this flame do occur. The calculated pressures of 0 and 0 2 , for instance, vary over 8 orders of magnitude within the range of oxidant-to-fuel ratios found in Figure 13. Furthermore, the temperature increases from 1800 K a t 02/H2 = 0.01 to 2950 K a t Oz/H2 = 0.50. Atomization Efficiencies. The atomization efficiencies for elements in the flame reflect these environmental changes. Metal-to-metal monoxide ratios for 3 of the 4 metals studied. as a function of 02/H2, are found in Figure 14. The data illustrated in this figure indicate that the maximum atomization for all elements should be greatest in the very fuel-rich flame. This conclusion confirms the experimental results discussed above. Atomic absorption for Na, for example, was found to be nearly independent of Oz/Hz in the oxygen-hydrogen flame. Maximum Fe absorption was found a t O2/H2 = 0.1 and decreased with increasipg OzjH2 to 40% of the maximum value a t O2/H2 = 0.5. Absorption by Cr, which has a monoxide dissociation energy close to that for Be [(DO cr0 = 4.4 eV (35)],also exhibited its absorption maximum in the very fuel-rich flame, but oddly enough, Cr absorption decreased by nearly a factor of 100 as the oxidant-to-fuel ratio is increased to 0.5. The calculated atomization efficiency (M/ MO) for Be is probably high because this element forms hydroxides (BeOH and Be(OH)2) as well as a monoxide in flames ( 4 2 ) . Beryllium, however, should be illustrative of elements with similar monoxide dissociation energies, that T,O = 7.2 eV) is not includdo not form hydroxides. Ti (DO ed in Figure 14 because the maximum calculated value of Ti/TiO was only 6 x 10-5 a t 0 2 / H 2 = 0.25. This value is consistent with experimental results since T i could not be observed spectroscopically, even at concentrations as high as 1000 pug Ti/ml. when aqueous solutions were nebulized. Comparison of the IL’20-CaH2 and 02-H2 Flames. Since the maximum calculated free-atom fraction for Ti
-
in the nitrous oxide-acetylene flame is 0.33-0.49 ( 4 2 ) , it is evident that elements that form very stable monoxides are atomized much more efficiently in this flame than in the premixed oxygen-hydrogen flame. The maximum values of M/MO calculated for Na, Fe, and Be in the oxygenhydrogen flame, on the other hand, are nearly identical with the free-atom fractions calculated for these elements in the nitrous oxide-acetylene flame ( 4 2 ) . Measured emission and absorption intensities indicate that all the elements examined except Na were atomized considerably less efficiently in the oxygen-hydrogen flame. Since the maximum M/MO occurred in the relatively cool (1800 K), very fuel-rich oxygen-hydrogen flame, the most likely explanation for this discrepancy is incomplete solute vaporization. The temperature of the carbon-rich nitrous oxide-acetylene flame is at least 1000 K higher than the temperature of the very fuel-rich oxygen-hydrogen flame. Fassel and Becker (46) have shown that solute vaporization in this flame is complete in most cases. Practical Applications. The results of these calculations indicate that atomization efficiencies for all elements depend upon all the equilibrium conditions that exist for a given set of experimental parameters and not, as implied by Ando et al. (58), on temperature alone. Indeed, the data in Figure 14 indicate that, for the elements in this figure a t least, the atomization efficiencies are more dependent upon the partial pressure of oxygen than on the temperature, since the maximum M/MO occurs in the cool, very fuel-rich flame. Received for review July 24, 1972. Accepted March 23, 1973. (58) A . Ando, K . Kuwa, and 6.L. Vallee. Anal. Chern.. 42. 818 (1970)
Polypeptides as a Permanently Bound Stationary Phase in Liquid Chromatography Eli Grushka Department of Chemistry, State University of New York at Buffalo, Buffalo,
N. Y. 14274
R. P. W. Scott Physical Chemistry Section, Hoffmann-La Roche, lnc., Nutley, N.J. 071 10
A new type of permanently bound stationary phase is discussed in this paper. This phase is polyglycine peptide bound to (a) resin coated glass beads, (ta) Porasil C, and (c) Corasil II. While untreated glass beads could not separpte a mixture of several amino acids, the peptide-glass beads did separate them using distilled and deionized water as the mobile phase. Indications are that the whole peptide chain takes part in the separation. With the Porasil and Corasil support, although the natural particle did separate some of the amino acids, the bound peptide not only improved the separation but also inverted the elution order of the amino acids. Mixtures comprising different substituted benzenes showed the elution order para, meta, and ortho isomers when the peptide bound phase
was present on all three supports. Plain resin coated glass beads did not separate the substituted benzenes at all while, with the uncoated Porasil C and Corasil II, the retention order was ortho, meta, and para isomers. Possible mechanisms are discussed.
One of the limitations of high-speed liquid-liquid chromatography results from the instability of the stationary phase when it is coated on the solid support. This often causes stationary phase “bleed” especially a t high mobile phase velocities. To minimize this effect, the mobile phase is usually presaturated with stationary phase before entering the column. Such a procedure, however, is not completely satisfactory, particularly if one wishes to pro-
1626 * ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
gram the flow rate, the mobile phase composition, or the temperature. Permanently bound stationary phases can eliminate these difficulties, providing the bond between the stationary phase and the support is sufficiently strong. Although known for about a decade, the chemically bonded stationary phase became popular with Halasz’s (1) description of the “brush” system, and several manufacturers offer various bonded phases commercially. Since the work by Halasz ( I ) , which described the esterification of the silanol groups (-SiOH) of silica gel, which acted as the solid support, with various alcohols, several papers have appeared in the literature describing permanently bound phases. Little et al. ( 2 ) described the properties of various “brush” phases in GC work. Aue et al. (3, 4 ) demonstrated the utility of chemically bound silicone polymers. Similar bonded phases on controlled surface porosity support were recently investigated by Kirkland (5-7). More recently, Locke and coworkers (8) described the preparation of various bonded phases via the Grignard reaction, and Saunders et al. (9) described the preparation and properties of cation exchange stationary phases bound to silica. In general, however, the permanently bound phases have not received the wide acceptance which they deserve. There are several reasons for this: ( a ) the alcohol ester phases cannot be used with polar solvents (in particular, water), ( b ) the cost of the commercially available supports is rather expensive, and (c) the most stable bound phases can be cumbersome to prepare. We wish to report here preliminary studies of a new type of bound phase which can be easily bonded on various solid supports and which has the potential of being designed for specific separations, particularly those arising in the biological science areas. Recently, using a modified Merrifield solid-phase synthesis procedure, Scott et al. (IO, 11) have reported the synthesis of peptides, using chromatographic methods, on resin coated glass beads. In addition, Parr and Grohmann (12, 13) reported similar peptide synthesis on a non-Merrifield type inorganic matrix such as Porasil. These workers, however, were interested in the actual preparation of the peptides; i. e. their purity, absence of sequence failure. and ease of detachment from the solid support. On the other hand, if one chooses to leave the peptide attached to the solid support, one can have a strongly bonded stationary phase with properties that can be altered by changing the sequence and type of amino acids present in the peptide. The work described here reports the behavior of polyglycine peptides (the simplest possible peptide), synthe( 1 ) I . Halasz and I . Sebestian, Angew. Chem.. Inf. Ed. Engl., 8, 453 (1969). (2) J. N . Little, W. A. Dark, P. W . Farlinger, and K . J. Bombaugh. J. Chromatogr, Sci., 8, 647 (1970). (3) W. A. Aue and C. R. Hastings, J. Chrornatogr., 42, 319 (1969). (4) C. R. Hastings, W. A. Aue. and J. N . Augl, J. Chromatogr., 53, 487 (1970). (5) J. J. Kirkland and J. J. DeStefano, J. Chromatogr. Sci., 8, 309 (1971). (6) J. J. Kirkland, J. Chromatogr. Sci., 9, 206 (1971). (7) J. J. Kirkland in “Modern Practice of Liquid Chromatography,’’J. J. Kirkland, Ed., Wiley, New York, N . Y . , 1971, pp 161-204. ( 8 ) D. C. Locke. J. T. Schmermund, and 6. Banner, Anal. Chem., 44, 90 (1972). (9) D. H . Saunders, R. A . Barford, P. Magidrnan, R. J. Bestsch, and H . L. Rothbart, 164th National Meeting of t h e American Chemical Society. New York, N . y., Aug. 28-Sept. l , 1972, paper No. Anal. 47. (10) R . P. W . Scott, K. K . Chan. P. Kucera, and S. Zolty, J. Chromatogr. Sci., 9, 577 (1971). (11) R. P. W. Scott, S. Zolty, and K . K . Chen, J. Chromatogr. Sci., 10, 384 (1972). (12) W . Parr and K. Grohmann. Tetrahedron Lett.. 28, 2633 (1971) (13) W. Parr and K . Grohmann. Angew. Chem., lnt. Ed. Engl., 11, 314 (1972).
sized on various chromatographic supports and used as a stationary phase for liquid chromatography. The solid supports investigated were resin coated glass beads, Porasil C, and Corasil 11. Further work is now being carried out to determine the effect of other peptide sequences, of different peptide length, and the role played by the mobile phase in such systems.
EXPERIMENTAL Apparatus. The liquid chromatographic system employed consisted of the following units: a Milton Roy minipump (1000 psi maximum pressure), a LDC UV detector having an 8-pl cell capacity and operated at 254 nm, and a Honeywell model Electronik 194 recorder. The column was 30 cm X 3 mm i.d. precision bore glass column and all connections were made from l/,e-in. Teflon tubing. Injections were made with a Hamilton 10-pl syringe through a Chromatronix injection port. The column was packed by adding small amounts of the solid phase-stationary phase matrix accompanied by both vertical and lateral tapping. The mobile phase used throughout was deionized distilled water ( p H 2 6 ) . Reagents. The following chemicals, obtained from various suppliers, were used in characterizing the peptide stationary phase: 0.025M phenylalanine, 0.002M tryptophan, 0.00625M tyrosine (saturated solution), 0.27M histidine, 0.18M phenol, 0.19M resorcinol, 0.18M hydroquinone, 0.18M catechol, 0.16M pyrogallol. 0.096M m-cresol, 0.12M o-cresol, 0.33M aniline, 0.048M p-toluidine, 0.093M o-toluidine, and 0.092M rn-toluidine. The solvent used for these solutions was distilled and deionized water ( p H of about 6). The following mixtures were used. Mixture A: 2 cm3 of 0.025M tryptophan, 10 cm3 of phenylndanine, plus 10 cm3 of tyrosine. Mixture B: equal volumes of solutions of histidine, phenylalanine, tryptophan, and tyrosine. Mixture C: equal volumes of solutions of hydroquinone, resorcinol, and pyrogallol. Mixture D: equal volumes of solutions of hydroquinone, resorcinol, and catechol. Mixture E: 2 cm3 of each solution of the toluidine plus 0.8 cm3 of aniline solution. Procedure. Preparation of Bonded Peptide. ( a ) Peptide on Resin Coated Glass Beads. The resin coated glass beads (120-140 mesh) were obtained from Northgate Laboratories. Hamden. Conn. The resin was designated as lot No. of SSS-312 and had a chlorine content of 0.57%/g of beads. The attachment of the first glycine (Gly) was carried out by adding to 25 g of the beads 697 mg of tertiary butyloxycarbonyl glycine, Boc-Gly, and 0.5 ml of triethylamine (TEA). The mixture was refluxed for 17 hr in 75 cm3 of dry ethanol. Elemental analysis of the product after reflux revealed a nitrogen content of 1.4 mg/g of beads. This corresponds to 0.1 mmol of Boc-Gly per g of beads. Additional Gly units were added by following the usual procedure ( I O ) . (b) Peptide on Porasil C and on Corasil 11. Both these solid supports were obtained from Waters Associates. The Porasil C used was 80-100 mesh (GC support) with 50-100 m2/g surface area and 200-400 A pore diameter. The Corasil I1 had a particle size range of 37-50 pm, and surface area of about 14 m2/gram. Unlike the resin coated glass beads, these two supports do not have chlorine on them. In order to introduce the required C1. the Porasil C and Corasil I1 were reacted with Union Carbide reagent No. Y-5918 [(l-trimethoxysilyl-2(4-chloromethylphenyl)ethane], which has the following structure
C~CHZ-C~H~-(CH~)~-S~(OCH~)~ This reagent reacts with the surface hydroxyl groups via the methoxy groups to give
R(S~~)~S~-(CHZ)~-C~H~-CH~C~ where R indicates the solid support particle. It must be noted that in all probability the silane reagent does not always react all three surface SiOH groups. In the reaction, 15.2 g of Porasil C were added to 60 cm3 of benzene and 8 cm3 of Y-5918, whereas with the use of Corasil 11, 10.1 g were added to 50 cm3 of benzene and 3.5 cm3 of Y-5918. The two mixtures were shaken a t room temperature for about 6 hr and then dried a t 80 “C in a vacuum oven overnight. Elemental analysis showed 1.5% Cl/g of Porasil C and 0.19% Cl/g of Corasil
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 9. AUGUST 1973
1627
Table I . Type of Packing Used in This Study 5.b Bound (w/w)
Support
coated glass beads Resin
(RCGB)
Porasil C Corasil I I
Size 120-140 mesh (181-212 w ) 80-100 mesh 37-50
'R indicates the solid support part
stationary phase 04
1.2 11
o
Most probable Composition of stationary phasen (Gly),-R
Boc-Gly-(Gly)n-R' BOC-GI~-(GI~)~-R'
R' indicates that between the pep-
tide and the support is the Y-5918 reaaent
11. The attachment of the amino acid groups is then carried out conventionally. The first Boc-Gly was placed on the solid support by adding 1.145 g of the protected amino acid and 0.82 cm3 of TEA to 15.2 g of Porasil C and in the case of Corasil, 90.7 mg of the Boc-Gly plus 0.068 cm3 of TEA to 10 g of the support. The two mixtures were refluxed for 17 hr in dioxane a t about I10 "C. Elemental analysis indicated that after reflux, the Porasil C contained 0.41% nitrogen and the Corasil I1 0.03% nitrogen, which corresponds to 0.293 mmol of Boc-Gly/g of Porasil and 0.0214 mmol of Bocy-Gly/g of Corasil 11. Seven additional Gly units were a t tached.
RESULTS AND DISCUSSION Throughout the study no attempt was made to optimize the chromatographic system. As mentioned previously, our immediate aim was to verify the utility of the peptide as a viable stationary phase. It should be mentioned here that the preparation of each bound stationary phase is limited by the kinetics of the actual coupling step. Each of the steps in the coupling procedure was carried out manually. Consequently, about 4 days were required to complete the entire synthesis described above. By using the automated system described by Scott ( I l ) , this time can be shortened considerably. The procedure of making the peptide stationary phase is rather simple (for a complete discussion on solid-phase synthesis of peptide, see ref 14). The Experimental section indicates that in the case of the resin coated glass beads and Porasil C about 1/3 to of the available chlorine was involved in the actual peptide synthesis. In the case of Corasil 11, only about one-sixth of the attached chlorine atoms were replaced by the amino aci.d. The same phenomenon of incomplete C1 utilization occurs in the usual Merrifield synthesis. Elemental analysis at the end of the procedure described above gave the following results: the resin coated glass beads contained about 0.4% Nz, the Porasil C support contained 1.2% Nz, and the Corasil I1 contained 0.1% N. These values were considerably lower than the expected values of about 1.1, 3.2, and 0.24% for the glass beads, Porasil C, and Corasil 11, respectively. A ninhydrin test was then performed on all three supports. Since the last attached amino acid should be protected by the Boc group, the test ought to be negative. With the Porasil and Corasil solid phases, the test indicated, a t least qualitatively, that 90% or more of the peptide was indeed protected. With the resin coated glass beads, the test indicated, by its intense blue color, that the majority of the terminal amino groups existed as the free amino. In other words, the coupling kinetics became very slow after the third amino acid was attached, and the deprotecting steps with the 4N HC1 in dioxane and the TEA leave the last amino acid in its amino form. (For some reason, some lots (14) J. M . Stewart and J. D. Young, "Solid Phase Peptide Synthesis," W. H. Freeman, San Francisco, Calif..1969.
1628
of the resin coated beads were found to be inferior to others as far as peptide synthesis was concerned. We chose this resin because of its high C1 content.) We decided to see how the peptide (most likely a tripeptide) affected the retention behavior of the several test compounds. The reason for the apparent low amino acid content on the Corasil I1 and on Porasil C might lie in the fact that when the elemental analysis was performed, the material was not completely dried from the last solvent used in the synthesis (methanol). Frequently in the course of the preparation, we checked for reaction completion with the ninhydrin test. The test always indicated a t least 90% completion. We did not, however, carry out a quantitative analysis after each coupling step. It is interesting to note, that after coupling three additional Boc-Gly to the Gly already on the support, elemental analysis showed that the nitrogen contents for Porasil C and Corasil I1 were about 0.8 and 0.06%, respectively. Repeating the coupling reaction four more times almost doubled the amount of amino acid on the Corasil (from 0.06 to 0.1%) and increased by a factor of 1.5 the amino acid content on the Porasil (from 0.8 to 1.2%). A summary of the packing used is given in Table I. Resin Coated Glass Beads (RCGB). The column was first packed with the RCGB only. Because of the large size of the beads, the efficiency, in terms of plate height, of the system was a mediocre 0.14 cm a t a velocity of about 0.5 cm/sec. At much lower velocities, we were able to obtain HETP values of less than 1 mm. A test mixture, mixture A, made of phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Try) failed to be resolved on the beads with distilled and deionized water as the mobile phase. The investigation was limited to these amino acids as a result of using the UV detection system. The peak shape indicated slight column overloading. The retention volume of the peak was about 0.78 which is about the void volume. Similarly, mixtures C and E showed only one peak with no resolution of the individual components (at a flow rate of about 18.8 cm3/hr). The peak due to mixture C was sharp and symmetrical having the retention volume of 0.78 cm3 which corresponded roughly to the dead volume of the system. The aniline and the toluidine peaks tailed to some extent and the retention volumes. roughly 0.88 cm3, were slightly higher than the dead volume. The column was then packed with the RCGB carrying the bound peptide. When mixture A was injected, a twopeak chromatogram resulted. The first peak was a composite of Phe and Tyr. The peak was symmetrical and sharp. The second peak, which was rather broad, was due to Try. The separation between the two peaks was complete and the whole analysis took about 6 min. Mixture B was then injected onto the column and the results are shown in Figure la. The first peak is due to histidine (His), the second to Phe, and the last to Try. The total analysis time was 80 min at a flow rate of about 2.97 cm3/hr. The HETP as measured from the Phe peak was about 0.096 cm. Note the large resolution between the Phe and Try. From a peak capacity point of view (15, 16) the system is inefficient as more components can, at least in theory, be separated between the above mentioned two peaks. The Try peak is quite broad, thus indicating some sort of interaction. The NH group in the indole side chain of Try is slightly acidic and, therefore. might interact with the free terminal amine group on the peptide or with ( 1 5 ) E. Grushka, Ana/. Chem., 42, 1142 (1970). (16) R . P. W. Sc0tt.J. Chromatogr. Sci., 9,449 (1971)
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 9, AUGUST 1973
~~~~
~
~
~
~
Table I I . Retention on the Various Solid Support-Peptide Systemsa RCG 6-Poly-Gly Solute
VI{, cm3
k' 0
His Phe Try TY r
0.79 1.1 2.9
...
...
Aniline
3.0 4.7 5.2 5.5 7.2 4.3 7.5
2.8 5.0 5.6 5.9 8.1 4.4 8.4
0.33 2.7
Porasil C-Bareb
Porasil-Poly-Gly'
Corasil-Bared
V H , cm3
k'
V R , cm3
k'
V H , cm3
4.5 1.9 1.9 1.5 2.4 5.1 2.6 3.1 1.6 1.6 1.6
5.0 1.5 1.5
1.1 1.7 2.7 1.7 2.1 2.0 3.1 2.7
0.47 1.3 2.6 I .3 1.8 1.7 3.1 2.6
1 .o 0.87 0.83 0.79 1.3 2.8 1.3 1.6 0.85 0.83 0.79 0.82
1.o 2.2 5.8 2.5 3.1 1.1 1.1 1.1
Corasil
k'
V H , cm3
0.45 0.26 0.20 0.14 0.88 3.1 0.88 1.3 0.23 0.20 0.14 0.19
0.75 0.85 1.2 0.85 1.1 1.3 1.4 1.4
,
+ Poly-Gly k'
0 0.13 0.57 0.13 0.48 0.68 0.92 0.92
p-To1uid ine o-Toluidine rn-To I uid i ne Phenol ... ... ... ... Hydroquinone 2.7 2.6 1.2 0.66 Resorcinol 3.6 3.8 1.6 1, I Catechol ... ... ... ... 3.7 3.9 1.6 1.1 Pyrogallol 5.2 5.6 1.6 1.1 3.4 3.5 ... ... 1.6 1. I o-Cresol 16.2 19.5 1.8 1.4 5.5 6.3 0.86 0.25 ... ... rn-Cresol 17.2 20.8 1.8 1.4 5.6 6.5 0.88 0.28 ... . . . a Mobile phase: distilled and deionized water. Due to pump limitation the retention data are accurate to 10% only. k' is based on the assumption that the column void volume is 0.75 cm3. k' is based on the assumption that the column void volume is 0.69 cm3 (see text).
--
the carbonyl oxygen on the backbone of the peptide. The His, on the other hand, has an imidazole side chain which is slightly basic and, consequently, it does not interact with the peptide phase. The Phe and Tyr which differ only by one OH group in the para position of the phenyl group are much too similar in their chromatographic properties to be separated by this particular peptide stationary phase. Two points of interest should be made here. (1) The separation was achieved with water as the mobile phase. The effect of different solvents or of different buffered solutions was not investigated as this aspect of the research is left for subsequent work. (2) The His peak was unusual in that it exhibited the shape of a first derivative of the Gaussian curve. This behavior occurred whenever the column was packed with the peptide support system irrespective of the type of solid support. On natural support, His exhibited the usual shaped peak. The reasons for this phenomenon are not clear. The behavior of mixture C is shown in Figure l b . The order of elution is hydroquinone, pyrogallol, and resorcinol, and the flow rate of the water mobile phase was about 8.73 cm3/hr. Because of the low efficiency, the peaks are not completely resolved. but lower flow rate would have improved the resolution considerably. Optimizing the system could not only increase the resolution but also decrease the analysis time. In any event, it became obvious that the poly-Gly peptide can be used in separating slightly acidic solutes while the natural RCGB could not. The low efficiency of the column should be noted. It, again, might indicate large resistance to mass transfer in the beads. To examine the effect of substituted phenols, phenol, o-cresol, and rn-cresol were injected separately onto the column. The retention volumes are shown in Table II. Since the mobile phase was water, this behavior indicated that the water-bound poly-Gly system behaves as a reverse phase. The introduction of the -CH3 group increases the retention noticeably. Mixture E was injected a t a flow rate of about 2.23 cm3/hr, and the first peak, aniline, was completely resolved from the toluidines. The toluidines, however, were not resolved although a shoulder due to p-toluidine could be observed. Again, introducing a methyl group causes the solute to be retained more strongly, thus again indicating a reverse-phase system.
10
Phe
His #",.SI
70
60
50
40
30
thin1
20
10
ib Pyr0qollol
1 Resorcinol
\
Hydroquinone A
l ,L , , , /
\
i",.Cl
eo
70
60
50
40
30
20
10
t (minl
Figure 1. (a) Mixture of His, Phe, and Try and (b) Mixture of hydroquinone, resorcinol, and pyrogallol (a) flow rate, 2.97 cm3/hr; 3 pl injection: mobile phase, distilled deionized water: peptide on RCGB. (b) flow rate, 8.73 crn3/hr: mobile phase, water: peptide on RCGB.
Table I1 summarizes the data obtained on the RCGB. With the exception of hydroquinone and pyrogallol, the hydroxyl benzenes are more retained than the basic aniline and toluidines. The data seem to indicate that the order of elution, at least for the aromatic system investigated so far on RCGB, ;.s para, ortho, and meta isomers. The reason for that seems to lie in steric or geometrical effects rather than electrostatic effects only. For example, the dissociation constant for phenol, hydroquinone, and resorcinol are much too close in value to account for the retention behavior. Porasil C. The column was first packed with unreacted Porasil C for comparison purposes. The particle size was too large (80-100 mesh) to yield efficient columns. The reasons for using such large particles were as follows: ( a )
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 9, A U G U S T 1973
1629
Phe t Tyr
75 50 25
t (mid Figure 2. Mixture B Flow rate, 3.63 crn3/hr; mobile phase, water; Porasil and Poly-Gly system.
the immediate availability in the laboratory, (b) an intention to use this support in investigating gas-liquid chromatographic systems, and (c) the unknown effect of the particle size on the coupling kinetics. The amino acids, except for His, gave fairly symmetrical peaks. His, on the other hand, gave a very broad and tailing peak having a much larger retention volume than Phe, Tyr, or Try. All the chromatograms showed a small extraneous peak which was probablji due to the solvent front (the same phenomenon is observed in Kirkland’s work (5, 6)) and which had the retention volume of about 0.75 cm3. This volume was taken as the void volume. The His could be separated from Try and Phe easily a t a flow rate of about 15.3 cm3/hr. The peak due to Phe and Try came at 1.9 cm3 while the His peak came a t about 4.5 cm3. The His peak was strongly skewed. The retention behavior of the His is clear, in view of the basicity of the imidazole and its interaction with the acidic sites on the surface of the Porasil (silica gel beads). At a much slower flow rate (about 1 cm3/hr), the chromatogram showed slight evidence that there is more than one component under the first peak; the Tyr causes a break in the smoothness of the front part of the peak. A more efficient system, at low flow rates, could most likely separate Tyr from the Phe and Try peaks. The capacity ratios of the amino acids are shown in Table II. The cy value between Tyr and Phe or Try is about 1.3. An efficient column system would without doubt separate these compounds. The retention behavior of some of the aromatic compounds is also shown in Table 11 and as expected the basic anilines are retained longer. On this chromatographic system, phenols and the individual anilines with the exception of p-toluidine could not be separated; however, it appeared that the order of elution was ortho, meta, and para. The system described here is, of course, adsorption chromatography, and it is to be expected that para isomers, being more linear, interact strongly with the adsorption sites on the silica surface. As the two substituent groups on the aromatic ring get closer, the interaction 1630
with the adsorption sites is weaker due to (a) steric hindrance and (b) fewer adsorption sites which are involved in the retention mechanisms (17). In addition, in the case of the anilines the relative basicity of the species affects the retention. p-Toluidine is the most basic of the anilines. After these initial studies, the column was repacked with the Porasil C-peptide system. Consistent packing was hard to achieve here since the solid support particles tended to agglomerate and also electrostatically adhere to the glass column wall well above the actual packed head and, as a result, the column produced was extremely inefficient. We continued experimentation with this column since, albeit inefficient, it showed some interesting phenomena, later substantiated by the more efficient Corasil columns. Figure 2 shows a chromatogram of the amino acids (mixture B) at a flow rate of about 3.63 cm3/hr. The peaks, in order of elution, are His, Phe + Tyr, and Try. Note (a) that again the peculiar “first derivative” shape of the His peak was shown and (b) the reversal of the elution order of the His and the Try. The elution order in Figure 2 is similar to that of Figure 1 where the solid support was RCGB. The reasoning behind the retention order here is similar to the RCGB-peptide system and it indicates that most of the available surface to the solute is “covered” with the bound peptide. Here the resolution between His and Phe + Tyr is just about unity and between the second and the third peaks is about 0.7, The anilines had the retention volumes shown in Table 11. Again, the order of elution among the toluidines is quite different from that obtained with the bare Porasil C and, in fact, the ortho and para seem to reverse their behavior. p-Toluidine is considerably more basic than aniline and the other two toluidine isomers which may explain its rapid elution. At the flow rate of about 4.86 cm3/hr, the mixture of the anilines (mixture E) showed two peaks separated by a small valley ( R , = 0.6). The first peak was due to p-toluidine and aniline, while the second was that of the other two isomers. With a more efficient system, the ortho and meta toluidine would most likely be separated from one another and from the aniline. In any event, the permanently bound peptide phase has a profound effect on the retention behavior of these compounds. Mixture C (hydroquinone, resorcinol, and pyrogallol) at the lowest possible flow rate (gravity flow) gave a system of a shoulder on the front side of a main peak (ie., resolution less than 0.5). The shoulder was due to hydroquinone, while the main peak was a combination of resorcinol and pyrogallol. The individual retention volumes are given in Table II. Similarly, mixture D (dihydroxybenzene isomers) yielded a chromatogram consisting of two partially resolved peaks ( R , = 0.6), the first due to hydroquinone and the second due to resorcinol plus catechol. The relative retention of these two peaks was 1.4. An efficient system would have little difficulty in separating these two peaks. With the RCGB, it should be remembered, most of the terminal amino groups were free. Here most of them are protected by the Boc. The retention behavior is very similar in both cases. Consequently, it can be said that the free amino group has little influence on the overall partitioning mechanism. In order to check this observation, the Boc group was removed from the peptide on the Porasil C. ( 17) L. R. Snyder, “Principles of Adsorption Chromatography,” Marcel Dekker, New York. N. Y . 1968.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
Mixture B gave the same chromatogram as on the protected peptide. The His peak still had the peculiar “first derivative” shape to it, and the anilines and the phenols had the same retention properties on the unprotected as on the protected peptide phase. They were eluted in the same order and had roughly the same retention volumes. This result has a very important implication; the whole peptide chain is involved in the separation and not only the terminal groups. The cresols had larger retention volumes than any of the phenols, thus again indicating a reverse-phase type system. Corasil 11. This solid support is particularly well suited for liquid chromatography. Since the outer shell of the pure packing is essentially silica gel, similar retention behavior should be observed here as compared to Porasil C. Indeed, with mixture B (the amino acids), the order of elution is similar on both supports (without the peptide bound phase). However, because of the much better efficiency of the Corasil I1 column, the Tyr was well sepaPhe peak. The His peak, which was rated from the Try also well resolved, tailed severely and, as with the Porasil, was retained the most. This is shown in Figure 3a Some trouble was experienced in keeping the flow rate constant (because of pump limitations) in these sets of experiments, and the flow changed by about 10% during the analysis. The two spurious peaks at the beginning of the chromatogram are due most likely to solvent front and some impurity. It was thought at first that the injection head was contaminated, but careful cleaning did not change the chromatogram. From these extraneous peaks we summarized that the dead volume is about 0.69 cm3. By running the individual components, the retention volumes shown in Table I1 were obtained. Because of difficulties in flow regulation, these values are correct to within 10% only. It seems, in fact, that pure Corasil 11 can have some potential in amino acid analysis via adsorption chromatography. An optimum system can separate all 4 amino acids, a t least partially resolving the Try-Phe pair, using deionized distilled water as the mobile phase. Mixtures C and D do not separate at all on pure Corasil 11. The retention volumes of the individual compound are much too close. The anilines, however, because of their basic nature, can be separated. Figure 3b shows the chromatogram of mixture E. The first peak to elute is aniline and o-toluidine, the second is rn-toluidine, and the last one is p-toluidine; this is the same order of elution as on Porasil C. The retention volumes were 1.19, 1.91, and 4.14 cm3, respectively. At a higher flow rate (16.73 cm3/hr). the retention volumes were 1.27, 1.63, and 2.75 cm3 for the o-, m-, and p-toluidine, respectively. The difference in the retention volumes a t high and low velocity can be attributed to either one of the two causes: ( a ) flow variations due to pump irregularities and (b) capacity ratio dependence on the flow rate. The latter was described by Kirkland ( 6 ) in his study on bound phases. The mobile phase velocity effect on the partition coefficient merits a more careful examination in light of Kirkland’s data and the present work. As expected, the bound peptide on the Corasil I1 u.fected the retention behavior of the various solutes. Figure 3c shows the amino acid mixture at a flow rate of 1.47 cm3/hr. The order of elution is again reversed and His came out first while Try was last. From the Try peak, we calculated an H value of 0.026 cm (1160 plates) which indicate an efficient system. The Phe and the Tyr peaks are again unresolved. As expected with the peptide phase on,
3b
r,lk,_,,o, &kw 60
40
20
140
120
t Iminl
3c
1
1I
3d
lw
80 t h”I
40
X
Pnilins
+
p-Taluidify
Phe t Tyf
60
1
i
Figure 3. ( a and c ) mixture B, ( b and c) mixture E (a) Mixture B: flow rate, 1.38 cm3/hr; mobile phase, water; pure Corasil II. (b) Mixture E: flow rate, 2.14 cm3/hr; mobile phase, w a k . pure [Cor. asil / I . (c) Mixture E: flow rate, 1.47 cm3/hr: mobile pnasc Hater; Corasil-poiy-Gly peptide. ( d ) Mixture E: same condition as c
His yielded the “first derivative” shape. The retention data are shown in Table 11. The anilines mixture (mixture E ) gave three peaks, which, in order of retention time, are aniline, p-toluidine, m-toluidine (see Figure 3 4 . Again, the p-toluiand 0dine retention was inverted in that it came before the other two isomers. The peaks showed slight overloading and the resolution between each pair of peaks was about 0.7 and 0.8. Some of the retention data is shown in Table
+
11. Mixtures C and D each gave a chromatogram made of two well separated peaks ( R , was at least 1.0). For mixture C, the flow rate was 1.40 cm3/hr. The first peak was that of hydroquinone while the other was due to resorcinol plus pyrogallol. In mixture D, the first peak was again due to hydroquinone while the second was resorcinol plus catechol. The peaks were symmetric and sharp. The plate height from all the peaks was about 0.021 cm. Even at higher flow rates, e . g . , 14.9 cm3/hr, we obtained H < 0.1 cm. Here, again, as with the previous two supports and peptide system, the hydroquinone eluted first. The retention data are shown in Table 11. Apparently, the hydroquinone does not interact as strongly with the peptide as the other dihydroxybenzenes or the pyrogallol. The reasons for that behavior are most likely due to geometrical factors rather than polar forces. The cresols as expected had higher retention volumes than any of the phenols. Although again trouble was encountered in keeping a constant flow with the pump used, it was obvious that the
A N A L Y T I C A L CHEMISTRY, VOL. 45, N O . 9, AUGUST 1973
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retention order was para, meta, and ortho, as observed with the other systems. However, the meta and ortho isomers were usually very close so that they were eluted as one peak.
CONCLUSIONS The permanently bound peptide can make an interesting stationary phase. It has a pronounced effect on the elution characteristics of the several classes of compounds which we discussed above. The fact that water was used as the mobile phase in all the studies should be emphasized. The effect of other solvents is being investigated. The poly-Gly peptide can be used in separation of amino acids and para isomers from ortho and meta of some classes of solutes. The fact that the retention behavior was the same on all three support-peptide phase systems seems to demonstrate that the peptide had the major role in the separation. In this connection, it would be of major importance to investigate the effect of the peptide chain length and, perhaps more important, the relative amount of peptide per unit weight of the support on the retention behavior and mechanism. In addition, it seems that the
whole peptide chain takes a direct part in the partitioning mechanism and not the terminal group only. This indicates that other polypeptides having different amino acid subunits can be tailored to different separations. These aspects of the peptide phases are now under study. Finally, by using amino acids having asymmetric centers as bound phase, and using, if needed, the recycling techniques recently described by Little (181, separation of D and L amino acids and peptides can be attempted. This is also currently under investigation.
ACKNOWLEDGMENT The authors wish to thank C. G. Scott and K. K. Chan for many helpful discussions and suggestions. We also thank J. A. Faucher of Union Carbide Corporation for the sample of Y-5918. Received for review January 19, 1973. Accepted March 19, 1973. (18) J. N. Little, 164th National Meeting of the American Chemical Society, New York, N. Y . , Aug. 28-Sept. 1, 1972, paper No. Anal. 9.
Rapid, Selective Method for Lead by Forced-Flow Liquid Chromatography M. D. Seymour1 and J. S. Fritz Ames Laboratory-USAEC and Department of Chemistry, lowa State University, Arnes, lowa 50010
Lead(l1) is retained on a small anion exchange column from 0.5M hydrochloric acid and separated from many other metal ions. Then it is eluted with 8M hydrochloric acid and the elution curve is recorded at 270 nm. The amount of lead is obtained from a plot of elution peak height YS. pg of lead. The entire separation sequence requires only 8 min. Several standard samples have been successfully analyzed for lead.
The literature abounds with methods for the determination of lead. This reflects the long-standing interest in the development of more rapid and selective methods for this element in a great variety of matrices. In general, these methods involve a separation step in which lead is removed from interferences and concentrated. Solvent extraction using sodium diethyldithiocarbamate or dithizone followed by photometric determination of the resulting lead complex in the organic phase has proved sensitive and selective (1-3). However, extraction methods often require addition of a number of reagents which require painstaking purification. The high p H values necessary for selectivity often preclude analysis of samples high in metal content and can lead to reagent instability. Ion ex1 Present address, T h e Procter a n d Gamble Company, M i a m i
Valley Laboratories, Box 39176, Cincinnati, Ohio 45239. (1) t i . H. Lockwood. Anal. Chem. Acta. 10, 97 (1954). (2) H . Bode, Fresenius' Z.Ana/. Chem.. 144, 165 (1955) (3) L J. Snyder. Anal. Chem.. 1 9 , 684 (1947).
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change methods have enabled separation of lead from bismuth, cadmium, and thallium, which often interfere with extraction techniques, as well as a large number of other metals ( 4 ) . Using strong-base anion exchange resin, separation from a t least thirty-nine ions can be effected in hydrochloric acid media ( 5 ) . This is the basis for several methods utilizing subsequent polarographic, spectrophotometric, and spectroscopic determinations (6-9). Again, much sample manipulation is involved, consuming time and sacrificing accuracy. Recently a forced-flow ion exchange separation of iron from metal ions was described ( I O ) . Iron(II1) was eluted with hydrochloric acid and measured using a UV detector; the amount of iron eluted was determined from a linear calibration plot of peak height us. amount of iron. In the present work, lead is separated from other metal ions by forced-flow chromatography and measured in a manner similar to that used for iron (10). Lead(I1) is sorbed on an anion exchange column from dilute hydrochloric acid and separated from most matrix elements. It is then eluted with more concentrated acid and estimated spectrophoto(4) 0. Samuelson. "Ion Exchange Separations In Analytical Chemistry," John Wiley and Sons, New York, N.Y., 1963, p 406. (5) K . A . Kraus and F. Nelson, "Metal Separations by Anion Exchange," in ASTM Spec. Tech. Pub/. No. 195. American Society for Testing a n d Materials, Phiiadelphia. Pa., 1958. J R. Nash and G. W . Anslow, Analyst (London). 88, 963 (1963) E. i Johnson and R. D. Pohill. Analyst (London), 82, 238 (1957). E. A . Wynne, R . D.Burdick. and L. H. Fine, Ana/. Chem., 33, 807 (1961) . N. G. Sellers, Anal. Chem., 44, 410 (1972). M D. Seymour, J . P. Sickafoose. and J . S. Fritz, Anal. Chem.. 43, 1T34 (1971).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
