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
|