ON HPLC COLUMNS
PeptidesProteins
Amino Acids
No.TI055E
TI055E-AI27
LC and FAB Mass Analyses of D-Amino Acid-containing Peptides
Courtesy of A. Sugihara, Osaka Municipal Technical Research Institute
LC
[LC]Cadenza CD-C18, 75 x 4.6 mm5-45% of methanol/acetonitrile (6:4) in 50 mM phosphate, pH 5.71 mL/min
LC
FAB-MS FAB-MS
No.TI056E
TI056E-AJ10
Peptides - BSA Tryptic Digest
150 x 2 mmA: water / TFA = 100 / 0.08B: acetonitrile / TFA = 100 / 0.055%B(0-2min), 5-40%B(2-20min), 40-90%B(20-25min), 90%B(25-30min), 90-5%B(30-33min)0.2 mL/minUV at 214 nm40 °CBSA Tryptic Digest, 18.5uL (ca.6.5ug)
min0 5 10 15 20 25
mAU
-50-25
0255075
100
min0 5 10 15 20 25
mAU
-50-25
0255075
100
min0 5 10 15 20 25
mAU
-50-25
0255075
100
min0 5 10 15 20 25
mAU
-50-25
0255075
100
Brand-A ODS
Brand-B ODS
Brand-C ODS
min0 5 10 15 20 25
mAU
-200
20406080
100
min0 5 10 15 20 25
mAU
-200
20406080
100
min0 5 10 15 20 25
mAU
-200
20406080
100
min0 5 10 15 20 25
mAU
-200
20406080
100
No.TI057E
TI057E-AJ10
Peptides - BSA Tryptic Digest
150 x 2 mmA: acetonitrile / 1-propanol / water / acetic acid / TFA = 1 / 1 / 98 / 0.1 / 0.02B: acetonitrile / 1-propanol / water / acetic acid / TFA = 70 / 20 / 10 / 0.09 / 0.025%B(0-2min), 5-40%B(2-20min), 40-90%B(20-25min), 90%B(25-30min), 90-5%B(30-33min)0.2 mL/minUV at 214 nm40 °CBSA Tryptic Digest, 18.5uL (ca.6.5ug)
Brand-A ODS
Brand-B ODS
Brand-C ODS
No.TI076E
TI076E-BI10
LC-MS Application of Myoglobin Tryptic Digest
Courtesy of D.Higo, Bruker Daltonics K.K.
UV at 215 nm
BPC 300-1500 + MS
0
50
100
150
200
0
1
2
3
4
5
0 5 10 15 20 25 30 35 40 45 Time [min]
mAU
Ints.
UV at 215 nm
BPC 300-1500 + MS
0
50
100
150
200
0
1
2
3
4
5
0 5 10 15 20 25 30 35 40 45 Time [min]
mAU
Ints. [MS]Esquire 3000plus( Ion Trap MS, positive mode )
Cadenza CD-C18, 150 x 2 mmA: 0.01% TFA in waterB: 0.01% TFA in acetonitrile2 - 50 %B ( 0 - 50 min )0.2 mL/min, 45 °CMyoglobin (Horse Heart), Tryptic digest
No.TI081E
TI081E-BI13
Unison UK-C18, 250 x 4.6 mm50mM H3PO4 + 10mM sodium heptanesulfonate0.8 mL/min, 37 °C, 12.8 MPa, 210 nm, 2-10uL (4-20ug)
Amino Acids - 1
1
2
3 4
5
6
min0 5 10 15 20 25 30
mAU
0
10
20
30
40
50
1 2 3
4 5 6
glycine (Gly / G)L-aspartic acid (Asp / D) L-asparagine (Asn / N)
L-serine (Ser / S) L-hydroxyproline L-glutamine (Gln / Q)
C OHO
H2N CH2 C C
H
OHOH2N
H2N CH2CO
C C
H
OHOH2N
OH
CH2C C
H
OHOH2N
CH2 CH2CO
H2NNH
HO
H
C OHO
C C
H
OHOH2N
CH2HO CO
No.TI082E
TI082E-BI13
Unison UK-C18, 250 x 4.6 mm50mM H3PO4 + 10mM sodium heptanesulfonate / ACN = 92.5/7.50.8 mL/min, 37 °C, 13.5 MPa, 210 nm, 2-5uL (4-10ug)
Amino Acids - 2
1
2
3
4
5
6
1 2 3
4 5 6
L-alanine (Ala / A)
L-valine (Val / V)
L-cysteine (Cys / C)
L-cystine L-glutamic acid (Glu / E)
L-threonine (Thr / T)
C C
H
OHOH2N
H3C C C
H
OHOH2N
HS CH2C C
H
OHOH2N
OH
CHH3C
C C
H
OHOH2N
CH2 CH2CO
HOC C
H
OHOH2N
S CH2
C CH
OHOH2N
S CH2
C C
H
OHOH2N
CH2H3C
H3C
min0 5 10 15 20 25 30 35
mAU
0
10
20
30
40
50
No.TI083E
TI083E-BI17
Unison UK-C18, 250 x 4.6 mm50mM H3PO4 + 10mM sodium heptanesulfonate / ACN = 85 / 150.8 mL/min, 37 °C, 14.2 MPa, 210 nm, 0.4-10 uL (4-20 ug)
Amino Acids - 3
1 2 3 4
5 6 7 8
9 10 11 12
L-phenylalanine (Phe / F)
3,4-dihydroxy L-phenylalanine
L-isoleucine (Ile / I)
L-leucine (Leu / L) L-tryptophan (Trp / W)
L-tyrosine (Tyr / Y)
L-proline (Pro / P)L-histidine (His / H) L-methionine (Met / M)
L-arginine (Arg / R)L-ornithine
L-lysine (Lys / K)
1
23
4
56
8 910
11 12
7
min0 10 20 30 40 50 60
mAU
0
20
40
60
80
100
120
140
C C
H
OHOH2N
CH2CH2
CH3
H3C C C
H
OHOH2N
CHC C
H
OHOH2N
H2N CH2 CH2CH2 CH2
C C
H
OHOH2N
H2N CH2 CH2CH2
C C
H
OHOH2N
CH2HO
N
NH
C C
H
OHOH2N
CH2NH H
C OHO
C C
H
OHOH2N
CH2HO
HOC C
H
OHOH2N
S CH2H3C
C C
H
OHOH2N
HN CH2 CH2CH2CHN
H2NC C
H
OHOH2N
CH CH2H3C
H3CN
C C
H
OHOH2N
CH2
No.TI097E
TI097E-BL02
Cadenza CD-C18, 250 x 1 mmA: 0.05% TFA in waterB: 0.05% TFA in acetonitrile5 - 45 %B ( 0 - 80 min )0.05 mL/min, 12 MPa, 37 °C, 210 nm5 uL
-CaseinPeptides, Tryptic Digest of
min0 20 40 60 80
mAU
0
200
400
No.TI098E
TI098E-BL02
Cadenza CD-C18, 50 x 2 mmA: 0.05% TFA in waterB: 0.05% TFA in acetonitrile5 - 35 %B ( 0 - 20 min )0.5 mL/min, 10 MPa, 37 °C, 210 nm5 uL
-CaseinPeptides, Tryptic Digest of
min0 5 10 15 20
mAU
0
50
100
150
200
No.TI100E
TI100E-BL02
Unison UK-C18, 50 x 2 mmA: 0.05% TFA in waterB: 0.05% TFA in acetonitrile5 - 35 %B ( 0 - 20 min )0.5 mL/min, 9 MPa, 37 °C, 210 nm5 uL
-CaseinPeptides, Tryptic Digest of
min0 5 10 15 20
mAU
0
50
100
150
200
No.TI101E
TI101E-BL02
Unison UK-C18, 250 x 2 mmA: 0.05% TFA in waterB: 0.05% TFA in acetonitrile5 - 45 %B ( 0 - 90 min )0.2 mL/min, 14 MPa, 37 °C, 210 nm5 uL
-CaseinPeptides, Tryptic Digest of
min0 20 40 60 80
mAU
0
50
100
150
No.TI153E
TI153E-DD01
A: water / TFA, B:acetonitrile / TFA0.5 mL/min, room temp., 220 nm
-CaseinPeptides, Tryptic Digest of
17.6 MPa
0 50 100 150min0 20 40 60 80min
116 peaks80 peaks
9.8 MPa
10-45%B ( 0-60min), 10µL 10-45%B ( 0-120min), 20µL
3 µmConventional ODS 5 µm500 x 4.6 mm250 x 4.6 mm
No.TI170E
TI170E-DF09
L-Carnitine
1
1
Unison UK-Silica, 150 x 3 mmacetonitrile / 50mM ammonium acetate = 65 / 35, 0.5 mL/min (7MPa)37 °C, 210 nm, 2µL(20µg)
Unison UK-C18, 75 x 4.6 mmwater / heptafluoro-n-butyric acid = 100 / 0.1, 1 mL/min (7MPa)37 °C, 210 nm, 2µL(20µg)
L-carnitine
C C
H
OH
O
O-CH2 CH2N+H3C
CH3
CH3
Unison UK-Silica
0 2 4 6 8 10min
0
10
20
mAU
Unison UK-C18
0
10
20
mAU
0 2 4 6 8 10min
No.TI188E
TI188E-DH20
Cadenza CL-C18, 250 x 3 mmwater / trifluoroacetic acid = 100 / 0.10.5 mL/min (15MPa), 37 deg.C, 210 nm
Theanine in Green Tea
0 5 10 15 min
0
50
100
mAU
L-theanine, 2ug (2uL)
green tea, 20 µL(theanine enriched)
C C
H
OHOH2N
CH2 CH2CH3 CH2 CO
NH
L-theanine
No.TI191E
TI191E-DI01
Unison UK-Silica, 250 x 3 mmacetonitrile / 50mM ammonium formate = 70 / 300.5 mL/min (10MPa), 37 deg.C, 210 nm, 2uL(20ug)
Betaine
beta
ine
CO
O-CH2N+H3C
CH3
CH3
0 5 10 min
0
50
mAU
No.TI282E
TI282E-FK08
Courtesy of Dr. Takeshi Fukushima, Univ. of Shizuoka
Ref.) M.Tomiya, T.Fukushima, J.Kawai, C.Aoyama, S.Mitsuhashi, T.Santa, K.Imai and T.Toyo'okaUniv. of Shizuoka, Univ. of Tokyo and Musashino Univ.
Biomedical Chromatography, 20, 628-633 (2006)
Cadenza CD-C18, 250 x 4.6 mmA) water /acetonitrile /2-propanol /TFA = 90 /10 /0.8 /0.08B) water /acetonitrile /2-propanol /TFA = 90 /10 /5 /0.08C) water /acetonitrile = 90 /10D) water /acetonitrile /TFA = 10 /90 /0.08100%A, 0%BCD (0-25min), 100-0%A, 0-100%B (25-32min), 100%B (32-35min),100-97%B, 0-3%D (35-39min), 97-0%B, 0-90%C, 3-10%D (39-45min),90-65%C, 10-35%D (45-60min), 0-15%B, 65-55%C, 35-30%D (60-63min),15%B, 55%C, 30%D (63-68min), 15%B, 55-50%C, 30-35%D (68-70min), 15%B, 50%C, 35%D (70-82min), 15-0%B, 50-40%C, 35-60%D (82-85min),40%C, 60%D (85-100min), 100%A, 0%BCD (100-115min)0.7 mL/min, 40 deg.CEx.470nm, Em.540nm
NBD-amino acids from rat plasma
A representative chromatogram of rat plasma amino acids derivatized with NBD-F. 1:NBD-Tau, 2:NBD-His, 3:NBD-Asn, 4:NBD-Gln, 5:NBD-Ser, 6:NBD-Arg, 7:NBD-Cit, 8:NBD-Asp, 9:NBD-Gly, 10:NBD-Glu, 11:NBD-Thr, 12:NBD-Ala, 13:NBD-Pro, 14:NBD-ε-amino-n-caproic acid (IS.), 15:NBD-Met, 16:NBD-Val, 17:NBD-Orn, 18:NBD-Lys, 19:NBD-Ile, 20:NBD-Leu, 21:NBD-Phe, 22:NBD-Tyr.
No.TI291E
TI291E-GA24
250 x 3 mmA: water /trifluoroacetic acid = 100 /0.1, B: acetonitrile /trifluoroacetic acid =100 /0.050.4 mL/min, 37 deg.C, 220 nm
Cadenza CD-C18 5-30%B (0-115min) 70%B(115-120min)158 peaks
Intrada WP-RP 0-30%B (0-115min) 70%B(115-120min)80 peaks
Peptides, Tryptic Digest of Hemoglobin (Human)
mA
U
0 50 100 min
0
40
No.TI311E
TI311E-GD13
Branched-chain Amino Acids
Unison UK-Amino, 150 x 2 mmacetonitrile / 10mM ammonium acetate = 85 / 15 0.2 mL/min (4MPa), 37 deg.C, ELSD0.5 uL (1.5ug)
0 5 10 min
0
100
200mV 1
2
3
3
L-valine
C C
H
OHOH2N
CH2H3C
H3C
1
L-leucine
C C
H
OHOH2N
CH CH2H3C
H3C
2
L-isoleucine
CH2
CH3
H3C C C
H
OHOH2N
CH
No.TI329E
TI329E-GF14
Unison UK-Amino, 250 x 4.6 mmacetonitrile / 100mM ammonium formate = 76 / 241 mL/min (10MPa), 37 deg.C, ELSD, 3uL (1ug)
Basic Amino Acids
0 5 10 15 20 min
0
20
40
60mV
L-arginine
C C
H
OHOH2N
HN (CH2)3CHN
H2N
L-lysine
C C
H
OHOH2N
H2N (CH2)4
L-histidine
N
NH
C C
H
OHOH2N
CH2
L-proline
NH H
C OHO
1
2
3
4
12
3 4
unkn
own
No.TI371E
TI371E-GJ31
HPLC assay of methylated arginines using NBD-F as a fluorescent reagent
Courtesy of Dr. TSUNODA, University of Tokyo, JAPAN
Journal of Chromatography A, 1066 (2005) 41-45
(A) Standard mixture
Unison UK-C18, 150 x 4.6 mmA: 50mM sodium phosphate buffer (pH 3.2) / acetonitrile = 91 / 9 (v/v)B: acetonitrile0%B (0-18 min), 0-30%B (18-28 min), 30%B (28-32 min)0.75 mL/min, 40 deg.C, Ex.470 nm / Em. 530 nm
Pre-column derivatization: 105 ul of 100mM borate buffer (pH 9.0) and 30 uL of 40mM NBD-F in acetonitrile were added to the 30 uL of sample. Then, the reaction was incubated at 40 deg.C for 3 min. To stop the reaction, 435 uL of 0.5% acetic acid (v/v) was added.A10 uL aliquot of sample was injected onto the HPLC.
min min
12
3
4
1
2 3 4
10 uM
10 uM 10 uM 5 uM
HO
O
HN
NH
HN CH3
NH2
1: NG-monomethyl-L-arginine (L-NMMA)
2: NG,NG-dimethyl-L-arginine (ADMA)
HO
O
HN
NH
NNH2 CH3
CH3
3: NG,NG'-dimethyl-L-arginine (SDMA)
HO
O
HN
NH
HN C3H7
NH2
4: N -propyl-L-arginine (N-PLA)
(B) Rat plasma sample (200 uL)
HO
O
HN
NH
N CH3
NH2
CH3
No.TI374E
TI374E-GK02
Angiotensins
1. Angiotensin IV (Human)
2. Angiotensin III (Human)
3. Angiotensin I (Human)
4. [Val5]-Angiotensin I (Bovine) 5. Angiotensin II (Human)
Unison UK-Amino150 x 3 mm,A: acetonitrileB: 50mM ammonium acetate10-35%B(0-15min)0.4 mL/min 37 deg.C220 nm, 2 uL (0.4ug)
Unison UK-C18150 x 3 mmA: water/ TFA = 100/ 0.1B: acetonitrile/ TFA = 100/ 0.120-30%B(0-15min)0.4 mL/min, 37 deg.C220 nm, 2 uL (0.4ug)
normal phase
reversed phase
1
2,5
34
mA
U
0 5 10 15 min
0
50
1 2
34
5
mA
U
0 5 10 15 min
0
50
No.TI377E
TI377E-GK26
Betaine, Carnitine, GABA
1 2 3
1
2
3
0 2 4 min
0
50
mV
Unison UK-Amino, 50 x 3 mmA: acetonitrileB: 10mM ammonium acetate15-30%B(0-5min)0.5 mL/min, 37 deg.C, ELSD, 1 uL (1ug)
betaine
CO
O-CH2N+H3C
CH3
CH3
H2NO
OH
GABAL-carnitine
C C
H
OH
O
O-CH2 CH2N+H3C
CH2
CH2
No.TI387E
TI387E-HB01
Calcitonin in Gelatin Aqueous Solution
Cadenza HS-C18, 30 x 3mmA: water /trifluoroacetic acid = 100 /0.1B: acetonitrile /trifluoroacetic acid = 100 /0.070-45%B (0-2min), 0.4mL/min (3MPa), 37deg.C, 220nm2uL (calcitonin: 1ug, gelatin: 10ug)
gelatin
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-
Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-
Gly-Ser-Gly-Thr-Pro-NH2 .CH3COOH
calcitonin acetate, salmon (MW 3492)
gelatin in water
calcitonin in water
calcitonin in gelatin aqueous solution
0 1 2 3 4 min0
50
100
mA
U
calcitonin
No.TI388E
TI388E-HB05
Calcitonin, Salmon
Unison UK-Amino, 100 x 3 mmA: acetonitrileB: 50mM ammonium acetate 5-100%B(0-5min)0.4mL/min (2MPa), 37deg.C, ELSD1.5uL (1.5ug)
100 x 3 mmA: water /TFA = 100 /0.1B: acetonitrile /TFA = 100 /0.0730-50%B(0-5min)0.4mL/min (4MPa), 37deg.C, 220nm1uL (1ug)
reversed phase
normal phase
mA
U
min0 2 4 6
0
100
150
50
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-
Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-
Gly-Ser-Gly-Thr-Pro-NH2 .CH3COOH
calcitonin acetate, salmon (MW 3492)
min0 2 4 6
0
mV
50
Cadenza CD-C18
Cadenza CL-C18
Cadenza CW-C18
Unison UK-C18
No.TI393E
TI393E-HB14
Glutathiones
1 2
12
mA
U
0 2 4 6 8 min
0
50
UK-Amino, 100 x 4.6 mmA: acetonitrile, B: 50mM ammonium acetate80-100 %B (0-8min), 1 mL/min37 deg.C, 220 nm, 5 uL(5ug)
glutathione reduced (GSH) glutathione oxidized (GSSG)
HN
O
O
NHO
HO OH
O
NH2H
HHS H
NO
ONH
O
HO OH
O
NH2H
HS
HN
O
O
NHO
HO OH
O
NH2H
HS
No.TI419E
TI419E-HG01
Microcystins
150 x 3 mm,A: water / TFA = 100 / 0.05B: acetonitrile / TFA = 100 / 0.0535-60 %B (0-10min)0.4 mL/min, 37 deg.C220 nm, 5 uL (5 ppm)
1
2
3
1
2 3
1
2 3
1
23
1
23
1
23
mA
U
0 5 10 min
0
20
40
CH2
NH
NH
O
NH
NH
HN
HN N
O
HN NH2
NHO OH
O
O
O
O OH
O
OCH3
O1
2
=
=
NH
H2N
NH
(CH2)3
HO
CH2
microcystin RR
microcystin YR
3=
H3C
H3C(CH2)2
microcystin LR
No.TI420E
TI420E-HG01
Unison UK-Amino, 150 x 3 mmA: acetonitrile, B: 50mM ammonium acetate, 20-30 %B (0-10min)0.4 mL/min (4MPa), 37 deg.C, 220 nm, 5 uL (50 ppm)
Microcystins
1
CH2
NH
NH
O
NH
NH
HN
HN N
O
HN NH2
NHO OH
O
O
O
O OH
O
OCH3
O
3
2
1
2
3mA
U
0 5 10 min
0
50
=
=
=
NH
H2N
NH
(CH2)3
HO
CH2
H3C
H3C(CH2)2
microcystin RR
microcystin YR
microcystin LR
No.TI423E
TI423E-HG08
NDA-amino acids
Cadenza CD-C18, 150 x 2 mmA: 50mM phosphate buffer (pH 5.8) /acetonitrile /methanol = 15 /2 /3B: 50mM phosphate buffer (pH 5.8) /acetonitrile /THF = 73 /18 /90-100%B (0-30min), 0.3 mL/min, 37 deg.C, FLS (Ex.420nm, Em.480nm)standard amino acids (10 pmol) derivatized with NDA
-100
0
100
200
300
400
500
600
700
0 5 10 15 20 25 30
Time (min)
Re
lative
Flu
ore
sce
nce
(mV
) Asp Glu
Asn His
Gln
SerArg
Gly
Thr
AlaTau
Tyr
GABA
CHO
CHO
NDA (naphthalene-2,3-dicarboxaldehyde)
No.TI425E
TI425E-HH06
Microcystins
CH2
NH
NH
O
NH
NH
HN
HN N
O
HN NH2
NHO OH
O
O
O
O OH
O
OCH3
O1
2
=
=
NH
H2N
NH
(CH2)3
HO
CH2
microcystin RR
microcystin YR
3=
H3C
H3C(CH2)2
microcystin LR
Unison US-C18, 50 x 2 mmA: water / TFA = 100 / 0.05B: ACN / TFA = 100 / 0.0530-40 %B (0-13min)0.4 mL/min37 deg.C, 240 nm
12
3
1
1
1
2
2
2
3
3
3
mA
U
0 1 2 3 4 min
0
5
10
20 ppb x 100 uL
40 ppb x 50 uL
100 ppb x 20 uL
1 ppm x 2 uL
No.TI469E
TI469E-HL26
Unison UK-Amino, 250 x 2 mmA: acetonitrile B: 10mM ammonium acetate10-30 %B (0-20min)0.16 mL/min (5MPa)37 deg.C, ELSD, 1 uL (0.5 ug)
Aliphatic Amino Acids
1
2
3
4
5
6
1
2
3
4
56
mA
U
0 10 20 min
0
20
40
glycine (Gly / G)
C OHO
H2N CH2
L-alanine (Ala / A)
L-valine (Val / V)
C C
H
OHOH2N
H3C
C C
H
OHOH2N
CH2H3C
H3C
L-isoleucine (Ile / I)
L-leucine (Leu / L)
L-proline (Pro / P)
CH2
CH3
H3C C C
H
OHOH2N
CH
NH H
C OHO
C C
H
OHOH2N
CH CH2H3C
H3C
No.TI470E
TI470E-HL26
Unison UK-Amino, 250 x 2 mmA: acetonitrile B: water /acetic acid = 100 /120-25 %B (0-20min)0.2 mL/min (8MPa)37 deg.C, ELSD, 0.4 uL (0.4 ug)
Hydrophilic Amino Acids
1
2
3
4
51
2
3
4
5
0 10 20 min
0
10
20
mA
U
glycine (Gly / G)
L-asparagine (Asn / N)
L-serine (Ser / S)
L-glutamine (Gln / Q)
C OHO
H2N CH2
C C
H
OHOH2N
H2N CH2CO
C C
H
OHOH2N
OH
CH2
C C
H
OHOH2N
CH2 CH2CO
H2N
L-threonine (Thr / T)
C C
H
OHOH2N
OH
CHH3C
No.TI471E
TI471E-HL26
Unison UK-Amino, 250 x 2 mmA: acetonitrile B: water /acetic acid = 100 /170-90 %B (0-20min)0.2 mL/min (16MPa)37 deg.C, ELSD, 2 uL (2ug)
Unison UK-Amino, 50 x 2 mmA: acetonitrile B: water /acetic acid = 100 /170-90 %B (0-5min)0.2 mL/min (3MPa)37 deg.C, ELSD, 1 uL (1ug)
Acidic Amino Acids
1
2
1
2
mA
U
0 5 10 15 20 min0
50
1
2
mA
U
0 5 min
0
100
L-aspartic acid (Asp / D)
C C
H
OHOH2N
CH2HO CO
L-glutamic acid (Glu / E)
C C
H
OHOH2N
CH2 CH2CO
HO
No.TI472E
TI472E-HL26
Unison UK-Amino, 250 x 2 mmA: acetonitrile B: 50mM ammonium acetate50-90 %B (0-20min)0.2 mL/min (7MPa)37 deg.C, ELSD, 5 uL (2-5ug)
Sulfur-containing Amino Acids
1
2
3
1
2
3
mA
U
0 10 20 min
0
50Cl-
L-cysteine (Cys / C)
L-cystine
C C
H
OHOH2N
HS CH2
C C
H
OHOH2N
S CH2
C CH
OHOH2N
S CH2
L-methionine (Met / M)C C
H
OHOH2N
S CH2H3C
No.TI473E
TI473E-HL26
Unison UK-Amino, 250 x 2 mmA: acetonitrile B: 10mM ammonium acetate20-50 %B (0-20min)0.2 mL/min (6MPa)37 deg.C, ELSD, 2 uL (1 ug)
Aromatic Amino Acids
1
2
3
1
2
3
mA
U
0 10 20 min
0
20
L-phenylalanine (Phe / F)
C C
H
OHOH2N
CH2
L-tryptophan (Trp / W)
L-tyrosine (Tyr / Y)
C C
H
OHOH2N
CH2HO
N
C C
H
OHOH2N
CH2
No.TI474E
TI474E-IA06
Normal Phase Separation for Amino Acids
0 10 20 30 min
0
20
40
mV
Phe
IleLe
u Trp
Met
Val
Pro
Tyr
Thr
Ala
Arg
, Ser
, Lys
His
Gly
Asn
Gln
Cys
Glu
Asp
Unison UK-Amino, 100 x 3 mmA: ACN*1 /THF*2 /50mM TFAA*3
= 46 /46 /8B: MeOH*4 /50mM HCOONH4 = 20 /80�0-5 %B(0-15min)8 %B(15-25min)8-60 %B(25-35min)0.4 mL/min, 37 deg.C, ELSD2 uL(1-4ug)
Unison UK-Amino, 100 x 3 mmA: ACN*1 /THF*2 /50mM TFAA*3
= 46 /46 /8B: MeOH*4 /50mM HCOONH4 /50mM TFAA = 20 /20 /60�0-5 %B(0-20min)5 %B(20-25min)30-60 %B(25-40min)0.5 mL/min, 37 deg.C, ELSD2 uL(1-2ug)
0 10 20 30 40 min
0
50
mV
Phe Ile
Leu
Trp
Met
Val
Pro
Tyr
Thr
Ala
Gly
, Ser
, His
, Gln
Asn
Cys
Glu
Asp
Arg
Lys
*1 : acetonitrile*2 : tetrahydrofuran*3 : ammonium trifluoroacetate*4 : methanol
*1 : acetonitrile*2 : tetrahydrofuran*3 : ammonium trifluoroacetate*4 : methanol
No.TI521E
TI521E-IF17
50 x 3 mmA: 5mM ammonium acetateB: 100mM ammonium acetate / ACN = 50 / 500-100 %B (0-5min), 0 %B (5-10min)0.4 mL/min (3MPa), 37 deg.C, ELSD2 uL (2ug)
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Glutathiones
1
glutathione reduced (GSH)
HN
O
O
NHO
HO OH
O
NH2H
HHS
2
glutathione oxidized (GSSG)
HN
O
ONH
O
HO OH
O
NH2H
HS
HN
O
O
NHO
HO OH
O
NH2H
HS
1
2
0 2 4 6 min
0
50
100
mV
ON
HN
N
N
OH
OK
PO
HO
KO OCH2
O
Inosine 5'-monophosphate dipotassium
N
NH
H2NO
OHHN
O
L- carnosine(beta-alanyl-L-histidine)
CH3
N
N
H2NO
OHHN
O
.HNO3
L- anserine nitrate(beta-alanyl-1-methyl-L -histidine nitrate)
150 x 3 mmA: 10mM ammonium acetateB: 50mM ammonium acetate / acetonitrile = 80 / 200-100%B (0-10min), 0.4 mL/min (9MPa), 37deg.CELSD (spray chamber 50 deg.C, drift tube 100 deg.C)5 uL (2-6ug)
No.TI524E
TI524E-IF22
Anserine and related compounds
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10
0
100
200
min
1
2 3K+
K+
1,3
2
1
2
3
mV
No.TI526E
TI526E-IG22
Amino acids: neutral pH condition
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10 15 min
0
200
400
mV
Trp
Met
His
Gln
Phe
Ile Leu
Val
Tyr
Arg
Lys
Gly
, Ala
, Pro
, Thr
, Ser
, Asn
, Asp
, Glu
, Cys
Scherzo SM-C18, 150 x 3 mmA: 5mM ammonium acetate B: 125mM ammonium acetate / acetonitrile = 20 / 800.4 mL/min (8MPa), 37 deg.CELSD (spray chamber 50 deg.C, drift tube 100 deg.C)2-5 uL (2.5-4ug)
basic amino acids0-10 %B (0-15min)
hydrophilic amino acids0-10 %B (0-15min)
aliphatic amino acids0-20 %B (0-15min)
branched-chain amino acids (BCAA)0-10 %B (0-15min)
0-10 %B (0-15min)
No.TI527E
TI527E-IG22
Amino acids: acidic condition
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Glu
Asp
Scherzo SM-C18, 150 x 3 mmA: water / acetic acid = 100 / 0.1B: acetonitrile / acetic acid = 100 / 10.4 mL/min (8MPa), 37 deg.C,ELSD (spray chamber 50 deg.C, drift tube 100 deg.C)2-5 uL (1-5ug)
Trp
Met
Gln
Phe
Ile Leu
Val
Tyr
Gly
, Ala
, Pro
, Thr
, Ser
, Asn
, His
, Lys
, Arg
, Cys
acidic amino acids0-30 %B (0-10min)
hydrophilic amino acids0-10 %B (0-10min)
aliphatic amino acids0-30 %B (0-10min)
branched-chain amino acids (BCAA)0-10 %B (0-10min)
0-10 %B (0-10min)
0 5 10 min
0
200
400
mV
No.TI528E
TI528E-JB15
Amino acids: normal phase
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Scherzo SM-C18150 x 3 mmA: acetonitrile / acetic acid = 100 / 0.5B: water / acetic acid = 100 / 0.52-50 %B(0-15min)0.4 mL/min (3MPa)37 deg.C,ELSD (spray chamber 50 deg.C, drift tube 100 deg.C)
mV
0 5 10 15 min0
500
Cys
ProGlnTrp
Ala
Ser
ArgAspGlu
Thr
HisAsn
Met
Phe
TyrValIle
Leu
Lys
Gly
No.TI597E
TI597E-JC30
RP analysis of intact amino acids using Corona CAD
Courtesy of Kyoko Fukushima, LMS CO., LTD. / JAPANImtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])
North America Other Countries
Cadenza CD-C18, 250 x 4.6 mmA: 0.15% TFAB: 0.15% TFA in acetonitrile0%B (0-5min), 0-40%B (5-20min), 40%B (20-23min)40-0%B (23-23.1min), 0%B (23.1-35min) 0.6 mL/min, 25 °CCorona CAD ( GAS=35psi, Filter: None )10 µL
5 10 15 20 min0
each 100 µg/mL
each 1 µg/mL
Tau G
ly Ser
Ala Ly
sA
spG
lu
Arg
Pro
Car
Val M
et IleLe
u Phe Trp
0 50 100 150 min
0
50
100
mA
UNo.TI605E
TI605E-JD14
Peptide mapping
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
250 x 3 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.150 deg.C, 220 nm, 5 uL
1-50 %B (0-150min)0.2 mL/min (25 MPa)
5-37 %B (0-150min)0.4 mL/min (13 MPa)
320 peaks
198 peaks
Tryptic digest of alpha-casein
No.TI646E
TI646E-KC14Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
250 x 4.6 mm�A: water / formic acid = 100 / 0.5B: acetonitrile / formic acid = 100 / 0.52-50 %B (0-60min)50 deg.C, ELSD(spray chamber 35 deg.C, drift tube 100 deg.C)tryptic digest of alpha-casein 20 uL
Peptide mapping (50°C, formic acid)
0 20 40 60 min0
5
10
15
mV
Presto FF-C18
Cadenza CD-C18
0.4 mL/min(22MPa)
1 mL/min(12MPa)
No.TI653E
TI653E-KD05Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Presto FF-C18, 250 x 4.6 mm�0.4 mL/min (22MPa), 50 deg.CELSD(spray chamber 35 deg.C, drift tube 100 deg.C)tryptic digest of alpha-casein 20 uL
The effect of TFA modifier on peptide mapping
A: water /formic acid = 100 /0.5B: acetonitrile /formic acid = 100 /0.52-50 %B (0-60min)
0 20 40 60 min
0
10
20
mV
A: water /formic acid /TFA = 100 /0.5 /0.02B: acetonitrile /formic acid /TFA = 100 /0.5 /0.022-50 %B (0-60min)
No.TI660E
TI660E-KD20
Collagenpeptide
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Presto FF-C18, 250 x 4.6 mmA: water / formic acid / TFA = 100 / 0.5 / 0.02B: acetonitrile / formic acid / TFA = 100 / 0.5 / 0.021-50 %B (0-90min)0.4 mL/min (21MPa), 50 deg.CELSD (spray chamber 35deg.C, drift tube 100deg.C)20 uL (20ug, water)
0 50 100 min
0
50
mV
collagenpeptide, acid hydrolyzed
collagenpeptide, enzyme hydrolyzed
N
NH
OH
O
NH2
N
NH
NH2
CH3
N
N
NH2
N
NH
O
OH
CH3
N
NO
OH
L-histidine
histamine
4-imidazoleacetic acid
N-methyl-4-imidazoleacetic acid
N-methylhistamine
No.TI682E
TI682E-KH09
Histamine related compounds
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Scherzo SS-C18, 150 x 3 mmA: methanolB: 100 mM ammnonium formate : 100 mM ammonium hydroxide = 95 : 5 (pH8.1)0-20 %B (0-20 min)0.4 mL/min (6 MPa), 60 deg.C, 230 nm, 1.6 uL ( 0.6-2.8 ug, MeOH)
0 10 20
0
50
100
mA
U
min
1
2
3
4
5
1
2
3
4
5
NH
HO
NH2
NH
OH
O
NH2
NH
OH
O
NH2
HO
NH
HO
OH
O
serotonin
L-tryptophan
5-hydroxy-L-tryptophan
5-hydroxyindole-3-acetic acid (5-HIAA)
No.TI683E
TI683E-KH10
Serotonin related compounds
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
150 x 3 mmA: water / formic acid = 100 / 0.1B: 100 mM ammnonium formate / acetonitrile = 70/ 300.4 mL/min, 37 deg.C, 260 nm, 1.2 uL ( 0.4 ug, 0.1% formic acid)
0 5 10 15
0
100
200
300
mA
U
min
15-50 %B (0-15 min)9 MPa
10-55 %B (0-15 min)9 MPa
25-90 %B (0-15 min)12 MPa
1
2
3
4
1
2 34
1
2
3
4
1
2
3
4
Presto FF-C18, Cadenza CD-C18, 150 x 3 mmA: water / formic acid / TFA = 100 / 0.5 / 0.02B: acetonitrile / formic acid /TFA = 100 / 0.5 / 0.0250 deg.C, 2uL (tryptic digest of alpha-casein)
FTMS (Thermo Scientific Exactive), ESI-positive (Full ms)
No.TI685E
Peptide mapping using LC-FTMS
0-40%B (0-60 min)0.2 mL/min
0-60%B (0-60 min)0.7 mL/min
TI685E-KH12
Courtesy of Dr. KUWATA Keiko, Kyoto University, JAPANImtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])
North America Other Countries
RT: 4.9 - 50.2
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
748.37634.36
523.22
598.34 412.72880.48
491.29 940.48457.21 1199.61669.34
1053.49905.48
796.42 486.25416.75 742.45755.87667.34415.73416.20 575.32 650.36 787.94801.73821.39473.77
1052.54692.87473.76 416.20
71081.02523.22 416.20
748.37
634.35
880.48416.19
1053.48598.34
412.72669.341199.61455.74 491.29
457.21 905.47494.31415.73
486.25742.45495.75 755.86623.82 473.77 787.94
692.87643.31 480.291081.02 684.20 684.20 684.20 684.20
Rel
ativ
e A
bund
ance
No.TI262E
TI262E-FA23
Interferon
mA
U
0 0.5 1.0 min
0
40
Intrada WP-RP, 30 x 2mmA: water / TFA = 100 / 0.1B: acetonitrile / TFA = 100 / 0.0720-45%B (0-0.5min), 45%B (0.5-1min)0.8 mL/min(10 MPa), 37deg.C, 280nm15uL (180ng)
Intrada WP-RP, 250 x 2mmA: water / TFA = 100 / 0.1B: acetonitrile / TFA = 100 / 0.0715-50%B (0-9min), 50%B (9-12min)0.2 mL/min(13 MPa), 37deg.C, 280nm10uL (120ng)
mA
U
0 5 10 15min
0
20
unkn
own
unkn
own interferon alfa-2b
(genetical recombination, 19,269 Da)
interferon alfa-2b(genetical recombination, 19,269 Da)
No.TI263E
TI263E-FA23
Interferon
Intrada WP-RP, 250 x 4.6 mmA: water / TFA = 100 / 0.1B: acetonitrile / TFA = 100 / 0.0715-50%B (0-9min), 50%B (9-12min)1 mL/min (14 MPa),37deg.C, 220nm8uL (72ng)
mA
U
0 5 10 15min
0
40
interferon alfa-2b(genetical recombination, 19,269 Da)
unkn
own
No.TI270E
TI270E-FG04
Intrada WP- RP, 250 x 4.6 mmA: water /TFA = 100 /0.1B: acetonitrile /TFA = 100 /0.0730-50%B (0-15min)1.0 mL/min (16 MPa)37 deg.C, 280 nm, 8 uL (42.4 ug)
Protein, beta-Glucosidasem
AU
0 5 10 15 20 min
0
20
40
bata-glucosidase(almond, 183 kDa)
No.TI271E
TI271E-FG04
Intrada WP- RP, 250 x 4.6 mmA: water /TFA = 100 /0.1B: acetonitrile /TFA = 100 /0.0710-95%B (0-15min)1.0 mL/min (16 MPa)37 deg.C, 280 nm, 8 uL (40 ug)
Protein, Thyroglobulinm
AU
thyroglobulin(porcin thyroid, 670 kDa)
0 5 10 15 20 min
0
20
40
No.TI272E
TI272E-FG04
Intrada WP- RP, 250 x 4.6 mmA: water /TFA = 100 /0.1B: acetonitrile /TFA = 100 /0.0720-65%B (0-15min)1.0 mL/min (16 MPa)37 deg.C, 280 nm, 8 uL (42.4 ug)
Protein, Hemoglobinm
AU
hemoglobin(bovin blood, 64 kDa)
0 5 10 15 20 min
0
20
40
No.TI273E
TI273E-FG04
Intrada WP- RP, 250 x 4.6 mmA: water /TFA = 100 /0.1B: acetonitrile /TFA = 100 /0.0720-50%B (0-15min)1.0 mL/min (16 MPa)37 deg.C, 280 nm, 3 uL (15.3ug)
Protein, Lactferrin
lactferrin(bovin milk, 93 kDa)
mA
U
0 5 10 15 min
0
20
40
No.TI274E
TI274E-FG04
Intrada WP- RP, 250 x 4.6 mmA: water /TFA = 100 /0.1B: acetonitrile /TFA = 100 /0.0725-50%B (0-40min)1.0 mL/min (16 MPa)37 deg.C, 220 nm, 0.4 uL
Bovin Serum
mA
U
0 10 20 30 40min
0
100
10 20 30
No.TI293E
TI293E-GB01
Bovine Milk
Intrada WP-RP, 250 x 3 mmA: water /trifluoroacetic acid = 100 /0.1, B: acetonitrile /trifluoroacetic acid =100 /0.050-100%B (0-25min), 100%B(25-30min)0.4 mL/min (12MPa), 37 deg.C, 5uL
mA
U
0 10 20 30 min
0
500
220 nm
280 nm
No.TI430E
TI430E-HI27
Courtesy of Dr.Thomas Dillon, AMGEN Inc., USA
Ref.) T. M. Dillon ., P.V. Bondarenko, D.S. Rehder, G.D. Pipes, G. R. Kleemann, M.S. RicciJournal of Chromatography A, 1120 (2006) 112-120
Intrada WP-RP, 150 x 2 mmA: 0.1% TFA, B: IPA /ACN /water /TFA = 70 /20 /9.9 /0.110%B (0-2 min), 25%B (2-4 min), 25-32%B (4-25 min)0.5 mL/min, 75 deg.C, 280 nm (upper) or 215nm (lower)
Intact IgG2 mAb
IgG2 mAb by reversed-phase analysis
Reduced IgG2 mAb
280 nm
215 nm
No.TI440E
TI440E-HJ15
Intrada WP-RP, 250 x 4.6 mmA: 0.15% TFA in (acetonitrile /isopropanol /water = 9 /1 /90)B: 0.15% TFA in (acetonitrile /isopropanol /water = 69 /1 /30)C: 0.20% TFA in (acetonitrile /isopropanol /water = 9 /1 /90)5%B,1%C(0-5min), 5-30%B,1-35%C (5-30min), 30%B,35%C(30-65min)30-35%B,35%C(65-70min), 35-38%B,35%C(70-130min)38-44%B,35-55%C(130-250min), 44%B,55%C(250-300min)44-47%B,55-53%B(300-330min), 47-60%B,53-40%C(330-480min)60-70%B,40-30%C(480-520min), 70-90%B,30-10%C(520-570min)0.55 mL/min, 60 deg.C, FLD( Ex.395nm, Em.505nm)
A proteomics study on human breast cancer cell lines by fluorogenic derivatization-liquid chromatography/tandem
mass spectrometry
Courtesy of Kazuhiro Imai, Ph.D. Dhc., Professor & Dean, Musashino University, Japan
Ref-1: Biomedical Chromatography, 2008, 22 (11), 1304-1314Ref-2: Journal of Proteome Research, 2007, 6, 2841-2849
DAABD-proteins
1
2
34
56
7 8
9
10
11
1213
Breast cancer cell _ 1
Breast cancer cell _ 2
Breast cancer cell _ 3
Breast cancer cell _ 4
Breast cancer cell _ 5
Breast cancer cell _ 6
Breast cancer cell _ 7
Normal mammary epitherial cell
No.TI289E
TI289E-GA16
Proteins
Intrada WP-RP, 150 x 4.6 mm, 1 mL/min, 37 degC, 280 nmA: water /TFA=100 /0.1, B: ACN /TFA=100 /0.1, 20-70%B (0-15min)
0 5 10 15 min
angiotensin II (1046 Da)
insulin (bovine pacreas, 5733 Da)
cytochrome c (horse heart, 12.4 kDa, pI=9.1)
alpha-lactalbumin (bovine milk, 14.2 kDa, pI=4.5)
lysozyme (chicken egg white, 14 kDa, pI=11.0)
papain (papaya latex, 21 kDa, pI=8.75-9.55)
alpha-chymotryosinogen A (bovine pancreas, 25.7 kDa, pI=8.97)
deoxyribonuclease I (bovine pancreas, 31 kDa, pI=4.78-5.2)
beta-glucosidase (almond, 183 kDa)
albumin (bovine, 66.3 kDa, pI=4.9)
albumin (human serum, 66.4 kDa, pI=4.7)
albumin (eggs, 45 kDa, pI=4.54-4.9)
hemoglobin (Human, 64.5 kDa, pI=6.8)
hemoglobin (Bovine, 66-68 kDa, pI=6.8)
xanthine oxidase (buttermilk, 360 kDa)
thyroglobulin (porcin thyroid, 670 kDa, pI=4.58)
lactoferrin (bovine milk, 80 kDa)
No.TI560E
TI560E-JA08
Presto FF-C18, 50 x 4.6 mmA: water / formic acid = 100 / 0.1B: acetonitrile / formic acid = 100 / 0.10-30%B (0-10min)0.4 mL/min (6MPa), 37 deg.CELSD (spray chamber 50 deg.C,drift tube 100 deg.C)2 uL (10ug, water)
Presto FF-C18, 150 x 4.6 mmA: water / formic acid = 100 / 0.1B: acetonitrile / formic acid = 100 / 0.10-30%B (0-15min)0.4 mL/min (16MPa), 37 deg.CELSD (spray chamber 50 deg.C,drift tube 100 deg.C)2 uL (10ug, water)
Polyglutamic acid
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10 min
0
400
200
mV MW: 1,500,000 - 2,500,000
MW: 4,000,000 - 6,000,000
Na+
Na+
0 5 10 15 min
0
200
mV
MW: 4,000,000 - 6,000,000
MW: 1,500,000 - 2,500,000
Na+
Na+
CO
HN CH2 CH2CH
HO CO n
poly-gamma-glutamic acid (sodium salt)
No.TI567E
TI567E-JA13
Gelatin
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10 15 min
0
50
mV
Presto FF-C18, 150 x 4.6mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.110-90 %B (0-15min), 0.4 mL/min (19MPa), 37 deg.CELSD (spray chamber 50deg.C, drift tube 100deg.C)2 uL (10ug)
gelatin (from bovine bone, ca.200 kDa)
No.TI580E
TI580E-JB12
Presto FF-C18, 250 x 4.6 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10-60 %B (0-20min)0.2 mL/min (13MPa), 37 deg.CELSD (spray chamber 15 deg.C, drift tube 80 deg.C)
Polyamino acids
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
poly-DL-alanineMW : 1,000-5,000
poly-DL-alanineMW : 1,000-5,0003 uL(12ug)
poly-L-lysineMW : 1,000-5,000
poly-L-lysineMW : 1,000-5,0002 uL(10ug)
nCH COH3C
NHn
CH CO
NH2
NH
1
2
0 10 20 30 min
0
100
200
mV
No.TI582E
TI582E-JB17
Presto FF-C18, 150 x 4.6 mm0-90 %B (0-15min)0.4 mL/min (14 MPa), 50 deg.CELSD (spray chamber 50 deg.C, drift tube 100 deg.C)
Lectin (concanavalin A)
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10 15 20
0
100
200mV
min
A: water /trifluoroacetic acid = 100 /0.1B: acetonitrile /trifluoroacetic acid = 100 /0.13 uL (7.5 ug)
A: water /formic acid = 100 /0.1B: acetonitrile /formic acid = 100 /0.12 uL (5 ug)
concanavalin A(from Jack been, 25.5 kDa)
concanavalin A(from Jack been, 25.5 kDa)
No.TI589E
TI589E-JB24
Presto FF-C18A: water / formic acid = 100 / 0.1B: acetonitrile / formic acid = 100 / 0.150 deg.C, ELSD (spray chamber 50 deg.C, drift tube 100 deg.C)
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
30 x 4.6 mm0-90 %B (0-2min)0.5 mL/min (4 MPa), 1 uL
150 x 4.6 mm0-90 %B (0-10min)0.4 mL/min (14 MPa), 1.4 uL
0 5 10 15 20
0
100
200mV
min
PEGylated protein
PEGylated protein, 60 kDa(20 kDa protein + 40 kDa PEG)
No.TI590E
TI590E-JB24
Presto FF-C18, 150 x 4.6 mmA: water /trifluoroacetic acid = 100 / 0.1B: acetonitrile /trifluoroacetic acid = 100 / 0.120-70 %B (0-30min)0.4 mL/min (15 MPa)1 uL
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
PEGylated protein (2)
0 10 20 30 min
PEGylated protein, 60 kDa(20 kDa protein + 40 kDa PEG)
280nm
ELSD
PEGylated protein, 60 kDa(20 kDa protein + 40 kDa PEG)
Presto FF-C18, 150 x 4.6 mmA: water /trifluoroacetic acid = 100 / 0.1B: acetonitrile /trifluoroacetic acid = 100 / 0.120-70 %B (0-30min)0.4 mL/min (19 MPa)1 uL 0 10 20 30 min
280nm
ELSD
50 °C
37 °C
No.TI591E
TI591E-JD08
Cadenza CW-C18, 250 x 3 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.150-57 %B (0-40min)0.4 mL/min, 280 nm, 5 uL
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
PEGylated protein (3)
PEGylated protein, 60 kDa(20 kDa protein + 40 kDa PEG)
50 °C10 MPa
37 °C13 MPa
0 10 20 30 40
0
20
40
mA
U
min
No.TI619E
TI619E-JG16
Hemocyanin and hemoglobin
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Presto FF-C18, 250 x 4.6 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min (22 MPa), 50 deg.C, 220 nm20 uL (100 ug, water or 0.02%TFA)
hemoglobin (from human)37-39 %B (0-180min)39-50 %B (180-300min)
hemocyanin (from limulus)35-50 %B (0-300min)
784 peaks
683 peaks
mA
U
0 100 200 300
0
20
40
60
min
No.TI620E
TI620E-JG26
Polyclonal IgG (from human serum)
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
00 100 200 300 min
10
20
30
mA
U
Polyclonal IgG (human serum)Oriental Yeast Co.,LTD1205 peaks
00 100 200 300 min
10
5
15
Polyclonal IgG (human serum)Sigma-Aldrich Corporation1168 peaks
Presto FF-C18, 250 x 4.6 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.133-38 %B (0-300min)0.4 mL/min (15MPa), 80 deg.C, 220nm10 uL (30-100 ug)
0 20 40 60 min0
10
20
mA
U
No.TI622E
TI622E-JG28
Gragient slopes affecting mAb separation
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
10-95 %B (0-20min)
30-45 %B (0-60min)
0 10 20 min
0
100
Presto FF-C18, 250 x 4.6 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min (15MPa), 80 deg.C, 220nm2 uL (anti-AFP, 2 ug)
No.TI623E
TI623E-JH02
Monoclonal and polyclonal antibodies
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Presto FF-C18, 250 x 3 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.132-40 %B (0-120min)0.2 mL/min (17MPa), 80 deg.C, 220nm2-10 uL (2-10 ug)
0 50 1000
10
20
30
mA
U
min
Monoclonal antibody(anti-Derf2)
Monoclonal antibody(anti-human LH)
Monoclonal antibody(anti-AFP)
Polyclonal antibody (human serum)
00 100 200 300
20
10
min
00 100 200 300
20
10
min
mA
U
0 20 40 60 min0
50
100
No.TI624E
TI624E-JH20
The effect of column length on resolution for polyclonal IgG
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Polyclonal IgG (human serum)
1205 peaks
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220nm, 10 uL (100 ug)
250 x 4.6 mm33-38 %B (0-300min)15 MPa
50 x 4.6 mm31-36 %B (0-300min)3.3 MPa
50 x 4.6 mm31-36 %B (0-60min)3.3 MPa
934 peaks
187 peaks
No.TI626E
TI626E-KB02
Gragient mixer volume affecting HSA separation
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
albumin (human serum, 66.4 kDa)
no mixer (T-union)
0.5mL mixer
1.7mL mixer
2.6mL mixer
Presto FF-C18, 250 x 4.6 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.130-40 %B (0-120 min)0.4 mL/min (18 MPa), 80 deg.C, 220 nm, 1.2 uL (12 ug)
0 20 100
0
10
20
30
min
mA
U
12040 60 80
40
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0
500
1000
mAU
0 5 10 min
0 40 120 min
0
10
20
80
0 20 40 60 min
0
200
150
100
No.TI627E
TI62E-KB02
The effect of gragient slope and column lengthfor HSA separation
albumin (human serum, 66.4 kDa)
50 x 4.6 mm0-80 %B (0-10 min)4 MPa
250 x 4.6 mm0-80 %B (0-60 min)16 MPa
250 x 4.6 mm30-40 %B (0-120 min)18 MPa
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220 nm, 1.2 uL (12 ug)
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 20 40 60 min
0
50
mA
U
0 40 120 min0
10
80
Presto FF-C18, 250 x 4.6 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min (16 MPa), 80 deg.C, 220 nm, 1 uL (1 ug)
No.TI629E
TI629E-KB02
The effect of gragient slope for angiotensin II separation
angiotensin II (human, 1046 Da)
2-52 %B (0-60 min)
2-12 %B (0-120 min)
No.TI630E
TI630E-KB02
The effect of gragient slope for insulin separation
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
insulin (bovine pancreas, 5733 Da)
2-52 %B (0-60 min)
21-31 %B (0-120 min)
0 20 40 60 min0
100
mA
U
0 40 120 min0
10
80
Presto FF-C18, 250 x 4.6 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min (16 MPa), 80 deg.C, 220 nm, 1.6 uL (1.6 ug)
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10 min
0
500
1000
mA
U
0 40 120 min
0
10
20
80
0 20 40 60 min
0
200
100
150
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220 nm, 0.6-1 uL (06-10 ug)
TI631E-KB02
No.TI631E
albumin (bovine serum, 66.3 kDa)
50 x 4.6 mm0-80 %B (0-10 min)4 MPa
250 x 4.6 mm0-80 %B (0-60 min)16 MPa
250 x 4.6 mm30-40 %B (0-120 min)18 MPa
The effect of gragient slope and column lengthfor BSA separation
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 40 120 min
0
10
20
80
0 5 10 min
0
500
mA
U
250
0 20 40 60 min
0
100
150
50
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220 nm, 1.6-4 uL (16-40 ug)
TI632E-KB02
No.TI632E
The effect of gragient slope and column lengthfor ovalbumin separation
albumin(chicken egg, 45 kDa)
50 x 4.6 mm0-80 %B (0-10 min)4 MPa
250 x 4.6 mm10-90 %B (0-60 min)17 MPa
250 x 4.6 mm40-50 %B (0-120 min)17 MPa
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10 min
0
200
400
mA
U
0 20 40 60 min
0
100
150
50
0 40 120 min
0
5
80
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220 nm, 2-5 uL (2-5 ug)
No.TI633E
TI633E-KB02
The effect of gragient slope and column lengthfor lectin separation
lectin (concanavalin A) (jack been, 25.5 kDa)
50 x 4.6 mm10-80 %B (0-10 min)4 MPa
250 x 4.6 mm10-80 %B (0-60 min)16 MPa
250 x 4.6 mm35-45 %B (0-120 min)16 MPa
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 20 40 60 min
0
50
75
25
0
500
250
mA
U
0 5 10 min
0 40 120 min
0
20
10
80
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220 nm, 10-20 uL (25-50 ug)
No.TI634E
TI634E-KB02
The effect of gragient slope and column lengthfor thyroglobulin separation
thyroglobulin (porcin thyroid, 670 kDa)
50 x 4.6 mm5-95 %B (0-10 min)4 MPa
250 x 4.6 mm5-95 %B (0-60 min)16 MPa
250 x 4.6 mm30-60 %B (0-120 min)18 MPa
TI635E-KB02Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 5 10 min
0
500
mA
U
0 20 40 60 min
0
100
150
50
0 40 120 min
0
10
20
80
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220 nm, 0.6-3 uL (6-30 ug)
No.TI635E
The effect of gragient slope and column lengthfor cytochrome c separation
cytochrome c (horse heart, 12.4 kDa)
50 x 4.6 mm0-80 %B (0-10 min)4 MPa
250 x 4.6 mm0-80 %B (0-60 min)16 MPa
250 x 4.6 mm21-31 %B (0-120 min)17 MPa
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
0 40 120 min
0
10
20
80
0
500
0 20 40 60 min
250
0
500
mA
U
0 5 10 min
250
Presto FF-C18A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.10.4 mL/min, 80 deg.C, 220 nm, 0.4-1.4 uL (4-14 ug)
No.TI636E
TI636E-KB02
The effect of gragient slope and column lengthfor alpha-lactalbumin separation
alpha-lactalbumin (bovine milk, 14.1 kDa)
50 x 4.6 mm0-80 %B (0-10 min)4 MPa
250 x 4.6 mm0-90 %B (0-60 min)16 MPa
250 x 4.6 mm30-38 %B (0-120 min)17 MPa
No.TI643E
TI643E-KB16Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Presto FF-C18, 75 x 4.6 mm�A: water / formic acid = 100 / 0.5, B: acetonitrile / formic acid = 100 / 0.510-50 %B (0-30min), 0.4 mL/min(10MPa)37 deg.C, ELSD(spray chamber 35, drift tube 80 deg.C), 0.4-14 uL(0.8-10ug)
lectin (concanavalin A, from Jack bean, 25.5 kDa)
myosin (rabbit muscle)
alpha-casein (bovine milk, 23-26 kDa)
alpha-amylase (Bacillus sp., 50-55 kDa)
mA
U
0 10 20 30 min0
50
100
cytochrome c (horse heart, 12.4 kDa)
alpha-lactalbumin(bovine milk, 14.2 kDa)
lysozyme (chicken egg white, 14 kDa)
papain (papaya latex, 21 kDa)
alpha-chymotryosinogen A(bovine pancreas, 25.7 kDa)
beta-glucosidase(almond, 183 kDa)
albumin (bovine, 66.3 kDa)
albumin (human serum, 66.4 kDa)
albumin (eggs, 45 kDa)
xanthine oxidase (buttermilk, 360 kDa)
thyroglobulin(porcin thyroid, 670 kDa)
lactoferrin (bovine milk, 80 kDa)
myoglobin(equine skeletal muscle, -17 kDa)
Protein separation on non-porous ODS column (37°C, formic acid)
No.TI644E
TI644E-KB16
Protein separation on non-porous ODS column (80°C, formic acid)
Courtesy of Dr. Naoki Komatsu, Kyoto Univ., JAPAN
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Presto FF-C18, 75 x 4.6 mm�A: water / formic acid = 100 / 0.5, B: acetonitrile / formic acid = 100 / 0.510-50 %B (0-30min), 0.4 mL/min(5MPa)80 deg.C, ELSD(spray chamber 35, drift tube 80 deg.C), 1-14 uL(2-14ug)
mA
U
0 10 20 30 min
0
50
100
lectin (concanavalin A, from Jack bean, 25.5 kDa)
cytochrome c (horse heart, 12.4 kDa)
alpha-lactalbumin(bovine milk, 14.2 kDa)
lysozyme (chicken egg white, 14 kDa)
papain (papaya latex, 21 kDa)
alpha-chymotryosinogen A(bovine pancreas, 25.7 kDa)
beta-glucosidase(almond, 183 kDa)
albumin (bovine, 66.3 kDa)
albumin (human serum, 66.4 kDa)
albumin (eggs, 45 kDa)
thyroglobulin(porcin thyroid, 670 kDa)
lactoferrin (bovine milk, 80 kDa)
myoglobin(equine skeletal muscle, -17 kDa)
monoclonal antibody (anti-AFP)
No.TI675E
TI675E-KG08
Amyloid beta-protein
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
100 x 4.6 mmA: water / formic acid = 100 / 0.5B: acetonitrile / formic acid = 100 / 0.537 deg.CELSD (spray chamber 35 deg.C, drift tube 70 deg.C)1.4 uL (1.4 ug, DMSO)
amyloid -protein(Human, 4329 Da)
20-40 %B (0-15min)0.2 mL/min (6MPa)
20-40 %B (0-15min)0.5 mL/min (4MPa)
5-25 %B (0-15min)0.5 mL/min (3MPa)
0 5 10 15 min0
60
mV
TI684E-KH09Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Presto FF-C18, 150 x 3 mmA: water / formic acid = 100 / 0.5B: acetonitrile / formic acid = 100 / 0.520-30 %B (0-60 min)0.2 mL/min (17 MPa), 50 deg.C, ELSD (spray chamber 50 deg.C, drift tube 100 deg.C)5 uL ( 50 ug)
No.TI684E
Cytochrome c ( 50°C, formic acid )
cytochrome c (horse heart, 12.4 kDa)
0 20 40 60
0
10
mV
min