oi CHARLES HISHTA, JOHN
P. MESSERLY,
s
and ROBERT F. WESCHKE
&isto/ Laboratories, Syracuse, N. Y .
B- As the amount of liquid phase on gas chromatography columns is decreased, retention times become shorter, and peaks become sharper without significant change in resolution. Hence, retention volume is a function of the amount of liquid phase as expressed in the fundamental equation V, = V , KV1. By using glass beads as the solid support, absorption is minimized and reduction of the liquid substrate to less than 0.3% is possible. With these columns, high boiling compounds can b e eluted rapidly a t lowered temperatures. A comparison is presented between conventional Celite columns and glass bead columns, using a polar mixture with a boiling point range of 56" to 156" C.,and a nonpolar mixture with a boiling point range of 174" to 253' C. Operation at low column temperatures retains high thermistor sensitivity and avoids decomposition and volatilization of the liquid substrate.
+
(9) has demonstrated that as the amount of liquid substrate on a conventional Celite column is decreased, retention times of given fractions become shorter and fraction peaks become sharper without loss of resolution. This is predicted by the fundamental equation, V, = V, $XVL, in which retention volume is a function of the amount of liquid phase. By substituting a less porous solid support for the usual Celite or firebrick, greatly reduced amoimts of liquid substrate are possible. Powdered glass (8) and glass beads (1, 5 ) have been used as solid supports in gas-liquid chromatography. Glass beads, because of their uniform shape and low porosity, are less susceptible to adsorption effects ( 7 ) , thus permitting the use of very small amounts of liquid phase. Littlewood (5) reported the preparation of columns packed with glass beads 0.1 mm. or smaller in diameter and coated with about 3% of tritolyl phosphate. I n this study the use of glass beads as a solid support was further explored. It was possible to reduce the amount of liquid substrate to less than 0.3% vvithout encountering appreciable adsorption effects. The columns provided chromatograms showing good resolution INO
1738
B
ANALYTICAL CHEMISTRY
and relatively short retention times at temperatures as much as 150" C. below the boiling point of the sample. To evaluate the columns, studies were made with mixtures of ketones (polar compounds) and mixtures of hydrocarbons (nonpolar compounds). APPARATUS
A Perkin-Elmer Model I54 B Vapor Fractometer equipped with a thermistor-type detector was modified with a Model 63 Thermistemp temperature controller to provide a more constant temperature. The injection block was insulated from the oven with glass wool batting, and its temperature was adjusted independently of the oven by a separate variable transformer to ensure rapid volatilization of the sample. The temperature of the vaporizer was indicated on a pyrometer connected to an ironconstantan thermocouple which was inserted in the injection block. The recorder was a 5-mv. Leeds &: Northrup Speedomax Type G with 1second response and a chart speed of 0.5 inch per minute. Helium was the carrier gas. Samples were measured and introduced into the injection port through a silicone rubber septum with a KO,701N Hamilton microliter syringe. The glass beads were purchased from Rficrobeads, Inc., Toledo, Ohio, and Jackson, Miss. They are 95% true spheres of soda-lime silica glass having alkaline properties and are available in 19 different sizes ranging from 20 to 400 mesh. The 60- to 80-mesh size (200 microns) provided reasonable g a s 4ow rates under the experimental conditions of operation. Since water will remove some surface alkali froni the beads, they were used as received from the supplier, without any pretreatment. Treating with hydrofluoric acid or chromic acid resulted in significant adsorption of the sample (increased tailing) even after many rinses with water and neutralization with sodium hydroxide. P R E ~ ~ R A T I 66NP COLUMNS
The columns were prepared from 2meter lengths of li4-inch stainless steel or soft copper tubing, with the exception of Apiezon L grease on Gelite, which m-as in a 0.45-meter stainleps steel column. ~a~~~~~~ 460 on Glass Beads. A solution of 500 mg. of Carboaax 400 in about 50 mi. of acetone was poured
over 200 grams of glass beads. Acetone was added to rinse the original container and to cover the glass beads. The mixture was then stirred so t h a t the beads were evenly coated while the acetone was evaporated on a steam bath. T h e last traces of acetone were removed a t 100' C. in an oven. This packing thus contained 0.25% liquid substrate. Apiezon
L
Grease on Glass Beads.
This column packing was prepared in a similar manner using 200 grams of glass beads, 250 mg. of Apiezon L grease, and warm chloroform as the solvent. After t h e chloroform was evaporated, much of t h e substrate was found on the sides of the beaker. Only t h e beads in t h e center of t h e beaker were used for t h e column packing, and these were assumed to have a coating of less than 0.125y0 Apiezon L grease.
Dow Corning High Vacuum Grease on Glass Beads. This column packing was prepared in the same way as 0.25% Carbowax 400 on glass beads,
except t h a t hot chloroform was the solvent for the Dow Corning high vacuum grease. Liquid Substrates on Celite. PackCarbowax 400 and 307, ings of 3QyO Apiezon L grease on 60- to 100-mesh Celite ( 4 ) were prepared in the usual manner. EXPERIMENTAL
A mixture of seven ketones boiling from 57" to 156" C. was chromatographed on the Carbowax 400 columns; the Apiezon L and Dow Corning high vacuum grease columns were used to study the separation of three hydrocarbons boiling from 174' to 253' C. The conditions under which the studies were carried out are given in Table I. DISCUSSION
The quantity of liquid substrate which can be applied successfully to the glass beads is dependent upon the nature of the substrate (6) and the size of the beads. 'i'arious amounts of Carbowax 400 were placed on 60- to 80-mesh beads. As judged by adequate resolution, the optimum l e ~ proved l to be 0.25"r,. During the first 10 minutes of elution, five inembers of the seven-component ketone mixture were resolved and measured. At a level of O.l25%, resolution of the ketone sample was poor under the conditions described above; at O.SY,, loss of the substrate was im-
mediately evident. Xpiezon L grease a t a level of 0.125% separated from the beads during preparation of the column packing described above. The resulting packing thus had a coating of less than 0.125% Apiezon L grease. Larger beads, since they have less surface area per gram, require less substrate, while the smaller sizes will permit application of slightly larger amounts to an equal weight of beads.
IO
9
KETONES
57
ACETONE b.METHYL ETHYL KETONE c DIETHYL KETONE d METHYL ISOBUTYL KEXXYE I METHYL C'TCLOPROPYL KETONE I CYCLOPENTANONE E CYCLOHEXANONE 0
8
025% CARBOWAX 4c3 ON UASS BEADS AT 40%.
80 102
ita
7
II 4 131 I56
6
6 4
3 2 I
RESULTS
Ketone Study. Figure 1 shows t h a t the first five components of t h e mixture were eluted from t h e glass bead column in less than 10 minutes. Cyclopentanone appeared in about 15 minutes and the sample was completely eluted in 33 minutes. TF7hen t h e sample was eluted from the Celite column a t 40" C., nothing appeared during t h e first 10 minutes; t h e fourth peak was recorded at 41 minutes. By using a larger sample and operating the recorder a t its maximum sensitivity to detect the broad peaks, elution was completed in 389 minutes or about 10 times as long as when the glass bead column was used. The specific retention volume per gram of liquid phase, Vg, was 364 cc. per gram for the glass bead column a t 40' C. and 447 cc. per gram for the Celite column a t 40" C. Under these conditions the number of theoretical plates (3, 6 ) for both columns was about 500. K h e n the Celite column was operated a t 105' C., the sample was completely eluted in about 35 minutes. This compares well with the elution time from the glass bead column,at 40' C., about 33 minutes. When the areas under the first two peaks were compared, those from the glass bead column a t 40" C. were on the average 2.1 times as large as those from the Celite column a t 40' C. Although the temperatures and column inlet pressures were identical, the gas flow figure for the glass bead column wab 32 cc. per minute, while that for the Celite column w-as 46 cc. per minute. This accounts for the differences in peak areas between the two columns. When the Celite column was operated at a flow rate of 32 cc. per minute, the sensitivities were identical. When the Celite column was operated at 105' C., a comparison of these areas Bhows that those from the glass bead column were on the average 2.7 times as large as those from the Celite column. I n this case the gas flow rate for the Celite column was 36 cc. per minute as compared to 32 cc. per minute for the glass bead column at 40" 6. This indicates that the larger ratio of 2.7 is due mainly to higher detector sensitivity at the lower temperature. The difference in the gas
0 30
20
30% CARBOWAX 400 ON MUTE
i3
ar I0l.C.
t
P
e
A
t 40
20
x)
Figure 1.
Elution of ketones
flow rates is only a minor contributing factor. The definition of Jones and Kieselbach (9)was used to evaluate resolution comparisons between the Celite and glass bead columns. I n this equation R = QXlZ; resolution, R, is equivalent to the product of the relative peak sharpness, Q, and the relative peak separation, XIZ. Q is further defined as Tx/W or the ratio of the elution time to the time width of the peak, where E7 equals width of peak as measured a t the base-line intercepts of the tangents to the peak. Relative peak separation is then defined as Tz-T1/T1, or the ratio of the difference in two
Table I.
10
nwwmms
elution times to the elution time of the first peak. The authors of this definition give 2.5 as a numerical value for complete separation. I n Figure 1 the numerical value of resolution, E , for the first two peaks, a and b, is 2.0 for the glass bead column a t 40' C., 3.7 for the Celite colunin at 40' C., and 2.2 for the Celite column a t 105' C. Although the Celite column a t 40' C. shows the best resolution value, the peak shapes are much sharper on the glass bead column; resolution is adequate, and the retention time is loffered by a factor of 10. For peaks c and d, resolution is 1.3 for the glass bead column a t 40' C., 1.2 for the Celite col-
Gas-Liquid Chromatography Operating Conditions
Hydrocarbon Study Ketone Study
0.25%
Apiezon L
D.C. Hi Vac grease
O
100
I00
O
200
200
0.2570
Carbowax
30% Carboaax
