatic, little time apart from that needed for loading is involved in running the apparatus. In the apparatus some drying out of the sample resin occurs, by evaporation rather than by drainage, but this does not lead to reduced recoveries for the amino acids most prone to oxidation--e.g., methionine (Table 111). Evaporation can be prevented for use with other analyzers such as that of Spackman, Stein and Moore, ( I I ) , where air must be excluded from the analytical system. However, this was not necessary with the Technicon analyzer because degassing of buffers is not required and gas segmentation is used in the analytical system. Coolant could be circulated through channels in the perspex block (Figure 1) if necessary. The design described accommodates twelve samples but is capable of considerable further development-e.g., the use of a circular sample block would have advantages. We have, however, used the simpler linear design. (Equipment with a 40-sample capacity based on the principle described here is available from the Technicon Chromatography Corporation, Ardsley Park, New York, N. Y.) Further, by using two identical columns, automatic replenishment of sample cartridges, and reciprocating drive, an indefinite number of sequential analyses can be envisaged. Some commercial Amino Acid Analyzers are based on a two-column system (11). The sample loader described here could in principle be used with this system either by having one for each column, or by having a double loader with two sample blocks in parallel with the appropriate eluate being diverted automatically to the analytical system. Recently, three alternative methods of fully automatic sample loading have been described. Two of these depend on the use of Teflon (Du Pont) rotary selector valves with the sample being held, in solution in narrow bore tubing, between two such valves (12), or between the inlet and outlet ports of one valve (13). The former device can operate at pressures up to 500 psi but for pressures greater than 120 psi
(11) D. H. Soackman, W. H. Stein, and S. Moore. ANAL.CHEM.. 30, 1185 (1'958). (12) K. Dus. S. Lindroth. R. Pabst. and R. M. Smith. A m / . Bio. &em., 18,'532 (1967). ' (13) A. L. Murdock, K. L. Grist, and C . H. W. Hirs, Arch. Biocheni. Biophys., 114, 375 (1966).
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ANALYTICAL CHEMISTRY
the latter, described by Murdock, Grist, and Hirs (13), must be placed at the low pressure side of the column pump. We have no experience with this type of valve but feel that the multiport versions required for automatic loading may present fabrication difficulties for the normal laboratory workshop, and because of the known tendency of Teflon to creep under pressure, there will be a recurring need to regrind the valves to keep them leak-free. Frequent making and breaking of tube couplings is also necessary to introduce samples into the sample tubes. In our system there have been no difficulties due to fluid leakage. We have also avoided the problem of changing samples in a system under pressure by allowing the pressure to fall to atmospheric uia the analytical system before sample change begins. No backflow of resin takes place from the column into the sample loader, and because the whole operation is automatic, there is minimal interference with the analysis. With this system, repouring of the column is seldom necessary and we have not observed either significant changes in resolution with time, or appreciable settling of the resin bed. This is perhaps partly due to the longer time taken for each analysis with our system [8 hours cf, 4 hours with the Dus system (12)], but more probably to the fact that after pouring, the column is packed at approximately twice the flow rate used for analysis and the ionic strength of the eluting buffers does not change significantly throughout the run. In addition, after each analysis, the column is regenerated with 0.2N NaOH. Most recently, a method of automatic loading has been described by Dymond (14). In this, the samples are presented by an automatic dipping device (similar in principle, to the normal Technicon Sampler), to a separate high pressure pump which is used to pump them onto the analytical column. The problems this equipment was designed to overcome are not experienced with the loader described in the present paper. The principle on which the present equipment is based is applicable to any analysis where the sample of interest can be held reversibly on a supporting material and can be eluted under conditions compatible with a subsequent separation process. Thus, the loader can be used for most ion-exchange analyses (including those employing ion-exchange celluloses) both organic and inorganic. It may also be used for gel filtration provided the samples to be analyzed can be reversibly adsorbed on some supporting material-e.g., ion exchange cellulose-from which they can be eluted under conditions (such as increased ionic strength) which do not interfere with subsequent gel filtration. Finally, a modified form of this type of device might well be of great value for automatic loading of samples on gas chromatographs. ACKNOWLEDGMENT
We acknowledge the technical assistance of S. G. Milsom, also helpful advice on design aspects from J. Grant and G. Shepherd, and expert workmanship of H. Rolls in the manufacture of the sample loader. Our gratitude is also due to Dr. H. Holy of Technicon Instrument Company, Ltd., Chertsey, Surrey, for the loan of equipment. RECEIVED for review October 28, 1968. Accepted January 21, 1969. (14) B. Dymond, ANAL.CHEM., 40, 919 (1968).
