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
Effect of column temperature on elution volume when RSpak KC-811 is used is shown.

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