Optimal Support for Heavily Loaded Columns in High Speed Liquid Chromatography Heinz Engelhardt and Norbert Weigand Lehrstuhl fur A n g e w a n d t e Physikahsche C h e m i e der Universitdt d e s S a a r l a n d e s , S a a r b r u c k e n , w e s t G e r m a n y
Experiments showed that optimum geometrical properties of the silica support are: surface area, 350-500 m*/g; pore volume, 1-1.3 ml/g; pore diameter, 100-200 A. For routine analysis more than 0.6 g liquid/g uncoated support is required. Efficiency, speed of analysis, and the influence of the active support in heavily loaded columns coated with ODPN are discussed. The advantages of these columns in routine work are demonstrated.
The use of heavily loaded columns (ie., more than 0.5 g stationary liquid phase/g uncoated support) in the field of gas chromatography was discussed in a preceding paper (1). The application of such column packing material in that field is more or less restricted. In a short ‘communication (Z), the advantages of heavily loaded columns in high speed liquid chromatography were pointed out: large sample sizes can be injected, the peak capacity is high. Further, the bleeding hardly influences the reproducibility of the retention of solutes if the eluent is presaturated with the liquid stationary phase. Last, but not least, much smaller volume flow rates are required for a given linear velocity because of the small total porosity ( t T = 0.40 to 0.55) of these columns. Some separations of steroids on heavily loaded columns had been shown recently (3). I t should be pointed out that heavily loaded columns have been used in classical liquid-liquid column chromatography as well as in the early stages of gas-liquid chromatography. In this paper the influence of the physical and chemical properties of the active supports on column performance will be evaluted by experiments. The influence of pore volume, pore size distribution, specific surface area, and the activity of the support on the column efficiency will also be discussed. In most experiments, silica was used a5 a support, because its physical properties can be easily adjusted to the chromatographic requirements ( 4 , 5 ) , but for comparison an alumina support was included in this study. EXPERIMENTAL Apparatus. The apparatus is the same as described previously (61 including the described device for pressure control and pulsation dampening 17). The detector was a differential refractometer (Waters Associates, Framingham, Mass., Model R 401) and the samples were injected a t pressures up to 150 atm with the aid of a syringe (Hamilton Corp., Whittier, Calif., Type H P 305 N ) . All measurements were made a t room temperature (22-25 “C). (1) J. Asshauer and I . Halasz, Anal. Chem., 45, in press, (2) I . Halasz, H . Engelhardt, J. Asshauer, and B. L. Karger, Anal. Chem.. 42, 1460 (1970). (3) 8.L. Karger and L. V . Berry, Clfn. Chem., 17, 757 (1971). (4) R. K. Iler, “The Colloid Chemistry of Silica and Silicates,” Cornell University Press, Ithaca, N . Y . , 1965. (5) K . Unger, Angew. Chem.. 84, 331 (1972); Int. Ed. Engl, 11, 267 (1972). (6) G. Deininger and I . Halasz, J. Chromatogr. Sci.. 9, 83 (1971). (7) G. Deiningerand I . Halasz,J. Chromatogr.. 60, 65 (1971).
Columns, Straight stainless steel tubes of 50-cm or 60-cm length and 2.0-mm inner diameter were used. Before filling, the columns were cleaned by rinsing with 2 M HC1, water, acetone, and dichloromethane. The stationary phase was packed as previously described by vibration and tapping 18). It was ascertained that the columns could be packed reproducibly with respect to the weight of stationary phase, and efficiency. Reagents. The eluent employed were either n-hexane or dichlaromethane purified through adsorptive filtration (91 before use. Formamide “for chromatography” (E. Merck, Darmstadt, West Germany), 3,3’-oxydipropionitrile (ODPN) (Schuchhardt, Munchen, West Germany). and distilled water were the stationary liquid phases. Eluents were saturated with the stationary liquids using presaturator columns of 50-cm length, packed with Kieselgur (E. Merck) 100-150 mesh and coated with approximately 3570 of the stationary liquid phases. The physical properties of the active supports are summarized in Table I. Most of the silicas described are research samples of E. Merck, Darmstadt, and are not commercially available. Other supports employed were “Porasil A” (Waters Associates. F’ramingham, Mass.) and neutral alumina W 200 (M. Woelm, Eschwege, Typ N 18). Coating. The organic stationary phases were dissolved in dichloromethane or acetone and the support was suspended in this solution. The solvent was removed in a rotary evaporator. The weight ratio of liquid stationary phase and the uncoated support (g/g) was predetermined and checked after the coating procedure by C,H analysis. When water was used as the stationary phase. it was added directly without solvent. The water-coated silica was kept in a closed flask for several hours, in order to get uniform coating. Solutes retentions remained unchanged. within experimental error by the use of special precautions, which will be discussed later. Consequently, the loss of the stationary phase (“bleeding”) during the use of the column was negligible which was confirmed after the experiments by a further C,H analysis.
RESULTS AND DISCUSSIONS Influence of the Amount of Liquid Stationary Phase. The silica SI 100 b R was coated with different amounts of ODPN and, as shown in Table I1,‘the height equivalent to a theoretical plate h for an inert peak increased by more than 50% when the amount of liquid stationary phase decreased from 1.0 g/g to 0.2 g/g. Similar effects were observed with silica coated with water ( I O ) . With decreasing h-values for the inert peak, the column porosity decreases ( I ) , and reaches values a t high liquid loadings. corresponding to those obtained with columns packed with gas impenetrable glass beads. For retarded peaks similar measurements were made. but these values are not included. Their discussion is sophisticated because of the mixed mechanisms of sorption (partition and adsorption). The capacity ratios are not a linear function of the loading and this will be discussed later.
(8) B. L. Karger, K . Conroe. and H . Engelhardt, J. Chromatogr. Sci.. 8 , 242 (1970) (9) H . Engelhardt, in “Ultrapurity,”M. Zief and R. Speights. E d . , Marcel Dekker, New York, N . Y . , 1972. (10) L. R . Snyder, Anal. Chem.. 39,698 (1967) A N A L Y T I C A L CHEMISTRY, VOL. 4 5 ,
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Table I. Physical Properties of the Active Supports Pore volume, cm31g
Surface area, m2/g
Mean pore diameter, A
Maximum loadability, g/g
Sieve fraction, fim
Permeability K , cm2 X 109
0.5 0.7
500
40 430 100 100 120 100 60
0.55
30-40 30-40 30-40 30-40 32-38 36-53 15-30
12
Silica SI 40 R Silica SI 500 R Silica SI 100 a R Silica SI 100 b R Merkogel SI 100 Porasil A Alumina N 1 8
1 .o 1.3 1 .o 1.1
65 400 520 300 480
0.25
200
Table II. Relative Peak Broadening h of an Inert Peaka g/g
h , mm
1 .o
0.75 0.95 1.08 1.12 1.17
0.8 0.6 0.4 0.2
aStationary phase: Silica SI 100 b R, coated with ODPN; linear velocity; 4 cm/sec; eluent; n-hexane; sample, n-heptane ( k = 0)
>/
41.
AP
Figure 1. Typical h vs. u curve on a heavily loaded column Column length, 50 cm; stationary phase, 0.8 g ODPN/g SI 100 b R; eluent, n-hexane; n-Ci. n-heptane ( k = 0 ) : Tol, toluene (0.28); AP, acetophenone (5.2); DMP, dimethylphthalate (27.5); BA, benzylalcohol (53); PH, phenol (129); sample size, 0.5-1 rng
$1
lSec"1 2.0
AP
TOL DMP
a4 PH
1 1
2
3
4
5
6
7
8
u~inlsoc)
Figure 2. Effective plates per second vs. velocity Stationary phase, 1.0 g ODPN/g SI 100 b R; eluent, n-hexane; Tol, toluene ( k = 0.3): AP, acetophenone (5.7); DMP, dimethylphthalate (30.0); EA, benzylalcohol (58); PH, phenol (144)
Efficiency. For different loadings the h us. u-curves were measured for samples with capacity ratios between zero and 150. The linear velocity of the eluent was changed from 0.5 to 8 cm/sec. The simple empirical relationship: h=A+Cxu 1150
(1)
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
0.60 0.95
1.o 1 .o
1 .o 0.25
9.1 8.5 10 8.5 19 7.4
is an excellent approach for this velocity range. A typical h us. u-curve is shown in Figure 1. The stationary phase was silica (SI 100 b R) coated with 0.8 g ODPN/g support. For a given velocity, the h-values increase with increasing capacity ratios and after going through a maximum ( k = 5.2), they decrease. Theory predicts (11) that this maximum should be a t k = 1, if the mass transfer in the stationary phase determines peak broadening alone. Unfortunately, we did not find samples having capacity ratios between 0.3 and 5.2. Efficiency and Speed of Analysis. The h-values in the velocity range discussed are comparable (especially for compounds with high capacity ratios) with those described in the literature for porous and non-porous supports (12). Of course, the sieve fractions must be similar in order to compare the results. The h/u-values are sometimes very impressive with heavily loaded columns. As shown in Figure 1 for phenol ( h = 129) at u = 5 cm/sec an h / u = 19 msec is calculable. The speed of analysis is moderate, if it is characterized by the number of theoretical plates generated per second ( n / t = 0.4 sec-1 for phenol). The resolution, however, is proportional to the square root of the number of effective plates. In heavily loaded columns, effective and theoretical plates are very similar because of the high capacity ratios. In Figure 2 the number of effective plates generated per second are plotted as a function of the velocity of the mobile phase. The stationary phase is the same as that used in Figure 1, only the liquid loading is higher (1 g/g). Optimal velocity is near 3 cm/sec. With increasing velocity the number of plates generated decreases and the pressure drop across the column increases. Consequently, the gain in the speed of analysis is sometimes over-compensated by the loss in efficiency. The speed of analysis with heavily loaded columns is acceptable but not comparable with those achieved with columns packed with porous layer beads (12), or porous materials, if the particle size is much smaller than that used in these experiments (13, 14). I t should be mentioned that no values are plotted in Figure 2 for the optimum capacity ratio of two. Influence of Pore Volume. The pore volume only indirectly influences the column characteristics. The higher the pore volume, the more stationary liquid phase can be added to the support without making the particles sticky. With a pore volume of 1 to 1.3 ml/g, a loading of 1.0 g/g can be obtained. With a pore volume of 0.5 ml/g, the beads become sticky with loadings higher than 0.5 g/g. The higher the amount of stationary liquid in the column, the less important and recognizable is the loss of stationS. Dal Nogare and R. S. Juvet, "Gas-Liquid Chromatography," Interscience, New York, N.Y., 1962. "Practice of High Speed Liquid Chromatography," J. J. Kirkiand. Ed., Wiley-lnterscience, New York. N.Y., 1971.
J. J. Kirkland, Lecture given at the 9th International Symposium on Chromatography 1972, Montreux, Switzerland, Oct. 1972. R. E. Majors,Anal. Chern.. 44, 1722 (1972).
0
1 LO
100
200
300
LOO
a'
-rii
Figure 3. Pore d i a m e t e r and efficiency Stationary phases: 40 A, 0.55 g O D P N / l g SI 40 R; 100 A, 1 g ODPN/ 1 g SI 100 b R; 430 A, 0.6 g ODPN/1 g SI 500 R ; eluent, n-hexane; u = 4 cm/sec; The capacity ratios are listed in Table I l l
m
*
N O O P N N O
*
o o * o m o
N * $
c'??
N m N r
*
oOui*coo
m w m
N
E
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c C
-C a
C
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C
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!
:
a U C
a C
c
-a
. c
W
ary phase through column bleeding and its effect on solute retention. Therefore silica with large pore volumes should be used for high speed liquid-liquid chromatography. Efficiency with Supports of Maximum Liquid Loading, and the Influence of Pore Diameter. It is desirable to coat the support with the maximum amount of liquid stationary phase, because of reasons to be discussed in this paper. In Table 111, capacity ratios h and the constants of Equation 1 are tabulated for different samples and different supports coated with the maximum possible amount of ODPN. The linear velocity of the eluent was 4 cm/sec. Regarding the h-dependance of h for a given support, the data in Table I11 confirm the previous discussion of Figure 1. It should be noticed that the average particle diameter of Porasil A is about 25% higher than that of the others. In our experience, the extrapolated A-term of Equation 1 is mainly determined by the properties of the tube and by the method of packing. From this point of view, on the column packed with SI 100, shown in Table I11 and mentioned in the discussion, the efficiency is worse. In the following, the influence of the pore diameter on the mass transfer term C will be mainly considered. It should be mentioned again, that SI 40 R and SI 500 R have average pore diameters of 40 and 430 A, respectively. The corresponding value for the other silicas of Table I11 was 100 A. The influence of the pore structure on the C term is of course minimal for the inert peak. If the pore diameter is as small as 40 p\, the C-terms become unusually high. From this point of view, there is scarcely a difference between silica with an average pore diameter of 100 and 500 A. The disadvantages of the silica with 40-x pore diameter are demonstrated in Figure 3, where h is plotted as a function of the average pore diameter for different samples. As shown in Figure 3, the efficiency h is not influenced significantly by the packing method, because the relative peak broadening of the unretarded peak is practically unchanged as the pore diameter changes. The interpolation between 100 and 500 A seems to b t reasonable since recent results in this laboratory (not described here) in this region are in good agreement with the plots of Figure 3. The pore diameter determines the stability of the liquid loading of the column. The liquid is kept constant by strong capillary forces if the pore diameter is 100 A. to 200 A or smaller. As the specific pore volume decreases, the maximum amount of liquid stationary phase decreases also with decreasing pore diameter. Thus the competition of the adsorption on the surface of the solid with the desired partition can increase. Larger mole1
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 7, J U N E 1973
*
1151
i
/
100-
8060LO
/
\ -
~
01
DMP
20 -
1
-
P AP
0.2
0.A
0.6
0.8
1:o
[+I
Figure 4. Capacity ratio and the amount of liquid loading Support, SI 100 b R; stat. liquid, ODPN; eluent, n-hexane; The abbreviations are the same as in Figure 1
K
u = 4 cm/sec;
I
600-
I 0A
Figure 5. Dependence of the chromatographic partition coefficient K on ~ / V L Conditions same as for Figure 4
cules, especially those with big solvation spheres may be excluded if the pore diameter becomes much smaller than 100 A (15). The silica (SI 500 R) with a pore diameter of 430 A was always slightly sticky regardless of the amount of liquid added. The column packing was more difficult in this case. At higher velocities, no stable base line could be obtained. With this support, the capillary forces were not (15) N. P. Lebedeva, I . I. Trolov, and Ya. I, Yashin, J. Chromatogr.. 58, 11 (1971)
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, J U N E 1973
02
03
OL
05
06
07
0.5
09
10
EE
Figure 6. Relative retentions 1y and liquid loading Conditions same as for Figure 4
great enough to prevent the liquid phase from being stripped off of the support, despite the use of a saturated mobile phase. The optimum pore diameter for heavily loaded silica supports seems to be in the region of 100 A. Retentions on Silica Support. Figure 4 shows the change of the capacity ratios as the amount of stationary liquid phase added to the silica (SI 100 b R) is increased. The capacity ratios on the uncoated silica were not added to this plot because, for the samples shown in Figure 4, the k-values were larger than 200, and the peaks were asymmetrical. The capacity ratios in Figure 4 decrease with increasing coating, and after going through a minimum a t a liquid loading of about 0.4 g/g, they increase again. This picture is typical for mixed adsorption and partition mechanisms in chromatography (16, 17) and has been discussed recently for gas chromatography on heavily loaded columns ( I ) . From the data of Figure 4, the chromatographic partition coefficients K for different samples with different amounts of liquid stationary phase were calculated. Plotting K us. l / V L , the slope of the curves increase with an increasing l/VL-value as expected for mixed sorption mechanisms (18, 19) and as shown in Figure 5 . The curves shown in Figure 4 are independent of the properties of the silica support if its specific surface area is larger than 350 m2/g and its pore volume is greater than 1 ml/g. These results can be of some interest when heavily loaded columns are used in routine analysis. Although the support was loaded with 1 g liquid/g uncoated silica, the partition properties of the liquid stationary phase were not approached. The partition coefficient K for phenol in the system ODPN/hexane was determined experimentally to be 39. From the chromatographic measurements (1 g/g loading), a K-value of 208 was calculated. A similar relationship was determined for dimethylphthalate with the K-values of 9 and 44, respectively. For less polar compounds (acetophenone) the influence of the silica decreased. The ratio of the static and the chromatographic partition coefficient decreased from 5 for polar samples to 2-2.5 for apolar ones. Figure 6 shows the relative retentions a of different pairs of samples as a function of the liquid loading. The support is identical with that shown in Figures 4 and 5. If column is coated with more than 0.6 g/g, the relative I . Halasz and E. E. Wegner, Nature. 189, 570 (1961). 6. L. Karger, H. Engeihardt, and I. Halasz, in "Gas Chromatography 1970," R. Stock, Ed., Institute of Petroleum, London, 1971 J. R . Conder, D. C. Locke, and J. H. Purnell, J. Phys. Chem.. 73,
700 (1969). D. F. Cadogan, J. R. Conder. D. C. Locke, and J. Phys. Chem.. 73, 708 (1969)
H. Purnell, J.
/I
Table IV. Efficiency of the Heavily Loaded Alumina Column A, c m X 103
k
Heptane Toluene Acetophenone Dimethyl phthalate Benzylalcohol Phenol
-
0
80
0.15 2.7 14 54 240
70 130 190 130
C, sec X 103 21 33 77 61
f
29
...
..
Velocity range, 0 5-8 cm/sec; eluent, n-hexane; stationary phase, 0.25 a ODPN/a alumina W 200 N Type 18; sieve fraction, 15-30 wrn
0
1
5
L
3
2
6
7
8t(rnin)
Figure 8. Separation of phenols and naphthols Conditions same as for Figure 7, 1. 2,3.5-TrimethylphenoI ( k = 0 . 9 ) ; 2. a-naphthol (2.1); 3. &naphthol (4.0); 4. phenol (13.5)
0
Figure 7.
1
2
3
4
5
6
7
t(rnin)
Separation of phenols
Stationary phase, 1 g Water/g Porasil A; eluent, n-hexane; column length, 60 c m (2.2 mm i.d.); u = 2.3 cm/sec, 1. Carvacrol (k = 0.5); 2. 1,3,4-xylenol (1.2); 3. 1,2,4-xylenol (3.0); 4. rn-cresol (5.2); 5. phenol (13.7)
retentions are in good approximation independent of the amount of coating. Column Bleeding. If the eluent is presaturated, liquid stationary phase is introduced or washed out by the saturated eluent. The long term change of the weight of the stationary liquid phase in the column is relatively small because of the heavy coating. This and the facts shown in Figure 6 assure the reproducibility of routine analysis. Mechanical erosion of the stationary liquid phase is the consequence of unusually high velocities of the eluent ( u > 6 cm/sec). On a column coated with 1 g ODPN/g SI 100 b R, the capacity ratio of 157 was measured for phenol a t u = 4.2 cm/sec. When the velocity was increased to 8.5 cm/sec, the k-values decreased with time. The curve levelled off after seven hours to a constant k-value of 120. As calculable, about 25% of the liquid stationary phase was washed out. After the velocity was reduced to 4.2 cm/sec the k-values increased again. The k-values measured after equilibration overnight corresponded to a loading of 0.9 g/g. Consequently the “equilibrium coating” of this support liquid-stationary phase system a t room temperature is about 0.9 g/g. Columns with smaller liquid loadings than the “equilibrium coating” cannot be used in routine analysis. With higher coatings, the routine work is possible, if extremely high velocities are avoided. (It should be mentioned, that the measurements on columns with liquid loadings smaller than this “equilibrium coating” were made immediately after a 30-minute equilibration of the freshly prepared column.) Retentions on Alumina. The pore volume of the alumina used in these experiments allowed a maximum liquid loading of only 0.25 g/g. The efficiency of this alumina column is characterized in Table IV. The constants A and C of Equation 1 are higher than with silica support. The strongly retained phenol peak tailed. The efficiencies in Table IV are poorer than for the silica/ODPN system or for the alumina-water system (20). One reason for the (20) H. Engelhardt and H. Wiedemann, Anal. Chern., 45, in press
0
1
Figure 9.
2
3
4
5
6
7
t h n )
Separation of steroids on formamide
Stationary phase, 1 g formamide/g Porasil A: eluent, dichloromethane; column length: 60 cm (2.2 mm i,d,); u = 1.42 cm/sec. 1. 5 a-Androstandion-3,17 (k = l 4 ) ; 2. A4-androstendion-3,17 (2.6); 3. testosteron (7.2)
poor efficiency could be that our experience in packing columns with ODPN/alumina material was not sufficient. Applications of Heavily Loaded Columns. A disadvantage of liquid-liquid separations is that the sample, if collected and isolated after separation, is always contaminated by the stationary liquid. Therefore, in the following separations, water was used as a liquid stationary phase because it could be removed easily from the sample. In the Figures 7 and 8, the separations of phenols and naphthols are shown. The support was the commercially available silica Porasil A coated with 1 g water/g support. The elution order corresponds to increasing solubility of the samples in water. With these separations, it is demonstrated that sometimes it is easy to transfer the results of classical column chromatography (21, 22) to high speed liquid chromatography. The relative retentions of these samples, but not the elution order, can be changed if the support is coated with buffered water solutions. The system of formamide/dichloromethane is typical for the separation of steroids in paper chromatography (23). In Figure 9, the separation of male sexual steroids is shown. Silica coated with formamide was the stationary phase and dichloromethane was the eluent. (21) “Chromatographie en Chimie Organique et Biologique,” Vol. i I, E. Lederer, Ed., Masson et Cie, Paris, France, 1960. (22) R. J. Zahner and W. B. Swann, Anal. Chem., 23, 1093 (1951). (23) F. Cramer, “Papier-chromatographie,” Verlag Chemie, Weinheim/ ’ Bergstr. 1958. A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 7, J U N E 1973
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0
1
2
Figure 10. Separation of
3
4
5
6
tlmn
steroids on buffered water
Stationary phase, 0.95 g phosphate buffer (pH 6 ) / g Porasil A, eluent, dichloromethane: column length, 60 cm (2 2 rnm i.d ) : u = 1 57 cm/sec, The samples are the same as in Figure
9
To overcome the difficulties of the separation of the samples from formamide after elution, in Figure 10 the same separation is shown with a phosphate buffer of p H 8 as stationary liquid. The same eluent (dichloromethane) was used. CONCLUSIONS Some advantages of heavily loaded columns in high speed liquid chromatography were pointed out in the introduction. The experimental data described above show that the optimal silica support has the following structure: specific surface area 350-500 m2/g; pore volume 1-1.3 ml/g; average pore diameter: 100-200 A. The amount of liquid stationary phase has to be greater than 0.6 gig. A minimum liquid loading corresponds to a eluent presaturated with the stationary liquid. It is possible to use liquid loadings higher than those of the steady state.
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ANALYTICAL CHEMISTRY, VOL. 45, N O . 7 , J U N E 1973
With less coating, no routine analysis can be made because the retentions change continuously. The liquid phase is washed out mechanically if the velocity of the eluent is higher than 6 cm/sec. Such a column can be regenerated, the steady state is reached by conditioning the column overnight a t low velocities of the presaturated eluent. The main advantage of the heavily loaded columns is the potential for large sample sizes. Separations of some interest can be made with water as stationary phase. Water has the big advantage that it can be easily separated from the eluted samples. Routine analysis is possible with heavily loaded columns, because the bleeding is small compared to the total change of the amount of stationary liquid in the column. The relative retentions are scarcely influenced by the amount of stationary phase, if the coating is higher than 0.6 g/g. Separations well-known in column liquid-liquid chromatography can be transferred to high speed work. The transfer from paper or thin layer chromatography is often much more complicated.
ACKNOWLEDGMENT The E. Merck A. G., Darmstadt, we thank for the preparation of the silica samples and for the measurements of their physical properties. We appreciated the helpful discussions with B. L. Karger, Northeastern University, Boston, Mass. Received for review November 22, 1972. Accepted January 16, 1973. This work was a part of the Diplomarbeit of N. Weigand, University Erlangen-Nurnberg, 1972. The authors thank the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 52, Analytik, Saarbrucken) for financial furtherance of this research work.
