The Sample as Its Own Stationary Phase in Gas Chromatography D. R. Deans I.C.I. Ltd., Petrochemicals Division, Billingham, Teesside, U.K. Experimental results are presented to show that overloading the column with sample can produce a phenomenon, at the inlet of the column, in which the sample acts as the stationary phase for a sample component eluted after the major component. The mechanism is briefly explained in mathematical terms. The effect is that some of the trace component accumulates on the back edge of the major component to produce the visible peak, but an appreciable amount spreads unsymmetrically forward under the major component peak. Normal measurement of the trace peak area can give results that seriously underestimate the quantity of trace component in the sample. Use can be made of this phenomenon to improve detection limits in trace analysis. By grossly overloading the first of two columns in series and heart cutting the peak of the trace component onto the second column, it is possible to obtain a separated peak for the trace component.
IT IS COMMON PRACTICE, in gas chromatography, to overload a column with sample to be able to measure trace components satisfactorily. When the column is overloaded, it is noticeable that the major peaks broaden but the peaks for trace components eluted after a major peak do not, although their retention time is normally increased. Trace components eluted before the major peaks tend to broaden and, if anything, their retention time decreases with increasing sample size ( I ) . The normal useful limit on increasing sample size is set by the spreading of the major peak over the trace components of interest or by the broadening of the rapidly eluted trace components. The heart cutting technique described by the author ( 2 ) has been used successfully to overcome the problems associated with spreading of the major peak. The aim of the work described in this paper was to postulate a mechanism for the phenomena described and to investigate the practical implications and limitations of overloading analytical columns. EXPERIMENTAL
To follow the peak of an unresolved trace component in, the presence of a gross overload of the major component requires a detector with a very high degree of selectivity as well as sufficient sensitivity. Many of the conventionally selective detectors, when used under these conditions, give a significant signal for the major component or suffer from other troubles. A flame ionization detector with water as the major component was chosen for the investigation, because, with certain precautions, it is possible to obtain a negligible signal for water. Carbitol (diethylene glycol monoethyl ether) which is completely miscible with water was used as the minor. A hot wire detector was used to follow the combined peak with the flame ionization detector in series to follow the carbitol peak. Excess water in the carrier gas going to a flame ionization detector can give negative signals and other problems associated with electrical shorting in the detector, so a continuous supply of diluent carrier gas was added to the effluent from (1) V. G. Berezkin, V. S. Tatarinskii, and L. L. Starbinets, Zh. Anal. Khim., 24, 600-4 (1969). (2) D. R. Deans, Chromatographia, 1/2,18-22 (1968). 2026
the column using the flow system shown in Figure 1. Inlet pressure control was used to keep the flow through the column constant when the sample was injected. Flow control to provide a constant flow of gas to the column was avoided because when a large sample is injected, the inlet pressure rises as the flow through the column increases to accommodate the volume of vapor from the sample. Preliminary ideas about the mechanism suggested that the introduction and vaporization of the sample played an important role. To investigate the contribution of the sample introduction and vaporization Teflon 6 (Du Pont) was used as a packing without adding stationary phase. That the packing is effectively inert in these experiments is demonstrated by the fact that a small sample of carbitol has the same retention time as methane. Scrupulous care was taken to ensure the cleanliness of all gases, the column, and the detector system, to avoid the elution of contaminant peaks when pure water was injected. The following chromatographic conditions were used for most of the work described in this paper. Some investigations of the effect of temperature and flow changes were made that are not described in detail here: Column, 40 cm X 2.4 mm i.d. stainless steel; packing, 30-60 mesh Teflon 6 (ex Phasep, 1.6 grams, 7.0 m*/gram); carrier gas, argon; inlet pressure, 27 psig; exit pressure, 20 psig; carrier velocity, 30 cm/sec; column temperature, 110 “C; and injection, on-column. Samples of pure carbitol, pure water, and 0.1 carbitol in water were injected. The sample size for carbitol was 0.2 pl, and for water and the solution of carbitol in water it was varied from 1 to 100 pl, Examples of the chromatograms obtained from the flame ionization detector are shown in Figures 2, 3, and 4, the corresponding chromatogram from the hot wire detector is shown superimposed, where appropriate. The chromatograms show that during the relatively symmetrical elution of water, which is not retarded by the column, carbitol is not eluted symmetrically, the major part of it is eluted as a peak on the back edge of the water peak and some of it is eluted at a steady level with the water. The peak for carbitol always coincided with the back edge of the water irrespective of the sample size and flow rate. The temperature affected the relative amounts of carbitol in the peak to that in the “escape zone” but, providing the temperature was below the boiling point of water at the inlet conditions, the same general shape was obtained. Confirmation that the zone under the water peak is due to carbitol is obtained from the fact that the total area is the same as the area obtained for the carbitol when it is all eluted as one peak, as described under Suggested Mechanism and shown in Figure 5 . SUGGESTED MECHANISM
When the liquid sample is introduced to the column, it spreads rapidly to produce a slug of liquid covering the support, through which the carrier gas can flow. The length of the slug will depend on the sample size, viscosity, and surface tension and on the column packing surface area, gas flow rate, etc. One of the main spreading forces will be the flow of carrier gas forcing paths through the sample. Consider a homogeneous sample of a major component in which a trace of minor component is dissolved. The major component is the more volatile of the two. If the column is
ANALYTICAL CHEMISTRY, VOL. 43, NO. 14, DECEMBER 1 9 7 1
COLUMN
Figure 1. Flow system used for most experimental work HOT WIRE DETECTOR
0
F.I.D.
held at a temperature below the boiling point of the major component, the sample will vaporize steadily into the carrier gas. Vaporization of the major component will take place only from the beginning of the slug, because as soon as the carrier gas is saturated no further vaporization can take place. The rate of vaporization will be proportional to its vapor pressure and the gas flow rate. If the sample obeys Raoult’s law, then the concentration of major component in the gas phase will be given by the following formula (deviation from Raoult’s law will not affect the mechanism suggested, but the concentrations calculated by the formulas would need correcting for the deviation).
Figure 3. Chromatogram of 40 pl water Hot wire detector response dotted
and the rate of removal in the gas stream will be
where the density of major component vapor in contact with pure liquid, grams/ml A = the quantity of major in the sample, grams B = the quantity of minor in the sample, grams mamb = molecular weights of the major and minor components, respectively Fi = gas flow rate, ml/min Da
=
If B is very small with respect to A , this approaches FiDa
gram/min
(3)
The time for A grams of major to vaporize will be AIFiDa
min
(4)
The minor component will vaporize from the slug of sample. As the gas flow carries the minor component over the slug, the liquid will act as a stationary phase. The movement of the band of minor component, which will be in equilibrium between the gas and liquid phase along the whole length of the slug, will be governed by normal gas chromatographic parameters. The rate of movement will be independent of concentration, providing this is low, and dependent only on the partition coefficient K , the phase ratio p, and the flow rate Fi. The concentration of minor component in the sample as injected will be B A + B
’
t
I--
Figure 2. Chromatogram of 0.2 pl carbitol
PL
gramiml
where P L is the density of the sample. The concentration at equilibrium of the minor component in the gas phase will be ANALYTICAL CHEMISTRY, VOL. 43, NO. 14, DECEMBER 1971
2027
-
...........
.
Figure 5. Chromatogram of 20 ~ 1 0 . 1 %carbitol in water plus 20 p1water
Figure 4. Chromatogram of 40 PI 0.05 carbitol in water
BPL ~.~ A+B
gram/ml
K+P)
If B is very small with respect to A , this approaches
The rate of removal of minor over the outlet edge of the slugLe., the rate of “escape” of minor will be BPL A(K
+ P) . Fi
gram/min
During the time of vaporization of the major component given in Equation 4 BpL _ . Fi~ A ( K P)
+
.
Agram DaFi (9)
of minor will have escaped, and (l
- ( K JLp)Da)
gram
will have been left behind. As the vaporizing edge of the slug moves along the column, the minor component accumulates in this edge as a narrow band. In other words, the rate at which the vaporizing edge of the slug moves along the column, toward the stationary outlet edge, is faster than that at which the minor component moves through the slug by chromatography. This will only apply when 2028
PL
(K
+ PWa is less than
[
one which is the
criterion for a peak to accumulate the back edge of a major component. It is clear that the phenomenon is independent of flow rate within the limits of normal chromatography, Equations 9 and 10; it is also apparent that the ratio of the amount of minor collected on the vaporizing edge to the amount that escapes is independent of both flow and initial concentration in the sample. Hence, the size of the peak accumulated on the tail of the major will be proportional to the concentration in the sample and to the sample size. If this mechanism is correct, it can be predicted that the escape of the minor component could be prevented by introducing a suitably sized additional slug of pure major in front of the sample slug. The front of the minor component escaping from the sample would enter the slug of major component and the vaporizing back edge of the major would catch up with this front before it reached the outlet edge of the pure major slug. This effect is shown in Figure 5 . The accumulation of the peak on the back edge of the major requires a number of theoretical stages (plates) to prevent the peak from spreading forward into the major peak. These plates are provided in the normal manner of a liquid phase spread over a support. The extension of the mechanism to columns containing normal stationary phase is straight forward. The effective stationary phase in the sample slug zone becomes a mixture of the normal stationary phase and the sample. The minor component on the back edge of the major will get further retarded after the vaporization zone by the normal effect of stationary phase. It is not necessary to assume that the column temperature is below the boiling point of the major component for the mechanism to operate when a stationary phase is present. Nor is it necessary to use on-column liquid injection to obtain the phenomenon. A sample injected through a vaporizer
ANALYTICAL CHEMISTRY, VOL. 43, NO. 14, DECEMBER 1971
Table I. Retention Times in Seconds of 100 ppm in N-Hexane Column, 1 m,7.5% Apiezon L, 10 cm/sec, 100 "C Vol injected, ~1 n-Octane n-Nonane n-Decane 625 1206 1 341 630 1215 10 343 648 1231 50 366 100 393 674 1259
&
'
-
"
Figure 6. Chromatogram of 400 MI 0.04 ppm 1,2-dimethylcyclohexane in isooctane Heart cutting between two 3-m10
APL column
will condense and dissolve at the beginning of the column to form the liquid sample slug. The viscosity of the vaporized sample is likely to be considerably less than that of the carrier gas. As it forms a significant fraction of the gas in the column, the gas velocity for a given inlet pressure will increase. Thus there are two factors that can have a major effect on the retention time of a minor component on the tail of a major. The retention time is shortened because of the increase in flow rate and it is lengthened because of the time delay of vaporization before it starts to move normally down the column. For peaks eluted before the major peak, spreading due to the length of injection slug occurs, together with speeding up due to the change in viscosity of the carrier gas. PRACTICAL IMPLICATIONS Quantitative Analysis. The measurement of the area of a peak on the tail of an overloaded major component, using the usual technique of interpolating the tail of the major peak as the base line for the minor, can give rise to serious errors. However, it should be possible to calibrate with
known synthetic mixtures of the same major and minor components to obtain an artificial response factor, for a given sample size and set of chromatographic conditions, that will give accurate analysis. The use of literature response factors or those determined under conditions not identical with the actual analysis could produce quantitative results seriously underestimating the quantity of minor component. Qualitative Analysis. The use of relative retention times of minor components, in the presence of major components, for identification purposes can give rise to errors, particularly when the major component concentration is not negligible and the reference retention times were not determined in the presence of the major component. Table I shows the change in retention time with sample size. Trace Analysis. The height of the trace component peak under the overload conditions described is proportional to the concentration in the sample and to the sample size. It should, therefore, be possible to improve the limits of detection by increasing the sample size. The ultimate limit will occur when the column becomes completely saturated with liquid sample. Unfortunately under these conditions the trace component peak is hidden in the tail of the major peak. It is comparatively easy to overcome this by the use of two columns in series in such a way that the effluent from the overloaded first column is diverted to atmosphere except during the period when the peak of the desired trace component is being eluted. The peak of the trace component, together with the tail of the major component associated with it, is passed into the second column. Separation on the second column of the trace component from the relatively small amount of major component is easy because this column is not overloaded. A simple method of performing this technique of heart cutting is described in Reference 2. Figure 6 shows a chromatogram of 0.04 ppm of 1,2dimethylcyclohexane in isooctane obtained using heart cutting between two 2.4 mm i.d. 10% Apiezon L columns 3 m each in length at 100 "C. The sample size was 400 111.
RECEIVED for review July 8, 1971. Accepted August 16, 1971.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 14, DECEMBER 1971
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