Peptide Separation in Normal Phase Liquid Chromatography

Anal. Chem. 1997, 69, 3038-3043

Peptide Separation in Normal Phase Liquid Chromatography Tatsunari Yoshida

Scientific Instrument Division, Tosoh Corporation, Tokyo Research Center, 2743-1 Hayakawa, Ayase-shi, Kanagawa-ken 252, Japan

A new method is established for separating peptides in normal phase liquid chromatography using TSK gel Amide-80, carbamoyl groups bonded to a silica gel matrix, and an acetonitrile-water solution containing 0.1% trifluoroacetic acid. Peptide retention time increased with acetonitrile concentration in the initial eluent. Hydrophilic peptides with no retention in a reversed phase column were retained and separated in the present method. Separation selectivities in the present and reversed phase methods differed significantly. Twodimensional separation of protein digest using reversed and normal phases was conducted, taking advantage of the differences in selectivities. All peptides obtained from the digest could be separated completely. The present method is useful for separating peptide mixtures in conjunction with reversed phase liquid chromatography. Peptide recovery from the Amide-80 column exceeded 80%, as with the reversed phase column, and repeatability and reproducibility were satisfactory. Reversed phase liquid chromatography (RPLC) on an octadecyl silica (ODS) column is indispensable for separating and purifying peptide mixtures but is not applicable to hydrophilic peptides with no retention on an ODS column. Normal phase liquid chromatography (NPLC), with the usual silica or alumina stationary phase, is widely used to separate polar compounds with a nonaqueous mobile phase, such as hexane or chloroform, under isocratic elution conditions. It is difficult to dissolve hydrophilic materials such as peptides in such nonaqueous mobile phases, and thus the biological applications1-4 of NPLC are limited, and this method is rarely useful for separating peptide mixtures. TSK gel Amide-80 for the NPLC column was prepared by chemically bonding nonionic carbamoyl groups on a silica gel matrix. In this method, an aqueous mobile phase can be used with gradient elution.1,5-9 The separation of peptides in NPLC (1) Haviicek, J.; Samueison, O. Anal. Chem. 1975, 47, 1845-1857. (2) Naider, F.; Sipzner, R.; Steinfeld, A. S.; Becker, J. M. J. Chromatogr. 1979, 176, 264-269. (3) Slebioda, M.; Kolodziejczyk, A. M. J. Chromatogr. 1985, 325, 282-286. (4) Lo, L. C.; Chen, S. T.; Wu, S. H.; Wang, K. T. J. Chromatogr. 1989, 472, 336-339. (5) Nomura, Y.; Tade, H.; Takahashi, K.; Wada, K. Agric. Biol. Chem. 1989, 53, 3313-3315. (6) Tomiya, N.; Awaya, J.; Kurono, M.; Endo, S.; Arata, Y.; Takahashi, N. Anal. Biochem. 1988, 171, 73-90. (7) Oku, H.; Hase, S.; Ikenaka, T. Anal. Biochem. 1990, 185, 331-334. (8) Higashi, H.; Ito, M.; Fukaya, N.; Yamagata, S.; Yamagata, T. Anal. Biochem. 1990, 186, 355-362.

3038 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

mode on this amide column with an acetonitrile-water solution as the mobile phase was examined in this study. EXPERIMENTAL SECTION Apparatus. The HPLC system was a Tosoh (Tokyo, Japan) liquid chromatograph equipped with an SC-8020 microcomputer, a CCPM-II pump, a UV-8020 detector, an AS-8020 sample autoinjector, and a CO-8020 column oven. Reagents. The Milli-Q system was used for water purification. Nearly all the peptides were purchased from the Peptide Institute (Osaka, Japan) or Sigma (St. Louis, MO). The others were obtained by cyanogen bromide degradation of myoglobin or tryptic degradation of concanavalin A. TSK gel ODS-80Ts (25 cm × 0.46 cm i.d.) and TSK gel Amide-80 columns (25 cm × 0.46 cm i.d.) were from Tosoh. Methods. In the present NPLC method, eluent A (initial eluent) was 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN)water (97:3), and eluent B was 0.1% TFA in ACN-water (55:45). The peptides were dissolved in 10 µL of ACN-water-formic acid (5:45:50), to which was then added 40 µL of initial eluent. The peptides were separated by a linear gradient from eluents A to B over 70 min (0.6% water/min). The flow rate was 1.0 mL/min. Four mobile phases, 0.1% TFA, 0.1% formic acid, 0.1% acetic acid, and one without acid, were used. Elution was monitored by UV absorption at 265 and 215 nm. In the RPLC method, eluent A (initial eluent) was 0.1% TFA in ACN-water (97:3), and eluent B was 0.1% TFA in ACN-water (55:45). The samples were dissolved in the initial eluent. The peptides were separated by linear gradient from eluents A to B over 83.3 min (0.6% ACN/min), as shown in Figures 3 and 4. The flow rate was 1.0 mL/min. The peptides were also separated by linear gradient from eluents A to B over 90 min (0.556% ACN/ min), as shown in Figure 6A. Elution was monitored by UV absorption at 215 nm. For identification of the separated peptides, each was subjected to amino acid analysis following hydrolysis. RESULTS AND DISCUSSION Effects of Mobile Phase Composition. The chromatographic behavior of peptides in the present NPLC method was studied by three methods. The effects of acid, concentration of ACN in the initial mobile phase, and modifier on retention are discussed in turn. In RPLC, ion exchange (IEX) interactions between peptide residues and residual silanol (SiOH) groups on the surface of the (9) Tomiya, N.; Lee, Y. C.; Yoshida, T.; Wada, Y.; Awaya, J.; Kurono, M.; Takahashi, N. Anal. Biochem. 1991, 193, 90-100. S0003-2700(97)00220-5 CCC: $14.00

© 1997 American Chemical Society

Table 1. Chromatographic Results for Peptides Obtained on TSK Gel Amide-80 at Various Concentrations of ACN in the Initial Mobile Phasea ACN concn (%) 75 tR FY

3.52

FGGF

3.52

FLEEI

3.52

DYMGWMDP-NH2

3.52

NFTYGGF

3.98

AGSQ

6.99

WAGGDASGE

9.94

a

80 Rs 0 0 0 2.3 11.4 9.0

tR 3.69 3.69 3.69 4.01 4.72 8.53

85 Rs 0 0 1.8 2.4 12.4 12.1

12.98

90 Rs

tR 4.03 4.33 4.56 6.31 7.44 12.53 19.57

1.8 0.9 5.1 2.8 13.1 17.0

tR 5.17 6.67 8.26 12.18 13.30 18.78 27.50

97 Rs 5.2 4.6 9.8 2.5 12.6 19.6

tR 11.67 15.53 19.02 23.63 24.61 29.88

Rs 9.3 9.2 11.5 2.3 11.8 20.3

39.10

tR, retention time, min; Rs, resolution.

Figure 1. Effects of acid components of eluents on the separation of peptides on TSK gel Amide-80. The peptide mixture was separated with 70-min linear gradients of water from 3 to 45% in (A) 0.1% TFA, (B) 0.1% formic acid, (C) 0.1% acetic acid, and (D) no acid. Eluted compounds were detected at 265 nm. Peak identification: 1, FY; 2, FGGF, 3, FLEEI, 4, DYMGWMDP-NH2, 5, NFTYGGF; 6, AGSQ; 7, WAGGDASGE; 8, YGGFMTSQKSQTPLVT.

silica gel matrix cause band tailing.10 Salts and organic acids such as TFA, formic acid, and acetic acid added to the mobile phase effectively prevent such interactions.10-13 The effects of acid in the mobile phase were examined using TFA,14,15 acetic acid, formic acid, and no acid. A peptide mixture was chromatographed using the four mobile phases as shown in Figure 1. The elution order of the peptides differed for each phase. NFTYGGF, WAGGDASGE, and YGGFMTSQTPLVT were strongly retained on the amide column. In the absence of acid, these peptides failed to be eluted from the column (shown in Figure 1D). With a weak acid such as formic or acetic acid, FY, FGGF, and FLEEI eluted with tailing (shown in Figure 1B,C). With a strong acid such as TFA, no tailing was observed in Figure 1A. TFA was effective for preventing IEX interactions. (10) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; John Wiley & Sons: New York, 1979; Chapter 7. (11) Verzele, M.; Damme, F. V. J. Chromatogr. 1986, 362, 23-31. (12) Brons, C.; Olieman, C. J. Chromatogr. 1983, 259, 79-86. (13) Verzele, M.; Simoens, G.; Damme, F. V. Chromatographia 1987, 23, 292300. (14) Dunlap, C. E., III; Gentleman, S.; Lowney, L. I. J. Chromatogr. 1978, 160, 191-198. (15) Mahoney, W. C.; Hermadson, M. A. J. Biol. Chem. 1980, 255, 11199-11203.

Figure 2. Effects of ACN concentration in initial mobile phase on TSK gel Amide-80. The peptide mixture was separated with a gradient slope of 0.6% water/min. ACN-water: (A) 75:25, (B) 80: 20, (C) 85:15, (D) 90:10, and (E) 97:3. Peak identification: 1, FY; 2, FGGF; 3, FLEEI; 4, DYMGWMDP-NH2; 5, NFTYGGF; 6, AGSQ; 7, WAGGDASGE; 8, YGGFMTSQKSQTPLVT.

The effects of ACN concentration in the initial mobile phase were studied, and the results are shown in Figure 2 and Table 1. Peptide retention time increased with ACN concentration in the initial eluent. At ACN concentrations above 85%, retention16-18 was sufficient. Water, methanol (MeOH), and ethanol (EtOH) were studied as modifiers, and the results are shown in Table 2. Specific solubility parameters19,20 have been prepoosed by Karger et al. and are shown as Table 3. They serve as a basis for assessing selectivity in chromatography. In consideration of the solvent properties in Table 3 and the results listed in Table 2, modifier strength for peptide eluting was in order of magnitude of protondonor and -acceptor: water > MeOH > EtOH. The separation mechanism of the present NPLC method is proposed here at the empirical level. Hydrophobic and IEX interactions were negligible, since a nonionic amide column was (16) Nikolov, Z. L.; Reilly, P. J. J. Chromatogr. 1985, 325, 287-293. (17) Carunchio, V.; Girelli, A. M.; Messina, A.; Sinibaldi, M. Chromatographia 1987, 23, 731-735. (18) Alpert, A. J.; Andrews, P. C. J. Chromatogr. 1988, 443, 85-96. (19) Kaliszan, R. Quantitative Structure-Chromatographic Retention Relationship; John Wiley & Sons: New York, 1987; Chapter 2. (20) Karger, B. L.; Snyder, L. R.; Eon, C. J. Chromatogr. 1976, 125, 71-88.

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Table 2. Effects of Mobile Phase Modifier on Elution Timea elution time (min) peptide

water modifier

MeOH modifier

EtOH modifier

FY FGGF FLEEI DYMGWMDP-NH2 NFTYGGF AGSQ

11.58 15.64 19.09 23.56 24.64 30.30

14.79 20.20 29.72 37.17 38.33 42.80

21.80 28.97 47.23 59.85 66.39 68.99

a Effects of mobile phase modifier of eluent on separation of peptides on TSK gel Amide-80. The peptide mixture was separated using 70min linear gradients of the modifier (water, MeOH, and EtOH) from 3 to 45% at a flow rate of 1.0 mL/min.

Table 3. Solvent

n-alkanes ACN EtOH MeOH water

Propertiesa

δt

δd

δo

δin

δa

δb

∼8.0 12.1 12.7 14.5 23.4

∼8.0 6.5 6.8 6.2 6.3

8.2 3.4 4.9

2.8 0.5 0.8

0 6.9 8.3 large

3.8 6.9 8.3 large

Figure 3. Comparison of chromatograms of peptides separated on (A) TSK gel ODS-80Ts and (B) TSK gel Amide-80. The peptide mixture was separated with (A) 83.3-min linear gradients of acetonitrile from 5 to 55% in 0.1% TFA (0.6% ACN/min) and (B) 70-min linear gradients of water from 3 to 45% in 0.1% TFA (0.6% water/min). Peak identification: 1, PG; 2, LG; 3, FG; 4, EHP-NH2; 5, VGSQ; 6, GGYR; 7, WAGGDASGE; 8, DSDPR.

a δ , total solubility parameter [(cal/cm3)1/2]; δ , the solubility t d parameter of dispersion; δo, the solubility parameter of orientation; δin, the solubility parameter of induction; δa, the solubility parameter of proton donor; δb, the solubility parameter of proton acceptor.

used. The high concentrations of the organic solvent (ACN) and acidic solution served as the mobile phase. Table 2 indicates hydrogen bonding21 to possibly be a factor of retention and the proton-donor and -acceptor features to possibly be related to peptide eluting from the amide column. NPLC and RPLC Selectivity. Differences in retention for the present NPLC and RPLC methods were studied using hydrophilic and hydrophobic peptides. The results for separating two typical peptides by the present NPLC method, together with those of the RPLC method, are shown, where hydrophilic peptides not retainable on an ODS column were strongly retained on the amide column, and hydrophobic peptides were weakly retained. EHP-NH2, VGSQ, and DSDPR (Figure 3A) containing hydrophilic side chains (P, D, N, Q, H, R, and K) eluted together in RPLC. These three peptides were well retained and separated completely in the present NPLC method (Figure 3B). AGSQ (Figure 4A) eluted at the void volume on the ODS column and was well retained on the amide column. FLIEEI and DYMGWMDP-NH2 (Figure 4), with hydrophobic side chains such as V, L, I, W, and F, were strongly retained on the ODS column, but they were only moderately retained on the amide column. A large peptide (peak 10 in Figure 4) was strongly retained on both columns. Separation selectivities for the present NPLC and RPLC methods differed significantly, as shown in Figures 3 and 4. The contribution22-26 of each amino acid residue to retention in the present NPLC method is presently being studied. (21) Fischer, J.; Jandera, P. J. Chromatogr. 1994, 684, 77-92. (22) Sasagawa, T.; Okuyama, T.; Teller, D. C. J. Chromatogr. 1982, 240, 329340. (23) Sasagawa, T.; Ericksson, L. H.; Teller, D. C.; Titani, K.; Walsh, K. A. J. Chromatogr. 1984, 307, 29-38. (24) Meek, J. L. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 1632-1636. (25) Parker, J. M. R.; Guo, D.; Hodges, R. S. Biochemistry 1986, 25, 54255432.

3040 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

Figure 4. Comparison of chromatograms of peptides separated on (A) TSK gel ODS-80Ts and (B) TSK gel Amide-80. The peptide mixture was separated with (A) 83.3-min linear gradients of acetonitrile from 5 to 55% in 0.1% TFA (0.6% ACN/min) and (B) 70-min linear gradients of water from 3 to 45% in 0.1% TFA (0.6% water/min). Peak identification: 1, FY; 2, FGGF, 3, FLEEI; 4, DYMGWMDP-NH2; 5, NFTYGGF; 6, AGSQ; 7, WAGGDASGE; 8, YGGFMTSQKSQTPLVT; 9, ASTTTNYT; 10, VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEM.

Recovery of Peptide. Recovery27 from the amide and ODS columns was assessed using the same peptides shown in Figure 4. The results are summarized in Table 4. Recovery in the preset NPLC method was essentially the same as that with RPLC, exceeding 80%. Compound 10 in Table 4 was recovered at 80%, compared to 62% with RPLC. Reproducibility and Repeatability. These parameters in the present NPLC method were determined using the same peptides shown in Figure 4. Chromatograms obtained with the new and used amide columns, following 500 peptides injections, are shown in Figure 5. The coefficient of variation (n ) 10) and all retention times, peak heights, peak areas, and resolutions on each column (26) Hopp, T. P.; Woods, K. R. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 38243828. (27) Kato, Y.; Nakatani, S.; Kitamura, T.; Yamasaki, Y.; Hashimoto, T. J. Chromatogr. 1990, 502, 416-422.

Table 4. Recovery of Peptides from TSK Gel Amide-80 and TSK Gel ODS-80Ts with Sample Injection of ∼1 µga peptide no. 1 2 3 4 5 6 7 8 9 10

sequence FY FGGF FLEEI DYMGWMDP-NH2 NFTYGGF AGSQ WAGGDASGE YGGFMTSQKSQTPLVT ASTTTNYT VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEM

A

recovery from recovery from Amide-80 (%) ODS (%) 96 101 98 90 90 96 85 92 94 80

96 89 93 74 95 65 96 96 89 62

a Peptides were separated with a 70-min linear gradient of water from 3 to 45% in 0.1% TFA on TSK gel Amide-80. Peptides were separated with a 83.3-min linear gradient of acetonitrile from 5 to 55% in 0.1% TFA on TSK gel ODS-80Ts.

B

Figure 5. Comparison of chromatograms of peptides separated on (A) used amide column (TSK gel Amide-80) and (B) new amide column. Peak identification: 1, FY; 2, FGGF; 3, FLEEI; 4, DYMGWMDP-NH2; 5, NFTYGGF; 6, AGSQ; 7, WAGGDASGE; 8, YGGFMTSQKSQTPLVT; 9, ASTTTNYT; 10, VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEM.

are listed in Table 5. Figure 5 indicates good reproducibility. For the used amide column, good resolution was noted, the same as for the new amide column. The amide column had a sufficiently long life span. Retention time on the used column was somewhat lower than that on the new column. Table 5 shows the repeatability of the present NPLC method. Two-Dimensional Separation of a Tryptic Digest of Concanavalin A. This separation was carried out on a tryptic digest of concanavalin A under RPLC by the present NPLC method. It was expected that two free amino acids (R and K) and 17 peptides would result from the tryptic cleavage of concanavalin A. The 17 peptides were obtained. The peptide mixture was initially separated by RPLC (Figure 6A). Each peak fraction collected was concentrated to one-third the original volume. Twenty microliters was then added to the same volume of formic acid, and the system was diluted with 50 µL of 97% ACN solution containing 0.1% TFA. Twenty-microliter aliquots of the sample solutions were subjected to NPLC (Figure 6B). The fractions not separated by RPLC could be separated by the present NPLC method (Figure 4). Fraction

Figure 6. (A) Chromatogram of tryptic digest of concanavalin A on TSK gel ODS-80Ts. The digest was separated with 90-min linear gradients of acetonitrile from 5 to 55% in 0.1% TFA (0.56% ACN/ min). Thirteen fractions were obtained. (B) Chromatogram of tryptic digest of concanavalin A on TSK gel Amide-80. The digest was separated with 70-min linear gradients of water from 3 to 45% in 0.1% TFA (0.6% water/min). Peak identification: 1, TAK; 2, DQK; 3, SK, 4, SVR; 5, VSSNGSPEGSSVGR; 6, VSSNGSPEGSSVGR; 7, SNSTHQTDALHPMFNQFSK; 8, ETNTILSWSFTSK; 9, VGTAHIIYNSVDK; 10, VGLSASTGKYK; 11, DILILQGDATTGTDGNLGLTR; 12, LLGLFPDAN; 13, ADTIVAVQLDTYDNTDIGDPSTPHIGIDIK; 14, SPDSHPADGIAFFISNIDSSIPSGSTGR; 15, LSAVVSYPNADATSVSYDVDLNDVLPEWVR; 16, SAVVSYPNADATSVSYDVDLNDVLPEWVR; 17, ALFYAPVHIWESSATVSAFEATFAFLIK.

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Table 5. Coefficients of Variation (n ) 10) for the NPLC Results Obtained for Peptide Retention Times, Peak Heights, Peak Areas, and Resolution Using the New and Used Amide Columnsa Retention Time (min) sample no. 1

2

3

CV (%)

11.58 11.58 11.55 11.56 11.57 11.56 11.58 11.58 11.56 11.58 0.10

15.49 15.49 15.47 15.48 15.48 15.48 15.48 15.48 15.47 15.48 0.05

18.98 18.98 18.96 18.98 18.98 18.97 18.97 18.98 18.96 18.97 0.03

CV (%)

10.90 10.93 10.92 10.92 10.90 10.90 10.95 10.91 10.95 10.89 0.18

14.86 14.89 14.86 14.87 14.86 14.87 14.88 14.87 14.90 14.84 0.11

18.56 18.56 18.53 18.54 18.55 18.54 18.54 18.54 18.56 18.53 0.05

4

5

6

7

8

9

10

New Amide Column 23.60 24.58 23.59 24.58 23.59 24.58 23.58 24.57 23.60 24.58 23.59 24.58 23.58 24.58 23.60 24.58 23.58 24.58 23.59 24.58 0.03 0.02

29.85 29.83 29.84 29.84 29.84 29.84 29.83 29.84 29.83 29.84 0.02

34.69 34.67 34.68 34.67 34.67 34.68 34.67 34.67 34.68 34.68 0.02

36.56 36.54 36.55 36.55 36.55 36.56 36.55 36.55 36.56 36.56 0.01

39.06 39.05 39.06 39.06 39.05 39.06 39.05 39.06 39.06 39.06 0.01

42.63 42.61 42.61 42.60 42.60 42.60 42.59 42.59 42.58 42.59 0.03

Used Amide Column 23.46 24.31 23.48 24.32 23.44 24.28 23.45 24.31 23.46 24.31 23.45 24.30 23.44 24.29 23.44 24.30 23.46 24.31 23.44 24.29 0.04 0.04

28.65 28.67 28.64 28.66 28.65 28.66 28.65 28.65 28.66 28.65 0.02

34.30 34.31 34.30 34.31 34.30 34.31 34.30 34.31 34.31 34.30 0.01

36.28 36.29 36.29 36.30 36.30 36.30 36.30 36.30 36.30 36.30 0.02

38.28 38.28 38.28 38.28 38.28 38.29 38.28 38.29 38.29 38.29 0.01

42.18 42.18 42.19 42.19 42.19 42.19 42.19 42.19 42.19 42.19 0.01

Peak Height (mV) sample no. 1

2

3

4

5

CV (%)

107 106 107 104 108 106 107 105 106 107 1.02

58 58 58 56 58 57 58 57 58 57 0.83

69 68 68 67 68 67 68 67 67 67 1.06

New Amide Column 83 111 83 110 82 109 80 106 82 109 81 107 82 108 80 107 80 107 81 107 1.31 1.24

CV (%)

117 116 115 113 115 111 115 115 114 111 1.76

65 65 64 62 64 61 64 64 63 62 1.84

70 70 68 66 68 64 67 66 66 66 2.47

Used Amide Column 82 110 81 110 81 109 78 105 80 108 76 102 79 106 79 106 78 105 77 104 2.34 2.38

6

7

8

9

10

43 43 42 42 43 42 42 42 42 42 1.17

90 89 89 87 89 87 88 87 87 88 1.32

41 41 41 40 41 40 40 40 40 40 1.53

100 99 98 96 98 96 97 96 96 96 1.45

63 63 63 62 64 62 64 63 64 64 1.16

44 44 44 42 43 41 43 43 42 42 2.06

93 93 92 89 91 86 90 89 89 88 2.59

39 39 38 37 38 36 37 37 37 36 2.78

103 103 102 98 101 95 99 98 98 96 2.66

51 47 49 48 51 48 51 50 53 52 3.35

6

7

8

9

10

789 776 776 758 773 761 765 758 753 760 1.40

1790 1765 1772 1742 1784 1760 1780 1758 1754 1775 0.81

1005 998 982 1006 1030 1008 973 963 961 965 2.26

1871 1862 1839 1801 1848 1800 1822 1803 1792 1795 1.54

2156 2118 2068 2102 2112 2090 2184 2165 2334 2274 3.71

Peak Area (mV‚s) sample no.

CV (%)

1

2

3

2442 2428 2459 2368 2533 2408 2436 2367 2392 2483 2.02

1122 1098 1083 1072 1086 1065 1094 1064 1077 1069 1.59

1065 1052 1048 1027 1051 1030 1042 1030 1029 1039 1.16

4

5

New Amide Column 1346 1909 1333 1902 1330 1891 1301 1843 1333 1898 1307 1863 1322 1878 1295 1852 1290 1851 1301 1865 1.39 1.20

3042 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

Table 5 (Continued) Peak Area (mV‚s) sample no.

CV (%) a

1

2

3

2609 2505 2560 2495 2547 2382 2432 2503 2426 2380 2.95

1102 1103 1100 1052 1097 1039 1065 1065 1062 1068 2.06

1040 1046 1024 989 1031 963 1005 1008 993 987 2.49

4

5

Used Amide Column 1371 1950 1370 1952 1363 1928 1306 1860 1347 1914 1276 1806 1333 1886 1325 1870 1319 1870 1294 1842 2.32 2.41

6

7

8

9

10

798 791 787 759 777 730 768 758 759 748 2.62

1757 1754 1742 1672 1731 1627 1696 1687 1686 1659 2.43

968 977 959 925 948 897 937 916 909 904 2.88

1887 1887 1867 1797 1844 1738 1814 1793 1788 1761 2.70

1853 1820 1880 1815 1963 1867 1874 1773 2037 1851 3.86

Peak identification is shown in Figure 4.

1 (RP1) obtained by RPLC was a mixture of hydrophilic peptides not retained without separation by RPLC. For this fraction (RP1), three peaks, TAK, DQK, and SK, were noted with the present NPLC method. Fraction 2 (RP2) obtained by RPLC was a mixture of hydrophilic peptides weakly retained without separation by RPLC and showed two peaks, LK and SVR, by NPLC. Figure 4B shows fraction 3 (RP3) by RPLC to contain impurities, which were separated by the present NPLC method. Fraction 8 (RP8) consisted of ETNTILSWSFTSK and SNSTHQTDALHFMFNFSK, which were not separated by RPLC. Their complete separation was achieved by NPLC. Seventeen peptides were isolated by twodimensional separation. The present NPLC method may thus be concluded to exceed the limits of RPLC. Its mobile phase is highly volatile, thus facilitating peptide purification with no need for desalting.17,18 Comparison with Other NPLC Methods. To the author’s knowledge, this paper is the first to present peptide separation using a normal phase column (amide column). The protected hydrophobic (tert-butoxycarbonyl-X-Met-OCH3) peptide2-4 has been reported to be separated on silica column by nonaqueous NPLC.28-30 Hydrophilic interaction chromatography (HIC)31,32 has been proposed as an alternative to the usual NPLC by Alpert. HIC was done using hydrophilic, strong cation-exchange (SCX) materials (28) Snyder, L. R.; Schunk, T. C. Anal. Chem. 1982, 54, 1764-1772. (29) Snyder, L. R.; Poppe, H. J. Chromatogr. 1980, 184, 363-413. (30) Snyder, L. R. J. Chromatogr. 1983, 255, 3-26. (31) Alpert, A. J. J. Chromatogr. 1990, 499, 177-196. (32) Alpert, A. J. J. Chromatogr. 1988, 444, 269-274.

and stationary phase and a hydrophobic, mostly organic mobile phase in conjunction. The separation of peptides, nucleic acids, and other polar compounds by HIC was previously reported.31 But with HIC, desalting is required in order to purify peptides. Basic peptides may be adsorbed on a HIC column (SCX). The present NPLC method using an amide column is thus shown to be superior to HIC for peptide separation. CONCLUSIONS A new method for the separation of peptides in aqueous NPLC is proposed. Separation selectivities for the present NPLC and RPLC methods differ significantly, and the use of the two methods in conjunction may give the best results. Peptide recovery by the present NPLC method was generally better than 80% and the same as that with RPLC. Reproducibility and repeatability of the present NPLC method were satisfactory. This method should prove quite useful for peptide separation and purification, as well as the separation of hydrophilic compounds, such as sugars, oligosaccharides, and polynucleic acids. ACKNOWLEDGMENT The author expresses appreciation to Dr. K. Tsukagoshi and Dr. T. Sasagawa for support and valuable comments. Received for review February 26, 1997. Accepted May 27, 1997.X AC9702204 X

Abstract published in Advance ACS Abstracts, July 1, 1997.

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