Some Aspects of Liquid-Sol id Vacancy Chromatography R . P. W. Scott, C. G. Scott, and Paul Kucera Chemical Research Department, Hoffman-LaRoche Inc. Nutley, N.J. 07110 Vacancy chromatography, which has been used hitherto mainly in gas-solid chromatography and gasliquid chromatography, has been applied to the separation of nucleic acid bases on an ion exchange column using a liquid mobile phase, Vacancy chromatography with a liquid-solid system can be used to evaluate differences in composition between a circulating mobile phase and an injected sample and to allow a single elution peak of some selected component to be observed even though it would be eluted partially unresolved under nonvacancy chromatography conditions. Because an essential part of the procedure under discussion involved injection of quite large samples, a theory is developed which allows the change in retention volume and the broadening of the elution band to be calculated for the case where large volumes of sample are plug injected into the chromatographic column. The calculation makes use of the data obtained from a normal sized injection. The theory is applied successfully to 600-pl plugs of nucleic acid bases in buffer injected into a column in which the total void volume i s only 800 p l ,
THETERM “vacancy chromatography” introduced by Zhukhovitski et al. ( I ) is used in this paper to describe the chromatographic technique in which the mobile phase consists of a solvent containing a mixture of components which are maintained at constant concentration. Upon injection of a “sample,” the resulting chromatogram may show negative and/or positive peaks depending on whether the components detected in the sample are present in lesser or greater concentration than they are in the mobile phase. Vacancy chromatography has been expounded for application t o gas-liquid and gas-solid systems in the context of monitoring process streams (l-3). The procedure can be operated in several ways, for example when it is difficult to obtain an accurate analysis from a process stream because the composition is changed (either by loss of more volatile components or by adsorption of more polar ones) during the manipulation of sampling and/or injection, then, with suitably heated transfer lines, it is not difficult to transfer such a stream continuously to the chromatograph and monitor composition by injection of plugs of pure carrier gas to produce negative chromatograms displaying all the components. Alternatively, if sample disproportionation is not a problem, the stream can be monitored by injection of plugs of reference sample taken from some suitable container to produce chromatograms showing up only the differences between the stream and the reference (referred t o as the difference technique). It seemed to us that liquid-solid vacancy chromatography, in addition to process stream applications, offered some potential for monitoring reactions in the laboratory using a system in which a reference mixture is pumped continuously through the column with the column effluent being recycled. I n this mode, samples can be analyzed in terms of their (1) A. A. Zhukhovitski and N. M. Turkel’taub, Dokl. Akad. Nauk USSR.,143, 646 (1961). (2) A. A. Zhukhovitski, “Gas Chromatography 1964“ A. Goldup, Ed., Institute of Petroleum, London, 1965, p 161. (3) C. N. Reilley, G. P. Hildebrand, and J. W. Ashley, ANAL. CHEW., 34, 1198 (1962). 100
differences from the cycled reference. Conversely, the reaction mixture can be cycled and reference samples injected. Another aspect of the vacancy technique which does not appear to have been exploited is the ability to manipulate the conditions t o reveal a single peak for a component when under normal elution conditions, the peak would be partially overlapped by another. This application could have significance in liquid chromatography until such time as the degree of sophistication in detector sensitivity and column efficiency has reached the same level as is achieved in gas chromatography. This unmasking technique could also be useful in looking for trace impurities and some information was required on the effect of sample overload on columns operating in the vacancy mode. In this paper, the theory for sample overload is developed and experimentally verified for some nucleic acid bases chromatographed on a pellicular cation exchange resin. The feasibility of vacancy chromatography in liquid chromatography is discussed on the basis of the experimental work carried out in this study. THEORY
If mobile phase, carrying a constant concentration of solute X , is fed continuously onto a chromatographic column and equilibrium is allowed t o become established, the eluant from the column will also contain the solute at a concentration X , . If a sample of the same solute dissolved in the mobile phase at a concentration X I is now injected onto the column where X I 5 X , then this will produce a perturbation on the concentration X , and from the plate theory, the equation for this perturbation when sensed by the detector at the end of the column will be
x,
” - li’*;2N
=
(Xl - X J
: d 2 N
where X N is the concentration of solute in the mobile phase in the Nth plate, u is the volume flow of mobile phase measured in plate volumes, N is the number of theoretical plates and W = u - N . It is obvious from Equation 1 that if X l > X , , a positive peak will be produced and if X I < X , , a negative peak will be produced. If the sample injected is only mobile phase carrying no solute, then X I = 0 X,e - W’/2N x, = (2)
d2nN
the actual concentration X , measured at the end of the column will thus be
At the peak maximum X E =
L’ =
Xo(l
N; thus from Equation 3
- 1/d2~N)
It is seen from Equation 3 that under the condition considered by the plate theory where the charge is placed on the first plate only, X N can never be equal to X,and pure mobile
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
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2
25-
0
c
2
20-
I
Figure 1. Negative elution curves for different injection volumes for a column of 500 theoretical plates
._w 15-
! '"1 P
-injection volume measured in p l a t e volumes
c
u d
i
5
J,
I
0
Flow of mobile phase
phase is never eluted from the column. However, the sample is rarely injected on the first plate but is dissolved in a specific volume of mobile phase which, when injected onto the column occupies a finite number of theoretical plates. If (p) plate volumes of pure mobile phase are injected onto a column equilibrated with mobile phase having a concentration of X, of solute, then on the application of the charge of pure mobile phase Forp
1 The change in the concentration of solute on the first plate will be X , (e-l - 1) p = 2 The change in the concentration of solute on the first plate will be X , (e-* - 1) p = p The change in the concentration of solute on the first plate will be X , - 1) =
Consider the injection of p plate volumes of pure mobile phase followed by u plate volumes of equilibrated mobile phase having a solute concentration of X,. After r plate volumes of charge have been fed onto the column, the concentration of solute on the first plate will be X,(e-' - 1). This concentration will subsequently be developed by u p r plate volumes of mobile phase. Thus the perturbation in concentration of the solute X N in the Nth plate due to the rth volume of charge will be -T
XES =
xa +
r=V/w
e - (y/a,+
(X,l - XJ(1 - e-')
r = V/u,
s=q
XS f
- ,V)2/2N -
(5)
- Xd x
(x8*
r=l
+
- (g/ua P / u s - r - N ) 2 / 2 N
(1 -
p-
V / u , --T
Thus the equation for the chromatogram for q solutes will be
S=l
It follows that the actual concentration of solute in the Nth plate ( X E ) resulting from an injection of p plate volumes of pure mobile phase followed by u plate volumes of mobile phase carrying solute at a concentration X, will be
volumes
d27N
r=l
XE =
-N ) 2 / 2 N
i n plate
803
concentration of the eluted peak does not fall to zero until the sample volume is greater than 100 plate volumes which is about 5 times the standard deviation of a normally loaded peak. From Equation 4, a general equation for a column equilibrated with q solutes of concentrations X I , X 2 , X I . . . X, . . . X, can be calculated. For any solute S if its normal retention volume is R,,then assuming a constant column efficiency for all solutes the plate volume of the column for solute S is D* = R,/N. If the sample injected is contained in volume V , then the charge measured in plate volumes for this solute will be V/u, and if there is a total volume of equilibrated mobile phase passed through the column of y ml, then this will be equivalent to y / c s measured in plate volumes. If the sample injected onto the column contains solute S at a concentration X e 1 , then from Equation 4, the concentration of this solute X E at the end of the column will be given by X E $as
+
o- ( U + P
600
400
xx)
(6)
d2aN If pure mobile phase is injected onto the column, then X,l and Equation 6 becomes
=
0
( u f p -r -N ) 2 / 2 N
(4) r =V/u8
By using Equation 4, the elution curves for different volumes of pure mobile phase injected onto a column that has been equilibrated with mobile phase carrying a constant concentration of solute can be calculated. Values of X E for such curves were calculated, using a computer, for a column of 500 theoretical plates and injections of 20, 50, 100, and 200 plate volumes, respectively. The results obtained are shown in Figure 1 as curves relating solute concentrations to plate volumes of mobile phase passed through the column. As the injection volume is increased, the retention volume of the peak is progressively increased and for small volumes of charge the retention volume of the negative peak will be equal to that for the solute E when X l > X,. Also the
~ +- r X, (e-' - ~ ) ~ - ( g / uV/ua
Er=l
-N)2/2N
d2nN
(7)
This equation is similar in form to that published by Reilley et al. (3) but the derivation is simpler as it utilizes the equation of the elution curve from the plate theory and not the more approximate binomial expansion. By using Equation 7, the chromatogram was calculated for three solutes (q = 3); having capacity ratios of 1, 1.2, and 2.0, respectively, and for sample injection volumes of 20, 50, 100, 200, and 400 plate volumes, respectively, based on the plate volume of the first peak. The first two peaks were taken as being approximately four standard deviations apart. The resulting calculated chromatogram is shown in Figure 2 . As the negative
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
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m
307
25
-valco
-
valve
column
20-
-mtcr
jacket
c
e s
c
a
'1
: I c
i
I zem concentration for solutes 1 + 2
6
200
Flow of mobile
400
600
u
800
phase i n plate volumes of solute 1
Figure 2. Negative elution curves for different injection volumes for a column of 500 theoretical plates and a mobile phase containing three solutes
peak depression increases with injection volume, the resolution of the first two peaks rapidly decreases. To obtain a maximum negative signal for the most retarded peak, the resolution of the first two peaks is completely lost and the two bands merge t o a single distorted negative peak. This shows that in vacancy chromatography the resolution of the early peaks may have to be sacrificed to obtain later peaks of a maximum size. This problem, however, would not arise if one were comparing the two mixtures of similar composition. As the sample volume increases, the retention volume for each solute increases. This phenomenon would also occur in normal chromatographic development. EXPERIMENTAL Apparatus and Procedure. The apparatus used for the investigation is shown as a block diagram in Figure 3. The reservoir was filled with a reference mixture of nucleic acid bases which was circulated through the column, detector, and back to the reservoir with a controlled volume diaphragm pump (Whitey, Model LP-10). The detector was a UV monitor (Laboratory Data Control Model 1205). The tap in the return line to the reservoir allowed column effluent to be directed t o waste at any time when recycling might change the overall composition ofthe reference. The column was a 1-meter straight length of 1.5-mm i.d. (0.125-in. 0.d.) stainless steel tubing, the top of which had been drilled out to a depth of 2 mm to allow a 5-cm long piece of 1.3-mm i.d. (0.0625-in. 0.d.) stainless tube to be let-in and silver soldered. This short length of narrower 0.d. tube enabled the end of the column t o be fitted directly t o the injection valve (Valco Instruments, Model VSV-6HPa). The column was packed dry with Pellicular Cation Exchange Resin (Northgate Laboratories, CP-122, 60 microequivalents g-' capacity. 270-325 mesh). The packing was retained in the column with a porous Teflon disk. Temperature control of the column was obtained by circulating water through the column jacket at the desired temperature. A 20-cm length of 0.25-mm i.d. tubing connecting the end of the column to the inlet of the detector was immersed in water at 20 "C t o reduce the temperature of the column effluent sufficient t o obtain good detector stability with the reference cell filled with stagnant mobile phase at room temperature. The mobile phase flow rate through the column was maintained throughout all the experimental work at approximately 0.3 ml min-l. The pressure drop across the column was measured with a 0-2000 lb in.-2 Helicoid gauge. The nucleic acid bases used were uracil, hypoxanthine, guanine, 102
Figure 3. Chromatographic equipment
cytosine, and adenine. Solutions were made up t o the desired p H and molarity as noted in the text. RESULTS AND DISCUSSION Effect of Strength of Reference Mixture on Retention Characteristics. The theory relating peak profile and re-
tention volume of the peak maximum is based o n the premise of Gaussian peaks and linear adsorption isotherms. It would be expected with any liquid-solid system that retention characteristics for eluted species would vary according t o the types and concentrations of components in the circulated mobile phase as these would control the degree of modification of the surface of the actjve solid. To examine this effect and t o ensure that the conditions used for experimental evaluation of the theory were such that reasonably Gaussian peaks were obtained, positive chromatograms were obtained while the following mobile phases were being circulated. (a) 0.07M potassium phosphate buffer solution at p H 3.0 (b) Buffer (a) conlaining a total of 7 mg l i t e r 1 of haw* (c) Buffer (a) containing a total of 14 mg liter-' of bases* (d) Buffer (a) containing a total of 28 mg liter-1 of bases*
* Uracil, hypoxanthine, guanine, cytosine, and adenine in the ratio 1 :1:4:4:4 In each case a positive chromatogram was obtained by injection of a n 8-pl plug of a mixture of the bases in buffer at concentrations a little higher than in the mobile phase being circulated. The chromatograms are shown in Figure 4. The retention volumes for each component decrease as the total concentration of bases in the mobile phase is increased. Asymmetry was measured for the last three peaks in each chromatogram as the ratio of the distances of the trailing and front edges of the curve from the perpendicular dropped to the base line from the peak maximum. Because of the overlapping of the fourth and fifth peaks in the later chromatograms, all the ratios were measured a t 0.5 of the peak height. The asymmetry ratio also decreases with increasing concentration of bases in the mobile phase as shown in Figure 5 which is a plot of peak asymmetry against retention volume at each of the concentration levels. With this mixture of components having high extinction coefficients, it was not possible to achieve further modification by increasing their concentration in the mobile phase because, even at a total of 28 mg liter1, it was found that the detector was being
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
0
10
20 30 minutes
0
40
10
20
30
minutes
Figure 4. Effect of strength of mobile phase on retention volume
I , , \: , ,
Column temperature, 60 "C Mobile phase buffered, pH 3.0 and 0.05M Mobile phase flow rate, 0.28 ml m h - ' Max. deflection (uracil) approximately 0.1 OD unit
0
1
I!
/
I;
.1.8-
s 0
>I
Guanine Cytosine Adenine
Buffer
7 mg I i t e r "
1.2-
1.0I
3.0
1
4.0 Retent ion
5.0 Volume
6.0
7.0
10
15
20
Figure 6. Calculated and experimental curves for different injection volumes Column temperature, 60 "C Mobile phase buffered, pH 4.0 and 0.14M Mobile phase flow rate, 0.30 ml min-' Max. deflection (cytosine), 0.1 OD unit The magnitude of the theoretical curves is based on solute concentration whereas the practical curves are modified by the extinction coefficients of the respective solutes
2.2
2.0-
5
Time-minutes
8.0
(ml)
Figure 5. Plot of asymmetry ratio us. retention volume
taken into its nonlinear region. Some slight reduction in peak asymmetry was obtained by using 0.14M potassium phosphate buffer at p H 4.0 and the verification of the theory for injection of large plugs was carried out using this buffer. To ensure that any retention volume changes observed during the vacancy work were not attributable-in part to activity factors (rather than overload factors), each evaluation was carried out under conditions to give both negative and positive peaks. Exact matching between the retention volumes of corresponding negative and positive peaks was taken as proof that, over the range of concentration change of the elutions, activity factors were negligible. Injection of Large Plugs of Sample. To verify the theory developed for injection of large plugs, volumes of 115, 300, and 600 ~1 of buffer were introduced into the column via loops inserted in the injection valve. The mobile phase being pumped was 0.14M buffer solution a t pH 4.0 containing
a total of 14 mg liter-1 of bases (uracil, hypoxanthine, guanine, and cytosine). At this point it is worth noting that the volume of the column occupied by mobile phase was about 800 pl and when a plug of 600 pl was injected, the front of the plug would have moved three quarters of the way down the column by the time the rear was about t o enter it. Figure 6 shows curves calculated from Equation 7 superimposed on the experimentally determined results. The plate numbers for the intrinsic efficiency of the column were taken individually for each peak from a n 8-pl injection of bases in buffer at a concentration t o give the positive chromatograms also shown in Figure 6. The retentions of the experimentally determined negative peaks were matched by positive peaks indicating linearity of the isotherm over the operating concentration ranges. The magnitude of the theoretical curves is based on solute concentrations whereas the practical curves are modified by the extinction coefficients of the respective solutes. Neglecting, therefore, the differences in quantitative response, the agreement between the calculated and experimental retention volumes is excellent. Further, the peak widths at 0.6 of the peak height are the same. There are differences in base-width due to a tailing o n the part of the experimental curves. Of particular interest is the reproduction of the inflection on the front of the first peak for the 300- and 6OO-pl injection volumes. Whereas this jnflection is reproduced for the calculated elutions as a gentle curve, there can be occasions in practice when it may show up as a sharp indentation in the
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
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front even for injection volumes as large as 1 ml. This inflection, which is equivalent to the profile of the breakthrough in frontal analysis, can be self-sharpening if the isotherm for the front peak of the pair is convex to the mobile phase concentration axis, Figure 7a shows the positive peak obtained for a 600-p1 injection of sample containing concentrations of uracil and hypoxanthine a t the same levels as in the mobile phase but with the concentration of cytosine increased from 4 mg liter-1 to 4.4 mg liter-I. There is no perturbation of the base line for the first three compounds because the mobile phase/stationary phase equilibrium is not disturbed by the injection. There is, however, a positive peak corresponding to the difference in concentration of 0.4 mg liter-l of cytosine. Figure 76 shows negative peaks obtained for 300- and 60O-pl injections of sample equivalent to the mobile phase except for the omission of hypoxanthine. In this case a negative peak corresponding to the difference in concentration of hypoxanthine (0.5 mg l i t e r 1 ) is obtained. Under these conditions, differences in concentration of hypoxanthine can be determined without any interference from uracil from which, under normal elution conditions, it is not completely resolved. Cycling Experiments. The concept of recycling column effluent was not a total success because of an instability of the column packing which gave rise to a gradual increase in pressure drop across the column from 500 lb in.-2 to 2000 Ib in.-2 over a period of a week or more of continuous running. While the chromatographic properties appeared t o be little changed, the pressure increase did lead to a reduction in the mobile phase flow rate which had to be adjusted daily, This behavior was contrary to our previous experiences with high pressure liquid chromatography using pellicular resins of lower capacity (about 10 microequivalents g-1) and with silica gels and aluminas. PROBLEMS ENCOUNTERED
It is a natural requirement of any chromatographic system that the system itself and the compounds t o be chromatographed should be stable. The conditions of vacancy chromatography are more severe on sample stability than conventional techniques because a Concentration of each compound is maintained on the surface of the active solid. Further, in the recycling mode, sample stability is of greater significance because the cycled material is heated up in the column, cooled, and returned to the reservoir a great number of times. Guanine was not sufficiently stable to be subjected t o this treatment. One criterion of a clean stable vacancy system is that a n injection of a large plug (600 pl) of mobile phase should not produce any perturbation of the base line. Frequently, after exploratory work involving injections of plugs of sample designed to give positive peaks, the valve assembly had to be well flushed to ensure that this criterion could be met. As is the case for any chromatographic work, the standard of the quantitative results is a function of the characteristics of the detection system and the manner in which it is used. The background signal seen by the detector under vacancy
104
0
,
0
I
5 10 15 T i me - mi n u t e s
20
Figure 7a. Injection showing cytosine divergence 6. Injection showing hypoxanthine divergence, conditions as for Figure 6 Deflection for cytosine = 0.02 OD unit Deflection for hypoxanthine = 0.02 OD unit
conditions is the sum of the signal for all the components in the mobile phase. With the UV monitor designed for conventional liquid chromatography, this background signal can take the detector out of its linear range even though the detector circuit is balanced by the reference cell. Thus with a background signal from 70 mg liter-' of bases (approximately 3.5 OD units), the detector becomes almost totally insensitive. With the instrument we were operating, the linearity appeared to be good up to a total background equivalent to 0.7 OD unit. Thus, contrary to conventional chromatography where it is possible to keep out of the nonlinear range by keeping sample mass low and increasing sensitivity, in vacancy, once the detector is put into the nonlinear region by the background, it is there for all samples however small the differences to be measured. CONCLUSION
Operation of a liquid-solid chromatographic system in the vacancy mode is a feasible proposition. The recycling procedure proposed places stringent requirements on sample stability. The characteristics of a detector such as a UV monitor can be affected by having t o operate with a high background signal. Advantages of the technique are the ability to use the difference method and to mask out unwanted peaks which could normally overlap and interfere with the determination of a component of special interest. Experience with plug injections as large as 100-plate volumes shows that the effect for components having capacity ratios greater than 5 is not too devastating and often such an injection or even larger could ameliorate to some extent the present limitation of restricted peak capacity.
RECEIVED for review July 16, 1971. Accepted August 20, 1971.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
