Anal. Chem. 1983, 55, 1390-1395
1390
pain and found to have LDH-1:2 levels >0.76 might be suspected of stroke or heart attack. Table IV shows also that certain other pathological conditions such as abdominal surgery and angina may also cause a LDH-l:LDH-2 ratio higher than normal. This work indicates that LDH isoenzymes in human serum samples or other bodily fluids can be fractionated completely with a pH-coupled salt gradient elution technique after initial absorption of a diluted sample (0.3 mL). The ratio of any pair of the isoenzymes can be obtained from the activities of the separated isoenzymes rather than from the percentages on the basis of electrophoretic method. Even small quantities of LDH-4 and LDH-5 present in normal serum samples have been measured by this technique. The sensitivity and precision of the assay using the chromatographic technique appear to be higher than that obtained by the electrophoretic method.
ACKNOWLEDGMENT We are grateful to R. M. G. Nair and Jana Johnson of VA Medical Center, Charleston, SC, Jane Jennings and Terry Best of Memorial Medical Center, and Geetha Bala of Candler General Hospital, Savannah, GA, for their assistance in obtaining human serum samples and their results. Laboratory assistance rendered by Jimmy Gregory is also appreciated. Registry No. Lactate dehydrogenase, 9001-60-9.
LITERATURE CITED (1) Cohen, M. D.; Djordjevlch, J.; Jacobsen, S. M e d . Clin. North Am. 1966, 50, 193-209. (2) Heln, R. C.; Grayback, J. T.; Goldberg, E. J. urology 1975. 113, 51 1-516. (3) Roe, C. R. Ann. Clin. Lab. Sci. 1977, 7 , 201-209. (4) Lott. J. A.; Stang, J. M. Clln. Chem. (Winston-Salem, N.C.) 1980, 2 6 , 1241-1250.
Talagerl, V. R.; Nadakarern. S. S.; Gollerken, M. P. Indlan J . Cancer 1977, 1 4 , 43-49. Wagner, 0. S.; Roe, C. R.; Llrnberd, L. E.; Rosali, R. A,; Wallace, A. C. Circulation 1973, 47, 263-269. Vasudevan, G.; Mercer, D. W.; Varat. M. A. Circulation 1978, 57 (E), 1055- 1057. Galen, R. S. Hum. Pathol. 1975, 6, 141-144. Wrlght, E. J.; Cawley, L. P.; Eherhardt, L. J . Clin. Pathol. 1966. 4 5 , 737-741. Wleme, R. J.; Macrche, Y. Ann N . Y . Acad. Sci. 1961, 9 4 , 898-901. Oliver, J. A.; Elhilal, M. M.; Belitsky, P.;Mackinnon, K. J. Cancer 1970, 25. - , 863-867. - .- - .. . Elhilall, M. M.; Oliver, J. A.; Sherwin, A. I.; Mackinnon, K. J. Cancer 1967. 98. 686-689. Mercer, b. W. Clh. Chem. (Wlnston-Salem, NC) 1975, 21, 1102-1 106. Kreutzer, H. H.;Eggels, P. H. Ciln. Chim. Acta 1965, 12, 80-88. Joseph, R. N.; Schulz, J. J . Lab. Clin. Med. 1962, 6 0 , 349-353. Springell, P. H.; Lynch, T. A. Anal. Blochem. 1976, 7 4 , 251-253. Hsu, Mei-Yung; Kohler, M. M.; Barolla, L.; Bondar, R. J. L. Clin. Chem. (Wlnston-Salem, N . C . ) 1979, 25 (E), 1453-1457. Wacker, W. E. C.; Ulrner, D. D.; Vallle, P. L. N . Engl. J . M e d . 1956, 295, 449-453. Wrobleswskl, F.; LaDue, J. S. Proc. SOC.Exp. Biol. M e d . 1955, 9 0 , 216-2 18. Technlon Method No. SG4-0021FH9, Technlcon SMAC System, Aug 1979. Dletz, A. A.; Lubrano, T. Anal. Biochem. 1987, 20, 246-257. Thlers, R. E.; Vallel, B. L. Ann. N . Y . Acad. Scl. 1958, 75. 214-218. Amador, E.; Dorfrnan, L. E.; Wacker, W. E. C. Cftn. Chem. (WlnstonSalem, N . C . ) 1983, 9 , 391-395. Buhl, S. N.; Jackson, K. Y.; Lublnskl, R.; Vanderllnde, R. E. Clln. Chem. (Winston-Salem, N . C . ) 1977, 23, 1289-1295. Leung, F. Y.; Henderson, A. R. Clin. Chem. (Winston-Salem, N.C.) 1979, 25, 209-211.
RECEIVED for review October 22, 1982. Accepted March 7, 1983. The financial support provided by the Division of Research Resources of the National Institutes of Health (MBRS Grant No. 5 SO6 RR08144-08, Project No. 5) is gratefully acknowledged. This paper was presented at the annual meeting of Georgia Academy of Sciences, Columbus, GA, April 24-25,1982.
Characterization of Substitution-Inert Cobalt(I 11) Complex Bonded-Phase Columns for Liquid Chromatography C. Allen Chang,’ Chen-Shl Huang,
and Cheng-Fan Tu
Department of Chemistry, The Universitv of Texas at El Paso, El Paso, Texas 79968
The preparation procedures for several substitution-inert cobalt( I I I ) complex bonded phases, [Co(en),]CI,, [Co(edda)(en)]CI, [Co(dmedda)(en)]CI, and [Co(deedda)(en)]Cl, are described where en = ethylenediamine, edda = ethylenediamlne-N,N’-dlacetate Ion, dmedda = N,N’-dimethylethylenedlamlne-N,N’-dlacetate ion, and deedda = N,N’dlethylethylenedlamlne-N,N’-dlacetateIon. All the complex bonded phases are found to have mlxed surface coverage of both diamine and the complexes. A method Is then deveioped to calculate the surface coverage of the complex. Besides the normal chromatographic characteristics such as the relationshlps of the capacity factor and the number of theoretical plates vs. sample size and flow rate, a simple thermodynamic approach to sort out lndlvldual contribution for the overall mlxed retention is reported. Knowing the AH value of solute transfer from the mobile phase to the stationary phase for the column, the AH value due to complex alone can be calculated provlded the percent surface coverage of the complex Is known. The AH values for all complex bonded phases are then compared and rationalized according to the structural nature of the complexes.
The use of metal ions or complexes to enhance separation has gained momentum only recently in modern high-performance liquid chromatography although the concept is not a new one. For example, it is known that early in 1903-1906, calcium carbonate was used by Tswett to separate plant pigments (I). In general, metal ions and their complexes can be roughly divided i n t ~ two classes, i.e., substitution inert and substitution labile, based on their kinetics of ligand displacement reactions. Many metal complexes are substitution labile and the use of them in liquid chromatography is generally called “ligand exchange chromatography” (2). Depending on whether the metal ion or complex is fixed on the stationary phase or whether it is moved along the column in the mobile phase, one can distinguish two types of ligand exchange chromatography: (1) the chromatography of ligands in which the metal ion is held by the stationary phase via strong complex formation or adsorption; (2) the chromatography of complexes in which the metal ion is bound more strongly toward the ligands in the mobile phase. One novel application of ligand exchange chromatography is the separation of enantiomers, particularly, d- and l-amino
0003-2700/83/0355-1390$01.50/00 1983 Arnerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
1391
concentrated hydrochloric acid was added to the purple complex acids (3-116). In most cases, the separations are based on the and the mixture was evaporated on a steam bath. The blue in situ stereoselective formation of the chiral diastereomeric complex, cis-H[Co(edda)Cl,], was collected by filtration and xeternary metal complexes. However, it is noted that in some crystallized in ethanol-water mixture (yield 6.9 9). cases, either the column "bleeds" or the mass transfer is poor To prepare [Co(edda)(en)]Clbonded phase materials, a 5.501-g in ligand esxchange chromatography. On the other hand, the sample of cis-H[Co(edda)Cl,] was dissolved in 50 mL of water mechanisms and stereochemistries of many reactions involving at pH 7.0. A 2.5-g portion of the ethylenediamine bonded silica substitution-labile metal complexes are not quite clear. These gel was added into the solution with stirring. The mixture was factors make the applications of substitution-labile metal then heated at ca. 70 "C for 4 h complexes in liquid chromatography complicated. Thus, it I I RSiOSi(CH2)3NH(CH[2),NH2 + H[Co(edda)Cl,] may be advantageous to use substitution-inert metal comI I plexes for liquid chromatographic separations. RbiOSi(CH2)3[Co(edda)(en)]C1.HC1 Up to the present, few chromatographic studies that use I I substitution-inert metal complexes have been reported. Chow The resulting pink cobalt complex bonded silica was filtered arid and Grushka have prepared a covalently bonded phase conwashed successively with 200 mL of an aqueous solution with a taining tris(ethylenediamine)cobalt(III) chloride, [C~(en)~]Cl:~, pH of 1.5 and 200 mL of absolute methanol and vacuum dried at 70 "C. to separate nucleotides and nucleosides (17). Inczedy and his Preparation of (N,N'-Dimethylethylenediamine-N,Wco-workers have used both [Co(en),13+ ion and [Co(NH3),13+ diacetato)(ethylenediamine)cobalt(III) Chloride, [Coion in the mobile phase to separate anions such as sulfate and (dmedda)(en)]Cl, amid (N,N'-Diethylethylenediamine-N,oxalate ions by using an anion exchange column (18). SepaN'-diacetato)(ethylenediamine)cobalt(III) Chloride, [Coration of optical isomers of aspartic acid and mandelic acid (deedda)(en)]Cl, Bonded Phases. The ligands dmedda and on a weaklly acidic cation exchange column loaded with Ddeedda were prepared according to the method of Legg and Cooke [Co(en),13' cations was also described (19). The mechanism (23). The dichlorocobdt complexes of dmedda and deedda were of separation has been ascribed to stereoselective outer-sphere prepared next using similar methods as that for cis-H[Coion-pair formation; however, detailed understanding of these (edda)C12]. The final bonded phase materials were prepared by refluxing cis-HICo(dmedda)Clz]or cis-H[Co(deedda)Cl,]with the systems stid1 awaits further studies. ethylenediamine bonded phase in an ethanol-water mixture and Recently, we have reported the liquid chromatographic then treated the same way as for [Co(edda)(en)]Clbonded phase. separations of dihydroxybenzenes with silica, ethylenediamine, Four cobalt(II1) complex bonded phase columns and an and two cobalt(IT1) complex bonded phase columns as an ethylenediamine bonded phase column (4.6 mm i.d. and 15 or 25 initial attempt to characterize these substitution-inert metal cm length) were packed by the up-flow method using Micromcomplex bonded phases in comparison with other stationary eritics Model 705 stirred-slurry column packer. 2-Propanol was phases (20). A similar study on the thermodynamic paramused as the solvent for column packing. eters for the separation of fructose, glucose and sucrose is also Chromatographic Procedures. Sample solutions of nitrocarried out (21). In this paper, we wish to report detailed anilines (2 mg/mL) were prepared in a 2-propanol-hexane mixchemical and physical characterizations of several substitu. ture. Pressure-Lok series (2-160 10-pL syringes (Precision Sampling Corp.) were used to inject 1-2 pL amounts of the samples. tion-inert cobalt(II1) complex bonded phases. Flow rates were generally either 1 mL/min or 2 mL/min with back pressures less than 1000 psi. All data points were collected EXPERIMENTAL SECTION by averaging more than four reproducible separations and treated Apparatus. A Micromeritica (Norcross,GA) Model 7500 liquid with normal statistical rmethods. m-Xylene or benzene was used chromatograph equipped with a Model 750 solvent delivery to determine tofor each column and it was found that toobtained system, Moldel 752 ternary solvent mixer, Model 731 column did not vary with different solvent composition and temperature. compartment with universal sample injector and variable temRESULTS AND DISCUSSION perature control from ambient to 150 "C, and Model 786 variable-wavelength (200-600nm) detector with a deuterium lamp was S u r f a c e Coverage of Bonded Phases. Chemically used. A Waters Associate Model 440 absorbance detector (254 amorphous silica consists of polysiloxane groups and several nm) was a h used. types of silanols which include free and hydrogen bonded ones Reagents. Partisil-10 solid silica support was obtained from The surface silanol groups are considered to be those chro. Whatman (Clifton, NJ). The 3- (2-(aminoethy1)amino)propylmatographically active. In order to prepare bonded stationary trimethoxysilane was purchased from Silar Laboratories (Scotia, phases, the very reactive silanol groups on the silica surface NY). Solvents used were all HPLC grade mainly from MCB are usually employed. In general, the silicas are heated at Manufacturing Chemists, Inc. (Cincinnati, OH) and Fisher Scibetween 125 and 175 OC: to liberate adsorbed water. The active entific Co. (Pittsburgh, PA). All other compounds and reagents silicas are then to react with the desired bonded phases. In were of reagent grade obtained from various sources. Elemental analyses were performed by Galbraith Laboratories, Inc., the present cases, ethylenediamine bonded phase is firstly Knoxville, TN, and Micanal Organic Microanalysis, Tucson, AZ. prepared and all the clobalt(II1) complex bonded phases are Preparation of Columns. The ethylenediamine and tristhen prepared by reacting the ethylenediamine with the (ethylenediarnine)ccsbalt(III)chloride, [Co(en),]C13,bonded phase cis-dichlorocobalt complexes. materials were prepared by using Partisil-10, 3-(2-(aminoIn order to account for the retention behavior with each ethyl)amino)propyltrimethoxysilane, and cis-[dichlorobisbonded phase, it is important to estimate the surface coverage (ethylenediamine)]cobalt(III) chloride, [ C~(en)~Cl,]Cl, according of each kind of bonded groups. In general, the method of to published methods with minor modification (17). Unger is used for the (calculation (24) Preparation of (Ethylenediamine) (ethylenediamine-N,N'-diacetato)cobalt(III) Chloride, [Co(edda)(en)]Cl, Bonded W Phase. The synthesis of hydrogen cis-dichloro(ethy1enediAexp = amine-N~'-diacetato)cobaltate(III), H[Co(edda)C12],was carried out by the mlodified procedures of Van Saun and Douglas (22). where Aerp = the surface concentration of bonded groups in To a solution of ethylenediamine-N,"'diacetic acid (10.0 g, 0.057 mol/m2, W = the weight of functional groups as grams per mol) and CoCl2.6H20(14.87 g, 0.063 mol) in water (150 mL) was gram of adsorbent, M = molar weight of the bonded functional added NaOH solution (6 N) to adjust the pH to ca. 8.0. A stream groups (g/mol), and S := specific surface area of the support, of air was bubbled into the mixture for 20 h and a purple complex corrected for the weight increase due to the modification formed gradually during the oxidation. After the solution was (m2/g). According to the manufacturer's guide the irregular concentrated, ethanol was added and the insoluble purple complex was separated by filtration (yield 7.39 9). Fifty milliliters of shaped Partisil-10 has a surface area of 396 m2/g. Also, it is
&
a
1392
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
Table I. Elemental Analysis Data for Diamine and Cobalt Complex Bonded Phases %Cod
%
bonded phases
%C
ethylenediamine
7.4Sa 6.80b 7.7Sa [Co(en)3 1c4 6.01 [Co(edda)(en)]Clb 6.06 [Co(dmedda)(en)]Cla 5.78 [Co(deedda)(en)]Cla 6.19
%N
Z H Coc LTOC
3.00 2.77 2.81 2.43 1.56 1.74 1.91
2.27 1.51 2.21 1.66 1.38 2.06 2.00
2.22 0.98 0.62 0.35
0.37 0.16 0.11 0.06
a Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN. Most data were averages of two independent measurements. Elemental analyses The cobalt were performed by Micanal, Tucson, AZ. content data were checked by the X-ray fluorescence technique and similar results were obtained ( 2 6 ) . If all the bonded ethylenediamine formed cobalt complexes, the % Co/% C ratio should be 0.49 for [Co(en),]Cl,, 0.41 for [Co(edda)(en)]Cl, 0.35 for [Co(dmedda)(en)]Cl, and 0.31 for [Co(deedda)(en)]Cl.
Table 11. Surface Coverage (pmol/m2)of Diamine Bonded Phasesa, if one CH30 reacted
if two CH,O reacted
if three CH,O
reacted _
1
2
C 2.69 2.42 N 3.40 3.08 H 3.91 2.37
3
1
2
2.83 3.14 3.78
3.13 3.26 4.63
2.81 2.97 2.81
3
1
l
l
2
due to the reason that the samples were not dried enough. In fact, if the samples were dried carefully the hydrogen percentages are generally low as found for the analysis performed by Micanal. On the other hand, it can be seen that the surface coverage values are more self-consistent if one assumes that two of the three methoxyl groups of the diamine have reacted. This is consistent with the report by Leydon et al. (25) and the averaged surface coverage value 3.1 pmol/m2 i5 also consistent with that reported by Chow and Grushka (17). This would mean that 80% of the surface silanol groups have reacted with the trimethoxysilane group of the diamine. In the cases of cobalt(II1) complex bonded phases, two additional factors should be considered in the calculation of the surface coverage. First, it is obvious that some of the bonded ethylenediamine has been washed away from the silica surface during the preparation. This is indicated by the lowered percent C and percent N data of cobalt complex bonded phase (Table I). Second, it is also observed that not all the diamines reacted with cobalt(II1) complexes which is demonstrated by the low values of cobalt content, i.e., the % Co/ % C ratios are lower than those assumed for complete reaction. A method to account for both factors has been established by us to calculate the surface coverage values. The surface concentration of bonded cobalt complex is first calculated from percent Co data. The percent C and percent N values are then modified by substracting the contribution due to the cobalt complex, Finally, the diamine surface concentrations are estimated from the remaining C, H, and N content. Table I11 lists the calculated surface concentration values for each cobalt complex bonded phase. It is observed that on an average 50% of the bonded diamine groups have been washed away during the reaction. Also, the surface concentration of the cobalt complex in decreasing order is [ C ~ ( e n ) ~ ]> C l[Co(edda)(en)JCl ~ > [Co(dmedda)(en)]Cl > [Co(deedda) (en)]C1. Complexes such as [Co(dmedda)(en)]Cl and [Co(deedda)(en)]Cl are difficult to be grafted on the silica surface which may be due to their lower solubility in water and the less reactivity in mixed solvent, i.e., ethanol and water. ChromatographicCharacterizations of Columns. In order to characterize each column, a mixture of nitroaniline isomers was injected into each column for separation. Table IV lists the capacity factors for these compounds using four cobalt complex bonded phase columns and the diamine column. It should be noted that the unreacted diamines in the columns have all been treated with dilute HCl solution so that the actual form is ethylenediammonium dichloride.
_
3
3.28 3.73 3.35 3.91 3.02 3.13 2.86 2.90 4.48 5.69 3.45 5.50
a 1, 2, and 3 are designations for each preparation. The numbers listed at the same row of C are calculated values Samusing % C data, and similarly for rows of N and H. ples of the first and the third preparations were analyzed by Galbraith Laboratories, Inc. The second preparation was analyzed by Micanal.
known that the surface silanol groups are usually 8.0 f 0.5 pmol/m2. Table I lists the elemental analysis data for each bonded phase material. Three preparations were made for the ethylenediamine bonded phase. Assuming one, two, or three methoxyl groups of (3-(Paminoethyl)aminopropyl)trimethoxysilane are reacted with the silica surface, the calculated values of surface coverage of diamines are listed in Table 11. It is observed that the surface coverages calculated from the hydrogen percentage data are unusually high which may be
-
~
Table 111. Surface Coverage Data (pmol/m2)for Each Cobalt Complex Bonded Phasea bound complex
a
0.95 [Co(en), IC13 0.42 [Co(edda)(en)]Cl 0.31 [Co(dmedda)( en) IC1 [ Co(deedda)(en)]Cl 0.18 [Bonded complex] + [free diamine] = [total diamine].
free diainine
total diamine
% diamine lost during reaction
0.52 0.93 f 0.30 1.21 0.20 1.69 f 0.20
1.47 1.35 i 0.30 1.52 0.20 1.87 0.20
53 57 52 40
* *
_+
____
lll.ll
_11_111~-_411_-
~_l--_lll_l_lllll-l-lllllll
Table IV. Capacity Factors of Nitroanilines from Five Bonded Phase Colutnns of k
columns
aniline
o-nitroaniline
diamine ICo(en), 1% [ Co(edda)(en)]C1 [Co(dmedda)(en)]CI [ Co(deedda)(en)]CL
0.95 +. 0.02
1.47 + 0.03 2 39 j. 0.07 1.71 + 0.02 1.82 t 0 0 6 1.42 t 0 03
1.90 ' 0.01
nz-nitroaniline 2.92 3 5 46 f 4.10 .t 4 31 f
3.77
j:
0.04 0.11 0.09
0.12 0 03
p-nitroaniline 6.16 r 0.05 f 0.17
12.64 7.81 8.84
+
0.14
i0.20
6.41 + 0 . 0 3
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983 3
A
-~ 0
2
1893
4
6
8
3
1
C
1
7
2
A
0
0
0
l
0
2
8
6
4
do
15
12
1C
o
i
L 00
I
2'0
10 Sample Size
io
(pi )
Figure 3. Plot of the nuimber of theoretical plates ( N )vs. sample size. Condltlons are the same as those given In Figure 2. Column length = 15 cm. .Table V. Separation Factors of Nitroanilines with o-Nitroaniline as the Referencea 3
2
4
6
8
1
0
1
a:(meta/ ortho)
2
Time ( min )
Figure 1. Chromatograms of nltroanlllne Isomers. Columns: (A) diamine bonded phase; (B) [Co(edda)(en)]CIbonded phase: (C) [Co(deedda)(en)]CIbonded phase. Solutes: (1) benzene, (2) aniline, (3) o-nitroaniline, (4) rn-nitroanlllne, (5) p-nltroaniline. Flow rate = 2 mL/mln. Solvent: 25% 2-propanol 75% hexane.
+
a
I
A
40 A
0 3.51l o o o
0
A 0
0
3.01
0
Figure 2. Plot of capacity factor (k)vs. sample size. Solute: [aniline] = 0.10% by volume. Flow rate = 2.0 mL/mln. Eluent = 20% 2-propanol -k 80% hexane. Columns: (0)[Co(dmedda)(en)]CIbonded
phase; (A)[Co(deedda)(en)]CIbonded phase.
Figure 1 shows several chromatograms, using some of the columns of interest. It is found in general that the peak asymmetry factors are in the range of 2.1-2.5. The retention time in decreasing order is para > meta > ortho, which is similar to that reported by using a hydroxylated silica (27). Figures 2 and 3 show the respective plots of capacity factors
a(para/ ortho)
1.73 4.18 diamine [Co(en),IC13 2.28 5.28 [Co(edda)(en)]Cl 2.40 4.57 [Co(dmedda)(en)]Cl 2.38 4.87 2.65 4.52 [Co(deedda)(en)]Cl Conditions are the same as those of Table IV.
and the number of tlheoretical plates as a function of sample size using [Co(dmedda)(en)]Cl and [Co(deedda)(en)]Cl columns as illustrations. The capacity factor decreases with increasing sample size when a very small amount of sample is used and it reaches a constant value when a slightly larger amount of sample is used. On the other hand, the number of theoretical plates first increases with increasing sample size and then decreases, i.e., a maximum is observed. The nature of this observation is not clearly known but it may be associated with a column having mixed retention mechanisms (vide infra). It is also found that the number of theoretical plates increases with increasing temperature as expected. In general, the numbers of theoretical plates for these columns are not as high as those reported for very eff,kient columns which may be due to the facts that the solute-stationary phase interactions are strong in the column and that mixed retention mechanisms are operative in the column. Retention Behaviors and Selectivities. One of the major purposes of the present work is to learn if there are significant differences among the diamine and cobalt complex bonded phase columns. Table V lists the separation factors of the three geometrical isomers of nitroaniline with o-nitroaniline as the reference. It is observed that all the five different bonded phases possess different selectivities. However, a simple trend is not obvious. This is understandable because for cobalt complex bonded phases, only a fraction of the bonded diamines reacted with the cobalt complexes which leads to a mixed-retention mechanism for the column, i.e., both the diamine and the cobalt complex on the silica will retain the solutes. Thus, in order to compare different bonded phases, it is important to separate individual contributions
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
1394
Table VI. Individual Contribution for the AH Values (kcal/mol) of Nitroanilines Transfer from the Mobile Phase to the Stationary Phasea$ bonded phase columns
ortho
meta
para
ortho
meta
para
ortho
meta
para
ortho
meta
para
silica diamine ICo(en),la, [Co(edda)(en)]Cl [Co(dmedda)(en)]Cl [Co(deedda)(en)]Cl
-0.75 -1.99 -2.46 -2.14 -1.76 -1.86
-1.91 -2.94 -3.61 -3.23 -2.72
-0.76 -2.64 -3.37 -2.65 -2.22 -2.22
-0.71 -1.37 -1.58
-1.05 -2.03 -2.34
-0.94 -1.82 -2.16 -2.39
-1.75 -0.77 -0.18 -0.06
-2.56 -1.20 -0.38
-2.43 -0.83 -0.12 t0.17
-2.71 -2.48 -0.88 -0.63
-3.96 -3.86 -1.86
-3.76 -2.67 -0.59 t1.79
AH'complex
diamine
a"be
AHOVerall
-1.80
AHcomp lex
a AH'keediamine + AH'complex. Data of ALHoverd were obtained by the van't Hoff plots of log h vs. 1/T, T = 2262 "C. b ~ c o , ~ l e is x the enthalpy change for the solute transfer from the mobile phase to the stationary phase if the surface is only covered with the complex bonded phase. AHcomplex = AH',^,^^,/% of surface coverage of the complex. Values of AHcomplex can best be used as measures of trend instead of absolute quantitative data because of the simple assumptions
moved=
made. for solute retention. A thermodynamic approach is then taken to sort out the individual retention contribution. Since the separation factor is defined to be a = k,/kb where k , and k b are respective capacity factors for two solutes, log a is then related to the free energy change for the separation (A(AG))
A(AG) = AGa - AGb = -RT In
ka = -RT Itb
In
CY
(1)
Furthermore, the temperature-dependent measurement of the retention time will give thermodynamic parameters, AH and AS, for the solute transfer from the mobile phase to the stationary phase I n k = In
(-)
= In
K
+ In v,
(2)
Vm
Ink=--
H + S + l n -
v,
RT
Vm
R
(3)
where k is the capacity factor, t , and to are retention times of the solute and a nonretained sample, respectively, K is the partition coefficient of the solute in the stationary phase and the mobile phase, and V , and V , are the volumes of the stationary phase and the mobile phase in the column, respectively. The difference of free energy change for the separation of two solutes (a and b) is then given by the next equation, where A(W = AHa - h H b and A ( M ) = A s a - M b
A(AG) = A ( m - A(AS)
(4)
Because a mixed-retention mechanism is expected, AG, AH, and A S values can all be separated into parts to account for different retention contributions
+ AGz + ... = C A G i AH = AH,4- A H 2 + ... = CAHi AG = AG,
(5)
1
(6)
I
and
A S = AS,
+ AS2 + ... = Ci A S j
(7)
where 1, 2, etc. are designated for different retention contributions. For the cobalt complex bonded phase columns, two contributions are apparently significant for the retention, i.e., that due to the diamine and that due to the complex. If one knows how many free diamines are present in the complex bonded phase, it is possible to estimate the retention contribution due to the diamine part. From the overall AH value and by difference, the contribution due to the cobalt complex can then be estimated. Table VI lists the calculated values of AH due to each contribution and a clear trend is in order. Appendix I gives an example of sample calculation. It should
be noted that the contribution due to free surface silanol groups is neglected because if it is taken into consideration, the -AH values are in an average more than 11 kcal/mol for metal complex contribution which is far greater than the normal accepted -AH values for hydrogen bonding, i.e., 3-5 kcal/mol. This would also mean that the fraction of solutes that can get into the silica surface is minimal in the presence of a large amount of polar solvent such as 2-propanol and polar bonded phases such as diamine and cobalt complex. For cobalt complex bonded phases this is even more likely because the larger-sized complex may block the interaction between solutes and surface silanol groups. An examination of the AHvalues due to complex interaction with the nitroanilines, it is found that the trend for the interaction in decreasing order is always as follows: [Co(en),]C13 > [Co(edda)(en)]Cl > [Co(dmedda)(en)]Cl > [Co(deedda)(en)]Cl. That the interaction is greater for [Co(en),]Cl, bonded phase is probably because that there are more hydrogen atoms available for hydrogen bonding and that the overall complex charge is +3. On the other hand, by substitution of alkyl groups on the nitrogens of ethylenediaminediacetate ion, it is observed that the alkyl groups not only reduce the number of hydrogen atoms for hydrogen bonding but they also provide steric hindrance to prevent hydrogen bonding formation. This argument is also confirmed by framework molecular model studies. Finally, it is noted again that if the retention contribution of the free surface silanol is taken into consideration, no simple trend is observed for the interaction between nitroanilines and cobalt complex bonded phases. This further suggests that the free surface silanol groups may not be important in the present chromatographic processes in which cobalt complex bonded phases and strong hydrogen bonded solvents are used. APPENDIX Calculation of Individual Contribution of AH for the m -Nitroaniline Transfer from the Mobile Phase to t h e Stationary Phase for t h e [Co(dmedda)(en)]Cl Bonded Phase Column. From Table VI, it is known for a 100% diamine bonded phase, the AHove,&value is -2.94 kcal/mol. The measured AH overall value for [Co(dmedda)(en)]Cl bonded phase is -2.72 kcal/mol. Also, the percentage surface coverage for diamine in this column is 79.6% and for the complex, 20.4% (calculated from the data given in Table 111). Thus, assuming AH'complex is the contribution due to the complex for the overall retention; the following relationship holds: moverdl
=
M'diamine
-2.72 = -2.94
X
+ AH'complex
79.6%
+ AH'complex
= -0.38 kcal/mol
Anal. Chem. 1983, 55, 1395-1399
AHcomplex= AH/complex/20.4%= -1.86 kcal/mol Registry No. [ C ~ ( e n ) ~ ] C13408-73-6; l~, [Co(edda)(en)]Cl, 56792-92-8;[Co(dmedda)(en)]Cl,85317-81-3;[Co(deedda)(en)]Cl, 85317-82-41,. LITERATURE CITED (1) Tswett. M. Ber. Dent. Bot. Grs. 1908, 2 4 , 313-328. (2) Davarikov, V. A.; Semechkin, A. V. J. Chromatogr. 1977, 141, 313-953. (3) Davanikov, V. A.; Rogozhin, S.V.; Semenchkin, A. V.; Sachkova, T. P. J. Chromafogr. 1973, 82,359-365, and papers thereafter. (4) Lefebvre, 6.; Audebert, R.; Qulvoron, C. Isr. J . Chem. 1878, 15, 69-73. ( 5 ) Boue, J.; Audebert, R.; Qulvoron, C. J . Chromafogr. 1981, 204, 185-193. (6) Faucault, A.; Caude, M.; Oliveros, L. J . Chromafogr. 1979, 185, 345-360. ( 7 ) Gubltz, G.; Jellsnz, 0.;Santi, W. J. Chromafogr. 1981, 203, 377-384. (8) Lindner, W.; LePage, J. N.; Davies, G.; Seitz, D. E.; Karger, 8. L. J . Chroniatoar. 1979, 185, 323-344. (9) LePage, J-N.; Lindner, W.; Davies, G.; Seltz, D. E.; Karger, B. L. Anal. Chem. 1979, 51, 433-435. (10) Tapuhi, Y.; Miller, N.; Karger. E. L. J . Chromafogr. 1981, 206, 326-337 - -- - -. . (11) Hare, P. E.; GII-Av., E. Science W79, 204, 1226-1228. (12) GII-Av., E.; Tlshbee, A.; Hare, P. E. J . Am. Chem. SOC. 1980, 102, 5115-5117. (13) Lam, S.; Chow, F.; Karmien, A. J . Chromafogr. W80, 199, 295-305.
1395
(14) Gllon, C.; Leshern. R.; Grushka, E. Anal. Chem. 1980, 52, 1206- 1209. (15) Gllon, C.; Leshem, R.; Tapuhl, Y.; Grushka, E. J . Am. Chem. Soc. 1979, 101, 7812-7613. (16) Gilon, C.; Leshemi, R.; Grushka, E. J. Chromatogr. 1981, 203, 365-375. (17) Chow, F. K.; Grushka, E. J . Chromafogr. 1979, 185, 361-373. (18) Halmos, P.; Inczedy, J. Talanta 1980. 2 7 , 557-560. (19) Gaal, J.; Inczedy, ,I. Ta/anfa 1978, 2 3 , 8. (20) Chang, C. A.; Tu, C.-F. Anal. Chem. 1982, 5 4 , 1179-1182. (21) Chang, C. A. Anal. Chem., in press. (22) Van Saun, C. W.; Douglas, B. E. Inorg. Chem. 1969, 8 , 115-1’18. (23) Legg, J. I.; Cooke, D. W. Inorg. Chem. 1965, 4 , 1576-1584. (24) Unger, K. K.; Becker, N.; Roumeliotis, P. J . Chromafogr. 1978, 125, 115-127. (25) Waddell, T. 0.; Leyden, D. E.; DeBello, M. T. J . Am. Chem. SOC. 1981, 103, 5303-6307. (26) Chang, C. A.; Huang, C. S.; Hoffer, J. M., unpubllshed results. (27) Klselev, A. V.; Aratskova, A. A.; Grozdovitch, T. N.; Yashin, Ya. I. J . Chromafogr. 1980, 195, 205-210.
RECEIVED for review October 20, 1982. Accepted March 1, 1982. Acknowledgment is made to the donors of the Petroleum Research Fund, Administered by the American Chemical Society, for support lof this research. Support of the Robert A. Welch Foundation of Houston, Texas, is also gratefully acknowledged.
Miniclolurnns for Affinity Chromatography Rodney kl. Waliers Departmeni of Ch@mistty,Iowa State University, Ames,
Iowa 5001 1
Concanavialln A was separated from albumln In 20 E and active trypsin was separated from inactlve trypsln In 118 s by using 8.35 mm long hlgh-performance afflnlty chromatographic “mlnlcolumns”. The adsorptlon capacities of the mlnlcolumris were suff lclently large for analytlcal appllcatlons even when low surface area supports were used. Band broadenlng was slgnlflcantly reduced compared to 5-cm columns and could be reduced further by decreaslng extracolumn band broadenlng. Mlnlcolumns were shown to be appllcable to separatlons In whlch the lmmoblllzed afflnlty llgand was of very hlgh speclflclty such that only one solute was retalned on the column. I n such cases, the use of mlnlcolumris resulted In improvements In analysis time and sensltlvlty of detection.
Chromatographic resolution of solutes 1 and 2 depends on the number of theoretical plates (N) and the capacity factors (123 of the solutes (1,2).If k’l = 0 and k’, > 20, it is simple to show that a column containing only a few plates will resolve the solutes. The length of such a column would typically be less than a millimeter. There are two major advantages in the use of columns which are very short. Separation times are reduced since retention times are directly proportional to column length. Since peak widths are proportional to the square root of column length (3),peak heights are larger and the sensitivity of detection is improved when short columnai are used. We will refer to columns of length less than 2 cm as “minicolumms”to differentiate them from columns which are
usually considered to be short columns, i.e., 2-5 cm columns. Minicolumns cannot be used for most analflical separations because solutes are present which have a range of k’values. If N is small, these solutes will not be resolved. Such columns can, however, be used for sample cleanup or concentration prior to analysis (for example, Waters Sep-PAK cartridges). Affinity chromatography is much more selective than other forms of chromatography. When “specific” affinity ligands, such as antibodies or enzyme inhibitors, are immobilized, only one solute should be adsorbed ( 4 , 5 ) . When “general” affinity ligands, such as lectinu or nucleotides, are immobilized, several solutes may be adsorbed ( 4 , 5 ) . In this case, efficient columns may be necessary to separate solutes which differ only slightly in 12’ (4-6).Sometimes, however, the adsorbed solutes can be eluted individually using specific substrates (4, 7,B)or by p l l or other changes which cause dissociation of one solute-affinity ligand complex but not the others (4, 9). Thus, in all separations with “specific” ligands and in some separations with “general” ligands, it may be advantageous to use minicolumns. In previous high-performance affinity chromatography (HPAC) separations, columns of length 4-12 cm were useld (6-8,10-16).In many of the separations, only one solute was adsorbed (6, 7,10,11, 14, 16). In other separations, several solutes were adsorbed, but the solutes could be eluted individually using biospecific elution (7,8,15). It is likely that only a few millimeters or less of the packing at the inlet af the column was used for adsorption of the solutes of interest in each of these cases. For example, in our previous work witlh immobilized glucosamine, 5 cm X 4.6 mm columns had adsorption capacities of up to 29 mg of concanavalin A (Con A) (16). Since the samples chromatographed contained 50 Mg of
0003-2700/83/0355-1395$01.50/00 1963 American Chemical Society
