Dual-Load Gas Chromatography Columns J. G. Nikelly Department of Chemistry. Philadelphia College of Pharmacy and Science, Philadelphia, Pa. 19104
The quantity, concentration, and distribution of the stationary phase in gas chromatography columns has been the subject of numerous studies throughout the two-decade history of the art. The interest in this parameter is not surprising in view of its critical role in determining column performance and the ease with which it can be studied. One way in which this parameter can be studied is to decrease the per cent of liquid phase along the column length in a continuous or stepwise linear or nonlinear gradient. Such a gradient distribution increases column performance as it permits a t the back section of the column length, the use of relatively low-liquid loadings and, consequently, a less significant liquid mass resistance term. At the front section, where the injected sample is not sufficiently separated into its components, there is a significant contribution to the plate height which is decreased if a higher liquid loading is used. Viewed from a different point, the injected sample volume is always sufficiently large to contribute to the plate height, and this contribution is reduced by using a larger liquid loading a t the front of the column. A decreasing distribution of liquid phase along the column was first suggested by Purnell ( I ) , who compared this with programmed temperature chromatography. Bunting, Locke, and Meloan (2, 3) have shown theoretically and experimentally that for solutes of low and intermediate retention, there is improved column performance when the partition ratio is decreased along the column length. Similar results were found by Duty in the case of preparative columns ( 4 ) and by Christophe who found that when deviating from optimal carrier velocity, and when large injection volumes are required, negative gradient columns are superior to uniformly loaded columns ( 5 ) . The efficiency of gradient columns was also studied by Kwok, Snyder, and Sternberg, who, in some cases, found
it to be higher than that of nongradient columns (6). Besides the effect of increased sample capacity on column performance, gradient columns may also show increased performance because of improved flow characteristics of the carrier gas. Because of compressibility, the flow rate and linear velocity of the carrier are not uniform along the column length. The linear velocity is considerably lower a t the front section of the column compared to the average value for the entire column length; this permits the use of larger liquid loadings without a significant increase in the liquid mass transfer term. A gradient distribution of liquid phase also provides the incidental advantage of reducing the sample-overloading effect that may normally damage the packing at the front end of the column (7-9). On the negative side, it should be explained that the advantage of gradient loading columns should be limited to low and intermediate partition ratios (2). As the peaks pass from the high- to the low-liquid loading, the front of the peak moves faster than the tail. This results in broader peaks (lower efficiency). At the same time, the peaks encounter a decreasing liquid mass transfer term (higher efficiency). The latter effect is dominant for peaks of low and intermediate retention, while the former effect is dominant for higher partition ratios. The preparation of gradient-loaded columns made with a continuous variation of loading is unlikely to be practical. Even columns of multistep stages may be impractical for widespread acceptance. It seemed possible, however, that the advantages of gradient loading would be largely retained if the number of stages is reduced to two, the dual-load column. A dual-load column was tested by Stewart and Keller (IO), who found that within the precision of the experiment the plate height was independent of gradient direction (positive or negative). Column Design Considerations. There are apparently many parameters, some possibly critical, that enter in the design and testing of dual-load columns. The length of dual-load columns is divided into two sections, each containing a packing of different liquid load, the higher load being in the front section of the column length. Among the important parameters to be considered are total liquid load (front and back), ratio of loads, and ratio of liquid concentrations, while other parameters such as sample composition, column temperature, column dimensions, choice of the liquid phase, and solid support are less important. Once the ratio of the loads and concentrations are selected, the ratio of the two column lengths is fixed. That there are many different ratios or combinations of lengths and liquids concentrations that produce the same gradient is shown in Table I where a particular linear gradient column equivalent to a 6-ft 5% nongradient column corresponds to three different combinations of lengths and
(1) H. Purnell. "Gas Chromatography,'' Wiley, New York, N.Y., 1963, p 388. (2) D.C. Locke and C. E. Meloan, A n a / . Chem., 36,2234 (1964). (3) T. Bunting and C. E. Meloan, A n a / . Chem., 42, 586 (1970) (4) R. C. Duty, J Gas Chromatogr.. 6, 193 (1968). (5) A . 8.Christophe, J. Chromatogr.. 58, 195 (1971).
(6) J. Kwok. L. R. Synder, and J. C. Sternberg, Ana/ Chem., 40, 118 (1968). (7) I . R. Hunter and M .K. Walden. A n a l Chem., 35, 1765 (1963). (8) L. Mikkelsen, F. J. Debbrecht. and A. J. Martin, J. Gas Chromatogr.. 4, 263 (1966). (9) A . B. Christophe, J . Chromatogr. Sci., 8, 614 (1970). (10) G. H. Stewart and R . A. Keller, J . Chromatogr., 12, 150 (1963).
The increased performance of gradient-load columns, in which the liquid phase has a linear or exponential gradient distribution, can be achieved to a large extent by dual-load columns in which the front section contains a packing of about twice the liquid concentration as the back section. Column performance, determined as the number of effective plates per second, is up. to 30% higher compared to equivalent nongradient (single-load) columns. In trace analysis, where the injected sample volume is necessarily large, the performance can be up to 8 0 % higher. The improvement levels off at high partition ratios ( k E 20), mainly because of the increased retention times rather than decreased column efficiency. For several reasons, the average overall concentration of liquid phase for dual-load columns is limited to between 3 and 7%.
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
Table I. Equivalent 6-Foot Columns Front length, ft X % Type 3 x 5 2.5 X 6 2 x 7.5 1 X 15
Nongrad ient Dual-Load Dual- Load Dual-Load ~
Back length, f t X 96 3 x 5 3.5 X 4.29 4 x 3.75 5 x 3
Table I I I. Analytical Samples Compd
~~~~
Table II. Dual- and Single-Load Columns Load distribution,ft
X %
1-Pentanola
Av %
Designation A B C D E F G H I
J R5 R7 R3.33
liquid phase
Front
Back
5.0 5.0 5.0 5.0 5.0 7.0 7.0 7.0 3.33 3.33 5.0 7.0 3.33
1 X 15.0 2 x 7.5 2 x 10.0 1.33 X 15.0 1 x 10.0 2 x 10.0 2 X 8.0 3 X 8.0 2 X 5.0 1 X 5.0 3 X 5.0 3 X 7.0 3 x 3.33
5 X 3.0 4 x 3.75 4 X 2.5 4.67 X 2.14 5 X 4.0 4 x 5.5 4 X 6.5 3 X 6.0 4 X 2.5 5 X 2.5 3 X 5.0 3 X 7.0 3 x 3.33
concentrations. T h e g r a d i e n t in a l l three d u a l - l o a d colu m n s i s t h e same; t h e f r o n t a n d b a c k section each c o n t a i n 50% o f t h e t o t a l liquid phase. A t t h e same t i m e , t h e r a t i o s o f section l e n g t h s a n d percentages of liquid phase are different. F o r separating a p a r t i c u l a r sample, one of these c o l u m n s m a y b e b e t t e r t h a n t h e others.
EXPERIMENTAL Apparatus. A Model 69-700 Gow-Mac instrument was used for all measurements. The injector and detector temperatures were maintained at 180 "C. All measurements were made with 1-mV McKee-Pedersen Instruments recorder, Model 1027. The chart speed was 0.5 in./min except in the case of very short retention times (such as for that of the nonretained component, methane) in which case the chart speed was 2 in./min. Flow rates were measured with flow meters (Brooks Instrument Company, Inc., Hatfield. Pa. 19440) which were calibrated with a soap bubble flow meter a t the column outlet. Column outlet pressures were always atmospheric. Column Dimensions. The column dimensions selected are 6-ft x ?b-in. 0.d. as these are practical and typical; furthermore, the l/g-in. rather than 1/4-in. 0.d. is particularly suitable because it is more likely to demonstrate the results of sample overloading. Stainless steel tubing (0.093-in. i.d.) was used without any pretreatment as supplied by Column Technology, Inc., Horseheads, N.Y. 14845. Solid Support Since column performance depends heavily on the solid support, the same lot of 60-80 mesh, High Performance Chromosorb-W (Supelco, Inc., Supelco Park, Bellefonte, Pa. 16823) was used throughout this study. The support was coated by the slurry technique since this is both convenient and at the same time probably the most widely used method of preparing coated packings. Liquid Phase. Carbowax 20M was selected because of its widespread popularity and suitability as a liquid phase. The total per column amount of liquid phase used ranged from 65 t o 137 mg. For equivalent nongradient columns, this range corresponds to packings of 3.33 to 7.00% liquid load which is representative of current practice. These limits are also determined, respectively, by the appearance of peak tailing and the reduction in column efficiency. T h a t is, for gradient (dual-load) columns, these percentages result in packings of as low as 2.15% and as high as 15% depending on the gradient selected. Thus the 3.33% low limit and the 7.00% high limit pertain to the average load of gradient columns. The selected liquid loads ranged up to 15% in order to determine whether the dual-load effect extends to the higher liquid loads where sample-overloading is less significant. Even a t higher column temperatures which result in lower k values, there may be
Partition ratio, k
%
Sample No. 1, 0.05 pl 2-Methyl-2-propanol 20 2-Methyl-1 -propanol 20 3-Pentanol 20 3-Methyl-I -butanoln 20 l-pentanolb 20
1-Hexanol 1-0ctanoP
Sample No. 2, 0.05 pI 33.3 33.3 33.3
Sample No. 3, 1 pI 3-Methyl-I -butanola 0.02 Chloroform 99.98 Sample No. 4, 1 ~l 0.02 l-Hexanola Chloroform 99.98
0.9-1.8 2.7-5.6 2.9-6.0 5.3-1 1.2 7.5-15.8 2.9-6.0 6.0-12.6 23.8-126 5.3-1 I .2 , . .
6-12.6 , . .
Peak used for calculating column performance. Peak used for estimating sample capacity. a liquid load above which there is no advantage accruing from the gradient effect. Accordingly, several columns were made with different gradients, total liquid loads, and ratios of per cent of the liquid phase (front to back section). The columns are listed in Table 11. The three columns, designated R5, R7, and R3.33 are the nongradient (single-load) columns which are equivalent to the gradient (dualload) columns and are thus used as reference standards of column performance. Because of subtle inadvertant variations in the slurry technique used in this work, it was necessary to make some columns in duplicate or triplicate and only the best one of these replicates was used in measuring column performance. In some cases, the front section of a reference (single-bed) column was unpacked and then filled with the higher liquid load packing, thus maintaining most variables constant and thereby increasing the validity of column comparison. Reagents. Four different analytical samples were used, composed of lower alcohols selected for their k values (Table 111).Depending on the total liquid load of the column and the column temperature (60 to 75 "C). these k values ranged from 0.9 for 2methyl-2-propanol to 126 for octanol and were thus suitable for testing the dual-loading columns over a relatively wide partition range and determining the upper limits of k beyond which the dual-loading columns do not offer an advantage. Two of the samples (No. 3 and S o . 4), one of low k and the other of high k , were designed to test the dual-load columns in trace analysis where the volume of injected sample must necessarily be large. Under this condition, the dual-loading column is expected to offer a n increased advantage. Procedure. After conditioning, each dual-load column was tested for column performance and sample capacity by comparison with a corresponding reference column. Column performance was calculated as the number of effective plates per second ( 1 1 )
where Neff
E
N [ rk k ] 2
and N = number of theoretical plates and k = partition ratio or capacity factor. N was calculated at optimum flow rate, usually 20 to 30 cm3/min. Sample capacity was determined by injecting up to 1 ~1 of sample and measuring the effect on plate number and on peak shape. Peak shapes were determined as symmetrical, slightly symmetrical, or unsymmetrical depending on the symmetry index as described by Kennedy and Knox ( 1 2 ) . ( 1 1 ) B. L . Karger, "Modern Practice of Liquid Chromatography, J. J. Kirkland, E d . , Wiley-Interscience, New Y o r k . N . Y . , 1971. pp 1 7 and 36. (12)
Gordon
J.
Kennedy and John t i K n o x .
J. Chromatogr. S o . . 1 0 , 549
(1972).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
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Table IV. Effect of Dual-Load as a Function of Total Load, Load Ratio, and Concentration Ratio
Table VI. Dual-Load Column Performance in Trace Analysisa
Ratio Column
AV Yo liquid phase
5.0 5.0 5.0 5.0
A 0
C D E F
G H I
J R5 R7
R3.33
5.0 7.0 7.0 7.0 3.33 3.33 5.0 7.0 3.33
Column performance for
Load
Concn
Column performance
1 1 1 2 0.5 0.9 0.9 1.3 1 0.4 1 1
5 2 4 7 2.5 1.8 1.2 1.3 2 2 1 1 1
5.83 6.52a 5.72 4.80 6.31 8.24O 4.91 5.35 1 1 .OOb 1 1 .32b 5.52 6.86 8.65
1
Column
6-ft, 3.33% (reference) 0.5-ft, 7.5% 5.5-ft, 3.33% 6-ft, 7% (reference) 1-ft, 1 1 % f 5-ft, 7 %
+
Peak shaDe Column A
B
D E Table V. Dual-Load Column Performance as a Function of k
R5
F
Column performance for kin
+ (4-ft x 3.75%)
9.52' 7.46
(6-ft X 5%) 2-Methyl-1-propanol, k = 4.2-4.4. increase in column performance.
G
k2'
H
3.67 3.84
1-Hexanol, k = 25-28.
R7 I
A N A L Y T I C A L CHEMISTRY, VOL.
R3
27% a
RESULTS AND DISCUSSION As shown in Tables IV through VII, in all cases where k is not too high (above 20), and the load-ratio (front to back) is about 2, there is a significant increase in column performance of dual-load over the corresponding singleload reference column. Since the column length is normally fixed, the increase in column performance means shorter analysis times (11) and in the case of trace analysis, increased sample capacity and consequently increased sensitivity. Flatness of Van Deemter Curves. Since the carrier gas velocity is lower a t the high-load front section of the column, dual-load columns are expected to have a flatter Van Deemter curve. However, such an improvement over single-load columns was not discerned in this work, possibly because the effect is smaller than the experimental errors in preparing the packings and determining the Van Deemter points. The curves are relatively flat for both column types, dual- and single-load, and the optimum flow rate was between 20 and 30 ml/min. Total Liquid Load, Load Ratio, and Concentration Ratio. The column performance values for 13 columns of various total loads, load-ratios, and concentration ratios are listed in Table IV from which the following conclusions may be drawn. (a) Column performance increases up to nearly 30% for low-total liquid loads and up to 20% for high-liquid loads. (b) The best column performances are obtained with load ratios near unity or less, i.e., with no more than half the total load in the front section. (c) The best column performances are obtained with concentration ratios near 2 , i.e., with the concentrations of liquid phase in the front equal to about twice the concentration in the back section of the column. Partition Ratio. For k values above 20, a dual-load is not better than a single-load column (Table V). In fact, 1266
7.02 6.88 5.12 5.81
Table VII. Sample Capacity
C
Column
k z 6
5.41 8.2!jb 4.30 7.70'
1-pl sample of 0.02% component in a chloroform solvent. 52% increase in column performance. 80% increase in column performance.
a Represents approximately 20% increase in column performance. Represents approximately 30% increase in column performance.
(2-ft x 7.5%)
k - 2
45, NO. 7, JUNE 1973
Na
1-Pentanol
700 1,320 1,020 690 960 880 1,170 1,160 1,170 1,040 730 500
SI unsyrn SI unsyrn SI unsyrn SI unsyrn SI unsyrn Unsyrn SI unsyrn SI unsyrn SI unsyrn Unsyrn SI unsyrn SI unsyrn
Plate number for 3-Methyl-1-butanol
there is a decrease in column performance shown for 1hexanol ( k N 26) while there is a 27% increase for 2methyl-1-propanol ( k N= 4.3). This decrease results mainly from the increased analysis time rather than from the plate number, which for both compounds was approximately 1,800 (1-mm plate height for 6-ft columns). Trace Analysis. In trace analysis wherein the volume of injected sample is necessarily large, the dual-load columns are superior to the single-load columns, but only for low k values (Table VI). In fact, the dual-load advantage falls off rapidly as k increases beyond 4. Fortunately, in biochemical and pharmaceutical trace analysis, the chromatographic conditions are often easily adjustable to low k values which makes dual-load columns particularly suitable for such applications. An additional advantage of dual-load columns in pharmaceutical analysis comes from the fact that pharmaceuticals are likely to show peak-tailing due to adsorption occurring mainly a t the head of the column. This effect would be reduced in the dual-load columns. Sample Capacity. Values of sample capacity which are listed in Table VI1 were determined by injecting 0.25 p1 of sample No. 1 and measuring the plate number of the fourth component (3-methyl-1-butanol). This sample volume was selected arbitrarily; other injection volumes ranging from 0.1 to 1.0 p1 gave equivalent results. However, the 0 . 2 5 ~ 1volume resulted in peak shapes for the fourth and fifth components that were particularly sensitive to column design and this was useful in further estimating sample capacity. Three conclusions may be drawn from Table VII. First, the plate numbers of all dual-load columns are significantly higher than that of the corresponding reference columns. Also, the peak shapes are more symmetrical. Second, the increase in capacity is about 50% for the low- or medium-load columns and only 10% for the high-load columns. Third, the largest increase is found when the ratio
Table V I I I . Effect of Increased Load at Column Front Column
Column performance
Modified R3.33a Original R3.33 Modified R7b Original R7
11.21 8.65 7.62 6.86
Increase in column performance, YO
30 11
a 6-in. of front packing (3.330/0) replaced with 7.5% packing. of front packing (7%) replaced with 11% packing.
12-in.
of liquid loads is near 2, a condition that limits the liquid load a t the front section to about 8% where the liquid mass resistance term is apparently not very high. All three observations are consistent with the plate theory of column efficiency and may be applicable to columns of larger diameter if to a smaller extent. Dual-Load from Single-Load Columns. Because of inadvertant and subtle variations in column packings, it
was sometimes necessary to remake columns to improve the reliability of column comparison. This reliability is further improved by converting single-load reference columns to dual-load through replacement of the packing a t the front with a higher load packing. This procedure is useful not only because it is simple but also because it rejuvenates the column by replacing the portion of the column packing that is most likely to be damaged even from normal use (7-9). As shown in Table VIII, typical single-load columns may be upgraded by as much as 30% in column performance by simply replacing the packing in the front 1 or 2 f t with a packing of about double the per cent of the liquid phase. The only requirements are that the partition ratios should not be high and that the original column packing contain no more than 7 to 10%of liquid phase. Received for review November 20, 1972. Accepted January 22, 1973.
NOTES
Chelometric Titrations of Metal Cations Using the Tungsten Bronze Electrode M. A. Wechter, P. B. Hahn, G. M. Ebert, P. R. Montoya, and A. F. Voigt Ames LahDratory-USA EC and the Department of Chemistry, Iowa State University, Ames, Iowa 50010
Metal tungsten bronzes, nonstoichiometric compounds of formula M,W03, have been found to function as indicating electrodes in a number of electrochemical systems ( I , 2 ) . Recent investigations in this laboratory demonstrate the utility of these electrodes as indicating electrodes in chelometric titrations with EDTA. Several potentiometric methods have been devised for end-point detection in chelometric titrations. One technique employs a mercury indicating electrode to detect the Hg(I1) displaced from an Hg(I1)-EDTA indicator by the metal being titrated ( 3 ) .Similarly, Cu(I1)-EDTA and a copper ion selective electrode have been used for the determination of several metals ( 4 ) . Other methods require back titrations or the use of auxiliary complexing reactions (5). The tungsten bronze electrode does not require the presence of an indicator ion or other auxiliary reaction. For the direct titrations of the divalent ions of Cu, Ni, Co, Mn, Pb, Zn, Ca, and Mg and for the back titration of Al(II1) it is necessary only that the solution be basic. Some ions, Fe(II1) for example, can be titrated in acid solution provided another oxidation state of the metal is present as an impurity in the solution. (1) M. A. Wechter, H. R. Shanks, G. Carter, G. M. Ebert, R. Guglielmino. and A. F. Voigt, Anal. Chem., 44, 850 (1972). (2) P. B. Hahn, M. A . Wechter, D. C. Johnson, and A . F. Voigt, Anal. Chem., 45, 1016 (1973). ( 3 ) A . I. Vogel, "Quantitative Inorganic Analysis," Wiley, New York, N.Y.. 1961, pp 959 ff. (4) E. W. Baumann and R. M. Wallace, Anal. Chem., 41, 2072 (1969). (5) F. Oehme and L. Dolezalova, Z. Anat. Chem., 251, 1 (1970).
EXPERIMENTAL Apparatus. Measurements of the potential between a bronze and a saturated calomel electrode were made with a Beckman "Zeromatic SS-3" p H meter. The tungsten bronze crystals used were sodium tungsten bronzes, NaxW03, with x = 0.65. Individual single crystals, 4 X 4 x 2 mm, were cut from a larger crystal with a diamond saw and then annealed a t 650°C for 48 hr to achieve a greater degree of homogeneity. Details of the single crystal growth and of the electrode fabrication are available elsewhere ( I , 6). Reagents. Solutions of Al(III), Zn(II), and Cu(I1) were made by dissolving the metals in a minimum volume of 6N "03 and diluting to 250 ml. The other ionic solutions were prepared by the dissolution of suitable reagent grade crystalline salts (Baker), Reagent grade disodium EDTA (Baker) was used as the titrant. Procedure. The general procedure was as follows. Twenty-five milliliters of the 0.1M metal ion solution was diluted to 150 ml and buffered to p H 10 with N H B / N H ~ C [I N H 4 N 0 3 in the case of Pb(II)]. The solutions were stirred magnetically and titrated potentiometrically with standard 0.1M EDTA solution. The procedure was modified slightly in the titrations of Al(I1Ij and Fe(II1). For the aluminum determination, a back titration was necessary. Excess EDTA was added to the solution containing the metal ion. The solution was boiled, cooled. buffered to p H 10 and rapidly titrated with Ca(I1). In the case of Fe(II1). the solution was not buffered and remained acidic. Calcium and magnesium were titrated in the same solution, made u p by mixing 15 ml of each 0.1M solution and diluting to 150 ml. The Mg(0H)Z was completely precipitated by adding 8M KaOH, t h e resulting solution was titrated for Ca(IIj, and a n additional 5 ml of titrant was added. The precipitate was dissolved (6) H. R. Shanks,J. Cryst. Growth, 13-14, 433 (1972).
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