The method can be easily scaled and run without major modifications on Agilent 1260 and 1290 systems. The chromatograms below demonstrate that using 4.6, 3.0 and 2.1 mm x100 mm columns can be easily used with only a change to the flow rate. Longer retention on the smaller diameter columns is due to the effect of instrument dwell volume.

An important step in any method validation is to examine the effect of lot to lot variability. As can be seen below, three lots were examined, some small variation is noted.

Lifetime of columns can be difficult to access. Typically columns are evaluated for 500 injections in the fastest way possible, i.e. continuous operation. In this case we chose to carry out a life-test in a manner similar to normal customer operation. In this work, columns are run for 100 injections (3 days) and flushed with mobile phase B for storage, and allowed to sit for 4 days . This is repeated for 1 month or 500 injections. As can be seen below, very minimal retention time loss or resolution change and no pressure gain.

Real samples such as beer or soy-sauce are typical of fermentation produced materials that are examined by HPLC amino acid methods. These application were carried out

using a 1290 Infinity UHPLC.

Poroshell HPH-C18 offers longer lifetime for Amino Acid

Analysis! It brings Poroshell Advantages of high resolution at

Low Pressure, Less risk of clogging. The new Poroshell HPH-

C18 is dissolution resistant. In short it can improve

productivity by reducing common causes of column failure,

reducing downtime and saving on costs per analysis.

Amino acid analysis (AAA) is commonly used food and fermentation testing as a tool to determine the precise amino acid (AA) makeup of samples. In order to do this in a timely fashion a short turnaround time is needed; to do it with limited sample amounts, a highly sensitive method is needed. And to maximize laboratory productivity, an automated method is desirable. In many cases these samples contain particulates which can clog many high resolution columns or necessitate additional sample clean-up, and the analysis is further complicated by the often rigorous conditions used, resulting in poor column lifetimes. This work describes methodology using a high pH stable superficially porous column with a 2.7 μm particle size. (Poroshell HPH C18) These particles have 90 % of the efficiency of sub two micron totally porous columns at 50 % of the pressure and offer longer lifetimes under the higher pH conditions typically used in this analysis. Additionally since they incorporate a 2 μm frit, they are as resistant to clogging as 3.5 and 5 μm columns. In this work, an automated pre-column OPA/FMOC amino acid method previously developed on 3.5 and 1.8 µm Zorbax Eclipse Plus C18 columns is expanded to include 2.7 µm superficially porous columns. This column shows good lifetime and transferability to different column dimensions which are shown in this work. Applications of this column to fermentation products will also be shown

Amino Acids are the building blocks of life. You can use the

analysis of Amino Acids to monitor complex chemical

processes. The Agilent/HP System allows a lab to bring the

work inside, using equipment They already have. Agilent ALS

has the capability within injector programming to derivatize

samples using OPA/FMOC. Lower costs in house capabilities

may already be present . In the past a major problem has

been identified in amino acid analysis Short column lifetime:

as columns age resolution decreases. Critical peak pair

resolution decreases Causes include: a) material gathering

on the frit (dirty samples, algae, etc.) b) insufficient starting

resolution c) silica dissolution Poroshell HPH-C18 has a large

frit, excellent resolution, and resists silica dissolution

resulting in log lifetime in amino acid analyses!

The primary amino groups react with ortho-phthalaldehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA) at about pH 10 to form an isoindole derivative. Secondary amino groups do not react. The OPA derivatized amino acid is then detected by UV at 338 nm. The secondary amino groups react with 9-fluorenylmethyl chloroformate (FMOC) at about pH 10 to form a secondary amide. The FMOC derivatized amino acid is then detected by

UV at 262 nm.

Benefits of Automated On-Line Derivatization: Simplified Operation: Just Add Samples and Reagents in separate vials, instrument, autosampler draws, mixes, does derivatization in sample loop, then injects. Automated derivatization reduces offline container transfer,

measurement, and operator errors.

High Speed Amino Acid Analysis on a New High pH Stable

2.7µm Column William Long, Anne Mack, Jason Link, Xiaoli Wang, Maureen Joseph, William Barber; Agilent Technologies, 2850 Centerville Rd, Wilmington, DE

Introduction

Conclusions

Column: Poroshell HPH C18 or ZORBAX Eclipse Plus C18 Column Temperature: 40 °C Mobile Phase A: 10 mM Na2HPO4: 10 mM Na2B4O7, pH 8.2: 5 mM NaN3 Mobile Phase B: MeCN: MeOH: Water(45:45:10, v:v:v) Injection Diluent: (0.25 mL H3PO4 + 100 mL H2O)

Two systems were used in this work. An Agilent 1260 HPLC System was used consisting of a Binary Pump (G1312B). A G1367C autosampler (ALS), a G1316B Thermostatted Column Compartment (TCC) and a G4212A Infinity (DAD) detector with a G4220-60007 flow cell. A second system, and Agilent 1290 UHPLC system, consisting of G4220A binary pump, a GG4226A 1290 Sampler, a G1316C TCC SL+ and a G4212A 1290 Infinity DAD detector was used for some applications

Using the same method conditions as outlined in published

application notes, the Poroshell HPH-C18 column is

compared with a Zorbax Eclipse Plus C18 totally porous

column. As can be seen in the above chromatograms and the

scatter-plot below the elution order on both columns is the

same and a high degree of correlation is found in the

retention times between both columns. The resolution

between Leucine and lysine looks better on the HPH, while

the separation between Lysine and Hydroxyproline and the

Sarcosine/Proline pair looks worse on the Poroshell HPH-

C18.

Improved Stability Results and Discussion

Background

Benefits of Automated OPA/FMOC Derivatization

Results and Discussion

min0 2 4 6 8 10 12

mAU

0

10

20

30

40

50

60

DAD1 C, Sig=338,10 Ref=390,20, TT (AMINO\EAA 2014-11-12 16-27-08LFS-RDWS022\POROSHELLHPH020.D)

0.8

70

1.2

93

2.4

57

2.6

28

3.0

71

3.2

38

3.4

37

3.6

02

4.1

99

4.5

31

5.5

63

6.7

69

7.0

24

7.1

94

7.4

58

7.8

97

8.1

90

8.3

56 8.8

69

9.3

49

9.5

48

11.2

70

11.7

81

min0 2 4 6 8 10 12

mAU

0

10

20

30

40

50

60

DAD1 C, Sig=338,10 Ref=390,20, TT (AMINO\EAA 2014-11-12 15-03-22LFS-RDWS022\ECLIPSEPLUS3516.D)

1.0

64

1.6

79

2.9

94

3.2

15 3

.659

3.8

34

4.0

69

4.2

13 4

.895 5

.104

6.0

17

6.9

60

7.4

19

7.5

74

7.8

36

8.2

05

8.5

08

8.6

61 9.1

50

9.4

21

9.5

17

10.0

50

10.9

53

11.6

97

12.1

78

Poroshell HPH C18

4.6 x 100 mm 2.7 µm

Eclipse Plus C18

4.6 x 100 mm 3.5µm

50

60

70

80

90

100

110

0 1000 2000 3000

% In

itial

Eff

icie

ncy

mL Stress Buffer

Silica SPP-C18

HPH SPP-C18

HPH SPP particles are synthesized by chemically modifying

the surface of the silica SPP particles with an organic layer,

resulting in 15x longer lifetime under these stressful

chromatographic conditions (columns are considered dead

after a 10% loss of efficiency. When High pH mobile phases

are necessary, the Agilent Poroshell HPH-C18 columns can

appreciably improve column lifetime.

Lifetime of SPP columns in phosphate buffer, pH 8, at

elevated temperature. Mobile phase: Premixed 60% 30 mM

sodium phosphate buffer at pH 8 and 40% methanol; Flow

rate 0.4 mL/min; UV absorbance 254 nm; 65 °C; Columns:

2.1 x 50 mm, 2.7 µm; Analyte: Naphthalene..

During product development it was revealed that the

selectivity of Poroshell HPH C-18 is similar to Poroshell 120

EC-C18 and Zorbax Eclipse Plus C18. This work

demonstrates the transferability of the Eclipse Plus C18 AAA

method to Poroshell HPH C-18

B14139

min2 4 6 8 10 12

mAU

0

20

40

60

80

0.44

0

0.59

0

0.97

9 1.50

3

1.66

6

2.75

1

2.96

3

3.38

9

3.55

1 3.80

1

3.95

8

4.50

0

4.86

1

5.80

1

6.47

3

6.61

2

6.85

26.

925

7.15

27.

256 7.

415

7.69

9

8.08

2

8.38

4

8.55

5 9.06

2

9.48

6

9.76

2

11.4

91

1 13

12

1110

9

8

7654

3

2

1918

1716

15

14

232221

20

min2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

0.45

9

0.59

9

1.02

9

1.60

2

1.75

5

2.92

6

3.13

9 3.57

8

3.75

0 3.99

5

4.15

3

4.37

9

4.71

3

5.06

6

6.01

1

6.68

4

6.81

7

7.05

07.

119

7.35

57.

442 7.

604

7.88

8

8.26

6

8.55

6

8.72

7 9.24

0

9.66

4

9.95

0

11.6

65

11.8

58

12.1

72

min2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

1.09

2

1.70

1

3.28

6

3.50

8

3.99

9

4.18

4 4.43

4

4.58

9

5.15

6

5.52

3

6.48

4

7.14

6 7.27

9

7.51

47.

613

7.81

37.

912 8.08

1

8.36

1

8.63

2

8.75

6

9.03

6

9.19

7 9.71

4

10.1

48

10.4

00

12.1

19

12.3

08

12.6

22

4.6 mm id

3.0 mm id

2.1 mm id

B14139

B14139

B14139

B14140

B14138

min2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

0.62

5

1.09

2

1.70

1

1.84

7

3.28

6

3.50

8

3.99

9

4.18

4 4.43

4

4.58

9

4.85

4

5.15

6

5.52

3

6.25

1

6.48

4

6.83

9 7.14

6 7.27

9

7.51

47.

613

7.81

37.

912 8.08

1

8.36

1

8.63

2

8.75

6

9.03

6

9.19

7

9.45

9

9.71

4

9.88

1

10.1

48

10.4

00

10.7

40

10.9

66

11.2

1811

.300

11.6

87

11.9

43 12.1

19

12.3

08

12.6

22

min2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

0.62

0

1.07

3

1.65

1

1.81

3

3.22

1

3.43

3

3.92

3

4.10

7 4.34

4

4.50

6

4.76

5

5.08

9

5.44

0

6.18

0

6.40

3

7.06

6 7.19

5

7.44

07.

533

7.73

67.

828 7.99

3

8.27

4 8.66

4

8.94

1

9.10

2

9.37

9

9.61

1

9.79

5

10.0

41

10.3

03

10.6

35

10.8

62

11.1

1011

.226

11.5

73

11.8

34 12.0

10

12.2

00

12.5

10

min2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

0.51

1

0.64

8

1.11

0

1.68

0

1.81

7

3.27

2

3.47

9

3.98

6

4.16

8 4.40

6

4.55

3

4.84

3

5.14

7

5.49

3

6.46

2

6.80

0

7.10

3 7.22

6

7.50

37.

617 7.86

6

8.04

3

8.31

3 8.72

0

8.98

5

9.13

8

9.38

9

9.64

4

10.1

07

10.3

10

10.6

52

10.8

78

11.1

3311

.230

11.5

83

11.8

34 12.0

10

12.1

62

12.5

02

Agilent 1260, G1367C ALSG4212A Infinity DAD, G4212-60008 Flow Cell,G1312B Binary Pump, No Pulse Damper, No Mixing Column, Red Tubing Throughout, 2.1 x100 mm, 2.7um

wjl

min0 2 4 6 8 10 12

mAU

0

20

40

60

80

DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-05 10-21-37LFS-RDWS022\WJL00010000025.D)

1.0

76

1.6

37

3.4

42

4.1

07

4.3

66

4.5

32

4.7

93

5.1

23

5.4

86

6.4

52

7.5

02

7.6

03 7

.884

8.0

51

9.0

08

9.1

66

9.6

71

10.1

04

11.1

53

min0 2 4 6 8 10 12

mAU

0

20

40

60

80

DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-15 16-13-54LFS-RDWS022\WJL00010000225.D)

1.0

81

1.6

56

3.4

21

4.0

91

4.3

42

4.5

03

4.7

63

5.1

05

5.4

48

6.4

17

7.4

72

7.5

68 7.8

42

8.0

11

8.9

65

9.1

16

9.6

22

10.0

70

11.1

20

min0 2 4 6 8 10 12

mAU

0

20

40

60

80

DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-18 14-02-17LFS-RDWS022\WJL00010000400.D)

1.0

61

1.5

89

3.4

03

4.0

73

4.3

19

4.4

81

4.7

42

5.0

79

5.4

15

6.3

88

7.4

41

7.5

39 7.8

07

7.9

76

8.9

32

9.0

84

9.5

86

10.0

41

11.0

83

min0 2 4 6 8 10 12

mAU

0

20

40

60

80

DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-25 12-56-57LFS-RDWS022\WJL00010000500.D)

1.0

70

1.5

46

3.3

81

4.0

49

4.2

90

4.4

51

4.7

15

5.0

60

5.3

85

6.3

64

7.4

20 7

.509

7.7

71

7.9

42

8.8

87

9.0

39

9.5

33

9.9

71

11.0

23

Injection 25

Injection 225

Injection 400

Injection 500

Similar Selectivity on Poroshell HPH C18 and Zorbax Eclipse Plus C18 using the same conditions

Old Dominion Cherry Blossom Lager

Kronenburg Blanc

Blue Moon Belgian Style Wheat

min 0 1 2 3 4 5 6 7

mAU

-50

0

50

100

150

200

DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000002.D)

0.6

88

1.1

04

2.1

70

2

.22

8

2.5

77

2.7

68

3.1

85

3.3

99

3

.51

6

4.0

51

4.7

93

4

.87

4

5.1

98

5.3

35

5.4

74

5.6

56

5

.74

4

5.8

47

min 0 1 2 3 4 5 6 7

mAU

-50

0

50

100

150

200

DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000004.D)

0.6

92

1.1

26

2.0

84

2.2

37

2.5

82

2.7

75

2.9

07

3.1

92

3.3

86

3.5

24

4.0

54

4.7

91

4

.88

4

5.2

18

5.5

48

5

.66

0

5.7

43

5

.86

0

min 0 1 2 3 4 5 6 7

mAU

-50

0

50

100

150

200

DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000006.D)

0.6

94

1.1

24

2.0

85

2.2

33

2.5

78

2.7

66

3.1

87

3.3

79

3.5

15

4.0

47

4.7

82

4

.88

1

4.9

78

5.2

18

5.5

35

5

.64

8

5.7

47

5

.84

6

6.0

71

min 1 2 3 4 5 6 7

mAU

0

25

50

75

100

125

150

175

DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000008.D)

0.4

84

0.7

05

1.1

23

1.2

75

1.8

93

2.2

25

2.5

79

2.7

75

2

.86

3

2.9

20

2

.99

3

3.1

92

3.4

00

4.0

62

4.7

92

4.9

78

5.2

24

5.5

38

5

.66

2

5.7

59

5

.84

6

6.0

65

6.6

65

min 1 2 3 4 5 6 7

mAU

0

10

20

30

DAD1 B, Sig=262,16 Ref=324,8, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000008.D)

0.3

68

0

.43

3

0.4

86

0

.53

1

0.5

47

0

.56

1

0.5

98

0

.67

3

0.6

93

1.1

23

1

.18

6

1.2

75

2.2

26

2.7

45

2

.77

8

2.8

63

2

.98

4

3.0

50

3.1

91

3

.26

0

3.3

99

3.5

72

4.0

62

4.5

89

4.7

92

5.0

09

5.5

39

5.5

65

5

.66

1

5.8

46

6.0

65

6.4

32

7.4

10

7

.46

0

7.6

32

Similar Selectivity on Poroshell HPH C18 and Zorbax Eclipse Plus C18 using the same conditions

Many other foods (such as

soy sauce) and pharmaceuticals that are

produced using fermentation processes

Reprint Requests

Diluted

1/100