eclipse plus c18 - chemical analysis, life sciences, … · amino acids are the building blocks of...
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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
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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
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