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, 54, 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
1396
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
Con A, columns of length 0.1-1 mm would have been adequate for total adsorption of the Con A. Columns of length 1-2 cm are adequate for conventional preparative affinity chromatography on agarose supports (17). In the separations cited above (6-8,10, 11, 14-16), the solutes adsorbed at the top of the column were eluted by a sharp increase in mobile phase strength. Since k’was sharply reduced, little or no interaction between the solutes and affinity ligands took place as the solutes passed through the rest of the column. The bulk of the column therefore served only to broaden the bands and increase analysis time. Short columns have been used previously for low pressure affinity and ion-exchange chromatography, typically in the form of a gel packed into the tip of a disposable pipet (18,19). We recently designed high-pressure stainless steel fittings for use with columns as short as a few millimeters (20). In this paper we will demonstrate the use of HPAC minicolumns of 6.35 mm length. Two model systems will be examined: immobilized glucosamine will be used to separate Con A from bovine serum albumin (BSA) (16);and immobilized soybean trypsin inhibitor (STI)will be used to separate active from inactive trypsin (21-23). Con A will be biospecifically eluted using methyl a-D-mannopyranoside. Active trypsin will be eluted by a pH change.
EXPERIMENTAL SECTION Reagents. Bovine pancreatic trypsin and all other biochemicals were obtained from Sigma (St. Louis, MO) and were the purest grades available. LiChrospher Si 500 and 4000 (10 pm) were obtained from Rainin (Woburn, MA). (3-Glycidoxypropy1)trimethoxysilane was from PetrarcR (Levittown, PA). 1,l’Carbonyldiimidazole was from Aldrich (Milwaukee, WI). HPLC grade H3P04,85%, was from Fisher (St. Louis, MO). Apparatus. The chromatographic system was described previously (16).A 3-pL loop was used for all injections. Minicolumns of 6.35 mm length and 4.1 or 4.6 mm i.d. were made as described previously (20). Methods. D-Glucosaminewas immobilized on 500 A pore size diol-bonded silica as described previously (16). The glucosamine content was determined by the alkaline ferricyanide method (24) to be 35 pmol/g of dry support. A 6.35 mm X 4.6 mm minicolumn was slurry-packed at 3000 psi (16,20).Chromatography using the glucosamine minicolumn was performed as previously described (16)except that the buffer contained sodium acetate instead of sodium phosphate. A 4000 A pore size diol-bonded silica was synthesized as before (16)except that 0.050 mL of silane/g of silica was used. The diol content was determined by periodate titration to be 33 pmol/g (25).The diol-bonded silica, 0.2 g, was activated with 0.08 g of 1,l’-carbonyldiimidazole(16). STI, 11 mg, was added to the activated silica in 1.0 mL of 0.1 M potassium phosphate buffer, pH 7.0. The mixture was gently shaken for 19 h at 4 O C , washed with buffer, and slurry-packed into a 6.35 mm X 4.1 mm minicolumn (16,20).The protein content of the silica was 2.8 mg/g as determined by the Lowry method, using BSA as standard (26). Chromatography using the STI minicolumn was performed at room temperature with a pH 7.0 potassium phosphate buffer, 0.1 M, as the weak mobile phase and a pH 2.5 buffer as the strong mobile phase. Both buffers were prepared from reagent grade KOH and HPLC grade H3P04. Trypsin (2.5 mg/mL in pH 7.0 buffer) was kept on ice prior to injection. Trypsin activity was assayed at room temperature in pH 7.0 buffer using benzoyl-Larginine ethyl ester (BAEE) as substrate (27). The assay mixture contained trypsin, 2.0 mL of pH 7.0 buffer, and 0.40 mL of 4.0 mM BAEE, RESULTS AND DISCUSSION Column Efficiency. To determine whether the minicolumns could be packed and operated as efficiently as longer columns, plate heights (If)were measured for several solutes. Equipment for measuring peak moments was not available, so all calculations were based on the width at half-height (2). Since considerable peak tailing was observed, the true plate
Table I. Comparison of Columns at 1.0 mL/min
solute
parameter
uracil
uv
N
(mL)
5 cm glucosamine column (16)
0.024 900 56
0.056 130 390 0.18 30 1600 H (pm)
glucosamine niinicolumn 0.017 59 110 0.025 20 320 0.042
STI minicolumn 0,019 27 230 0.021 21 300
13
490 0.019 37 170
a In stron mobile phase (0.02 M methyl a-D-mannopyranoside). gb In strong mobile phase (pH 2.5 buffer).
heights were larger than those calculated (28)by factors of up to 3-fold. Plate heights for uracil and BSA were measured in the appropriate weak mobile phases on the glucosamine and STI minicolumns, while the strong mobile phases were used for Con A and trypsin. Table I shows the data for the two minicolumns and for the 5-cm glucosamine column of ref 16, which contained the same packing material as the glucosamine minicolumn. All of the columns were operated a t a flow rate of 1.0 mL/min. Since the STI minicolumn had a smaller internal diameter, the linear velocity of this column was somewhat higher than for the other two columns. The small, nonretained solute uracil was used to assess packing efficiency. The plate heights in Table I were observed to be considerably larger on the minicolumns than on the 5-cm column. The standard deviation of each peak, u,, calculated from the width a t half height (l), should be proportional to the square root of the column length, as pointed out in the introduction. Eased on the 5-cm column, uv of the minicolumns should have been 0.009 mL. The larger measured uv values were caused primarily by extracolumn band broadening (28). Without a column, a, was found to be 0.014 mL. This is similar to the band broadening of most commercial UV detectors (29,30).Since variances):a( are additive, the expected value of u,, including extracolumn band broadening, should have been 0.017 mL. This was in good agreement with the measured values and indicated that the minicolumns were packed as efficiently as the longer column. Plate heights and peak widths were larger for BSA (Table I) than for uracil due to the smaller diffusion coefficient of the protein (16). From the 5-cm column, the expected value of a, for the minicolumns was 0.020 mL in the absence of extracolumn band broadening or 0.024 mL with extracolumn broadening. The measured data were in good agreement. Note that since the BSA peak was naturally broader than the uracil peak, extracolumn band broadening caused much smaller increases in the BSA plate heights compared to uracil. It is clear that to take full advantage of the sensitivity and speed of minicolumns, one needs to decrease extracolumn band broadening. This could be achieved by using smaller volume injectors and detectors as in microbore liquid chromatography. Such equipment would be necessary for studies of packing methods and band broadening but less important for quantitative analysis since protein bands are naturally rather broad due to diffusional and other kinetic effects (16). We previously showed that the Con A peak on an immobilized glucosamine column was broadened due to slow adsorption-desorption kinetics, even in the presence of high concentrations of competing sugar in the mobile phase which
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
012 r
(b)
0
I-I
1
2
0 1 2 Time (rnin)
I
(Cl
L 0
6397
0
1
2
Flgure 1. Separation of 15 pg of Con A from 15 pg of BSA at 1 mL/min on a 6.35 mm X 4.6 mm column containing immobilized glucosamlne. I n (a), only Con A was applied. I n (a) and (b), a step change in mobile phase approximately 1 min after injection was used to elute thle Con A. The time of the step change was adjusted in (c) to meet the resolution requirement given In the text.
reduced It’ to 0.4 (16). Table I shows that the plate height and peak width were also large on the minicolumn, but not as large as expected from the 5-cm column. We were unable to explain this discrepancy. The Con A plate heights were only slightly affected by injection volume and Con A concentration. Peak asymmetry factors (2) were large on both columns (3.1 on the minicolumn, 2.4 on the 5-cm column), but the discrepancy remained even when the true plate heights were estimated by graphical (31) and mathematical (32) methods. The plate height and peak width for trypsin (Table I) were smaller tlhan for BSA, reflecting the larger diffusion coefficent of the smaller trypsin molecule (33) and the effectiveness of the pH 12.5 buffer in eliminating retention (k’ = 0.1) and adsorption-desorption kinetic effects. Both olf the minicolumns contained less than 40 theoretical plates for the proteins tested, but, as shown below, were able to separate protein mixtures with base line resolution. Separation Time and Sensitivity of Detection. Since the void %volumes of the minicolumns were less than 0.13 mL, very rapid separations were obtained. Figure 1 shows the separation of Con A from BSA on the glucosamine minicolumn. In Figure la, 15 Mg of Con A was injected at time zero. A small impurity peak eluted at the void volume of the column. The Con A was strongly retained in the weak mobile phase but was eluted by a step change to the strong mobile phase. The time of this step change could be varied, as shown in Figure 1b,c, to achieve any desired resolution between the nonretained BSA and retained Con A peaks. It was pointed out in the introduction that separation time is proportional to column length. This is true only under isocratic conditions. Of course, the resolution also decreases when the length decreases. Since gradient elution is used in most affinity chromatographic separations and the retention time of the retained peak can be varied at will, it is convenient to measure the separation time at constant resolution. Separation time will not be directly proportional to column length under such conditions. We previously defined separation time as the retention time of the second peak at a flow rate of 1 mL/min when the height of the valley between the peaks was 10% of tlhe average height of the two peaks (16). This definition is convenient for tailing peaks and provides a resolution of about 1.25. Figure ICshows that the separation of Con A from BS14 required only 20 s. In comparison with a 5-cm column (.I@, the 7.9-fold decrease in column length resulted
1
2
0
1
2
0
1
2
Time lrnin)
Figure 2. Analysis of a commercial trypsin sample (7.5 pg) at 1 mL/min on a 6.35 mtm X 4.1 mm column containing immobiiitzed soybean trypsin inhibitor. A step change in pH from 7.0 to 2.5 caused a background peak (a) and caused the active trypsin to elute (b, c). The step changes were made at approximately the same times as in Figure 1.
in a 3.8-fold decrease in separation time. The separation tnme would be reduced further if extracolumn band broadening were smaller. Injections could be made at intervals of 1-1.5 min. As discussed in the introduction, peak width under isocriitic conditions should bo proportional to the square root of column length. Thus, peak height and the sensitivity of detection (detector response/mg analyte) should be inversely proyortional to the square coot of column length. Ln comparison with the 5-cm glucosamhe column (16),an improvement in sensitivity of 2.8-fold was expected for the minicolumn. The measured improvement for BSA, 2.4-fold under isocratic conditions, was probably slightly decreased by extracolumn band broadening. A 3.8-fold improvement was measured for Con A under noniriocratic conditions, but this was cawed partially by the anomalous improvement in Con A plate height noted earlier for thle minicolumn. Figure 2 shows the separation of active trypsin from inactive trypsin and other impurities on the STI minicolumn. The chromatogram of Figure 2a was a blank run. The two spurious peaks occurred when the mobile phase was changed from pH 7.0 to 2.5. The sizes of these peaks varied with each buffer solution used but could not be eliminated entirely. Exctept for the first few elution cycles, the peaks did not appear to be caused by a loss of immobilized STI. When a 7.5 Mg trypsin sample was chromatographed (Figure 2b), the retained trypsin peak coeluted with the second spurious peak. The separation time for this column was 18 s, as shown in Figure 2c. Here the first spurious peak coeluted with the unretained trypsin peak. Injections could be made at intervals of 1-1.5 min, i.e., as quickly as the mobile phase pH could be returned to ‘7.0. Adsorption Capacity. When very sharp changes in mobile phase strength are used in HPAC, the linear adsorption capacity (2) of the column is unimportant. Even if the analyte is adsorbed throughout the entire column, when the strong mobile phase front passes through the column the analyte band is sharpened as it desorbs along the front. Of more importance in HPAC are the total static and dynamic adsorption capacities. If the equilibrium is favorable, the static adsorption capacity is a measure of the number of active affinity ligands in the column (23,34,35).The total dynamic adsorption capacity, measured under nonequilibrium conditions, may be smaller than the static capacity due to slow adsorption kinetics or slow diffusion into the support (13). If the dynamic capacity is exceeded, some of the analyte will elute without retention. Since minicolumns contain very little support material, the capacities of the two minicolumns wlere
1398
.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
80 x
0 0
s 20 0 '
20
LO 60 Time ( m i d
80
100
Figure 3. Loss of trypsin actlvlty due to autovsis at roam temperature. Aliquots of a 2.5 mg/mL trypsin solution were assayed by the HPAC method (0)and by enzyme assay (0).
measured to see if low capacity might limit the utility of the columns. The static adsorption capacity of the glucosamine minicolumn was estimated to be 2.7 mg of Con A, based on breakthrough curves measured previously (16). The dynamic capacity at 1 mL/min was estimated to be at least 0.5 mg of Con A by injecting increasingly larger volumes of 10 mg/mL Con A until some of the Con A began to elute isocratically. This Con A was weakly retained, however, and may actually have been "nicked" Con A fragments (36). Hence, the dynamic capacity may have been greater than 0.5 mg. The static capacity of the STI column was determined to be 74 pg of trypsin by pumping 1 mL of 10 mg/mL trypsin through the column at 0.1 mL/min, washing the column with pH 7.0 buffer, and measuring the peak area of trypsin eluted with pH 2.5 buffer. This value indicated that approximately 60% of the immobilized STI molecules were active. The dynamic capacity at 1mL/min was found to be 5 pg of trypsin by injecting 3 pL of trypsin solutions of various concentrations. The nonretained peaks were collected and immediately assayed with BAEE as substrate. The dynamic capacity was calculated from the largest trypsin sample in which all the activity was retained. The calculated value of 5 pg includes a correction for the inactive trypsin in the sample. The 5 pg dynamic capacity of the STI minicolumn was small, but certainly large enough for analytical purposes, as shown in Figure 2. It may be possible to increase the capacity by improving the immobilization procedure. Silica of pore size 4000 A was chosen for the STI minicolumn because of the low surface area of this support (6 m2/g, as given by the manufacturer). Thus even low surface area supports appear to be adequate for HPAC minicolumns, although it remains to be seen whether nonporous supports will have sufficient capacities. Trypsin Analysis. One advantage of HPAC for quantitative analysis is that the results of the analysis can be expressed in absolute units, i.e., mass or concentration of analyte. Many biochemical assays give results in units of activity and thus tend to be quite sensitive to the assay conditions. The results of HPAC and activity assays should be proportional if the affinity ligands bind only to the sample molecules which have biological activity. In the case of trypsin, STI binds only to catalytically active trypsin molecules @ I ) , so the HPAC method should give results comparable to the enzyme assay method. HPAC and enzyme assay methods were compared by allowing a trypsin solution to degrade by autolysis (37) at room temperature and assaying the solution periodically by both methods. A plot of percentage of initial activity vs. time was
prepared, as shown in Figure 3. The percentage of initial activity was calculated from the change in absorbance per unit time in the enzyme assay and from the height of the retained peak in the HPAC method. Both methods gave similar results, in agreement with the expected specificity of STI. The results were in better agreement than those obtained by comparing enzyme assay and size-exclusion chromatography (38). The HPAC assay was much faster than high-performance sizeexclusion (38) or reversed-phase (39) chromatographic methods.
CONCLUSIONS It was demonstrated that the use of minicolumns improved the speed and sensitivity of HPAC separations in cases where a strongly retained analyte was separated from nonretained solutes. There are several other potential advantages in the use of HPAC minicolumns: (1)Low cost. The minicolumns used here contained less than 0.05 g of packing material and less than 0.3 mg of immobilized affinity ligand. (2) Decreased nonspecific adsorption. It was pointed out earlier that in long HPAC columns frequently only a small portion of stationary phase at the inlet is used for the separation. The unused packing material, in addition to increasing analysis time and decreasing sensitivity, also provides a surface where nonspecific adsorption of the analyte or other solutes can take place. Nonspecific adsorption can cause interference with the analysis if the nonspecifically adsorbed solutes coelute with the analyte. The problem can be minimized by using minicolumns containing supports of low surface area. (3) Coupled columns. If more than one solute is adsorbed, either specifically or nonspecifically, on the HPAC column, an additional separation step may be necessary to resolve the components. Since the peaks eluting from the minicolumn are of very low volume (see Table I), it would be simple to switch the analyte peak into a second analytical column, such as a size-exclusion or ion-exchange column. (4) Concentration of dilute solutions. Many biochemicals of analytical interest are present in very low concentrations. The analyte in a large sample could be adsorbed on an HPAC minicolumn and eluted as a concentrated peak. Samples could easily be concentrated to volumes of 0.5 mL or less in a few minutes. (5) Low pressure operation. From our experience with 5-cm columns, we expected the 6.35-mm minicolumns to have column pressures (including injector and detector) of 45-70 psi at 1mL/min. At these pressures, it would be possible to perform HPAC separations with peristaltic pumps. This would be very convenient for automating clinical or other analytical procedures based on HPAC. However, we observed pressures in the range of 50-300 psi. Perhaps refinement of the slurry packing method will decrease the pressures to acceptable levels. Registry No. Concanavalin A, 11028-71-0;trypsin, 9002-07-7. LITERATURE CITED (1) Karger, B. L.; Snyder, L. R. Hoivath, C. "An Introduction to Separatlon Sclence"; Wlley: New York, 1973;,,Chapter 5. (2) Snyder, L. R.; Klrkland, J. J. Introduction to Modern Llquid Chromatography"; Wlley: New York, 1979; Chapters 2 and 5. (3) Novak, J.; Janek, J.: Wlcar, S. I n "Liquid Column Chromatography"; Deyl, Z., Macek, K., Janek, J., Eds.; Elsevier: Amsterdam, 1975; Chapter 3. (4) Turkova, J. "Affinity Chromatography"; Elsevier: Amsterdam, 1978; Chapters 6 and 10; (5) Scouten, W. H. "Affinlty Chromatography"; Wiley: New York, 1981; Chapter 5. (6) Borchert, A.; Larsson, P.-0.; Mosbach, K. J . Chromatogr. 1982, 244, 49-56. Mosbach, K. FEBS Left. (7) Qhlson, S.; Hansson, L.; Larsson, P. 0.: 1978, 93, 5-9. (8) Lowe, C. R.; Glad, M.; Larsson, P. 0.; Ohlson, S.; Small, D. A. P.; Atkinson, T.; Mosbach, K. J . Chromatogr. 1981, 275, 303-316. (9) Martin, L. N. J . Immunol. Meth. 1882, 52, 205-212.
Anal. Chem. 1983, 55, 1399-1402 (10) Sportsman, ,J. R.; Wilson, G. S. Anal. Chem. 1980, 52, 2013-2018. (11) Glad, M.; Ohison, S.; Hansson, L.; Mansson, M. 0.; Mosbach, K. J. Chromatogr. 1880, 200, 254-260. (12) Small, D. A. R.; Atklnson, T.; Lowe, C. R. J. Chromatogr. 1981, 276, 175-190. (13) Kasohe, V.; Buchholz, K.; Galunsky, B. J. Chromatogr. 1981, 276, 169-174. (14) Coupek, J. I n “Affinity Chromatography and Related Techniques”; Gribnau, T. C. J., Visser, J., Nlvard, R. J. F., Eds.; Elsevier: Amsterdam. 1982: nD 165-179. (15) Nllsson, K.; Mosbach, K. Blochem. Blophys. Res. Commun. 1S81, 102, 449-457. (16) Walters, R. R. J. Chromatogr. 1982, 249, 19-28. (17) “Affinity ChromatograDhy”; Pharmacia Fine Chemicals: UDDsaia, .. . 1970; p 55. (18) Proffl, R. T.; Sankaran, L.; Pogell, B. M. Anal. Blochem. 1978, 8 8 ,
___
236--244. -
(19) Mercer, b. W.; Varat, M. A. Clin. Chem. (Winston-Sakm, N . C . ) 1975, 2 7 , 1088-1092. (20) Waiters, R. R. Anal. Chem. 1983, 55, 591-592. (21) Mosolov, V. V.; Lushnlkova, E. V. Biokhim/ya 1970, 35, 440-447. (22) Avrameas, S.; Guiibert, B. Biochimie 1971, 53, 603-614. (23) Porath, J.; Sandberg, L. Nature (London) New Biol. 1972, 238, 261-262. (24) Rob)rt, J. F.; Ackerman, R. J.; Keng, J. G. Anal. Blochem. 1972, 45, 5 17-524. (25) Siggha, S.;Hanna, J. G.; Stengle, T. R. I n “The Chemistry of the Hydroxyl Group”; Patai, S.,Ed.; Interscience: New York, 1971; pp 309-310. ... . . (26) Lowly, 0.H.; Rosebraugh, N. J.; Farr, A. L.; Randall, R. d. J. Blol. Chem. 1951, 793, 165-275.
1399
(27) Bergmeyer, H. U. ”Methods of Enzymatic Analysis”; Academic Press: New York, 1974 pp 515-516. (28) Kirkland, J. J.; Yau, W. W.; Stoklosa, H. J.; Dllks, C. H. J. Chrotnatogr. Sci. 1977, 75, 303-316. (29) Kok, W. T.; Brinkiman, U. A. T.; Frei, R. W.; Hanekamp, H. B.; Nooitgedacht, F.; Poppe, H. J. Chromatogr. 1982, 237, 357-369. (30) Knox, J. H. Anal. Proc. 1982, 79, 166-170. (31) Barber, W. E.; Carr, P. W. Anal. Chem. 1981, 53, 1939-1942. (32) Foley, J. P.; Dorsey, J. G. Anal. Chem. W83, 55, 730-737. (33) Green, N. M.; Nelurath, H. I n “The Proteins”; Neurath, H., Ed.; Academlc Press: New York, 1954; Vol. 11, Part B; p 1081. (34) Turkova, J.; Blahia, K.; Adamova, K. J. Chromatogr. 1982, ;?36, 375-383. (35) Amneus, H.; Gabel, D.; Kasche, V. J. Chromatogr. 1976, IW, 39 1-397. (36) Sitrin, R. D.; Anttrll, L.; Griswold, D. E.; Bender, P. E.; Grelg, R. G.; Poste, G. Blochlm. Biophys. Acta 1962, 777, 175-178. (37) Gabel, D.; Kasche, V. Acta Chem. Scand. 1973, 27, 1971-1981. (38) Buchholz, K.; Godelmann, B.; Molnar, 1. J. Chromatogr. 1982, 238, 193-202. (39) Strlckien, M. P.; Gemski, M. J.; Doctor, B. P. J. Li9. Chfomatogr. 1981, 4 , 1765-1775.
RECENEDfor review January 25,1983. Accepted March 21, 1983. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemiical Society, and to the Research Corporation for support of this research.
Two-.Dirnensional Liquid Chromatographic Determination of (3-Methoxy-4-hydroxyphenyl)glycol in Urine George M. Anderson* Department of Laboratory Medicine, Yale Unlverslty School of Medlclne, 333 Cedar Street, New Haven, Connecticut 065 10
Karin R. Schllcht and Donald J. Cohen The Child Study Center, Yale University School of Medicine, 333 Cedar Street, New Haven, Connectlcut 06510
The important norepinephrine metabollte (3-methoxy-4hydroxypheny1)giycoi (MHPG) Is determined In human urine after a two-dimensional HPLC separation. Enzymatically hydrolyzed urine Is flrst separated on a CI8 reversed-phase column using an acetate buffer moblle phase with fluorometric or UV absorbance monitoring. The eluent volume contalnlng MHPG ( k ‘ N 1.0) is collected. A portion of the collected sample Is reinjected on a reversed-phase column and separated with a phosphate buffer-methanol mobile phase. MHPG elutes In 6-6 min (k’ N 3) and Is detected by use of hydrodynamic amperometry. Recovery of added MHPG is quantitative and samples are determined with a coefficient of variation of 6-7 %.
The advantages of multidimensional chromatography are readily apparent when viewing a two-dimensional thin-layer chromatogram, and the theoretical and practical benefits of multidimensional high-performance liquid chromatography (HPLC) have been pointed out ( I , 2). Two-dimensional HPLC selparations have been performed by altering mobile phase or stationary phase selectivity and have permitted trace organic analysis in complex sample matrices (3-10). Twodimensional reversed-phase HPLC systems should be especially applicable to determinations in aqueous physiological
samples. Direct injection of samples would be possible and transfer of analyte bietween columns, achieved by peak-cutting or fraction collection and reinjection, should be facilitated due to mobile-phase compatibility. Selectivity changes and mode-sequencing in reversed-phase HPLC can be accomplished by altering mobile phase pH, molarity, and ion-pairing agent concentration or by changing bonded phase type and loading. We have applied the technique to the determination of (3-methoxy-4-hydroxyphenyl)glycol(MHPG) in human urine. MHPG is an important metabolite of the catecholamiine neurotransmitter nlorepinephrine and urinary MHPG is of clinical relevance in the diagnosis of catecholamine secretjng tumors (11). Levels also have been widely studied as an index of noradrenergic activity in neuropsychiatric disorders (12-14). Gas chromatographic methods with electron capture (15-1 7) or mass spectrometric (GC/MS) (18-21) detection often have been employed to determine MHPG in urine. Recently HPlLC methods with amperometric (LE-EC) (22-26) or fluorometric dection (27)have been developed. All of the methods require an organic solent extraction with its attendant drawbacks in terms of time, expense, and analyte recovery. The GC/MS methods are able to use deuterated MHPG as an internal standard; however, the other methods are compromised by a lack of suitable internal standards. In addition, most of the HPLC methods require long chromatographic runs to resollve
0003-2700/83/0355- 1399$01.!50/0 0 1983 Amerlcan Chemlcai Soclety
