Organic Acids
Effect of flow rate on column efficiency when RSpak KC-811 is used for the analysis of organic acids is shown.
Sample : 1% Acetic acid, 10micro-L
Column : Shodex RSpak KC-811 (8.0mmID*300mm) Eluent : 0.1% H3PO4 aq. Detector : Shodex RI Column temp. : 40deg-C
Effect of injection volume on column efficiency when RSpak KC-811 is used for the analysis of organic acids is shown.
Sample : 0.5% Acetic acid
Column : Shodex RSpak KC-811 (8.0mmID*300mm) Eluent : 0.1% H3PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Effect of sample load on column efficiency when RSpak KC-811 is used for the analysis of organic acids is shown.
Sample : Acetic acid
Column : Shodex RSpak KC-811 (8.0mmID*300mm) Eluent : 0.1% H3PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Effect of column temperature on column efficiency when RSpak KC-811 is used for the analysis of organic acids is shown. Effect of temperature changes with kinds of organic acid. By changing the temperature of a column, the improvement of separation is possible.
Sample : Acetic acid
Column : Shodex RSpak KC-811 (8.0mmID*300mm) Eluent : 0.1% H3PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
As the pH of the eluent and the column temperature affect the dissociation of organic acids, they are important for the adjustment of separation when RSpak KC-811 is used.
| Substance | Elution Volume (mL) | |||
|---|---|---|---|---|
| 1mM HClO4 |
2mM HClO4 |
3mM HClO4 |
5mM HClO4 |
|
| Oxalic acid |
9.27
|
9.41
|
9.62
|
9.72
|
| Maleic acid |
9.70
|
10.15
|
10.51
|
11.12
|
| alpha-Ketoglutaric acid |
9.96
|
10.44
|
10.87
|
11.37
|
| Oxalacetic acid |
10.48
|
11.22
|
11.77
|
12.50
|
| Pyruvic acid |
10.51
|
11.24
|
11.76
|
12.48
|
| 2-Ketogluconic acid |
10.59
|
11.13
|
11.52
|
11.73
|
| Phosphoric acid |
10.61
|
10.95
|
11.27
|
11.58
|
| Citric acid |
10.84
|
11.32
|
11.71
|
11.81
|
| Tartaric acid |
11.06
|
11.66
|
12.14
|
12.31
|
| Isocitric acid |
11.10
|
11.54
|
11.88
|
11.89
|
| Glucuronic acid |
11.16
|
11.44
|
11.71
|
11.76
|
| trans-Aconitic acid |
11.47
|
12.43
|
13.11
|
13.48
|
| Malonic acid |
11.54
|
12.36
|
12.96
|
13.40
|
| Galacturonic acid |
11.83
|
12.12
|
12.41
|
12.39
|
| Gluconic acid |
12.29
|
12.53
|
12.78
|
12.48
|
| Glyoxylic acid |
12.35
|
12.86
|
13.26
|
13.86
|
| Malic acid |
12.50
|
12.99
|
13.37
|
13.42
|
| alpha-Hydroxygltaric acid |
13.47
|
13.91
|
14.02
|
14.15
|
| Ascorbic acid |
13.53
|
13.69
|
13.88
|
13.80
|
| Fumaric acid |
14.47
|
16.09
|
17.16
|
17.75
|
| Succinic acid |
15.30
|
15.52
|
15.79
|
15.69
|
| Glycolic acid |
15.60
|
15.92
|
16.26
|
16.22
|
| Itaconic acid |
15.92
|
16.42
|
16.80
|
16.79
|
| Lactic acid |
16.02
|
16.39
|
16.67
|
16.68
|
| 2-Hydroxyisobutyric acid |
16.52
|
16.82
|
17.05
|
17.05
|
| Adipic acid |
16.65
|
19.84
|
20.17
|
20.01
|
| beta-Hydroxypropionic acid |
16.94
|
17.04
|
17.18
|
17.12
|
| Formic acid |
17.18
|
17.55
|
17.86
|
17.92
|
| Mesaconic acid |
17.20
|
19.20
|
20.47
|
21.06
|
| Uric acid |
17.89
|
17.93
|
18.31
|
18.00
|
| Pyroglutamic acid |
18.59
|
20.25
|
20.99
|
21.65
|
| Acetic acid |
19.19
|
19.29
|
19.41
|
19.36
|
| Levulinic acid |
20.01
|
20.19
|
20.27
|
20.23
|
| Propionic acid |
22.33
|
22.43
|
22.54
|
22.49
|
| CO32-,HCO3– |
24.72
|
24.72
|
24.50
|
24.68
|
| Isobutyric acid |
25.35
|
25.47
|
25.59
|
25.55
|
| n-Butyric acid |
27.32
|
27.46
|
27.61
|
27.57
|
| Isovaleric acid |
31.89
|
32.07
|
32.28
|
32.20
|
| n-Valeric acid |
38.36
|
38.57
|
38.89
|
38.75
|
Columns : Shodex RSpak KC-811 (8.0mmID*300mm) x 2 Eluent : HClO4 aq. Flow rate : 1.0mL/min Column temp. : 50deg-C
| Substance | Elution Volume (mL) | |||||
|---|---|---|---|---|---|---|
|
30deg-C
|
40deg-C
|
50deg-C
|
60deg-C
|
70deg-C
|
80deg-C
|
|
| Oxalic acid |
9.66
|
9.63
|
9.62
|
9.56
|
9.51
|
9.47
|
| Maleic acid |
10.74
|
10.65
|
10.51
|
10.53
|
10.47
|
10.41
|
| Phosphoric acid |
11.01
|
11.14
|
11.27
|
11.41
|
11.53
|
11.70
|
| alpha-Ketoglutaric acid |
11.08
|
10.95
|
10.87
|
10.77
|
10.70
|
10.64
|
| 2-Ketogluconic acid |
11.56
|
11.52
|
11.52
|
11.47
|
11.44
|
11.42
|
| Glucuronic acid |
11.67
|
11.69
|
11.71
|
11.69
|
11.68
|
*
|
| Pyruvic acid |
11.91
|
11.82
|
11.76
|
11.72
|
11.70
|
11.71
|
| Citric acid |
11.93
|
11.81
|
11.71
|
11.57
|
11.46
|
11.36
|
| Oxalacetic acid |
12.00
|
11.86
|
11.77
|
11.76
|
11.73
|
11.70
|
| Isocitric acid |
12.14
|
11.99
|
11.88
|
11.72
|
11.60
|
11.48
|
| Tartaric acid |
12.36
|
12.23
|
12.14
|
11.99
|
11.90
|
11.81
|
| Galacturonic acid |
12.48
|
12.44
|
12.41
|
12.31
|
12.26
|
12.20
|
| Gluconic acid |
12.79
|
12.78
|
12.78
|
12.76
|
12.90
|
12.91
|
| Malonic acid |
13.26
|
13.09
|
12.96
|
12.81
|
12.71
|
12.61
|
| trans-Aconitic acid |
13.99
|
13.46
|
13.11
|
12.74
|
12.47
|
12.24
|
| Glyoxylic acid |
13.30
|
13.27
|
13.26
|
13.19
|
13.14
|
13.09
|
| Malic acid |
13.69
|
13.51
|
13.37
|
13.19
|
13.04
|
12.91
|
| Ascorbic acid |
14.13
|
13.99
|
13.88
|
13.72
|
13.59
|
*
|
| alpha-Hydroxygltaric acid |
14.58
|
14.25
|
14.05
|
13.83
|
13.98
|
13.91
|
| Succinic acid |
16.53
|
16.11
|
15.79
|
15.40
|
15.10
|
14.82
|
| Glycolic acid |
16.55
|
16.39
|
16.26
|
16.06
|
15.89
|
15.74
|
| Lactic acid |
16.83
|
16.75
|
16.67
|
16.51
|
16.37
|
16.21
|
| 2-Hydroxyisobutyric acid |
17.02
|
17.06
|
17.05
|
16.94
|
16.82
|
16.66
|
| beta-Hydroxypropionic acid |
17.53
|
17.34
|
17.18
|
16.96
|
16.76
|
16.57
|
| Itaconic acid |
17.83
|
17.26
|
16.80
|
16.29
|
15.87
|
15.48
|
| Formic acid |
18.23
|
18.03
|
17.86
|
17.64
|
17.46
|
17.28
|
| Fumaric acid |
19.23
|
18.02
|
17.16
|
16.39
|
15.79
|
15.30
|
| Acetic acid |
19.92
|
19.66
|
19.41
|
19.11
|
18.82
|
18.55
|
| Uric acid |
20.71
|
19.28
|
18.31
|
17.40
|
16.75
|
16.22
|
| Levulinic acid |
22.06
|
21.07
|
20.27
|
19.53
|
18.87
|
18.27
|
| Pyroglutamic acid |
22.91
|
21.80
|
20.99
|
20.33
|
19.77
|
19.29
|
| Adipic acid |
23.10
|
21.41
|
20.17
|
19.03
|
18.11
|
17.34
|
| Propionic acid |
23.53
|
23.02
|
22.54
|
22.01
|
21.49
|
20.69
|
| Mesaconic acid |
24.02
|
21.93
|
20.47
|
19.15
|
18.14
|
17.31
|
| CO32-,HCO3– |
24.29
|
24.48
|
24.50
|
24.51
|
24.35
|
24.15
|
| Isobutyric acid |
27.10
|
26.35
|
25.59
|
24.77
|
23.95
|
23.14
|
| n-Butyric acid |
29.76
|
28.66
|
27.61
|
26.53
|
25.49
|
24.48
|
| Isovaleric acid |
35.65
|
33.92
|
32.28
|
30.60
|
29.00
|
27.46
|
| n-Valeric acid |
45.05
|
41.77
|
38.89
|
36.12
|
33.60
|
31.27
|
* : cannot be detected
Columns : Shodex RSpak KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Flow rate : 1.0mL/min
Effect of column temperature on elution volume when RSpak KC-811 is used is shown.
Sample :
1. Oxalic acid
2. Citric acid
3. Tartaric acid
4. Malonic acid
5. Succinic acid
6. Lactic acid
7. Formic acid
8. Acetic acid
9. Pyroglutamic acid
Column : Shodex RSpak KC-811 (8.0mmID*300mm) Eluent : 0.1% H3PO4 aq. Flow rate : 1.0mL/min Column temp. : 28, 40, 60, 80deg-C
Effect of acetonitrele in eluent on elution volume when RSpak KC-811 is used is shown. By adding 10% acetonitrile to the eluent, the elution time of organic acids with strong hydrophobicity can be shortened.
| Substance | Elution Volume (mL) | |
|---|---|---|
| 3mM HClO4 aq./CH3CN =90/10 |
3mM HClO4 aq. | |
| Oxalic acid |
9.28
|
9.62
|
| Maleic acid |
9.91
|
10.51
|
| alpha-Ketoglutaric acid |
10.39
|
10.87
|
| Citric acid |
10.99
|
11.71
|
| Isocitric acid |
11.05
|
11.88
|
| 2-Ketogluconic acid |
11.18
|
11.52
|
| Glucuronic acid |
11.29
|
11.71
|
| Oxalacetic acid |
11.40
|
11.77
|
| Pyruvic acid |
11.42
|
11.76
|
| Phosphoric acid |
11.43
|
11.27
|
| Tartaric acid |
11.53
|
12.14
|
| trans-Aconitic acid |
11.57
|
13.11
|
| Galacturonic acid |
11.90
|
12.41
|
| Malonic acid |
12.10
|
12.96
|
| Gluconic acid |
12.24
|
12.78
|
| Malic acid |
12.37
|
13.37
|
| Glyoxylic acid |
12.70
|
13.26
|
| alpha-Hydroxygltaric acid |
12.71
|
14.02
|
| Ascorbic acid |
12.71
|
13.88
|
| Succinic acid |
13.77
|
15.79
|
| Itaconic acid |
14.08
|
16.80
|
| Fumaric acid |
14.14
|
17.16
|
| Uric acid |
14.99
|
18.31
|
| Glycolic acid |
15.12
|
16.26
|
| Adipic acid |
15.16
|
20.17
|
| Lactic acid |
15.23
|
16.67
|
| 2-Hydroxyisobutyric acid |
15.26
|
17.05
|
| Mesaconic acid |
15.42
|
20.47
|
| beta-Hydroxypropionic acid |
15.61
|
17.18
|
| Levulinic acid |
16.44
|
20.27
|
| Formic acid |
16.59
|
17.86
|
| Acetic acid |
17.32
|
19.41
|
| Pyroglutamic acid |
18.02
|
20.99
|
| Propionic acid |
18.82
|
22.54
|
| Isobutyric acid |
20.13
|
25.59
|
| n-Butyric acid |
20.87
|
27.61
|
| Isovaleric acid |
22.16
|
32.28
|
| CO32-,HCO3– |
23.36
|
24.50
|
| n-Valeric acid |
24.64
|
38.89
|
Columns : Shodex RSpak KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq./CH3CN=90/10, 3mM HClO4 aq. Flow rate : 1.0mL/min Column temp. : 50deg-C
When analyzing organic acids using RSpak KC-811, the elution time of organic acids with strong hydrophobicity can be shortened by adding 10% acetonitrile to the eluent.
Sample :
1. Succinic acid
2. Lactic acid
3. Formic acid
4. Acetic acid
5. Propionic acid
6. Isobutyric acid
7. n-Butyric acid
8. Isovaleric acid
9. n-Valeric acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Reagent : 10-fold diluted ST3-R(for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagant); 0.5mL/min Detector : VIS(430nm) Column temp. : 47deg-C
Connection of the reversed phase column (RSpak DE-613) to RSpak KC-811 makes it possible to separate phosphoric acid, which is eluted earlier, from other organic acids.
| Substance | Elution Volume (mL) | ||
|---|---|---|---|
|
DE-613
|
KC-LG
+ KC811 x 2 |
KC-LG
+ DE-613 + KC-811 x 2 |
|
| Oxalic acid |
3.0
|
10.5
|
13.5
|
| Maleic acid |
4.2
|
11.4
|
15.6
|
| Phosphoric acid |
3.0
|
11.4
|
14.4
|
| alpha-Ketoglutaric acid |
3.2
|
11.5
|
14.7
|
| Pyruvic acid |
4.0
|
12.3
|
16.3
|
| Citric acid |
5.0
|
12.3
|
17.3
|
| D-Glucuronic acid |
3.3
|
12.3
|
15.6
|
| Isocitric acid |
5.0
|
12.6
|
17.6
|
| Tartaric acid |
3.8
|
13.0
|
16.8
|
| Gluconic acid |
3.4
|
13.5
|
16.9
|
| Malonic acid |
5.3
|
13.6
|
18.9
|
| trans-Aconitic acid |
15.7
|
13.7
|
29.4
|
| Glyoxylic acid |
3.7
|
14.0
|
17.7
|
| Malic acid |
5.1
|
14.1
|
19.2
|
| Succinic acid |
8.6
|
16.8
|
25.2
|
| Glycolic acid |
4.3
|
17.0
|
21.3
|
| Lactic acid |
5.5
|
17.5
|
23.0
|
| Itaconic acid |
15.7
|
17.6
|
33.3
|
| Fumaric acid |
16.9
|
17.8
|
34.7
|
| beta-Hydroxypropionic acid |
5.0
|
18.3
|
23.3
|
| Formic acid |
4.7
|
18.7
|
23.4
|
| Uric acid |
6.9
|
19.4
|
26.3
|
| Acetic acid |
6.3
|
20.4
|
26.7
|
| Mesaconic acid |
43.4
|
20.7
|
64.1
|
| Adipic acid |
30.2
|
21.1
|
51.3
|
| Levulinic acid |
11.5
|
21.2
|
32.7
|
| L-Pyroglutamic acid |
5.5
|
21.4
|
26.9
|
| Propionic acid |
12.8
|
23.9
|
36.7
|
| CO32-,HCO3– |
7.8
|
25.7
|
33.5
|
| Isobutyric acid |
32.3
|
26.5
|
58.8
|
| n-Butyric acid |
32.6
|
28.5
|
61.1
|
| Isovaleric acid |
Not eluted
|
32.9
|
Not eluted
|
| n-Valeric acid |
Not eluted
|
39.7
|
Not eluted
|
Columns : Shodex RSpak DE-613 (6.0mmID*150mm)
Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2
Shodex RSpak KC-LG (8.0mmID*50mm) + DE-613 (6.0mmID*150mm) + KC-811 (8.0mmID*300mm) x 2
Eluent : 2mM HClO4 aq.
Column temp. : 50deg-C
RSpak NN-814, which has a higher polarity gel than that of KC-811 is suitable for the analysis of aromatic organic acids. The ion exclusion and reversed phase modes contribute to the separation of aromatic organic acids.
| Substance | Elution Volume (mL) |
|---|---|
| Shikimic acid |
10.1
|
| DL-Mandelic acid |
19.2
|
| Phthalic acid |
19.8
|
| Trimellitic acid |
23.0
|
| m-Hydroxybenzonic acid |
46.5
|
| Vanillic acid |
47.1
|
| p-Hydroxybenzoic acid |
47.6
|
| o-Hydroxybenzoic acid |
48.8
|
| 3-Phenylpropionic acid |
49.7
|
| Substance | Elution Volume (mL) |
|---|---|
| Benzoic acid |
51.0
|
| Caffeic acid |
66.9
|
| Phenylpropionic acid |
68.4
|
| Trimesic acid |
69.0
|
| Salicylic acid |
71.4
|
| m-Toluic acid |
86.4
|
| Ferulic acid |
87.0
|
| p-Coumaric acid |
90.3
|
Column : Shodex RSpak NN-814 (8.0mmID*250mm) Eluent : 0.1% H3PO4 aq. Column temp. : 60deg-C
Elution volume of organic acids using RSpak NN-814 is shown.
| Substance | Elution Volume (mL) |
|---|---|
| Oxalic acid |
7.6
|
| Maleic acid |
8.8
|
| D-Glucuronic acid |
8.9
|
| Gluconic acid |
9.0
|
| alpha-Ketoglutaric acid |
9.2
|
| Tartaric acid |
9.2
|
| Pyruvic acid |
9.4
|
| Citric acid |
9.5
|
| Glyoxylic acid |
9.7
|
| Malic acid |
9.8
|
| Glycolic acid |
9.8
|
| Malonic acid |
10.0
|
| Pyroglutamic acid |
10.0
|
| Substance | Elution v Volume (mL) |
|---|---|
| Lactic acid |
10.2
|
| Formic acid |
10.2
|
| Citraconic acid |
10.7
|
| Succinic acid |
10.8
|
| Acetic acid |
10.8
|
| Levulinic acid |
11.1
|
| Glutaric acid |
11.7
|
| Itaconic acid |
12.3
|
| Adipic acid |
12.9
|
| Fumaric acid |
13.4
|
| trans-Crotonic acid |
15.2
|
| n-Capronic acid |
30.8
|
| L-Glutamic acid |
37.8
|
Column : Shodex RSpak NN-814 (8.0mmID*250mm) Eluent : 0.1% H3PO4 aq. Column temp. : 60deg-C
| Substance | Elution Volume (mL) |
|---|---|
| Oxalic acid |
1.75
|
| Glyoxylic acid |
1.91
|
| DL-Tartaric acid |
2.17
|
| Glycolic acid |
2.22
|
| beta-Hydroxypropionic acid |
2.61
|
| Formic acid |
2.68
|
| Malic acid |
2.72
|
| Pyruvic acid |
2.74
|
| Lactic acid |
2.98
|
| L-Pyroglutamic acid |
3.07
|
| Citric acid |
3.12
|
| Malonic acid |
3.18
|
| Substance | Elution Volume (mL) |
|---|---|
| Maleic acid |
3.36
|
| Acetic acid |
3.37
|
| alpha-Ketoglutaric acid |
3.55
|
| Succinic acid |
4.70
|
| Levulinic acid |
6.64
|
| Propionic acid |
7.12
|
| Itaconic acid |
8.94
|
| trans-Aconitic acid |
10.80
|
| Fumaric acid |
11.09
|
| Adipic acid |
16.38
|
| Mesaconic acid |
28.48
|
Column : Shodex RSpak DE-413 (4.6mmID*150mm) Eluent : 10mM H3PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp.: 50deg-C
Comparison of Analysis of Organic Acids using RSpak KC-811 with 4 Kinds of Detectors was shown. When analyzing organic acid satndards, 4 kinds of detectors are usable. However, when analyzing actual sample, we recommend to use the post-column reaction method which allows high-selective analysis of organic acids.
Sample :
1. 800ppm Citric acid
2. 900ppm Tartaric acid
3. 800ppm Malic acid
4. 700ppm Succinic acid
5. 1100ppm Lactic acid
6. 600ppm Formic acid
7. 700ppm Acetic acid
8. 1500ppm Levulinic acid
9. 1500ppm Pyroglutamic acid
Columns : Shodex RSpak KC-G (6.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Flow rate : 1.0mL/min Column temp. : 50deg-C
Comparison of Analysis of Organic Acids using RSpak KC-811 with 4 Kinds of Detectors was shown. When analyzing organic acid satndards, 4 kinds of detectors are usable. However, when analyzing actual sample, we recommend to use the post-column method which allows high-selective analysis of organic acids. For example, soy sauce is a complicated mixture of organic acids, amino acids, saccharides, NaCl, ions and organic compounds. With UV detector (210nm), too many organic substances, organic compounds and organic acids are detected. With a conductivity detector, organic acids cannot be quantified satisfactory, as the NaCl peak is appearing at around 10 minutes is large. Also it is difficult to quantify organic acids with an RI detector, as almost all the components including saccharides are detected.
Sample : Soy sauce
1. Citric acid
2. Pyruvic acid
3. Malic acid
4. Succinic acid
5. Lactic acid
6. Formic acid
7. Acetic acid
8. Pyroglutamic acid
Columns : Shodex RSpak KC-G (6.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Flow rate : 1.0mL/min Column temp. : 50deg-C
The post-column method of organic acids uses the reagent (Shodex ST3-R), the mixing module and the visible detector of 430nm wave length . The detector detects the color which comes from organic acids. Organic acids in a sample are separated by the column for organic acid analysis (RSpak KC-811) using acidic solution (ex. perchloric acid) as the eluent. The pH indicator in the reagent (ST3-R) is adjusted to be dissociated state (I–+H+). When the pH indicator and the eluted solution from the column are mixed in the mixing module, the acidic substances (organic acids) will be free state (AH->A–+H+) and the pH indicator generate color by the reaction of I–+H+->IH.
Right figure shows the spectrum of the pH indicator.
Organic acid standards were analyzed using RSpak KC-811.
Sample :
1. Oxalic acid , 2. alpha-Ketoglutaric acid
3. Pyruvic acid, 4. 2-Ketogluconic acid
5. Citric acid, 6. Tartaric acid
7. Malonic acid, 8. Galacturonic acid
9. Gluconic acid, 10. Malic acid
11. Ascorbic acid , 12. Fumaric acid
13. Succinic acid, 14. Formic acid
15. Acetic acid, 16. Pyroglutamic acid
17. Levulinic acid, 18. Propionic acid
Columns : Shodex RSpak KC-811 (8.0mmID*300mm) x 4 Eluent : 1mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : 1.0mL/min Detector : VIS(430nm) Column temp. : 40deg-C
Organic acid standards were analyzed using RSpak KC-811.
Sample :
1. alpha-Ketoglutaric acid
2. 2-Ketogluconic acid
3. Citric acid
4. Tartaric acid
5. Glyconic acid
6. Malic acid
7. Succinic acid
8. Lactic acid
9. Acetic acid
10. Levulinic acid
11. Pyroglutamic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagant); 0.7mL/min Detector : VIS(430nm) Column temp. : 40deg-C
Organic acid standards were analyzed using RSpak KC-811.
Sample :
1. Citric acid
2. Tartaric acid
3. Pyrubic acid
4. Malic acid
5. Succinic acid
6. Glyconic acid
7. Lactic acid
8. Fumaric acid
9. Acetic acid
10. Levulinic acid
11. Pyroglutamic acid
12. Propionic acid
13. Isobutyric acid
14. n-Butyric acid
Columns : Shodex RSpak KC-811 (8.0mmID*300mm) x 2 Eluent : 6mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : 1.0mL/min Detector : VIS(430nm) Column temp. : 50deg-C
When water or 100% buffer is used as the eluent, the column life of ODS columns is usually shorten. Since the packing material of RSpak DE-413 is chemically stable, long column life and stable analysis results can be obtained even when Water or 100% buffer was used as the eluent.
Sample : 5micro-L
1. Glyoxylic acid, 1.78mg/mL
2. Tartaric acid, 1.95mg/mL
3. Malic acid, 2.06mg/mL
4. Lactic acid, 2micro-L/mL
5. Malonic acid, 1.95mg/mL
6. Acetic acid, 2micro-L/mL
7. Succinic acid, 2.05mg/mL
8. Levulinic acid, 1.95mg/mL
9. Propionic acid, 2micro-L/mL
Column : Shodex RSpak DE-413 (4.6mmID*150mm) Eluent : 10mM H3PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 50deg-C
4 kinds of organic acids were analyzed by RSpak DE-213 which is packed in 2mmID semi-micro column. In 4 minutes, sharp peaks could be obtained. For the detection, RI detector with small cell volume type was used.
Sample : 1mg/mL each , 1micro-L
1. Glyoxylic acid
2. Tartaric acid
3. Malic acid
4. Lactic acid
Column : Shodex RSpak DE-213 (2.0mmID*150mm) Eluent : 10mM H3PO4 aq. Flow rate : 0.15mL/min Detector : Shodex RI (small cell volume) Column temp. : 50deg-C
The elution pattern of RSpak DE-413 and that of ODS column were compared. For di-carboxylic acids, the elution times of ODS column were faster than those of RSpak DE-413. To the contrary, for suberic acid whose carbon number of alkyl chain is six, the elution time of RSpak DE-413 is faster that of ODS column.
Sample : 5micro-L
1. Malonic acid (n=1) 0.7mg/mL
2. Succinic acid (n=2) 0.9mg/mL
3. Glutaric acid (n=3) 0.9mg/mL
4. Adipic acid (n=4) 1.2mg/mL
5. Pimelic acid (n=5) 2.0mg/mL
6. Suberic acid (n=6) 1.5mg/mL
Columns : (above); Shodex RSpak DE-413 (4.6mmID*150mm)
(below); ODS column from (other manfacturer)
Eluent : 20mM H3PO4 aq./CH3CN=92/8
Flow rate : 1.0mL/min
Detector : UV(210nm)
Column temp. : 40deg-C
Organic acid standards were analyzed using RSpak KC-811. The elution time of organic acids which have long carbon chain such as butyric acid or valeric acid can be shortened adding cyclodextrin in the eluent.
Sample : 5mM each , 20micro-L
1. Fumaric acid
2. Succinic acid
3. Lactic acid
4. Formic acid
5. Acetic acid
6. Propionic acid
7. n-Butyric acid
8. Isobutyric acid
9. n-Valeric acid
10. Isovaleric acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 2mM HClO4 + 4mM alph-cyclodextrin(pH3.0) Reagent : 10-fold diluted ST3-R (for post column method) Flow rate : (Eluent); 1.0mL/min, (Reagent); 0.5mL/min Detector : VIS(430nm) Column temp. : 47deg-C
Succinic acid and fumaric acid were analyzed using RSpak KC-811. The elution time of succinic acid and fumaric acid largely changes according to eluent pH and column temperature. And, sometimes their peaks interfere the peaks of other substances. (Please refer to Effect of Column Temperature on Elution Volume (1))
Comparing (1), (2) and (3), it was confirmed that the elution order of succinic acid and fumaric acid changes according to the conditions.
Sample :
1. Succinic acid
2. Fumaric acid
3. Lactic acid
4. Formic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2
Eluent : (1)2mM HClO4 aq.
(2)2mM HClO4 aq./CH3CN=90/10
(3)4mM HClO4 aq./CH3CN=90/10
Reagent : 10-fold diluted ST3-R (for post-column method)
Flow rate : 1.0mL/min
Detector : VIS(430nm)
Column temp. : 60deg-C
Citric acid in phosphoric acid was analyzed using RSpak DE-613 and KC-811.
Sample :
1. Phosphoric acid
2. Citric acid
3. Malic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + DE-613 (6.0mmID*150mm) x 2 + KC-811 (8.0mmID*300mm) x 3 Eluent : 2mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagent); 0.5mL/min Detector : VIS(430nm) Column temp. : 50deg-C
Citric acid in phosphoric acid was analyzed using RSpak DE-613 and KC-811.
Sample :
1. Phosphoric acid
2. Citric acid
3. Malic acid
4. Lactic acid
5. Succinic acid
6. Fumaric acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + DE-613(6.0mmID*150mm) x 2 + KC-811 (8.0mmID*300mm) x 3 Eluent : 3mM HClO4 aq. Flow rate : 1.0mL/min Detector : Non-suppressed coductivity Column temp. : 60deg-C
Organic acids in beer are analyzed with 4.8 mM perchloric acid as an eluent using RSpak KC-811. When four KC-811 columns are connected in line, phosphoric acid, citric acid and pyruvic acid can be separated from each other completely.
Please refer to the right figure.
Sample : Beer, 100micro-L
1. Phosphoric acid etc., 2. Citric acid, 3. Pyruvic acid, 4. Gluconic acid, 5. Malic acid
6. Succinic acid, 7. Lactic acid, 8. Fumaric acid, 9. Acetic acid, 10. Pyroglutamic acid, 11. Carbonic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 4.8mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagent); 1.1mL/min Detector : VIS(430nm) Column temp. : 63deg-C
Organic acids in soy source were analyzed using RSpak KC-811. Generally, comparing the two types of soy source, full body type and light body type, the latter has smaller content of organic acids, especially, the contant of lactic acid is small. Another type of soy source which is produced by chemical method and not by fermentation method is characterized by its large content of leuvlinic acids.
Sample : Soy sauce
1. Phosphoric acid, etc.
2. Citric acid
3. Pyruvic acid
4. Malic acid
5. Succinic acid
6. Lactic acid
7. Formic acid
8. Acetic acid
9. Levulinic acid
10. Pyroglutamic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 4.8mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagent) ; 1.1mL/min Detector : VIS(430nm) Column temp. : 50deg-C
Organic acids in vinegar were analyzed using RSpak KC-811.
Sample : Vinegar, 20 micro-L
(1) :
1. Citric acid , 2. Tartaric acid
3. Malic acid, 4. Succinic acid
5. Acetic acid
(2) :
1. Phosphoric acid, 2. Citric acid
3. Tartaric acid, 4. Gluconic acid
5. Malic acid, 6. Succinic acid
7. Lactic acid, 8. Fumaric acid
9. Acetic acid, 10. Propionic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagent) ; 0.7mL/min Detector : VIS(430nm) Column temp. : 50deg-C
The main organic acids and Vitamin C contained in fruit are separated using RSpak KC-811 with 1mM perchloric acid as an eluent.
Sample : Fruit juice
1. Cl–
2. Oxalic
3. Maleic
4. Citric
5. Malic
6. Vitamin C
7. Succinic
8. Formic
9. Acetic
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 1mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagent); 0.4mL/min Detector : VIS(430nm) Column temp. : 40deg-C
The relative hydrophilicity of gluconic acids with similar molecular structures can be decreased due to their suppressed dissociation in the acidic eluent. Thus, they are retained in RSpak DE-613 column by reversed phase mode and separated.
Sample :
1. HCl
2. 2-Ketogluconic acid
3. Gluconic acid
4. 5-Ketogluconic acid
5. Tartaric acid
6. Glycolic acid
Column : Shodex RSpak DE-613 (6.0mmID*150mm) Eluent : 2mM HClO4 aq. Reagent : 1/10 ST3-R (for post-column method) Flow rate : (Eluent); 0.5mL/min, (Reagent); 0.2mL/min Detector : VIS(430nm) Column temp. : 30 to 32deg-C
Four types of sake are analyzed using RSpak KC-811. As shown here, differences were observed in the total quantity and composition of organic acids. the differences are thought to reflect the production methods and flavours.
Sample : Sake , Japanese rice wine
1. Phosphoric acid
2. Citric acid
3. Pyruvic acid
4. Malic acid
5. Succinic acid
6. Lactic acid
7. Fumaric acid
8. Acetic acid
9. Pyroglutamic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 4.8mM HClO4 Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagent); 1.1mL/min Detector : VIS(430nm) Column temp. : 63deg-C
Organic acids in white wine were analyzed using RSpak KC-811. Fruit contains a large amount of malic acid. For wine, malic acid is transformed to lactic acid by enzymes during malolactic fermentation. The result of this transformation is said to give wine a mild taste. For high quality white wine, the chromatogram shows almost all the malic acid has been transformed to lactic acid.
Sample : Wine
1. Citric acid
2. Tartaric acid
3. Malic acid
4. Succinic acid
5. Lactic acid
6. Fumaric acid
7. Acetic acid
8. Pyroglutamic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : (Eluent); 1.0mL/min, (Reagent); 0.7mL/min Detector : VIS(430nm) Column temp. : 45deg-C
Although oxalic acid cannot be separated from chloride (from NaCl) under normal eluent conditions, the quantification of oxalic acid is made possible by using 50mM perchloric acid as an eluent using RSpak KC-811. Under such conditions, oxalic acid should be detected by RI or UV (at 210nm) detector.
Sample :
1. Anionic Compounds (Cl–)
2. Oxalic
3. Citric
4. Tartaric
5. Phosphoric
6. Malic
7. alpha-Ketoglutaric
8. Malonic
9. Pyruvic
10. Succinic
11. Lactic
12. Fumaric
13. Acetic
14. Pyroglutamic
Columns : Shodex RSpak KC-811 (8.0mmID*300mm) x 2 Eluent : 50mM HClO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 50deg-C
Organic acids in liquid for permanent wave were analyzed using RSpak NN-814.
Sample :
1. Thioglycolic acid
2. Dithioglycolic acid
3. Cysteine
4. Thioglycolic acid and cysteine mixture disulfide
Column : Shodex RSpak NN-814 (8.0mmID*250mm) Eluent : 0.06% H3PO4 + 0.138% NaH2PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 45deg-C
Camphor and benzoic acid were analyzed using RSpak NN-814.
Sample :
1. Ethanol
2. Camphor
3. Benzoic acid
Column : Shodex RSpak NN-G (6.0mmID*50mm) + NN-814 (8.0mmID*250mm) Eluent : 10mM H3PO4 aq./CH3CN=80/20 Flow rate : 0.8mL/min Detector : Shodex RI Column temp. : 40deg-C
Soybean paste was analyzed using RSpak KC-811.
Sample : Soybean paste extract, 100micro-L
1. Citric acid
2. Malic acid
3. Lactic acid
4. Formic acid
5. Acetic acid
6. Pyroglutamic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : 1.0mL/min Detector : VIS(430nm) Column temp. : 40deg-C
Soy sauce for noodles was analyzed using RSpak KC-811.
sample : Soy sauce for noodles, 2micro-L
1. Citric acid
2. Succinic acid
3. Lactic acid
4. Formic acid
5. Acetic acid
6. Levulinic acid
7. Pyroglutamic acid
Columns : Shodex RSpak KC-LG (8.0mmID*50mm) + KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Reagent : 10-fold diluted ST3-R (for post-column method) Flow rate : 1.0mL/min Detector : VIS(430nm) Column temp. : 40deg-C
Organic acids were analyzed using RSpak KC-811.
Sample :
1. Citric acid
2. Malonic acid
3. Succinic acid
4. Lactic acid
5. Formic acid
6. Acetic acid
Column : Shodex RSpak KC-811 (8.0mmID*300mm) Eluent : 3mM HClO4 aq. Flow rate : 1.0mL/min Detector : Non-suppressed coductivity, UV(210nm) Column temp. : 60deg-C
Organic acids in black-vinegar were separated using organic-acids-analysis column, RSpak KC-811.
Sample : Black vinegar, 20micro-L
1. Gluconic acid
2. Lactic acid
3. Acetic acid
4. Pyroglutamic acid
Columns : Shodex RSpak KC-811 (8.0mmID*300mm) x 2 Eluent : 3mM HClO4 aq. Flow rate : 1.0mL/min Detector : UV(210nm), Shodex RI Column temp. : 50deg-C