The Effect of Buffer Concentration and Cation Type in the Mobile Phase on Retention Of
The effect of buffer concentration and cation type in the mobile phase on retention of amino acids and dipeptides in hydrophilic interaction liquid chromatography
KvětaKalíková*,1, Milan Boublík1,Gabriela Kučerová1,Petr Kozlík2,$
1Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43 Prague 2, CzechRepublic
2Department ofAnalyticalChemistry, Facultyof Science, Charles University, Hlavova 8, 128 43 Prague 2, CzechRepublic
$Department ofOncology, LombardiComprehensiveCancer Center, Georgetown University, 3970 ReservoirRoad NW, Washington, DC 20007, UnitedStates
*Corresponding author, e-mail:
Table S1Retention times/retention factors and their standard deviations of amino acids and their N-blocked analogues on the amide and silica columns using different buffers. MP ACN/buffer 90/10 (v/v).
Amide column / Silicacolumnc (mM) / Tyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe / c (mM) / Tyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe
Tris / tR (min)
(SD) / Tris / tR (min)
(SD)
5 / 73.102 (0.004) / 6.011 (0.009) / 40.434 (0.009) / 4.604 (0.008) / 38.382 (0.008) / 4.472 (0.008) / 5 / 49.684 (0.012) / 5.312 (0.012) / 40.553 (0.011) / 4.882 (0.012) / 44.734 (0.012) / 4.923 (0.012)
10 / 74.773 (0.006) / 7.263 (0.012) / 40.503 (0.011) / 5.453 (0.010) / 38.451 (0.009) / 5.164 (0.011) / 10 / 51.692 (0.010) / 6.363 (0.009) / 41.124 (0.013) / 5.744 (0.011) / 45.610 (0.009) / 5.792 (0.009)
20 / 78.764 (0.009) / 8.571 (0.006) / 41.796 (0.008) / 6.290 (0.007) / 39.595 (0.010) / 5.900 (0.010) / 20 / 53.180 (0.009) / 7.291 (0.013) / 41.362 (0.013) / 6.444 (0.013) / 46.223 (0.015) / 6.520 (0.010)
25 / 81.700 (0.007) / 8.994 (0.009) / 42.522 (0.007) / 6.552 (0.008) / 40.360 (0.011) / 6.110 (0.009) / 25 / 53.683 (0.012) / 7.480 (0.009) / 41.973 (0.012) / 6.543 (0.012) / 46.392 (0.011) / 6.620 (0.011)
50 / 98.881 (0.008) / 10.263 (0.007) / 47.131 (0.009) / 7.150 (0.007) / 45.412 (0.011) / 6.673 (0.010) / 50 / 62.910 (0.010) / 8.832 (0.013) / 44.680 (0.009) / 7.382 (0.011) / 49.394 (0.013) / 7.374 (0.013)
TEA / TEA
5 / 72.800 (0.004) / 2.871 (0.005) / 40.221 (0.010) / 2.644 (0.008) / 37.522 (0.007) / 2.502 (0.006) / 5 / 46.042 (0.013) / 5.183 (0.013) / 37.594 (0.013) / 4.893 (0.014) / 40.703 (0.010) / 4.793 (0.010)
10 / 70.430 (0.008) / 3.180 (0.004) / 38.663 (0.008) / 2.853 (0.009) / 35.221 (0.007) / 2.683 (0.007) / 10 / 45.430 (0.009) / 6.032 (0.008) / 36.962 (0.015) / 5.682 (0.013) / 39.774 (0.011) / 5.952 (0.009)
20 / 68.211 (0.010) / 3.510 (0.007) / 37.633 (0.008) / 3.091 (0.011) / 33.973 (0.012) / 2.880 (0.010) / 20 / 42.164 (0.009) / 6.570 (0.010) / 34.233 (0.011) / 6.164 (0.010) / 36.490 (0.012) / 6.040 (0.012)
25 / 66.892 (0.009) / 3.612 (0.004) / 37.084 (0.009) / 3.170 (0.010) / 33.220 (0.009) / 2.954 (0.007) / 25 / 41.030 (0.010) / 6.724 (0.009) / 33.530 (0.012) / 6.330 (0.011) / 35.515 (0.013) / 6.141 (0.011)
50 / 64.593 (0.007) / 3.851 (0.006) / 35.301 (0.007) / 3.363 (0.007) / 31.592 (0.010) / 3.110 (0.006) / 50 / 40.574 (0.010) / 7.053 (0.011) / 32.884 (0.013) / 6.632 (0.010) / 33.882 (0.013) / 6.981 (0.014)
NH4+ / NH4+
5 / 74.550 (0.010) / 4.432 (0.004) / 40.990 (0.007) / 3.612 (0.008) / 38.502 (0.009) / 3.440 (0.009) / 5 / 47.224 (0.014) / 5.021 (0.008) / 37.592 (0.014) / 4.640 (0.010) / 41.612 (0.012) / 4.723 (0.011)
10 / 73.873 (0.008) / 5.243 (0.004) / 40.262 (0.012) / 4.125 (0.007) / 38.073 (0.010) / 3.933 (0.007) / 10 / 49.724 (0.008) / 5.953 (0.007) / 38.634 (0.012) / 5.433 (0.009) / 43.204 (0.013) / 5.491 (0.012)
20 / 74.482 (0.006) / 5.960 (0.009) / 39.861 (0.007) / 4.593 (0.006) / 37.920 (0.010) / 4.382 (0.011) / 20 / 50.132 (0.007) / 6.883 (0.010) / 39.220 (0.010) / 6.122 (0.010) / 43.233 (0.012) / 6.204 (0.014)
25 / 74.936 (0.009) / 6.191 (0.006) / 39.904 (0.009) / 4.750 (0.012) / 38.514 (0.008) / 4.510 (0.009) / 25 / 51.221 (0.008) / 7.060 (0.011) / 39.593 (0.012) / 6.290 (0.013) / 43.710 (0.014) / 6.340 (0.012)
50 / 79.795 (0.009) / 6.924 (0.007) / 40.410 (0.006) / 5.221 (0.009) / 39.362 (0.010) / 4.913 (0.009) / 50 / 54.940 (0.009) / 7.842 (0.010) / 40.060 (0.011) / 6.744 (0.011) / 45.084 (0.010) / 6.762 (0.011)
Li+ / Li+
5 / 73.182 (0.010) / 5.542 (0.004) / 40.313 (0.009) / 4.412 (0.006) / 38.804 (0.014) / 4.110 (0.009) / 5 / 44.302 (0.011) / 4.723 (0.009) / 35.661 (0.016) / 4.442 (0.011) / 39.630 (0.015) / 4.461 (0.013)
10 / 74.083 (0.007) / 6.581 (0.007) / 39.890 (0.010) / 5.081 (0.007) / 39.132 (0.011) / 4.834 (0.013) / 10 / 44.693 (0.009) / 5.302 (0.011) / 36.764 (0.015) / 4.902 (0.009) / 40.274 (0.016) / 4.954 (0.011)
20 / 76.136 (0.011) / 7.446 (0.008) / 39.894 (0.009) / 5.600 (0.008) / 38.610 (0.009) / 5.260 (0.012) / 20 / 44.884 (0.011) / 5.690 (0.012) / 37.213 (0.012) / 5.213 (0.013) / 40.880 (0.013) / 5.233 (0.011)
25 / 78.334 (0.007) / 7.894 (0.006) / 40.290 (0.005) / 5.884 (0.010) / 39.271 (0.010) / 5.495 (0.009) / 25 / 48.140 (0.008) / 6.073 (0.011) / 37.400 (0.012) / 5.450 (0.011) / 42.733 (0.014) / 5.520 (0.012)
50 / 85.642 (0.009) / 8.390 (0.005) / 41.582 (0.006) / 6.110 (0.011) / 41.860 (0.011) / 5.762 (0.008) / 50 / 49.811 (0.12) / 6.381 (0.009) / 37.911 (0.013) / 5.604 (0.011) / 42.772 (0.013) / 5.631 (0.012)
Amide column / Silicacolumn
c (mM) / Tyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe / c (mM) / Tyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe
Tris / k
(SD) / Tris / k
(SD)
5 / 25.683 (0.001) / 1.191 (0.002) / 13.761 (0.003) / 0.681 (0.001) / 13.010 (0.003) / 0.631
(0.001) / 5 / 15.562 (0.004) / 0.771 (0.002) / 12.521 (0.003) / 0.631 (0.002) / 13.913 (0.004) / 0.642 (0.002)
10 / 26.491 (0.002) / 1.672 (0.003) / 13.891 (0.004) / 1.002 (0.002) / 13.140 (0.003) / 0.902
(0.002) / 10 / 16.231 (0.003) / 1.122 (0.002) / 12.712 (0.004) / 0.913 (0.003) / 14.200 (0.003) / 0.931 (0.001)
20 / 28.173 (0.003) / 2.170 (0.002) / 14.483 (0.003) / 1.330 (0.001) / 13.663 (0.003) / 1.190
(0.002) / 20 / 16.910 (0.003) / 1.450 (0.003) / 12.931 (0.004) / 1.173 (0.002) / 14.562 (0.005) / 1.200 (0.002)
25 / 29.370 (0.003) / 2.341 (0.002) / 14.810 (0.002) / 1.431 (0.002 / 14.000 (0.004) / 1.270
(0.002) / 25 / 17.201 (0.004) / 1.540 (0.002) / 13.231 (0.004) / 1.221 (0.002) / 14.731 (0.003) / 1.240 (0.002)
50 / 36.893 (0.003) / 2.933 (0.002) / 17.060 (0.003) / 1.740 (0.002) / 16.402 (0.004) / 1.561
(0.002) / 50 / 20.040 (0.003) / 1.951 (0.003) / 13.940 (0.003) / 1.470 (0.002) / 15.523 (0.004) / 1.463 (0.003)
TEA / TEA
5 / 25.570 (0.001) / 0.050 (0.000) / 13.68 (0.003) / X / 12.691 (0.002) / X / 5 / 14.191 (0.004) / 0.711 (0.002) / 11.412 (0.004) / 0.611 (0.002) / 12.431 (0.003) / 0.582 (0.001)
10 / 24.702 (0.003) / 0.160 (0.000) / 13.111 (0.003) / 0.040 (0.000) / 11.850 (0.002) / X / 10 / 14.040 (0.003) / 1.001 (0.001) / 11.241 (0.005) / 0.881 (0.002) / 12.172 (0.003) / 0.970 (0.001)
20 / 23.892 (0.004) / 0.280 (0.001) / 12.731 (0.003) / 0.131 (0.000) / 11.402 (0.004) / 0.050
(0.000) / 20 / 13.012 (0.003) / 1.180 (0.002) / 10.371 (0.003) / 1.052 (0.002) / 11.120 (0.004) / 1.010 (0.002)
25 / 23.411 (0.003) / 0.321 (0.000) / 12.532 (0.003) / 0.162 (0.001) / 11.120 (0.003) / 0.082
(0.000) / 25 / 12.590 (0.003) / 1.232 (0.002) / 10.100 (0.004) / 1.100 (0.002) / 10.763 (0.004) / 1.031 (0.002)
50 / 22.571 (0.002) / 0.410 (0.001) / 11.880 (0.002) / 0.230 (0.000) / 10.530 (0.003) / 0.140
(0.000) / 50 / 12.482 (0.003) / 1.341 (0.002) / 9.922 (0.004) / 1.201 (0.002) / 10.261 (0.004) / 1.321 (0.003)
NH4+ / NH4+
5 / 26.210 (0.004) / 0.620 (0.001) / 13.960 (0.002) / 0.321 (0.001) / 13.050 (0.003) / 0.260
(0.001) / 5 / 14.692 (0.004) / 0.671 (0.001) / 11.491 (0.004) / 0.540 (0.001) / 12.821 (0.004) / 0.571 (0.001)
10 / 25.962 (0.003) / 0.911 (0.001) / 13.691 (0.004) / 0.503 (0.001) / 12.892 (0.003) / 0.432
(0.001) / 10 / 15.522 (0.002) / 0.982 (0.001) / 11.832 (0.004) / 0.802 (0.001) / 13.352 (0.004) / 0.820 (0.002)
20 / 26.281 (0.002) / 1.180 (0.002) / 13.600 (0.002) / 0.682 (0.001) / 12.890 (0.003) / 0.601
(0.002) / 20 / 15.601 (0.002) / 1.282 (0.002) / 11.990 (0.003) / 1.031 (0.002) / 13.311 (0.004) / 1.052 (0.002)
25 / 26.654 (0.003) / 1.280 (0.001) / 13.721 (0.003) / 0.750 (0.002) / 13.213 (0.003) / 0.660
(0.001) / 25 / 15.960 (0.002) / 1.340 (0.002) / 12.112 (0.004) / 1.080 (0.002) / 13.470 (0.004) / 1.100 (0.002)
50 / 28.662 (0.003) / 1.572 (0.002) / 14.020 (0.002) / 0.940 (0.002) / 13.630 (0.003) / 0.831
(0.002) / 50 / 17.190 (0.003) / 1.601 (0.002) / 12.260 (0.003) / 1.232 (0.002) / 13.931 (0.003) / 1.241 (0.002)
Li+ / Li+
5 / 25.711 (0.004) / 1.020 (0.001) / 13.712 (0.003) / 0.611 (0.001) / 13.162 (0.005) / 0.500
(0.001) / 5 / 13.671 (0.003) / 0.561 (0.001) / 10.811 (0.005) / 0.470 (0.001) / 12.120 (0.005) / 0.480 (0.001)
10 / 26.142 (0.002) / 1.411 (0.002) / 13.610 (0.003) / 0.860 (0.001) / 13.331 (0.004) / 0.772
(0.002) / 10 / 13.751 (0.003) / 0.751 (0.002) / 11.132 (0.005) / 0.620 (0.001) / 12.292 (0.005) / 0.633 (0.001)
20 / 27.092 (0.004) / 1.752 (0.002) / 13.722 (0.003) / 1.070 (0.002) / 13.250 (0.003) / 0.940
(0.002) / 20 / 13.812 (0.003) / 0.880 (0.002) / 11.281 (0.004) / 0.720 (0.002) / 12.490 (0.004) / 0.732 (0.002)
25 / 28.011 (0.003) / 1.921 (0.001) / 13.920 (0.002) / 1.182 (0.002) / 13.541 (0.003) / 1.033
(0.002) / 25 / 14.940 (0.002) / 1.011 (0.002) / 11.380 (0.004) / 0.800 (0.002) / 13.151 (0.004) / 0.830 (0.002)
50 / 31.320 (0.003) / 2.170 (0.001) / 14.690 (0.002) / 1.310 (0.002) / 14.800 (0.004) / 1.171
(0.002) / 50 / 15.490 (0.004) / 1.110 (0.002) / 11.550 (0.004) / 0.850 (0.002) / 13.161 (0.004) / 0.861 (0.002)
X - elutionwithdeadtimemarker
Table S2Retention times/retention factorsand their standard deviations of dipeptides and their N-blocked analogues on the amide and silica columns using different buffers. MP ACN/buffer 90/10 (v/v).
Amide column / Silicacolumnc (mM) / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp / c (mM) / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp
Tris / tR (min)
(SD) / Tris / tR (min)
(SD)
5 / 30.033 (0.011) / 27.312 (0.009) / 16.973 (0.011) / 21.133 (0.010) / 4.264 (0.009) / 4.473 (0.014) / 5 / 28.942 (0.009) / 26.172 (0.015) / 23.320 (0.011) / 25.123 (0.013) / 4.353 (0.011) / 4.404 (0.017)
10 / 32.162 (0.010) / 29.713 (0.013) / 18.592 (0.012) / 22.154 (0.012) / 5.033 (0.011) / 5.204 (0.012) / 10 / 31.160 (0.010) / 28.204 (0.013) / 25.333 (0.012) / 27.332 (0.011) / 4.922 (0.009) / 5.093 (0.012)
20 / 35.140 (0.009) / 32.810 (0.010) / 20.301 (0.010) / 23.420 (0.013) / 5.741 (0.010) / 5.933 (0.001) / 20 / 32.973 (0.011) / 30.932 (0.013) / 26.584 (0.011) / 30.192 (0.011) / 5.510 (0.008) / 5.711 (0.016)
25 / 36.264 (0.009) / 33.974 (0.011) / 20.924 (0.011) / 23.932 (0.009) / 5.940 (0.007) / 6.132 (0.012) / 25 / 33.660 (0.013) / 31.550 (0.012) / 26.941 (0.010) / 30.731 (0.013) / 5.590 (0.011) / 5.790 (0.013)
50 / 41.840 (0.009) / 39.510 (0.009) / 22.870 (0.012) / 25.611 (0.011) / 6.282 (0.010) / 6.481 (0.010) / 50 / 36.784 (0.012) / 34.353 (0.011) / 27.772 (0.009) / 30.904 (0.012) / 6.253 (0.012) / 6.464 (0.012)
TEA / TEA
5 / 19.512 (0.013) / 16.442 (0.011) / 10.492 (0.009) / 14.682 (0.014) / 2.350 (0.007) / 2.442 (0.010) / 5 / 24.981 (0.009) / 24.153 (0.010) / 22.543 (0.013) / 23.270 (0.014) / 4.733 (0.013) / 4.904 (0.019)
10 / 19.090 (0.012) / 16.320 (0.012) / 10.511 (0.010) / 14.313 (0.013) / 2.562 (0.012) / 2.663 (0.011) / 10 / 24.794 (0.008) / 23.951 (0.011) / 22.392 (0.012) / 23.132 (0.012) / 4.710 (0.012) / 4.783 (0.014)
20 / 19.013 (0.009) / 16.455 (0.009) / 10.605 (0.008) / 14.191 (0.010) / 2.793 (0.009) / 2.905 (0.013) / 20 / 24.730 (0.010) / 23.553 (0.013) / 22.100 (0.010) / 22.911 (0.010) / 4.684 (0.009) / 4.605 (0.014)
25 / 18.984 (0.010) / 16.393 (0.010) / 10.563 (0.009) / 13.940 (0.009) / 2.864 (0.009) / 2.974 (0.014) / 25 / 24.422 (0.011) / 23.244 (0.012) / 22.081 (0.011) / 22.813 (0.013) / 4.510 (0.013) / 4.554 (0.011)
50 / 20.000 (0.008) / 16.570 (0.009) / 10.560 (0.010) / 13.813 (0.010) / 3.084 (0.010) / 3.172 (0.010) / 50 / 23.883 (0.012) / 23.020 (0.012) / 21.690 (0.008) / 22.634 (0.013) / 4.473 (0.014) / 4.513 (0.012)
NH4+ / NH4+
5 / 24.730 (0.008) / 21.893 (0.013) / 13.674 (0.011) / 17.923 (0.009) / 3.264 (0.011) / 3.385 (0.012) / 5 / 26.870 (0.011) / 24.193 (0.009) / 20.912 (0.010) / 23.683 (0.015) / 4.032 (0.014) / 4.334 (0.014)
10 / 25.734 (0.011) / 23.080 (0.011) / 14.532 (0.010) / 18.262 (0.012) / 3.735 (0.012) / 3.894 (0.009) / 10 / 27.974 (0.008) / 24.782 (0.008) / 22.293 (0.010) / 24.234 (0.013) / 4.563 (0.015) / 4.745 (0.016)
20 / 27.065 (0.010) / 24.484 (0.013) / 15.260 (0.009) / 18.621 (0.011) / 4.183 (0.009) / 4.351 (0.011) / 20 / 29.472 (0.012) / 26.464 (0.010) / 23.804 (0.012) / 25.770 (0.011) / 5.021 (0.013) / 5.253 (0.017)
25 / 27.380 (0.012) / 24.850 (0.010) / 15.540 (0.008) / 18.760 (0.009) / 4.330 (0.012) / 4.513 (0.012) / 25 / 29.770 (0.012) / 27.632 (0.011) / 24.181 (0.013) / 26.772 (0.013) / 5.180 (0.013) / 5.393 (0.018)
50 / 29.672 (0.008) / 27.192 (0.009) / 16.523 (0.010) / 19.444 (0.011) / 4.723 (0.013) / 4.910 (0.010) / 50 / 31.373 (0.011) / 29.281 (0.010) / 24.850 (0.009) / 27.902 (0.012) / 5.552 (0.015) / 5.782 (0.019)
Li+ / Li+
5 / 24.471 (0.013) / 22.210 (0.014) / 14.122 (0.013) / 17.562 (0.010) / 3.863 (0.012) / 4.023 (0.016) / 5 / 23.102 (0.014) / 21.004 (0.010) / 18.340 (0.012) / 20.763 (0.010) / 3.833 (0.016) / 4.085 (0.018)
10 / 26.543 (0.012) / 24.433 (0.010) / 15.581 (0.011) / 18.624 (0.011) / 4.612 (0.010) / 4.824 (0.012) / 10 / 23.403 (0.012) / 21.503 (0.009) / 19.303 (0.011) / 21.020 (0.011) / 4.294 (0.014) / 4.343 (0.017)
20 / 28.250 (0.009) / 26.402 (0.010) / 16.705 (0.010) / 19.402 (0.013) / 5.084 (0.009) / 5.232 (0.015) / 20 / 23.732 (0.013) / 21.893 (0.011) / 19.432 (0.012) / 21.214 (0.011) / 4.442 (0.015) / 4.514 (0.013)
25 / 28.404 (0.010) / 26.574 (0.009) / 16.790 (0.009) / 19.110 (0.009) / 5.251 (0.013) / 5.420 (0.012) / 25 / 23.951 (0.012) / 22.160 (0.011) / 19.660 (0.011) / 21.652 (0.011) / 4.602 (0.014) / 4.743 (0.015)
50 / 30.963 (0.008) / 29.060 (0.009) / 17.563 (0.010) / 19.763 (0.011) / 5.462 (0.014) / 5.611 (0.011) / 50 / 24.491 (0.013) / 22.764 (0.011) / 19.721 (0.009) / 21.982 (0.013) / 4.783 (0.012) / 5.602 (0.013)
Amide column / Silicacolumn
c (mM) / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp / c (mM) / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp
Tris / k
(SD) / Tris / k
(SD)
5 / 9.961 (0.004) / 8.971 (0.003) / 5.192 (0.003) / 6.711 (0.003) / 0.553 (0.001) / 0.631 (0.002) / 5 / 8.651 (0.003) / 7.721 (0.004) / 6.770 (0.003) / 7.372 (0.004) / 0.452 (0.001) / 0.473 (0.002)
10 / 10.821 (0.003) / 9.922 (0.004) / 5.831 (0.004) / 7.142 (0.004) / 0.852 (0.002) / 0.912 (0.002) / 10 / 9.390 (0.003) / 8.402 (0.004) / 7.442 (0.004) / 8.111 (0.003) / 0.641 (0.001) / 0.701 (0.002)
20 / 12.010 (0.003) / 11.150 (0.003) / 6.520 (0.003) / 7.670 (0.004) / 1.130 (0.002) / 1.201 (0.000) / 20 / 10.102 (0.003) / 9.411 (0.004) / 7.953 (0.003) / 9.161 (0.003) / 0.860 (0.001) / 0.920 (0.003)
25 / 12.482 (0.003) / 11.633 (0.004) / 6.783 (0.004) / 7.901 (0.003) / 1.210 (0.001) / 1.280 (0.003) / 25 / 10.410 (0.004) / 9.690 (0.004) / 8.130 (0.003) / 9.420 (0.004) / 0.890 (0.002) / 0.960 (0.002)
50 / 15.030 (0.003) / 14.140 (0.003) / 7.760 (0.004) / 8.810 (0.004 / 1.411 (0.002) / 1.480 (0.002) / 50 / 11.303 (0.004) / 10.491 (0.003) / 8.291 (0.003) / 9.333 (0.004) / 1.092 (0.002) / 1.162 (0.002)
TEA / TEA
5 / 6.121 (0.004) / 5.001 (0.003) / 2.831 (0.002) / 4.361 (0.004) / X / X / 5 / 7.241 (0.003) / 6.971 (0.003) / 6.442 (0.004) / 6.680 (0.004) / 0.562 (0.002) / 0.622 (0.002)
10 / 5.970 (0.004) / 4.960 (0.004) / 2.841 (0.003) / 4.222 (0.004) / X / X / 10 / 7.213 (0.002) / 6.930 (0.003) / 6.411 (0.003) / 6.661 (0.003) / 0.560 (0.001) / 0.581 (0.002)
20 / 5.942 (0.003) / 5.003 (0.003) / 2.873 (0.002) / 4.180 (0.003) / 0.020 (0.000) / 0.062 (0.000) / 20 / 7.220 (0.003) / 6.821 (0.004) / 6.340 (0.003) / 6.610 (0.003) / 0.553 (0.001) / 0.533 (0.002)
25 / 5.933 (0.003) / 4.982 (0.003) / 2.852 (0.002) / 4.090 (0.003) / 0.040 (0.000) / 0.081 (0.000) / 25 / 7.091 (0.003) / 6.702 (0.003) / 6.311 (0.003) / 6.552 (0.004) / 0.490 (0.001) / 0.513 (0.001)
50 / 6.300 (0.003) / 5.050 (0.003) / 2.850 (0.003) / 4.041 (0.003) / 0.121 (0.000) / 0.160 (0.001) / 50 / 6.931 (0.003) / 6.650 (0.003) / 6.210 (0.002) / 6.523 (0.004) / 0.492 (0.002) / 0.501 (0.001)
NH4+ / NH4+
5 / 8.030 (0.003) / 6.992 (0.004) / 3.992 (0.003) / 5.541 (0.003) / 0.192 (0.001) / 0.233 (0.001) / 5 / 7.930 (0.003) / 7.041 (0.003) / 5.951 (0.003) / 6.872 (0.004) / 0.341 (0.001) / 0.442 (0.001)
10 / 8.392 (0.004) / 7.420 (0.004) / 4.301 (0.003) / 5.661 (0.004) / 0.363 (0.001) / 0.422 (0.001) / 10 / 8.293 (0.002) / 7.231 (0.002) / 6.412 (0.003) / 7.053 (0.004) / 0.512 (0.002) / 0.573 (0.002)
20 / 8.913 (0.003) / 7.973 (0.004) / 4.590 (0.003) / 5.820 (0.003) / 0.531 (0.001) / 0.590 (0.001) / 20 / 8.761 (0.004) / 7.762 (0.003) / 6.883 (0.003) / 7.530 (0.003) / 0.660 (0.002) / 0.742 (0.002)
25 / 9.100 (0.004) / 8.170 (0.003) / 4.730 (0.002) / 5.920 (0.003) / 0.600 (0.002) / 0.661 (0.002) / 25 / 8.860 (0.004) / 8.151 (0.003) / 7.010 (0.004) / 7.861 (0.004) / 0.720 (0.002) / 0.781 (0.003)
50 / 10.031 (0.003) / 9.111 (0.003) / 5.141 (0.003) / 6.232 (0.004) / 0.751 (0.002) / 0.830 (0.002) / 50 / 9.392 (0.003) / 8.700 (0.003) / 7.230 (0.003) / 8.241 (0.004) / 0.841 (0.002) / 0.911 (0.003)
Li+ / Li+
5 / 7.930 (0.004) / 7.110 (0.004) / 4.151 (0.004) / 5.411 (0.003) / 0.411 (0.001) / 0.471 (0.002) / 5 / 6.651 (0.004) / 5.953 (0.003) / 5.070 (0.003) / 5.872 (0.003) / 0.271 (0.001) / 0.353 (0.002)
10 / 8.722 (0.004) / 7.951 (0.003) / 4.710 (0.003) / 5.822 (0.003) / 0.690 (0.001) / 0.772 (0.002) / 10 / 6.722 (0.003) / 6.102 (0.003) / 5.372 (0.003) / 5.940 (0.003) / 0.423 (0.001) / 0.431 (0.002)
20 / 9.420 (0.003) / 8.741 (0.003) / 5.163 (0.003) / 6.160 (0.004) / 0.872 (0.002) / 0.931 (0.003) / 20 / 6.831 (0.004) / 6.221 (0.003) / 5.411 (0.003) / 6.003 (0.003) / 0.471 (0.002) / 0.492 (0.001)
25 / 9.523 (0.003) / 8.842 (0.003) / 5.220 (0.003) / 6.080 (0.003) / 0.940 (0.002) / 1.010 (0.002) / 25 / 6.930 (0.003) / 6.340 (0.003) / 5.510 (0.003) / 6.171 (0.003) / 0.520 (0.002) / 0.571 (0.002)
50 / 10.682 (0.003) / 9.970 (0.003) / 5.631 (0.003) / 6.461 (0.004) / 1.061 (0.003) / 1.120 (0.002) / 50 / 7.110 (0.004) / 6.542 (0.003) / 5.530 (0.003) / 6.280 (0.004) / 0.581 (0.001) / 0.850 (0.002)
X - elutionwithdeadtimemarker
Table S3Linearregressionanalysisofthedependenciesof log kofaminoacidsandtheirN-blockedanalogues on log c ofdifferentbuffersobtainedforthe amide andsilicacolumns. MP ACN/buffer 90/10 (v/v).
Amide column / Silica columnBuffer / Analyte / Slope / Intercept / R2 / Buffer / Analyte / Slope / Intercept / R2
Tris / Tyr / 0.1467 / 1.2846 / 0.8190 / Tris / Tyr / 0.1011 / 1.1103 / 0.8610
t-BOC-Tyr / 0.3893 / -0.1801 / 0.9915 / t-BOC-Tyr / 0.3987 / -0.3711 / 0.9854
Trp / 0.0875 / 1.0621 / 0.7959 / Trp / 0.0451 / 1.0601 / 0.8894
t-BOC-Trp / 0.4095 / -0.4285 / 0.9766 / t-BOC-Trp / 0.3659 / -0.4308 / 0.9689
Phe / 0.0932 / 1.0321 / 0.7724 / Phe / 0.0457 / 1.1079 / 0.9441
t-BOC-Phe / 0.3931 / -0.4547 / 0.9845 / t-BOC-Phe / 0.3584 / -0.4145 / 0.9613
TEA / Tyr / -0.0543 / 1.4466 / 0.9950 / TEA / Tyr / -0.0651 / 1.2013 / 0.8975
t-BOC-Tyr / 0.9208 / -1.8349 / 0.8980 / t-BOC-Tyr / 0.2738 / -0.3062 / 0.9214
Trp / -0.0589 / 1.1781 / 0.9853 / Trp / -0.6871 / 1.1087 / 0.9238
t-BOC-Trp / 1.0716 / -2.3809 / 0.9060 / t-BOC-Trp / 0.2886 / -0.3774 / 0.9213
Phe / -0.0791 / 1.157 / 0.9926 / Phe / -0.0906 / 1.1642 / 0.9583
t-BOC-Phe / 1.0027 / -2.5622 / 0.9634 / t-BOC-Phe / 0.3167 / -0.4086 / 0.8668
NH4+ / Tyr / 0.0356 / 1.3836 / 0.6179 / NH4+ / Tyr / 0.0615 / 1.1229 / 0.9041
t-BOC-Tyr / 0.4044 / -0.4647 / 0.9794 / t-BOC-Tyr / 0.37812 / -0.4109 / 0.9669
Trp / 0.0008 / 1.1388 / 0.0035 / Trp / 0.0279 / 1.0428 / 0.9696
t-BOC-Trp / 0.4702 / -0.7959 / 0.9763 / t-BOC-Trp / 0.3573 / -0.4807 / 0.9452
Phe / 0.0182 / 1.0961 / 0.4827 / Phe / 0.0315 / 1.0878 / 0.8872
t-BOC-Phe / 0.5077 / -0.9061 / 0.9638 / t-BOC-Phe / 0.3404 / -0.4497 / 0.9427
Li+ / Tyr / 0.0818 / 1.3409 / 0.8490 / Li+ / Tyr / 0.0558 / 1.0877 / 0.7399
t-BOC-Tyr / 0.3298 / -0.1981 / 0.9682 / t-BOC-Tyr / 0.2992 / -0.4409 / 0.9673
Trp / 0.0277 / 1.1101 / 0.6213 / Trp / 0.0283 / 1.0158 / 0.9754
t-BOC-Trp / 0.3348 / -0.4213 / 0.9563 / t-BOC-Trp / 0.2642 / -0.4907 / 0.9490
Phe / 0.0436 / 1.0804 / 0.6379 / Phe / 0.0395 / 1.0535 / 0.8271
t-BOC-Phe / 0.3666 / -0.5186 / 0.9427 / t-BOC-Phe / 0.2629 / -0.4814 / 0.9350
Table S4Linearregressionanalysisofthedependenciesof log kofdipeptidesandtheirN-blockedanalogues on log cofdifferentbuffersobtainedforthe amide andsilicacolumns. MP ACN/buffer 90/10 (v/v).
Amide column / Silica columnBuffer / Analyte / Slope / Intercept / R2 / Buffer / Analyte / Slope / Intercept / R2
Tris / H-Tyr-Phe / 0.1735 / 0.86554 / 0.9617 / Tris / H-Tyr-Phe / 0.11548 / 0.85605 / 0.9993
H-Phe-Tyr / 0.19245 / 0.80784 / 0.9728 / H-Phe-Tyr / 0.13682 / 0.79178 / 0.9946
H-Phe-Trp / 0.17245 / 0.59305 / 0.9977 / H-Phe-Trp / 0.09003 / 0.77653 / 0.9352
H-Trp-Phe / 0.11596 / 0.74025 / 0.9803 / H-Trp-Phe / 0.11359 / 0.79806 / 0.8735
Z-Trp-Phe / 0.40613 / -0.50427 / 0.9554 / Z-Trp-Phe / 0.38611 / -0.59466 / 0.9787
Z-Phe-Trp / 0.3748 / -0.43385 / 0.9649 / Z-Phe-Trp / 0.39471 / -0.57626 / 0.9657
TEA / H-Tyr-Phe / 0.00826 / 0.77159 / 0.0780 / TEA / H-Tyr-Phe / -0.01789 / 0.87531 / 0.7492
H-Phe-Tyr / 0.00434 / 0.69348 / 0.3262 / H-Phe-Tyr / -0.02246 / 0.86068 / 0.9125
H-Phe-Trp / 0.00481 / 0.4487 / 0.5687 / H-Phe-Trp / -0.01599 / 0.82172 / 0.9358
H-Trp-Phe / -0.03269 / 0.66061 / 0.9542 / H-Trp-Phe / -0.01133 / 0.83362 / 0.9267
Z-Trp-Phe / 1.95184 / -4.19589 / 0.9444 / Z-Trp-Phe / -0.06883 / -0.19207 / 0.6938
Z-Phe-Trp / 1.03702 / -2.55822 / 0.9817 / Z-Phe-Trp / -0.10196 / -0.13948 / 0.9448
NH4+ / H-Tyr-Phe / 0.09477 / 0.83218 / 0.9684 / NH4+ / H-Tyr-Phe / 0.07331 / 0.84661 / 0.9974
H-Phe-Tyr / 0.1126 / 0.76028 / 0.9795 / H-Phe-Tyr / 0.09588 / 0.77249 / 0.9586
H-Phe-Trp / 0.10875 / 0.52394 / 0.9969 / H-Phe-Trp / 0.0876 / 0.71779 / 0.9692
H-Trp-Phe / 0.59851 / -1.08668 / 0.9564 / H-Trp-Phe / 0.08377 / 0.77254 / 0.9635
Z-Trp-Phe / 0.04962 / 0.70518 / 0.9567 / Z-Trp-Phe / 0.39205 / -0.70998 / 0.9593
Z-Phe-Trp / 0.54665 / -0.96585 / 0.9587 / Z-Phe-Trp / 0.32471 / -0.57086 / 0.9835
Li+ / H-Tyr-Phe / 0.1247 / 0.8122 / 0.9893 / Li+ / H-Tyr-Phe / 0.02905 / 0.80006 / 0.9519
H-Phe-Tyr / 0.14308 / 0.75323 / 0.9939 / H-Phe-Tyr / 0.04022 / 0.74519 / 0.9818
H-Phe-Trp / 0.13059 / 0.53511 / 0.9785 / H-Phe-Trp / 0.03618 / 0.68638 / 0.8463
H-Trp-Phe / 0.37192 / -0.53889 / 0.9602 / H-Trp-Phe / 0.02959 / 0.74567 / 0.8964
Z-Trp-Phe / 0.4098 / -0.62133 / 0.9071 / Z-Trp-Phe / 0.32545 / -0.75698 / 0.9165
Z-Phe-Trp / 0.07411 / 0.68589 / 0.9785 / Z-Phe-Trp / 0.36538 / -0.73412 / 0.9310
Table S5 Results of t-test. pq - p-value obtained for testing similarity of intercepts of dependences of log kvs log c; pk-p-value obtained for testing similarity of slopes of dependences of log kvs log c; Fq - F value obtained for testing similarity of interceptsof dependences of log kvs log c; Fk - F value obtained for testing similarity of slopes of dependences of log kvs log c; Fcrit - critical F value. If p-value α (α = 0.05) or FFcritthe null hypothesis is accepted. The values in bold indicate that null hypothesis is accepted. The values in italics means that null hypothesis is rejected.
TrisTyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp
R2 amide c. / 0.819 / 0.991 / 0.795 / 0.978 / 0.771 / 0.771 / 0.961 / 0.972 / 0.998 / 0.980 / 0.954 / 0.963
R2silicac. / 0.862 / 0.986 / 0.891 / 0.971 / 0.944 / 0.959 / 0.999 / 0.995 / 0.936 / 0.872 / 0.978 / 0.967
pq / 0.025 / 0.005 / 0.975 / 0.988 / 0.095 / 0.000 / 0.734 / 0.533 / 0.000 / 0.139 / 0.327 / 0.123
pk / 0.362 / 0.819 / 0.168 / 0.422 / 0.166 / 0.002 / 0.028 / 0.029 / 0.001 / 0.929 / 0.701 / 0.811
Fq / 8.872 / 18.292 / 0.001 / 0.000 / 3.932 / 487.280 / 0.126 / 0.438 / 101.915 / 2.912 / 1.140 / 3.228
Fk / 0.973 / 0.057 / 2.458 / 0.742 / 2.483 / 26.065 / 8.366 / 8.083 / 32.989 / 0.009 / 0.162 / 0.062
Fcrit / 5.987
TEA
Tyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp
R2 amide c. / 0.995 / 0.907 / 0.986 / 0.903 / 0.992 / 0.999 / nonlinear / nonlinear / nonlinear / 0.953 / 0.975 / 0.997
R2silicac. / 0.898 / 0.919 / 0.924 / 0.961 / 0.958 / 0.864 / 0.727 / 0.921 / 0.948 / 0.922 / nonlinear / 0.794
pq / 0.000 / 0.000 / 0.004 / 0.003 / 0.645 / 0.003 / - / - / - / 0.000 / - / 0.002
pk / 0.446 / 0.011 / 0.442 / 0.024 / 0.366 / 0.022 / - / - / - / 0.004 / - / 0.002
Fq / 229.366 / 46.988 / 20.224 / 38.467 / 0.234 / 329.302 / - / - / - / 873.105 / - / 648.605
Fk / 0.666 / 13.324 / 0.678 / 12.401 / 0.957 / 43.420 / - / - / - / 21.408 / - / 308.172
Fcrit / 5.987 / 7.709 / 5.987 / 18.512 / 5.987 / 18.512
NH4+
Tyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp
R2 amide c. / nonlinear / 0.981 / nonlinear / 0.979 / nonlinear / 0.972 / 0.967 / 0.979 / 0.997 / 0.954 / 0.956 / 0.959
R2silicac. / 0.904 / 0.967 / 0.970 / 0.945 / 0.886 / 0.947 / 0.997 / 0.957 / 0.969 / 0.963 / 0.963 / 0.984
pq / - / 0.428 / - / 0.008 / - / 0.002 / 0.320 / 0.543 / 0.000 / 0.003 / 0.014 / 0.004
pk / - / 0.655 / - / 0.137 / - / 0.054 / 0.086 / 0.308 / 0.070 / 0.025 / 0.057 / 0.017
Fq / - / 0.721 / - / 14.843 / - / 27.260 / 1.173 / 0.416 / 253.944 / 22.289 / 11.648 / 20.684
Fk / - / 0.221 / - / 2.939 / - / 5.722 / 4.207 / 1.241 / 4.839 / 8.799 / 5.531 / 10.697
Fcrit / 5.987
Li+
Tyr / t-BOC-Tyr / Trp / t-BOC-Trp / Phe / t-BOC-Phe / H-Tyr-Phe / H-Phe-Tyr / H-Phe-Trp / H-Trp-Phe / Z-Trp-Phe / Z-Phe-Trp
R2 amide c. / 0.848 / 0.968 / nonlinear / 0.957 / nonlinear / 0.942 / 0.990 / 0.994 / 0.978 / 0.961 / 0.916 / 0.907
R2silicac. / 0.739 / 0.965 / 0.976 / 0.946 / 0.826 / 0.934 / 0.949 / 0.980 / 0.844 / 0.900 / 0.910 / 0.936
pq / 0.000 / 0.007 / - / 0.382 / - / 0.655 / 0.292 / 0.388 / 0.000 / 0.004 / 0.308 / 0.122
pk / 0.383 / 0.541 / - / 0.217 / - / 0.163 / 0.000 / 0.000 / 0.001 / 0.005 / 0.376 / 0.964
Fq / 52.286 / 16.181 / - / 0.890 / - / 0.220 / 1.335 / 0.867 / 68.770 / 20.571 / 1.239 / 3.238
Fk / 0.888 / 0.420 / - / 1.904 / - / 2.522 / 131.324 / 195.327 / 43.097 / 18.353 / 0.915 / 0.002
Fcrit / 5.987