expensive method of analysis. The widespread interest in the possible harmful effects of ingesting an edible oil that contains dimers and/or higher polymers due to heat abuse, as discussed by Melnick (3, Firestone (8),Friedman (9),Nolan (IO), and many others, coupled with the relatively few ways of measuring polymer content in edible oils (Ferren ( I I ) ] , should lead others to improve our present GPC method (see Figure 3).
ACKNOWLEDGMENT The technical assistance of Leonard Maltese and the staff of Stillwell Gladding Testing Laboratories, 130 Cedar Street, New York, N.Y., is gratefully recognized. Received for review April 20, 1973. Accepted May 22, 1973.
Figure 3. GPC-column and Buchler linear fraction collector
(7) D. Melnick, J. Amer. OilChem. Soc., 34,578 (1957). (8) D. Firestone, W. Horowitz. L. Friedman, and G. M. Shue, J. Amer. OBChem. Soc.. 38,253 (1961). (9) L. Friedman, W. Harowitz. G. M. Shue. and D. Firestone, J. Nutr.. 73.85 (1961). (10) G. A. Nolan, J. C. Alexander. and N. R. Artman, J. Nut,., 93, 337 (1967). (11) W. P. Feirenand R. E. Morse,FoodTechnol., 17,112 (1963).
Dynamically Coated PLOT Columns J. G. Nikelly Department of Chemistry, Philadelphia College of Pharmacy and Science. Philadelphia, Pa. Porous layer open tubular (PLOT) columns, which were first proposed by Golay (I) and demonstrated by Halasz and Horvath (Z),have been commercially available since 1966. Their dimensions are typically 0.02-in. i.d. and 50 ft in length and are conventionally made by a static coating procedure requiring a specialized apparatus and technique (2). Their advantages and uses have been described extensively (34). Recently, PLOT columns have also been made by a more convenient dynamic coating procedure (7,9, IO) resulting in columns that are nearly as good as those made by the static procedure (8). However, dynamically coated PLOT columns tend to have relatively low partition ra(1) M. J. E. Golay, "Gas Chromatography. 1960,"R. P. w. Scott. Ed., Butterworihs, Washington. D.C.. 1960, p 139. (2) I. Halasz and C. Howath. Anal Chem., 35. 499 (1963):U.S. Patent 3,295,296(1967). (3) L. S. Ettre, J. E. Pureell and S. D. Norem. J. Chromatogr. ScL, 3, 181 (1965). ( 4 ) J. E. Purcsll and L. S. Ettre, J. Chromatogr. ScL, 4,23 (1966). (5) L. S. Eltre. J. E. Pureell. and K. W e b , J. Chromatogr., 24, 335 (1966). (6) L. S . Ettre, J. E. Purcell. and K. Billeb. Separatlon S c i , 1, 777
(1966). (7) J. G. Nikeily. Anal. Chem., 44,623 (1972). (8) J. G. Nikeliy. Anal. Chem., 44, 625 (1972). (9) Max Blumer. Anal. Chem., 45,980 (1973). (10) R. Kaiser. Chromatographfa. 1-2, 34 (19681, 2280
19104
tios, k, because the coating mixture must be sufficiently thin (low concentration of liquid phase and solid support) in order to reduce the risk of column-clogging during the coating step. These columns also tend to have lower efficiencies, mainly because of the larger inside diameter that is required to prevent clogging; however, adequate efficiencies are still possible as demonstrated by Blumer, who achieved 1.2-mm plate heights by using Silanox 101 as the solid support (9). This Note describes the preparation and evaluation of improved dynamically coated PLOT columns that are, in some ways, equal to the conventional PLOT columns. The improved columns are made by the same coating technique described earlier (3, except that they are coated in shorter lengths (25 t o 35 ft). While shorter columns are generally less efficient, in this case they have increased partition ratios and, as expected, less analysis time compared to similarly coated 50-ft columns. The increased partition ratios result from the fact that shorter columns of relatively large inside diameter permit the use of higher viscosity coating mixtures a t higher coating rates. These factors favor increased thickness of the porous layer, as found experimentally (8). Specifically, coating mixtures could be used that contained as much as 20% liquid phase and 30% solid support, with coating rates as high as 2 ft per see at only 10 psig inlet pressure.
ANALYTICAL CHEMISTRY. VOL. 45, NO. 13. NOVEMBER 1973
7
a Figure 1. Separation of
methyl esters
Column: 25-ft. X 0.030-in. i.d., DEGS. Temp.: 180 "C. Carrier: 1 2 ml/ min. Sample vol: 0.1 pl. Peaks: 1 , chloroform (solvent); 2, palmitate; 3. stearate; 4, oleate; 5, linoleate; and 6, linolenate
EXPERIMENTAL Apparatus. The same column-coating apparatus described earlier (7) was used. The columns were made from type 304 stainless steel tubing, 0.03-in. i.d. and 0.062-in. o.d., available from Superior Tube Company, Norristown, Pa. 19404. All separations were made on a Gow-Mac gas chromatograph, model 69-700 (Gow-Mac Instrument Company, Madison, N.J. 07940). The l/g-in. fittings were modified to take lh6-h. columns by using, a t the inlet, a l/g to Y~s-in.adapter (made locally) and, a t the outlet, a Teflon reducing ferrule (RF 200/lOO, Alltech Associates, Arlington Heights, Ill. 60004), which allowed the column to extend 4 cm into the detector housing. Recordings were obtained with a 1-mV strip chart recorder, model 1027 (McKee-Pedersen Instruments, Danville, Calif. 94526). The chart speed was 0.5 in. per min., except in the case of very short retention times, such as for a nonretained component, methane, in which case the chart speed was 2 in. per min. Flow rates were measured with flow meters (Brooks Instrument Company, Inc., Hatfield, Pa. 19440) which were calibrated with a soap bubble meter a t the column outlet. The average linear gas velocity a, was calculated from the equation
u
=
LJt,
(1)
where L is the column length and t , is the retention time for methane. Sample injections were made (without a stream splitter) using a 0- to 1-p1 syringe (Catalog No. 140011, Precision Sampling Corporation, Baton Rouge, La. 70815). Reagents. The coating mixtures were prepared using, as solid support, Chromosorb Rb470-1, a finely divided diatomaceous silica available from Johns-Manville, Manville, N.J. 08835. The support was used as received-ie., without pretreatment such as sizing or drying. Procedure. A 25-ft length of 0.03-in. i.d. tubing is coiled, connected to the filling tube, and flushed with a few milliliters of CHC13 using air (or nitrogen) pressure. The coating suspension is prepared by mixing and shaking in a small capped vial 2 ml of 7 to 10% chloroform solution of liquid phase and 0.3 g solid support. About 1 ml of coating mixture is then added to the filling tube and forced through the tubing under 10 to 20 psig air pressure, which is allowed to continue flowing for 5 to 10 minutes after the excess coating mixture emerges from the column. The tubing of PLOT columns may be reclaimed by removing the coating. This can be done by alternately connecting each end of the column to the filling tube and flushing with solvent under air pressure of up to 100 psig. For stubborn cases, a manually op-
2 4 6 RETENTION, MIN.
Figure 2.
8
Separation of lower normal primary alcohols
Column: 24-ft X 0.30-in. i.d., Carbowax-1540. Temp.: Programmed 60 to 140 "C at 20 "C per min. Carrier gas (He) flow rate: 13 ml/min. Sample vol.: 0.06 PI. Peaks 1 to 7 correspond to the respective alcohols erated high pressure oil pump can be used which is capable of exerting over 1000 psig (Catalog No. 777, Grove Pressure Tool, Wabash Corporation, Chicago, Ill. 60621).
RESULTS AND DISCUSSION Typical Separations. Several columns were made and evaluated by comparing their separation characteristics with published separations using similar samples. The criteria of comparison were completeness of resolution, analysis time, and sample capacity. Figure 1 shows the separation on a DEGS column, of some C16-C18 methyl esters typical of linseed oil (AOCS Reference Mixture RM-2, Supelco, Inc., Bellefonte, Pa. 16823). The critical stearate-oleate separation is achieved in 8 min., even though the injected sample size is 0.1 pl. Figure 2 shows the separation of some lower primary alcohols on a Carbowax-1540 PLOT column. The complete resolution of the MeOH-EtOH pair requires only 1.5 min., which compares favorably with that of 50-ft PLOT columns (8, 1 1 ) . Owing to its low partition ratio, k < 2, this pair is normally considered a difficult separation for open tubular columns. Figure 3 shows the separation of lower free fatty acids on a DEGS column containing some stearic acid added to prevent peak-tailing due to absorption. The separation time is lower than for 50-ft PLOT columns, valeric acid being eluted in 2.5 min, instead of 4.5 min (12). This performance may be explained by the increased amount of liquid phase that can be coated in the 25-ft columns, thereby nearly doubling the partition ratio k . According to the Purnell resolution equation
(where R is the resolution, N is the number of theoretical (1 1 ) Perkin-Elmer Corporation, Norwalk, Conn.. Gas Chromatography Application, Report No. GC-AP-008, 1966. (12) Perkin-Elmer Corporation, Norwalk, Conn., Chromatogr. Newslett., 1, 27 (1972).
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2281
5
4
3 HETP, mrn
2
1
c
4
8
FLOW
RATE,
12
ML/MlN.
Figure 5. Flow rates Column as in Figure 4. Injected sample: 0.1 pI 20% benzene in CHC13
bl
RETENTION,
4, MIN.
Figure 3. Separation of lower fatty acids Column: 25-ft X 0.030-in. i.d., DEGS with added stearic acid. Temp.: 125 "C. Carrier: 13 ml/min. Sample vol.: 0.1 pl. Peaks: 1, acetic; 2, propanoic: 3, isobutanoic and n-butanoic, 4 , pentanoic; 5, hexanoic: and 6, heptanoic
n4
RETENTION, M I N .
Figure 4. Separation of benzene homologs Column: 50-ft. X 0.030-in. i.d.. m-bis(rn-phenoxyphenoxy)benzene with added lgepal Co-880. Temp.: 75 "C. Carrier: 8 m l per min. Sample: 0.08 PI. Peaks: 1, benzene; 2, toluene; 3, 4 , and 5, xylenes
plates, cy is the relative retention, and k is the partition ratio), a 50% reduction in column length, which reduces resolution by 30%, can be partially compensated by a 30% 1). For samples of low k increase in the factor k / ( k values, this increase of the capacity factor can be achieved by roughly doubling k - i e . , doubling the liquid phase load (amount of liquid phase per unit column length). Column Characteristics. Characteristics of a typical 25-ft PLOT column are listed in Table I, which shows that the amount of liquid phase per column length is about double the conventional r a n g e 4 e . , the total weight of liquid phase in the 25-ft columns is about the same as that in a typical 50-ft PLOT column (11). At the same time, the gas phase volume is a little more than
+
2282
Table I . Characteristics of a Typical Dynamically 9 Coated PLOT Column Column length 25 f t Tubing inner diameter 0.03 in Column volume 5 . 2 ml Geometric surface areaa 180 cm2 40-60 p m Thickness of porous layerb,h Liquid phase DEGS a0 mg Volume of liquid phaseC 50 Phase ratio, p Weight of liquid phase per column length 3.2 mg/ft
*
a Calculated from tubing dimensions. Estimated from the volume of liquid phase and solid support retained in the column from the coating step; the result is in good agreement with that calculated by Ettre from the true and apparent densities of the support ( 1 3 ) . Determined experimentally (7)
double because of the larger column diameter, 0.03 in. us. 0.02 in. The phase ratio, p, is, therefore, not much different than that of typical 50-ft x 0.02 in. i.d. columns. There is an optimum volume ratio of liquid phase and solid support and this was determined experimentally by studying its effect on resolution and analysis time. The test sample selected for this purpose was an aromatic mixture containing the xylene isomers which are difficult to resolve. The alpha value for meta and para isomers is approximately 1.06 on rn-bis(rn-phenoxyphen0xy)benzene liquid phase, and the partition ratio is about 15. Accordingly, the best results were obtained when the concentration of liquid phase in the coating mixture was between 7 and 10 v/w%. Figure 4 shows this separation where the xylenes are essentially completely resolved in 18 minutes. Because of the very low separation factor for the two xylene isomers ( a = 1.06), it was necessary to use a 50-ft column made up with two separately coated 25-ft sections. Carrier Flow Rate. Figure 5 shows that the optimum (minimum HETP) flow rate is 7 to 9 cm3 per min. In practice, as shown in Figures 1-4, a suitable flow rate is 10 to 13 cm3 per min since the reduction in resolution is relatively small while the saving in analysis time is significant. Sample Capacity. In addition t o producing adequate resolution per unit analysis time, the increased partition ratio that is achieved with coating shorter columns pays a dividend in increased sample capacity, thus obviating the requirement of a sample splitter. Received for review February 26, 1973. Accepted May 31, 1973. (13) L. S. Ettre, Encyciopedia of Industrial Chemical Analysis, Port Chester, N.Y., 10573, personal communication, 1972.
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