Peptides Proteins Amino Acids - MZ-AT · BSA Tryptic Digest, 18.5uL (ca.6.5ug) 0 5 10 15 20 25 min...

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


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


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


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


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


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


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


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


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


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


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


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


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







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


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


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


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)


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)


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


PEGylated protein (2)

0 10 20 30 min


280nm

ELSD


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


PEGylated protein (3)


50 °C10 MPa

37 °C13 MPa

0 10 20 30 40

0

20

40

mA

U

min

No.TI619E

TI619E-JG16

Hemocyanin and hemoglobin


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)


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


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


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


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


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


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


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)


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


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)


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


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


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


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


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


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


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


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 (eggs, 45 kDa)

xanthine oxidase (buttermilk, 360 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


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)


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 (eggs, 45 kDa)



myoglobin(equine skeletal muscle, -17 kDa)

monoclonal antibody (anti-AFP)

No.TI675E

TI675E-KG08

Amyloid beta-protein


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 )


0 20 40 60

0

10

mV

min

Peptides Proteins Amino Acids - MZ-AT · BSA Tryptic Digest, 18.5uL (ca.6.5ug) 0 5 10 15 20 25 min...

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Transcript of Peptides Proteins Amino Acids - MZ-AT · BSA Tryptic Digest, 18.5uL (ca.6.5ug) 0 5 10 15 20 25 min...