Accordingly, a 3.5-meter column of ’/?-inch aluminuni tubing nas filled with a packing prepared using a ratio of 10 grams of diphenyl phthalate t o 100 grams of solid. This column was connected in a series to 3.5 meters of l/r-inch aluminum tubing filled with a packing prepared using a ratio of 10 grams of NTE t o 100 grams of solid. The ratio of 10 grams of substrate t o 100 grams of solid for each column was chosen t o give sharper peaks than those obtained for columns using a greater substrate-solid ratio. The columns were maintained a t 130’ i 0.5’ C. and a flow rate of helium of 90 ml. per minute was used. Under these conditions, the 14 components of the text mixture were completely resolved, as shown in Figure 4. The series arrangement of columns had a n efficiency of 1890 theoretical plates measured on the 2,6-tiimethylpyridine peak.
ACKNOWLEDGMENT
Thanks are extended to the American Petroleum Institute Research Project 52 on the h’itrogen Constituents of Petroleum for supplying several of the pyridines. The authors are indebted to R. C. Petterson and Lawrence A. Gemel, Purex Corp., South Gate, Calif., who performed many analyses on the new solid support and provided much useful information. LITERATURE CITED
(1) Brooks, V. T., Collins, G. A , Chem.
& Ind. (London) 38, 1021 (1956). (2) Desty, D. H., “Vapour Phase Chromatography,” pp. xi-xiii, Academic Press, New York, 1957. (3) Desty, D. H., Harbourn, C. L. A , , ASAL. CHEW31, 1965 (1959). (4) Epton, S. R., Trans. Faraday SOC. 44,226 (1948).
( 5 ) Haines, W. E., Helm, R. V., Bailey,
C. W., Ball, J. S., J. Phys. Chem. 58, 270 (1954). (6) . . James, A. T., ANAL. CHEM.28, 1564 (1958). (7) \ , James. A. T.. Biochem. J . 52. 242 (1952). (8) James, A. T., Martin, A. J. P., Analyst 77, 915 (1952). (9) James, A. T., Martin, A. J. P., Biochem. J . 50, 679 (1950). (10) James, A. T., Martin, A. J. P., Smith, G. H., Ibid., 52, 238 (195’2). (11) Murray, W. J., Williams, A. F., Chem. & Ind. (London).38, 1020 (1956). (12) Stupel, H., “Synthetische Wasch- und Reinigungsmittel,” p. 278, Konradin Verlag, Robert Kohlhammer G. M. b. H., Stuttgart, 1954. RECEIVED for review January 27, 1959. Accepted October. 26, 1959. Division of Analytical Chemistry, 134th Meeting, dC9, Chicago, Ill., September 1958. Work done under a cooperative agreement bet-xeen the University of Wyoming and the U. S. Department of the Interior, Bureau of Mines.
Factors Affecting the Use of Gas-Liquid Chromatography for the Separation of Large Samples Sample Inlet System, Distribution Coefficient of Solute, and Amount of Liquid in Stationary Phase W. J. d e WET,’ and VICTOR PRETORIUS Departments o f Agriculfure and Physical Chemistry, Universify o f Preforia, Pretoria, South Africa
b Column efficiency decreases as the sample volume i s increased. The effects of a number of factors on this decrease have been studied both theoretically and experimentally. Highest column efficiencies are obtained for large samples b y introducing the sample in the form of a concentrated plug, by choosing a stationary phase in which the distribution coefficient of the solute i s small, and by using relatively large amounts of liquid in the stationary phase.
G
chromatography has established itself as a powerful technique for the separation of small (milligram) amounts of volatile chemical compounds. The extension of the technique to handle larger (gram) quantities of material offers a number of attractive possibilities. The conipoAS-LIQUID
1 Present address, Central Tobacco Research Station, Department of Igriculture, Kroondal, South Africn.
nents of n mixture could be isolated in sufficient quantitics to enable auxiliary methods of analysis t o be used to identify each component. Pure samples of volatile compounds which are required for numerous physicochemical studies could be obtained a t relatively low cost from impure mixtures. Although large-scale chromatography is being used at present ( 1 , 2, I O ) , no systematic study of the optimum conditions under which such columns should be operated appears to have been undertzken. This is the purpose of the present investigation. APPARATUS
A schematic diagram of the apparatus is shown in Figures 1 and 2. Columns mere constructed from lengths of straight glass tubing and were packed with a mixture of Celite 545 and dibutylphthalate (30% of the liquid phase) to a density of 0.53 gram per nil., unless otherwise stated. The packing was held in position by plugs of glass ~ 0 0 1 ,J , one of which, J1, served to promote rapid evaporation of the liquid samples. The columns were supported
on pressed asbestos rir!gs, K , in a length of glass tubing (75-mni. inner diameter) around which two sections of resistance wire were wound-one serving to heat the packed portion of the column and the other being used as a preheater a t the column inlet. The temperature of each heated section could be controlled independently and measured on thermometers, I . The flow rate of the nitrogen carrier gas n a s measured on a rotameter, A . X manometer (not shown) was used t o measure the gas pressure a t the column inlet. After passing through a n electrically heated furnace, F , packed n.ith granular copper oxide to remove traces of oxygen, the nitrogen was preheated to a suitable temperature in the electrical heater, B. All heated portions of the apparatus were lagged with asbestos, D , to minimize heat loss. Liquid samples were placed in a vessel, H, and were srrept into the column preheater by bypassing the nitrogen through the stopcock, G. The nonreturn valve, Ez, consisted of a small metal ball fitted onto a ground seat and prevented the escape of carrier gas while H was open. Because of the rapid evaporation of the sample, it vias VOL. 32, NO. 2, FEBRUARY 1960
169
necessary to place a nonreturn valve, El, a t the column inlet so that the sample could not be forced back into the carrier gas preheater. A small portion of the gas eluted from the column was passed through one arm of a katharometer, &, details of which are shown in Figure 2. The amount of gas bypassed through the katharometer was controlled by a needle valve, AT, and was measured on a bubble-type flowmeter, R. The katharometer formed two arms of a Wheatstone bridge, the out-of-balance potential of which was fed directly onto a recording potentiometer with 5-mv. full-scale deflection.
number of theoretical plates for infinitely small samples u = the effective plate volume A similar equation derived by Porter, Deal, and Stross (7)is: no = the
where
Figure 2. Katharometer A. Tungsten wire 6. Silver solder C. Platinum wire (0.1 -mm. diameter)
and
xi = x,+
RESULTS AND DISCUSSION
Equations relating the column efficiency to the sample volume may be derived as follows. Consider the simple case where the sample is introduced into the column as an undiluted plug. The equation of the elution curve is given (9) by:
-
V,
By using Equation 1 or 2, elution curves may be drawn for various values of V a and the corresponding column efficiency may be calculated from these curws by means of the following equa: tion (5,Q)
may be obtained from the theory of van Deemter et al. (9) as vRI =
V'Rf f
va
(4)
where = limiting
final retention volume for infinitely small samples
From Equations 3 and 4,
_where n
= effective number of theoretical
liR/ =
TI'
where
Cl,,, = concentration of the solute in the mobile phase of the last plate C, = initial solute concentration of the plug when entering the column S = carrier gas volume parameter V8 = volume of undiluted vaporized sample 170
ANALYTICAL CHEMISTRY
=
plates final retention volume peak width equivalent volume (distance between intersection points of tangents drawn through inflection points of elution curve and base line, corrected to a n equivalent eluent gas volume)
A more direct relation between column efficiency and sample volume
Furthermore (9), VOR,
and
nDv=
','z
V, f
'/2
W
(6)
v,+ V I . K
where u
= effective plate volume
V o = volume of mobile phase per theoretical plate phase per theoretical plate K = distribution coefficient of the solute 1'1
= volume of liquid
Table I.
Influence of Sample Volume on Column Efficiency Calculated from Equations 8, 9, and 10
Sample Volume, Ml. Liquid Vapor 2 3 5 7 15
372 558 930 1,302 1,860 2,790
2 3 5 7 10 15
744 1,116 1,860 2.604 3 1720 5,580
10
TIME(min.)
2.5 3.5 5.0 7.5
Figure 3. Experimental elution curves for various volumes of toluene Liquid toluene a. 2 ml. b. 3 ml. C. 5 ml. d. 7 mi. Column diameter, 38.2 mm.; column length, 2 3 0 cm.; column temperature, 9OoC.; preheoter temperature, 130OC.; linear gas velocity at column outlet, 2.5 cm./second; column packing, Celite and dibutyl phthalate 130% b y weight of liquid)
=
5,500 5 9,300 7 13,020 Height equivalent per 3
concentration of solute in gas per concentration of solute in liquid
From Equations 5 and 6, n =
(T-) 4 + 2V, not)
(7)
where u, = -ItV d n ,
The parameter ZL: is related to the sample volume by Equation 1: WE--"
5.'
VdZ
+
where 8 is giwn by : 6e - ' / t
2,325 3,255 4,650 6,975
-
'
21
I
s
d/no
Dilution Factor K , = 1 0.119 4.00 12,536 4.00 12,536 0.179 0.296 4.01 12,567 4.03 12; 630 0.415 4.05 12,693 0,593 0.890 4.15 13,006 Dilution Factor K , = 2 0,237 4.01 12,567 4.01 12,567 0,356 4.03 12 630 0.593 0.831 4.11 12;881 1.186 4.18 13,100 4.64 14,542 1.780 Dilution Factor K , = 5 0,7417 4.01 12,849 1,0380 4.15 13,006 1.4830 4.38 13,727 2,2250 4.90 15,357 Dilution Factor K , = 10 1. 780 4.54 14,228 2.9176 5.50 17 ,237 4.153 6.68 20,935 theoretical plate.
impossible to avoid a certain amount of mixing of the sample with the carrier gas. If the amount of mixing remains constant while the sample enters the column or if no further dilution takes place, T', may be replaced by Ti,' where V,' = K,V. and K , is a dilution factor represrnting the degree of dilution of the sample. If the introduction of sample into the column is accompanied by turbulent mixing with the carrier gas, the concentration of the sample will decrease as the sample enters the column and Equations 8, 9, and 10 will no longer hold. Porter, Deal, and Stross ( 7 ) have shown that if the decrease in the sample concentration is assumed to be exponential-Le.,
where
C(S) = instant concentration of solute entering the column after a volume of carrier gas S has passed through the column V,' = K,V, = initial simple volume before exponential dilution takes place then, Cl.n = G O
The sample volume, V,, in the above equations refers to an undiluted plug. I n practice, however, it is virtually
J;
W
W
e-x2dx
(11)
n
H.E.T.P.,a Cm.
383 383 383 383 382 372
0.600 0,600 0.600 0.600 0.600 0.618
383 383 383 378 372
311
0,600 0.600 0.600 0.608 0.618 0,739
379 375 344 285
0.608 0.613 0.668 0.807
305 234 168
0.754 0.980 1.369
where
x, = and
The derivation of equations analogous to Equations 8, 9, and 10 is excessively difficult for exponential sample injection. It is much simpler to draw elution curves for various values of V,' from Equation 11 and to calculate the corresponding column efficiencies from these curves by means of Equation 3. Column efficiencies calculated in this manner will only be approldmately correct, Equation 3 is strictly valid for plug sample injection only. The accuracy of Equations 1 to 3 and 8 to 10, and the relative merits of plug and exponential sample injection for the separation of large samples have been tested by the folloFving experiments. Various volumes of an equimolar mixture of benzene and toluene were separated on the apparatus described above. The column parameters -Le., column temperature, linear carrier gas velocity, and packing density-were adjusted to give optimum efficiency for small samples. The experimental conditions and elution curves obtained for toluene are given in Figure 3. The column efficiency in each case was calculated from the elution curves by means of Equation 3. The theoretical elution curves for VOL. 32, NO. 2, FEBRUARY 1960
171
plug and exponential sample injection have been calculated from Equations 2 and 11 for various values of K,. Typical examples of these elution curves are shown in Figures 4 and 5 and the corresponding column efficiencies cslculated from these curves by means of Equation 3 are graphically represented in Figure 6. The column efficiencies were slso calculated from Equations 8, 9, and 10 for plug injection (diluted and undiluted) and these values rre given in Table I, from which the corresponding curves in Figure 6 have been drawn. For the purpose of these calculations the experimentally determined value no = 383 was used. The limiting retention volume, Vi, by V i = 61,340 ml., was determined from the experimentzl elution curves ( 7 ) . The liquid volume of the sample was converted to the vaporized volume a t the pressure End temperature a t the column inlet bj. the v m der Waals equation. The curves in Figure 6 show that higher column efficiencies may be obtiined for large samples by introducing the sample in as concentrated a form as is possible. Although both exponential and plug injection methods appear to be almost equally efficient for undiluted samples-Le., for K , = 1-this is not entirely true, as in the calculation of the column efficiency from the elution curves the tailing effect in the elution curves obtained for exponential injection (Figure 5 ) is ignored. Consequently. plug injection is more efficient for large samples than is exponential injection. Several methods of introducing the sample in the form of a concentrated plug have been investigated. The technique described above, by which the sample is smoothly and rapidly vaporized on a large surface of hot glass wool, has given the best results. The technique differs from the commonly used flash heater ( 2 ) in that nonreturn valves have been incorporated to confine the sample to the column. I n this way the vapor pressure of the sample itself is used to force the solute into the column and dilution of the sample with the carrier gas is greatly reduced. By comparing the experimental elution curves in Figure 3 with the calculated curves for plug and exponential sample injection (Figures 4 and 5 ) , it is evident that the present method does indeed closely approximate diluted plug injection. For maximum efficiency the temperature of the preheater must be set a t a value somewhat higher than the optimum column temperature (Figure 7 ) . In the present experiments the optimum preheater temperature has been found t o be roughly independent of the sample volume, provided the column is not too h e a d y overloaded or a suitably large
172
ANALYTICAL CHEMISTRY
TIM E
Figure 4. Typical elution curves calculated for plug injection according to Equation 2 with K, = 10 Liquid toluene a.
2 mi.
c.
b.
3 ml.
d.
column is used for large samples (Figure S). Under these conditions the sample injection closely approximates a diluted plug n i t h K , = 7 (Figure 6). The d u e of K , is roughly constant for a column of given dimensions. S o particular significance is, however, attached to the values of K , and to the optimum preheater temperature, because both depend primarily on the physical characteristics of the preheater. The results in Figure G shon that excellent agreement is obtained between the experimental curve and the curves calculated from the theories of van Deenitcr et aZ. and Porter et aZ. for large samrlcs. For smaller samples, however, the curve calculated from Equation 2 deviates from the expcriniental results. One of the main assumptions made in the derivation of Equations 8, 9, and 10 is that the distribution coefficient] K , remains constant for all values of the sample volume 8,’ (9). Under these conditions symmetrical elution curves result ( 4 ) . Where the above assumption does not hold, the theoretical elution curves shon- marked asymmetry ( 4 ) . The elution curves ‘iThich have been obtained in this investigation (Figure 3) may be taken as evidence that Equations S, 9, and 10 are valid in the range of sample volunies which have been used. The rate of decrease of the column efficiency with an increase in the sample volume depends on the value of the distribution coefficient, K , of the solute as is shown by the experimental results in Figure 9. Similar results may be obtained theoretically as follows: ?V = K,V, -r v.\/2= for
v’,> 3cdZ
and 1.t’ = 4 vv%
for Ti,’
TIME (mi n.)
5 mi. 7 mi.
< 0.5 vd
