Large Diameter Porous Layer Open Tubular Gas Chromatography Columns John G. Nikelly Department of Chemistry, Philadelphia College of Pharmacy and Science, Philadelphia, Pa. 79 104
Large diameter, 0.04-in. i.d., porous-layer open-tubular (PLOT) columns, made by the dynamic coating procedure, are compared with similar columns of smaller Ld., 0.03 in. As expected, the large i.d. columns have somewhat lower efficiency but are better than the 0.03-in. i.d. columns in terms of performance expressed as effective plates per second, sample Capacity, and tolerance to extra-column volume effects. These advantages are due to the larger surface area, intra-column volume and cross-section (permeability or lower pressure-drop in the dynamic coating procedure), which allow an increased thickness of the porous layer (decreased phase ratio, p ) and, consequently, increased capacity factor, k‘.
where C is the v/v% concentration of the solution used, r is the column tubing radius, u is the average velocity of the liquid plug, 7 is the viscosity of the liquid, and u is its surface tension; of course, the intra-column area (geometric area of cylinder wall), a , increases linearly with the i.d. In short, as i.d. is increased, sample capacity and capacity factor should increase while extra-column volume effects should decrease and the dynamic coating procedure should be simpler because of lower pressure drop. Alternatively, the lower pressure drop should permit the use of thicker coating mixtures ( v ) , and higher coating rates ( u ) which result in increased thickness ( h )of the porous layer (5,Ei).
EXPERIMENTAL Both types of open-tubular gas chromatography columns, support-coated and wall-coated, have low sample capacity, low capacity factors, are susceptible to extra-column volume effects, and are not as easily made as conventional, packed columns. These limitations are a t least partly due to the smallness of the inside diameter (i.d.) of these columns which usually ranges from 0.01 in. for the wall-coated (WCOT) to 0.03 in. for the porous-layer (PLOT) columns. I t follows that open tubular columns that are made with larger diameter tubing and which, therefore, have larger surface area, intracolumn volume and cross section (permeability), would therefore have larger sample capacity, less susceptibility to extra-column volume, and lower pressure drop. The advantage of low pressure drop in the dynamic coating procedure permits the use of thicker coating mixtures and, consequently, higher capacity factors, k’. Unfortunately, the larger i.d. columns are less efficient, i.e., have a smaller plate number. According to the Golay equation for WCOT columns ( 1 ) which was later modified for PLOT columns (21, the theoretical maximum value (at optimum carrier velocity) for column efficiency or plate number is approximately inversely linear with column i.d. I t was, therefore, interesting to determine whether an increase in i.d. could be practical and useful in the case of dynamically coated PLOT columns. The question is, whether a modest increase in i.d. is sufficient to increase the sample capacity and the capacity factor, reduce the effect of extracolumn volume, and make it easier to prepare PLOT columns by the dynamic coating method, without a significant reduction in column efficiency. A factor that may favor this question is the fact that while the intra-column geometric area increases linearly with i.d., both the volume and cross section increase exponentially, Le., a modest increase from 0.03-in. to 0.04-in. i.d. nearly doubles the volume and cross section. In order to prevent a significant reduction in the column efficiency of PLOT columns, it is important that the increase in sample capacity and capacity factor be achieved by increasing the thickness h and the area a of the porous layer. Both h and a increase linearly with i.d.: as shown by Guichon ( 3 )and verified experimentally by Bartle ( 4 ) ,the liquid film thickness of dynamically coated columns is proportional to r C h = -(uv/u)1’qr) (1) 100
Apparatus. PLOT columns in lengths from 30 to 50 feet were made from type 304 seamless stainless steel tubing supplied by Handy and Harman Tube Company, Norristown, Pa. 19404. (A convenient method of measuring capillary column lengths is by weight: regardless of origin or type of steel, the per cm. weight of all Y16-in. 0.d. tubing is consistently 0.24 and 0.094 g for 0.030-in. and 0.040-in. i.d. tubing, respectively.) All separations were made on a Gow-Mac gas chromatograph, Model 69-700 (Gow-Mac Instrument Company, Madison, N.J., 07940). The %-in. fittings were modified to take Y16-h. columns by using a t the inlet, a %- to %6-in. adapter (made locally) and, at the outlet, a Teflon reducing ferrule ( R F 200/100, Alltech Associates, Arlington Heights, Ill. SOOOS), which allowed the column to extend about 4 cm into the detector housing. With these changes, the extracolumn volume was less than 0.3 cm3, (see Extra-column Volume under Results and Discussion). Carrier flow rates were determined by dividing the calculated volume of the chromatographic system by the “retention” time of an unretained component (methane). Carrier flow rates were optimized to minimum plate height while the column temperature was judiciously adjusted (between 60 and SOOC),so as to time-normalize the retention of the peak in question t o about 3 min. Recordings were made with a 1-mV strip chart recorder, Model 1027 (McKee-Pedersen Instruments, Danville, Calif. 94526). The chart speed was 0.5 in./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./min. Reagents. The coating mixtures were prepared using, as solid support, Chromosorb R6470-1, a finely divided diatomaceous silica (Johns-Manville,Denver, Cola., 80217), which was used as received, Le., without pretreatment such as sizing or drying. Procedure. PLOT columns of 0.03- and 0.04-in. i.d. were made (coated) using the dynamic coating procedure (5, 6 ) . The columns were evaluated by determining the total weight of coating (weightgain) and the net weight of liquid phase (calculated from the ratio of liquid phase to solid support in the original coating mixture). The phase ratio, 0,was then calculated and used as one of the criteria for comparing columns. For columns of equal length, the area and volume of 0.04-in. columns are, respectively, 1.3 and 1.8times larger than that of 0.03-in. columns; consequently, in order that the 0.04-in. columns be at least as good as the 0.03 columns, it is necessary that the former have a considerably higher, possibly doubled, liquid phase load (wt per length) as the latter. Further evaluation consisted of comparing the 0.04-in. i.d. columns t o equal length 0.030-in. columns, using as criteria of comparison, the resolution of a “difficult” pair of components, plate heights (HETP) and column performance (7) defined as:
N (+)Z
t k + l where k‘ is the partition ratio or capacity factor and t is the retention ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
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D
1 4 -
t
a
z 1 2 L I-
1.0
-
CARRIER
FLOW
R A T E , ML/MIN
Figure 1. Plate height vs. carrier flow rate (A) 0.03 in., 5 % liquid phase concn. (6)0.03 in., 10% liquid phase concn. ( C ) 0.04 in.. 5 % liquid phase concn. (D) 0.04 in., 10% liquid phase concn.
Table I. Coating Characteristics of 50-ft PLOT Columns: Weight of Porous Layer and Phase Ratio
I
l
_ _ 1 1 1
. 0
Weight of porous layer, g. Concn. of liquid phase in coating suspension
.
.
.
2 RETENTION
. 4 0 2 TIME, MIN.
4
Figure 2. Separation of a difficult pair, 2-propanol and 2-methyl-2-
0.03-in. i.d.
0.04-in. i.d.
5% ov-17
0.27 0.29 0.27 (0 = 115)
0.66 0.60 0.71 (P = 85)
10%ov-17
0.44 0.35 0.40
0.83 0.80 0.71 ( P = 40)
( P = 44)
propanol Separation conditions: 304 PLOT columns coated with Carbowax 20 000; column temp. 60 O C ; 0.04-$1 sample volume; columns a and b. 0.04- and 0.03-in. i d , respectively
Table 11. Column Comparison, Effective Plates per Second
E sec (+)2k + 1 time in seconds. The values of N , t , and k' were based on the 2methyl-1-propanolpeak, one of four components of the test sample, consisting of lower alcohols ( 8 ) ,injected in volumes ranging from 0.02 to 0.2 pl using a Hamilton microsyringe or an inlet sample splitter made locally (9).
Column id., in.
k'- 2
0.03 0.04
21 22
k'
N
6
16 14
RESULTS AND DISCUSSIONS Weight of Porous Layer a n d Phase Ratio. The weight of the porous layer and liquid phase in the 0.04-in. columns is indeed consistently higher than that in the 0.03-in. columns. The comparison was made by using coating suspensions of the same concentration, 5 to 10 wt/vol % of liquid phase (OV-17 or Carbowax 20,000). Typical results are listed in Table I. As expected, the data show that the larger diameter columns can be coated with thicker porous layers and, consequently, higher liquid loads (weight of liquid phase per unit column length). The results are reproducible to better than 10%. The data in Table I also show that, despite the nearly doubied column volume, larger diameter columns can be made with a lower phase ratio, p. This is, of course, important as a lower 6 value means increased column capacity h' and increased capacity to handle a larger sample injection volume. C a r r i e r Flow Rate. Since the open tubular volume of 0.04-in. i.d. columns is nearly double that of 0.03-in. columns, it follows that the optimum (minimum H E T P ) carrier flow rate should be about 10 cm3/min. However, the optimum flow rate appears to be somewhat lower, near 8 cm3/min, not much higher than for 0.03-in. columns, Figure 1.This may arise from the fact that the porous layer of 0.04-in. columns being thicker than that of 0.03-in. columns, requires a longer diffusion time or lower linear flow rate. For the same reason (resistance to mass transfer in the liquid phase), Figure 1 also shows that the optimum flow rate 988
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(minimum HETP) depends on the phase ratio, the lower @ values (higher liquid loads) requiring lower flow rates for minimum HETP. Column Efficiency a n d Performance. As expected, the 0.04-in. columns have slightly lower efficiency or higher HETP compared to the 0.03-in. columns (Figure 1).The practical significance of this difference can be assessed by calculating and comparing the number of effective plates or, better, the number of effective plates per second (column performance) as shown in Table 11: compared on the basis of effective plates per second, there is found little, if any, difference between the two column diameters, i.e., if retention time and capacity are considered together with the plate number, the two column diameters are about equal in performance. The comparability of the two column diameters is further supported by looking a t the separation of a difficult pair of components on two Carbowax 20 000 PLOT columns, Figure 2. The two components, 2-propanol (bp 82 "C) and 2methyl-2-propanol (bp 83 "C) have a relative retention of 1.15; considering the low capacity factors (h' < 0.9), this is not an easy separation. There is, however, a 100%resolution on the 0.04-in. column because its lower efficiency is overcompensated by its higher capacity factor. Sample Capacity. Sample capacity was determined by measuring H E T P as a function of sample injection volume. As shown in Table 111, sample capacity for both the 0.03- and
Table 111. Column Comparison, Sample Capacity
HETP 0.03 in.
0.04 in.
Inj.vol.,pl
k’= 2
k’= 6
h’= 2
0.02
1.1 1.2
1.2 1.3
1.3 3.0
1.3 3.0 3.5
1.4 1.5 1.5 1.7
3.5
4.0
0.04 0.06 0.10
0.15 0.20
1.8
2.0 2.0
h’= 6
1.3 1.5
1.8 2.8 2.5 3.0
0.04-in. columns is limited to only few hundredths of a microliter. But a t injection volumes higher than 0.1 pl, the 0.04-in. columns are better in that their H E T P values are significantly lower than those of 0.03-in.. columns. That 0.04-in. columns have a significantly higher sample capacity was also shown by combining the PLOT columns with packed precolumns ( I O ) : in the case of 0.02- or 0.03-in. columns, there was an appreciable increase (doubling) of sample capacity while, in the case of the 0.04-in. columns, no increase in sample capacity was observed even for injection volumes as large as 0.1 pl. I t is interesting to note parenthetically that the larger increase in plate height found for late peaks (h’ N 6), may suggest that the capacity is limited by the liquid phase (liquid volume) rather than by the gas phase (column volume). I t follows that there may be very little, if any, advantage in further scaling-up the column i.d.; any further increase in column capacity would have to come from scaling-up the thickness of the porous layer. Extra-Column Volume. The extra-column volume of the instrument used for this work (Gow-Mac,model 69-700) was
determined experimentally by plotting the “retention” time of an unretained peak (methane) vs. the corresponding column volumes of lo-, 30-, and 50-ft capillary columns. The resulting straight line was extended to zero “retention” time and the extra-column volume was thus found to be 0.3 cm3. It has been shown that in order to prevent a significant decrease in the expected column efficiency, the extra-column volume of the chromatographic system should be no more than 5 to 10%of the intra-column volume (11).It follows that the efficiency of a typical PLOT column-0.02-in. i.d., 50-ft length, 3.1 cm3 intra-column volume-may decrease significantly if the extra-column volume is much larger than 0.1 cm3. On the other hand, by using a 0.04-in. i.d. column and operating at a higher flow rate, a much larger extra-column volume can be tolerated. Stated differently, this means that there is no advantage in using small bore PLOT columns with gc instruments that are not designed for open-tubular columns, while 0.04-in. columns may be used with about the same column efficiency as that obtained with instruments specifically designed for open tubular columns. LITERATURE CITED (1) M. J. E. Golay, “Gas Chromatography 1958”, D. H. Desty, Ed., Butterworths, London, 1958, pp 36-55. (2) M. J. E. Golay. Anal. Chem., 40, 382 (1968). 13) G. Guichon. J. Chromatoo. Sci.. 9. 512 (1971). ’ (4) K. D. Bartle, Anal. Chem: 45, 1831 (1973). (5) J. G. Nikelly, Anal. Chem., 45, 2280 (1973). (6) J. G. Nikeliy and M. Blumer, Am. Lab., 6, 12 (1974). (7) G. L. Karger, “Modern Practice of Liquid Chromatography”, J. J. Kirkland, Ed., Wiley-Interscience. New York, N.Y., 1971, pp 17 and 36. (8) J. G. Nikelly, Anal. Chem., 46, 290 (1974). (9) J. G. Nikeily, PhiladelphiaCollege of Pharmacy and Science, unpublished work, 1975. (10) J. G. Nikelly, Anal. Chem., 47, 168,(1975). (1 1) F. Baumann, “Capillary Columns”, Tech. Bull. No. 123-66, Varian Aerograph, Walnut Creek, Calif., 1966.
RECEIVEDfor review July 21, 1975. Accepted February 24, 1976
Thin Layer Chromatographic Method for Monitoring the Purity of S-2-(5-Am nopenty lamino)ethy Iphosphorothio ic Acid Robert S. Rozman Division of Medicinal Chemistry, Walter Reed Army Institute of Research, Washington, D.C. 200 12
A quick sensitive TLC method using water as the developing solvent and ninhydrin for visualization has been described. Quantitative separation of two aminoalkylaminoethylphosphorothioic acids from their respective dephosphorylated thiols and their respective symmetrical disulfides has been achieved.
The a-adrenergic blocking agent, S-2-(5-aminopentylamino)ethylphosphorothioic acid (WR 2823) ( I ) , has evoked much interest recently with reports that the drug protects animals such as dogs, monkeys, and sheep from otherwise lethal regimens of induced hemorrhagic shock ( 2 ) ,and endotoxic shock ( 3 ) .The intravenous use of WR 2823 for treatment of hemorrhagic shock is of sufficient therapeutic promise that a Notice of Claimed Investigational Exemption for a New Drug (No. 8345) has been filed with the Food and Drug Administration. Human Phase I intravenous tolerance studies have been successfully carried out ( 4 ) .
The degradation of WR 2823 has been shown to result in a symmetrical disulfide, possibly via either the mercaptan or other dephosphorylated intermediate. To facilitate further clinical investigation of WR 2823, a quick sensitive method for determining the purity of the drug and for estimating these products after aqueous solubilization was developed. This procedure was also used to compare the radioprotectant WR 2721 and its analogous degradation products. Molecular formulas are presented in Table I. Studies on the aqueous stability of WR 2823 were performed to determine time limits for use of the drug after solubilization of the sterile lyophylized powder. Analyses were carried out on freshly prepared aqueous solutions and on solutions aged a t room temperature (ca. 25 “C) for seven weeks. EXPERIMENTAL A procedure u t i l i z i n g thin layer chromatography (TLC) was developed t h a t effected r a p i d m u t u a l separation o f WR 2823, i t s deANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
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