15 10-1-3. 1. HPLC 1M KH 2 PO 4 ph 3.0 50 ml Anthranilic acid: AnA 600 ml 1-1. 350 ml AnA 1.0 ml / min Anthranilic acid=137.14 PRESSURE140 kgf / cm 2 TOSOH TSK-GEL ODS- 80 Ts 1) mg / ml AnA in (φ4.6 250 mm) AnA 0.0019 g 1.9 40 ml SHIMADZU RF 10AXL 2) 50 1) 0.1 ml SHIMADZU CR- 8A 4.9 ml 340 nm 3) 2) 410 nm ε333nm= 3990 0.2742 / 3990 = 6.87 1-4. 10-5 M 1) AnA 1.3710-6 M 20 µl 4) 50 2) 0.02 ml 27.4 pmol 1 0.98 ml 50 pmol 1.3710-6 1048111 / 27.4 = 38252 /pmol M20 µl 2) AREA / 1 pmol (ml) / 0.02 ml 10-3 = nmol / day 0.02 ml= 1M KH 2 PO 4 (ph 3. 0) 3 =136.09 g 965815 / 3825225 / 0.0210-3 = 32 nmol/day 136.09 g 800 ml ph ph 2. HPLC Kynurenic 3.0 1000 ml acid: KA 2-1. KA 1-2. Kynurenic acid= 207.19 1) 0.1 mg / ml KA in 0.6 M PCA 2000rpm5 min KA 0.0004 g 0.6 M PCA 4 ml LC-122 0.45 µm 2) 20 1) 0.2 ml 0.6 M HPLC PCA 3.8 ml 94
3) 3) 2) 0.6 M 1 M 1 ml / min PCA ε243nm= 42500 HPLC 20 µl 0.3889 / 42500 = 9.15 10-6 M 2-4. 1) KA 9.15 10-6 M 20 µl 0.6 M PCA 183 pmol 1 Perchlonic acid= 100.46 pmol 1537325 / 183 = 8401 / pmol 70% PCA11.65 M1.0 ml 18.4 2) AREA / 1 pmol ml (ml) / 0.02 ml 10-3 = nmol / day 0.02 ml= 0.5 M ph 4.5 Sodium Acetate Trihydrate= 136.08 2439925 / 840125 / 0.0210-3 = 363 nmol / day 1) 68.04 g 800 ml 3. HPLC 3-1 L 3-Hydroxyanthranilic acid: 3-HA, 0.5 M ) 2) 17.4 M1 ml 33.8 ml 0.5 M 3) 1) 2) ph 4.5 Xanthurenic acid: XA 3-1. 3-HA 3-Hydroxy anthranilic acid= 153.14 1 M Zinc Acetate Dihydrate= 219.51 1) 0.1 mg / ml 3-HA in 3-HA 0.0014 g 14.0 219.51 g ml 800 ml 2) 5 1) 1.0 ml 1 L 2-2. 4.0 ml 2) ε340nm= 3860 0.5774 / 3860 = 1.5010-4 2000rpm5 min M 4) 100 3) 0.01 ml, LC-122 0.99 ml 100 0.45 µm 1.5010-6 M 20 µl 2-3. 0.5 M - XA ph 4.520 ml 980 ml Xanthurenic acid= 205.17 52.6 ml 1) 0.1 mg / ml XA in XA 1.0 ml / min PRESSURE112 kgf / cm 2 TOSOH ODS-80Tsφ4.6 250 mm 40 HITACHI F - 1050 HITACHI D-2500 0.0008 g 8.0 ml 2) 1) ε340nm= 8648 20 µl 0.5645 / 8648 = 6.5310-5 M 344 nm 398 nm 1 M KH 2 PO 4 (ph 3. 0) 95
=136.09 12 136.09 g 1489526 / 1966225 / 0.0210-3 = 95 nmol / 800 ml ph day ph XA 3.0 1000 ml 1) XA 6.5310-5 M 20 l 3 mg / ml EDTA-2Na 1.31 nmol 1 nmol 13 EDTA-2Na= 372.24 325136 / 1.31 = 248195 / nmol 2) AREA / 1 nmol EDTA-2Na 0.0162 g 5.4 (ml) / 0.02 ml= nmol / day ml 0.02 ml= 14 3-2. 45165 / 24819525 / 0.02 = 227 nmol / day 4. HPLC Quinolinic 2000rpm5 min acid: QA LC-122 4-1. 0.45 µm QA HPLC Quinolinic acid= 167.13 3-3. 1) 0.1 mg / ml QA QA 0.0019 g 1 M KH 2 PO 4 (ph 3.0) 50 ml 19.0 ml 949 ml 3 mg / ml EDTA-2Na 1 ml 2) 5 1) 1.0 ml 100 ml 4.0 ml 1.0ml / min 3) 2) ε275nm= 4040 PRESSURE85kgf / cm 2 STR ODS- φ4.6 250mm 20 µl 0.4173 / 4040 = 40 1.0310-4 M 15 3-HA SHISEIDO NANOSPACE SI 2 0.2 M XA SHIMADZU SPD 10AVVP = 210.14 2.10 g 3-HA SHIMADZU CR 8A 50 ml XA SHIMADZU CR 6A 1 M KH 2 PO 4 (ph was adjusted 3.8 by 0.2 M 3-HA citric acid) +500 mv = 136.09 XA UV 340 68.045 g nm 400 ml ph 3-HA XA HPLC 0.2 M 10 ph 3.8 500 ml 3-4. 3-HA 4-2. 1) 3-HA 1.5010-6 M 20 l 30 pmol 1 pmol 11 2000rpm5 min 589861 / 30 = 19662 / pmol LC-122 2) AREA / 1 pmol 0.45 µm (ml) / 0.02 ml 10-3 = nmol / day HPLC 0.02 ml= 96
4-3. 1 M KH 2 POpH 4 was adjusted to 3.8 by 0.2 M citric acid 50 ml 40 ml 15% TMA Tetramethylammonium hydroxide solution30 µl 1 L 0.6ml / min PRESSURE56kgf / cm 2 Unisil Q C 81φ4.6 250 mm 30 SHIMADZU RF 10AXF SHIMADZU CR 6A 326 nm 380 nm 4-4. 1) QA 1.0310-4 M 20 µl 2.06 nmol 1 nmol 16 1205640 / 2.06 = 585262 / pmol 2) AREA / 1 nmol (ml) / 0.02 ml = nmol / day 0.02 ml= 17 191815 / 58526225 / 0.02 = 410 nmol / day 97
UV AnA HPLC 98
HPLC UV 99
HPLC 100
KA HPLC HPLC 101
3- UV UV 102
103-HA XA HPLC 103
3-HA 11 3- HPLC 12 3- HPLC 104
XA 13 HPLC 14 HPLC 105
15 UV QA 16 HPLC 106
17 HPLC 107