The Use of Lightly Loaded Columns in Gas Chromatog raphy DAVID H. FREDERICK,' BlENVENlDO T. MIRANDA? and W. D. COOKE Baker laboratory, Cornell University, Ithaca, N. Y.
b A study has been made of the advantages of using lightly loaded columns for the separation of highboiling compounds. lntercomparisonsof column performance are more difficult than superficial consideration would reveal, and it is felt that many of the conclusions appearing in the literature are drawn from insufficient data. Lightly loaded columns of high efficiency can b e prepared with both glass beads and Chromosorb P as supports. However, under the conditions studied, the efficiency of Chromosorb W columns deteriorated as the amount of liquid phase was decreased.
A
prominent trend in the development of gas chromatography iq the widespread interest in extending the working range of the technique to include higher and higher boiling compounds. I n such methods, the main concern is the reduction of the retention time to some reasonable value. Such a reduction can be accomplished in the following ways: increasing the operating temperature of the column; increasing the carrier gas velocity; deerpasing the percentage of liquid loading; decreasing the length of' the column; and choosing a liquid phase which results in smaller partition coefficients. The first of these methods of reducing retention times has been employed b y a number of investigators ( 1 , 3, I S ) . In most cases conventional columns containing 10 t o 307, of liquid phase have been uqed. The numerous problems associated with high temperature operations have been discussed in these papers. The second method of decreasing retention times, namely, increasing carrier gas velocity, is limited by the finite rate of mass transfer and the allowable pressure at the sample injection system. Decreasing the amount of liquid phase, a third alternative, results in smaller retention volumes, but adsorption effects a t the surface of the solid support and other peak RCCCST
Present address, Dept. of Chemistry, Lycoming College, Williamsport, Pa. a Present address, Dept. of Chemistry, University of the Philippines, Quezon City, P. I.
broadening processes may complicate operation of lightly loaded columns. Shorter columns can be used to obtain reasonable retention times but only a t a sacrifice in column efficiency. Concerning the fifth method, the large amount of work reported in the literature has been aimed a t increasing separability but not reducing retention times. This study is mainly concerned with lowering retention times by decreasing the amount of liquid phase and using high carrier gas velocities. I n particular, the role of the solid support in establishing minimum liquid loading has been investigated. EXPERIMENTAL
Apparatus. T h e succemful duplication of gas chromatographic columns a n d t h e reproduction of d a t a obtained from them greatly depends upon accurate, detailed accounts of t h e preparation and operation of the original columns. It is for this reason t h a t a full account follows here of t h e treatment given in this laboratory to instrumentation, t o t h e preparation of materials for the columns, the procedures of packing them, to sample materials used, and to the determination of -optimum operating conditions for each column. A Perkin-Elmer hIodel 154C Vapor Fractometer designed for oneration from room tmper&ure to about'250" C. was modified for use in this work. Helium carrier gas was connected directly from the tank source to a preheater consisting of a 24-inch length of 1/4 inch 0.d. copper tubing coiled in the bottom of the oven chamber and thence to the liquid sample injection block. A second 22-watt cartridge heater, identical to that within the injection block, was installed in the gas stream immediately ahead of the injection block to act, with the copper coils, as a preheater of the carrier gas. About 15 feet of B and S No. 32 Nichrome wire was insulated, wound around the injection block, and covered with glass wool batting t o insulate the block from the oven. All three heaters were powered independently of the oven heater b y a n auxiliary voltage supply. The resulting surface temperature of the block was above 300' C. and the internal temperature was estimated as close t o 400' C. No port septa were found which withstood these temperatures continuously; therefore, the
voltage supply was reduced from 110 to about 70 volts for most work, resulting in a n internal temperature of about 300" C. The instrument's carrier gas regulator valve, pressure gage, and rotanieter were by-passed to make gas flow rate entirely controlled b y the tank reduction valves. Pressures up to 100 p.s.i.g. have been used with c,olumns installed with lead "0" rings and Swagelock fittings. To decrease drift in the detector signal, stray voltage noise w s minimized b y separating B.C. and d.c. wiring n-ithin the instrument, shielding the detector leads, and properly grounding instrumental components. Baqe line drift could be maintained to + l o pv. per hour. Samples were approximately 50 pg. per component and were injected in solution using a 10-pl. syringc. The specifications and sources of materials used in preparing the columns for this study are given in Table I. GLASSBEADS. llagnification under a microscope shons beads from both sources to be spherical b u t of a wide range of diameters. Eacept for a single column to be discussed later, these beads were used without further sizegrading. .%ppreciable quantitie:. of iron filings. some free and some fused t o glass, are present in these beads and mere partially remowd by poui ing the beads over a magnet. OTHER SOLIDStmoRra. Except as noted, columns were preparecl from conventional supports nithout slmial treatment. Size-grading of C-22 firebrick and stainless steel beads ~wts necessary to get a narroiv range of particle sizes. I n preparing colunins the uwal 1110cedure of dissolx-ing liquid substrate in excess solvent (acetone in most of the present nork) and mixing n i t h solid support n as followed. Solvent from Celite and firebrick column prcparations was stripped off by infrared htlat and inanual stirring to prevent crushing of the fragile particles. The same procedure nas usually used for the hard sphere packings although a rotary evaporator was sometimes employed without fear of fracturing the beads. All Celite and firebrick preparations were dry and freely flowing b u t those of the nonabsorbent glass and stainles steel beads were damp and cohesive nith as little as 0.0670 silicone oil. Celite and firebrick colunins m r e packed by the usual methods of tapping or vibrating the column until no more VOL. 34, NO. 12, NOVEMBER 1962
1521
material \vas accepted. Beads could be packed by tapping the column vertically or by using a ramrod. Because of the cohesiveness of these latter packings, vibration tends to form voids through the column leading to channeling. The tapping method, if used after each small portion of packing is added t o the column, avoids this problem. With liquid loadings of 3% by weight or less, satisfactorily packed columns contain, on the average, 1.5 grams of Celite, 2.4 grams of firebrick. 8.0 grams of glass beads, or 25 grams of stainless steel beads per foot of 0.18 inch i d . stainless steel tubing. OBJECTIVES
The objective of the work presented here stemmed from interest in chromatographing high boiling compounds. Decreasing the percentage of liquid loading, if adsorption of samples on the solid support did not occur, and increasing the carrier gas velocity, if column efficiency could be maintained, appeared to offer more advantages in chromatographing such compounds than raising the operating temperature. Hishta. Messerly, and Reschke (8) in 1960 demonstrated these effects in separating Come relatively high boiling compounds a t lowered temperatures usinq columns of wetted glass head. to avoid adqorption problem.. Using both conventional supports as well as glasq beads, the present work waq undertaken to find the loner limits of liquid loading \rhich were permissible for the several types of solid supports.
t o investigate the effects of other variables such as carrier gas velocity, column length, and temperature, and t o devise a method of comparing the performance of the columns. PROBLEMS IN INTERCOMPARING COLUMNS
One of the major objectives of this study was the evaluation of the suitability of various solid supports for use v i t h small quantities of liquid phases. Interconiparison of column performance is more difficult than superficial considerations would reveal. It is insufficient and misleading to compare t i r o different columns b y operating both under conditions of equal temperature. particle size of solid support. liquid loading, and carrier gas velocity. If two different solid supports are being compared for use with a particular liquid phase. all of the above variable. should he separately optimized for each column. For evample. each should be operated a t the optimum carrier gas velocity which varies with the choice of solid support. Such an approach vastly multiplies the amount of evperimental work but is necessary to draw reasonably valid conclusions. Some idea of the magnitude of the work which iq necessary can be dranm from the fact that 70 columns were prepared and about 2000 chromatographs were obtained in evaluating only five different solid supports for use with a single liquid phase and a particular sample. The conclusions drawn from this work apply
strictly only to the liquid phase used in the study and the type of samples chosen for the comparison. Another important consideration in column comparison is the normalization of the analysis time (6). It is impossible to compare the ultimate, relative efficiencies of t\ro columns if the elution times of the sample are markedly different. For euample, one column may just resolve a pair of components in 1 hour, and another column, because of a smaller partition coefficient, may elute the component unresolved in one-half hour. The operating temperature of the second column could be decreased until its retention time was also an hour and in most cases, the reqolution would be increased. A further complication arises in intercomparing columns which is brought about by the euperimental limitation of the particular apparatus. For evample, in this study the practical pressure limit on the inlet system \vas approyitnately 100 p. the gas velocity that could be used with glasq head columns because of their low permeabilitv Conceivahly different conclusions would be obtained with other apparatus that did not have this experimental limitation Detector sensitiritv i. also an experimental parameter that can eFect the comparison of columns. II'ith lightly loaded columns it is necessary to aroid overloading of the column and in some cases the loadinq could be decreased if a more sensitiJ-e detector n a r used. EFFECT OF COLUMN LOADING O N
SEPARABILITY
Table I.
Class Glnss heads
Sources and Specifications of Solid Supports Type Mesh range Supplier 80-140 A . S . LaPine Co.,
70-80 140-230 325.. 100-140
1014M-P
Celite
1723
170-230
2332.5
230-325
Chromosorb IT'
30-60
Chromosorb 11-
60-100
.. .......
-
Chromosorb P Stainless steel beads Fluorocarbon
1522
316
Fluoropak 80
ANALYTICAL CHEMISTRY
Chicago, Ill. -4.S. LaPine Co., Chicago, Ill. A . S. LaPine Co., Chicago, Ill. A. S. LaPine Co.. Chicago, Ill. hlicrobeads Inc., Jackson, Miss. 1Iicrobeads Inc.. Jackson, Miss. hlicrobeads Inc., Jackson, Miss. Johns-Manville Co.. Kew 'I-ork, S . T. Johns-l\Ianville Co., 3'ew York, E.T. Johns-Manville Co..
rlvondale, Pa.
80- 100
1 5 s . ..
An important relationship dcwloped by Purnell (14) has been helpful in evplaininq the difference in efficiency of variouq columns. This relationship shows that the number of plates in a column necessary to resolve ti\-o solutes a t con*tant temperature is a function of their relatire volatility. CY, the dead volume of the column. T 7 ~ , and the retention volume of the less volatile component. T ' p as follows:
.
F 8- A1 Scientific Gorp .Ivondale, Pa. Federal Mogul Division.
.Inn Arbor, 3lich. Fluorocarbon Co , Anaheim, Calif.
.is Purntxll iioints out, a t constant temperature the number of plates, S.required to effect a given separation decreases as T'R is increased if CY and T7d are held constant. This increase in V R is proportional to the increase in liquid loading on the column; hence. best separations should occur on heavily loaded columns. If. however. both liquid loading is decreased; column temperature is appropriately lowered, TIR can remain unchanged, a: will probably increase, and separations \rill require fewer plates.
Figure 1 . Number of plates required for exact separation of isomeric octanes a t different temperatures VR'
-
VRI
= 1.5W
An increase in CY a t lower temperatures, in particular between members of a honiologous series, is related to the frequently observed divergence of Clausius-Clapeyron plots of log p us. 1/T as CY is the ratio of vapor pressures of two solutes, or more precisely, the ratio of their Henry's law coefficients at a given temperature. Thus. separations of solutes at temperatures well below their boiling points may be enhanced because of increased CY values. This so-called Clausius-Clapeyron advantage is not very large for members of the naphthalene mixture used in this study. I n the case of the isomeric octanes, however, for which vapor pressure-temperature data are available (15), there is a noticmble divergence of log p us. 1,'T plots and an increaied separation of components at loner temperature. Figure 1 shows actual peak positions of a niivture of three octane isomers on various columns and the improved separation reflecting the increased 01, which occurs with decreased liquid loading and lon-ered temperature. T h e number of plates necessary for complete separation of each pair of isomers is shown on the diagram. Further evidence of the importance of the increased 01 value a t lower temperatures is illustrated in Figure 2. The retention T-olunies (determined euperimeiitally) for various normal hydrocsrbons are shoirn for a heavily loaded and a lightly loaded column. As predicted by the Clausius-Clapeyron equation, the slopes of these plots increase as the temperature is ion-ered. -1 steeper slope indicates that the peak3 of the chromatogram are more 1% idely separated (increased CY'S). K i t h the more heavily loaded coluiiin, decreasing the operating temperature to obtain better separation cxn be acconipli~hed only a t the eyiense of a greatly increased retention volume. For evample. n-decane TI-ouldbe eluted a t 90 nil (1.9 minutes) a t 236" C. but only a t 1750 ml. (34.2 minutes) a t 91" C. With the lightly loaded coluninq, however, it is possible to operate a t a lower temperature with the resulting steeper slope and yet maintain
the retention volumes a t reasonable values. The great difference between these two columns is illustrated b y the fact that the retention volume of the normal octane on the 0.06% column at 37" C. is less than that on the 30% column operated a t 236" C. The increased slope of the plots of retention volume shown in Figure 2 does not necessarily result in better resolution between the chromatographic peaks. The resolution will be increased only if it is possible to maintain the required column efficiency with lightly loaded columns operating a t lower temperature. Efficiency here is defined b y the usual expression (10) A' = 16 (d/w)2and is readily converted to the height equivalent t o a theoretical plate, HETP, by dividing column length, L, by S.
I
30%Silicone oil 710 on Chrornororb w --.06%Silicone oil 710 on Gloss Beodr
8
IO I2 No o f Carbon Atoms
I
14
Figure 2. Retention volumes v5. carbon number and column loading
EFFECT OF COLUMN LOADING O N EFFICIENCY
Clearly there is a lower liniit t o the amount of liquid loading permissible for gas chromatography columns. This limit is determined by the adsorptive properties of the solid support which will always decrease column efficiency as well as other peak broadening phenomena. Quantitative knowledge of such peak broadening processes is not available and i t is only by esperiment that an evaluation of tlie problem can be ascertained. One of the main purposes of this research was to determine the minimum loading which could be tolerated for various suppoi ts 11itliout significant loss in efficiency. Qualitatively, the minimum amount of liquid which can be tolerated varies n-ith the solid support and its pretreatment, the chosen liquid phase, the nature of the samples to be separated, and the operating temperature. The niinimum loading will be dependent on the polarity of the liquid which affects its ability t o react with or cover up the active adsorption sites on the solid matrix and thus prevent or reduce solute adsorption. If the solutes to be separated are very polar, it n ould probably be necesqary t o use a iiiore heavily loaded column to decrease peak broadening. Minimum loading could probably be attained with a polar liquid phase and nonpolar samples. The last effect, that of operating temperature, is important because of the great dependence of adsorption on temperature. The choice of liquid phase for the separation of a given sample mixture is a subject which has received widespread attention in the development of gas chromatography but v hich has been largely ignored in the present work. K i t h the exception of two or three columns, all of the comparisons made in this study have dealt n-ith columns containing Dow Corning Silicone Oil 710 as liquid phase, thus fixing one of tlie experimental variables. This
should not be construed as indicating trivial importance of the choice of liquid phase. Indeed, as one of tlie primary objectives of this project was the reducing of solute retention volumes, i t is clear that the choice of a liquid phase nhich n ould lead to smaller values of the partition coefficient, k , nhile still producing the desired resolution and column efficiency, would permit operation a t still loner tcniperatures. Each liquid phase represents a u-hole new system requiring optimization of all other parameters for a given separation. It is hoped that iiiany of the conclusions of the present work ~ 1 1 reduce 1 the necessary labor in evaluating other systems. The above discussionq have, so far, been devoted to the benefits of decreasing the amount of liquid phase. There are two notable eweptions to the desirability of this procedure. First, when resolution is not a problmi-i.e., when separation is easily obtained b y large differences in solute properties-a heavier liquid loading (but still n i t h its attendant required teniperature increa3e) nil1 permit laiger sample charges to a column before over-loading is observed and will lead to preparative-scale performance. It is doubtful, under the particular esperimental conditions used so far, t h a t preparative-scale I\ ork will be possible with very lightly loaded columns because of the sinall sample charges required. For this reayon, preparativescale gas chroniatographj of \ iwy high boiling materials is unlihcly to benefit froni the adxantages of the present method.. The second exception involves the separations of solutes whose cllau&x+ Clapeyron plots con1 erge at low temperature. For such systems, which are rare for similar compounds, a decrease in temperature results in a smaller VOL. 34, NO. 12, NOVEMBER 1962
1523
CHOICE OF OPTIMUM CONDITIONS
Table II. Components of Naphthalene Mixture Used; listed in Order of Elution from Columns of Dow-Corning Silicone Oil 710
Boilins point, Component C. Benzene (Solvent 1 80 Naphthalene 218 2-hIethv1nsphthalene 245 1-Meth~~lnaphthalene 240 Ttttrahydroacenaphthene 260/719 min. 2, ~-Diniethylnaphthalene 262 1.6-Diniethylnaphthalene263 1,7-Dimethylnaphthalene 261 2,3-Dimethylnaphthalene Sublimes 1,2-Dimethylnaphthalene I40/15 mm. Acenaphthene 278 Table 111. Efficiency of Glass Bead Columns of Different Particle Size
Mesh range 70 180 Optimum flow nil. /inin. 70 Temp. 74” c. Silicone oil, 0.16 HETP cni. Saphthalene 0 30 Tetrahydroacenaphthene 0 14 Acenaphthene 0 11
140/230 69
7 4 O c. 0 16
0 13
0 08 0 04
relative volatility and would lead to poorer resolution on lightly loaded columns. Cwept for these two caqes. the authors believe that for most efficient operation of gas chromatography column< used to separate high boiling compounds, liquid loading should be decreased as much as possible consistent with high efficiency. and the temperature lowered to maintain desirable retention times. If the analysis time is normalized as previously discussed, the second term of Equation 1 is essentially the same for both heavily and lightly loaded columns. K i t h the lightly loaded column, 01 is probably larger reculting in a smaller N.
Table IV.
RESULTS
The parameters evaluated in this work are mainly concerned with the solid support. Particle size. pretreatment, minimum loading, and absorptive effects mere studied for the qolid supports listed in Table I. Particle Size. The choice of t h e
Some Characteristics of Most Efficient Columns Prepared for Each Type of Solid Support
Support NBS niesh range Av. particle diameter, microns Liquid loading (Silicone 0.1 DC 710) wt.-wt.90 Grams/5-foot column Surface area R q . meters/gram Sq. meters/5-foot colnmn Liquid distribution G r a m liquid X loa,/ Sq. meter surface Cross sectional area void, yo Calcd. as spheres.
1524
T o compare the various solid supports used in gab chromatography i t was necessary to decide what is meant by the “ b e d bupport”. I n this particular study the “best support” was defined as that support which would give the best resolution of a particular mixture within a time limit of 30 minutes. The mixture chosen for the experiment mas the group of aromatic hydrocarbons listed in Table 11. The liquid phase used throughout was Dow Corning Silicone Oil 710 nhich fixed one of the variables in the comparison. Other parameters not varied in the comparison were column length ( 5 feet) and diameter (0.25 inch). The optimum operating conditions for each column v a s then defined as those conditions n Iiicli would give the maximum reaolution of the sample mixture in the specified 30 minutes. To accomplish this end chroniatograms were obtained for different liquid loadings at a variety of temperatures, and the effect of flow rate v-aq determined a t each temperature. I n each ea-e, the temperature and flon rate finally chosen resulted in the l a d component of the mixture being eluted in 30 i 1.5 minutes. For eunmple, if under particular operating conditions, the last component wa? eluted in 50 minute., it would be necessarv to either increaqe the temperature or carrier gas velocity or decrease liquid loading to reduce the analysis time. That procedure resulting in the best rvolution would be chosen as optimum.
ANALYTICAL CHEMISTRY
Chromosorb W 60-100 200 30 3.21 1.41 (6) 10.6 304 53
C-22 Firebrick 60-100 200 3.0 0.40 4.14 (6) 53.8 7.4 71
Glass beads 200-230 65 0.16 0.060 O.04la 1.55 39
36
most
