Supplementary data for

Tubing modifications for countercurrent chromatography (CCC): Investigation of geometrical parameters

Michael Englert, Walter Vetter*

University of Hohenheim, Institute of Food Chemistry, Garbenstrasse 28, D-70599 Stuttgart, Germany

* Corresponding author:

Walter Vetter

Phone: +49 711 459 24016

Fax: +49 711 459 24377

e-mail:

Table S-1. Nine different geometrical options of crimping depth and distance between the crimping of the tubing modifications used in this study

Option / Crimping depth
[mm] / Distance between crimping
tubing modification 1 / 0.3 / 0.50
tubing modification 2 / 0.3 / 1.79
tubing modification 3 / 0.3 / 3.07
tubing modification 4 / 0.6 / 0.50
tubing modification 5 / 0.6 / 1.79
tubing modification 6 / 0.6 / 3.07
tubing modification 7 / 0.9 / 0.50
tubing modification 8 / 0.9 / 1.79
tubing modification 9 / 0.9 / 3.07

Table S-2. Design of experiments for the investigation of 4 parameters (crimping depth, distance between crimping, β-value and flow rate) using a full factorial design. The stationary phase retention Sfwas measured in a hydrophobic (HAcn) andanmedium (HTWM) polarity solvent system.

Solvent system / HAcna / HTMWb
No. / crimping depth
[mm] / distance between crimping
[cm] / ß value / flow rate
[mL/min] / Sf (HT) / Sf (TH) / Sf (HT) / Sf (TH)
1 / 0.3 / 3.07 / 0.54 / 0.5 / 0.86 / 0.87 / 0.77 / 0.81
2 / 0.9 / 0.50 / 0.62 / 0.5 / 0.85 / 0.86 / 0.85 / 0.84
3 / 0.6 / 3.07 / 0.62 / 1.0 / 0.84 / 0.86 / 0.76 / 0.81
4 / 0.6 / 0.50 / 0.58 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
5 / 0.9 / 3.07 / 0.54 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
6 / 0.9 / 1.79 / 0.62 / 2.0 / 0.80 / 0.83 / 0.78 / 0.83
7 / 0.6 / 0.50 / 0.62 / 3.0 / 0.81 / 0.82 / 0.67 / 0.79
8 / 0.9 / 0.50 / 0.58 / 2.0 / 0.85 / 0.85 / 0.76 / 0.76
9 / 0.3 / 1.79 / 0.58 / 4.0 / 0.63 / 0.68 / 0.57 / 0.58
10 / 0.6 / 3.07 / 0.58 / 2.0 / 0.78 / 0.83 / 0.76 / 0.81
11 / 0.6 / 3.07 / 0.54 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
12 / 0.9 / 0.50 / 0.58 / 4.0 / 0.66 / 0.71 / 0.61 / 0.62
13 / 0.3 / 3.07 / 0.62 / 2.0 / 0.74 / 0.80 / 0.75 / 0.80
14 / 0.9 / 3.07 / 0.58 / 4.0 / 0.61 / 0.66 / 0.57 / 0.57
15 / 0.3 / 0.50 / 0.58 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61
16 / 0.9 / 3.07 / 0.62 / 2.0 / 0.77 / 0.80 / 0.75 / 0.80
17 / 0.9 / 0.50 / 0.54 / 2.0 / 0.77 / 0.80 / 0.75 / 0.80
18 / 0.9 / 0.50 / 0.62 / 3.0 / 0.83 / 0.84 / 0.70 / 0.83
19 / 0.6 / 0.50 / 0.54 / 0.5 / 0.86 / 0.85 / 0.76 / 0.80
20 / 0.3 / 3.07 / 0.62 / 3.0 / 0.69 / 0.69 / 0.56 / 0.66
21 / 0.6 / 0.50 / 0.58 / 4.0 / 0.63 / 0.68 / 0.58 / 0.59
22 / 0.6 / 1.79 / 0.58 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
23 / 0.3 / 3.07 / 0.62 / 4.0 / 0.62 / 0.67 / 0.62 / 0.63
24 / 0.6 / 3.07 / 0.54 / 3.0 / 0.81 / 0.85 / 0.72 / 0.74
25 / 0.6 / 3.07 / 0.62 / 3.0 / 0.75 / 0.76 / 0.62 / 0.73
26 / 0.9 / 1.79 / 0.54 / 3.0 / 0.80 / 0.82 / 0.71 / 0.73
27 / 0.6 / 0.50 / 0.58 / 2.0 / 0.81 / 0.84 / 0.72 / 0.72
28 / 0.9 / 3.07 / 0.54 / 4.0 / 0.61 / 0.66 / 0.57 / 0.57
29 / 0.9 / 3.07 / 0.62 / 0.5 / 0.85 / 0.86 / 0.75 / 0.80
30 / 0.6 / 1.79 / 0.58 / 4.0 / 0.63 / 0.68 / 0.58 / 0.58
31 / 0.9 / 1.79 / 0.58 / 0.5 / 0.85 / 0.86 / 0.75 / 0.80
32 / 0.9 / 3.07 / 0.62 / 3.0 / 0.79 / 0.80 / 0.65 / 0.77
33 / 0.3 / 3.07 / 0.58 / 0.5 / 0.85 / 0.86 / 0.75 / 0.80
34 / 0.3 / 0.50 / 0.58 / 0.5 / 0.86 / 0.87 / 0.77 / 0.81
35 / 0.9 / 0.50 / 0.54 / 3.0 / 0.83 / 0.86 / 0.74 / 0.76
36 / 0.3 / 1.79 / 0.54 / 2.0 / 0.71 / 0.77 / 0.65 / 0.65
37 / 0.3 / 1.79 / 0.58 / 3.0 / 0.68 / 0.71 / 0.60 / 0.61
38 / 0.6 / 3.07 / 0.58 / 0.5 / 0.85 / 0.86 / 0.75 / 0.80
39 / 0.9 / 3.07 / 0.62 / 4.0 / 0.73 / 0.78 / 0.72 / 0.73
40 / 0.6 / 0.50 / 0.62 / 4.0 / 0.76 / 0.80 / 0.62 / 0.76
41 / 0.6 / 1.79 / 0.54 / 0.5 / 0.86 / 0.88 / 0.75 / 0.80
42 / 0.3 / 0.50 / 0.58 / 1.0 / 0.82 / 0.84 / 0.73 / 0.75
43 / 0.3 / 0.50 / 0.58 / 4.0 / 0.59 / 0.64 / 0.54 / 0.54
44 / 0.9 / 1.79 / 0.54 / 0.5 / 0.90 / 0.90 / 0.80 / 0.82
45 / 0.3 / 0.50 / 0.54 / 0.5 / 0.85 / 0.86 / 0.76 / 0.80
46 / 0.6 / 0.50 / 0.58 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61
47 / 0.3 / 1.79 / 0.54 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
48 / 0.9 / 3.07 / 0.54 / 3.0 / 0.78 / 0.81 / 0.69 / 0.71
49 / 0.3 / 3.07 / 0.54 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
50 / 0.3 / 1.79 / 0.62 / 0.5 / 0.86 / 0.87 / 0.77 / 0.81
51 / 0.9 / 3.07 / 0.54 / 2.0 / 0.79 / 0.84 / 0.72 / 0.72
52 / 0.3 / 3.07 / 0.58 / 2.0 / 0.71 / 0.77 / 0.69 / 0.74
53 / 0.3 / 3.07 / 0.54 / 3.0 / 0.68 / 0.71 / 0.60 / 0.61
54 / 0.3 / 3.07 / 0.58 / 4.0 / 0.59 / 0.64 / 0.54 / 0.54
55 / 0.3 / 0.50 / 0.62 / 0.5 / 0.86 / 0.88 / 0.79 / 0.82
56 / 0.9 / 0.50 / 0.54 / 1.0 / 0.82 / 0.84 / 0.74 / 0.78
57 / 0.6 / 0.50 / 0.62 / 1.0 / 0.85 / 0.86 / 0.77 / 0.81
58 / 0.9 / 3.07 / 0.58 / 0.5 / 0.88 / 0.89 / 0.81 / 0.83
59 / 0.9 / 1.79 / 0.58 / 4.0 / 0.75 / 0.80 / 0.61 / 0.75
60 / 0.6 / 0.50 / 0.54 / 2.0 / 0.73 / 0.76 / 0.71 / 0.76
61 / 0.3 / 0.50 / 0.54 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61
62 / 0.6 / 1.79 / 0.62 / 1.0 / 0.81 / 0.86 / 0.76 / 0.81
63 / 0.6 / 0.50 / 0.54 / 4.0 / 0.66 / 0.70 / 0.62 / 0.62
64 / 0.3 / 0.50 / 0.58 / 2.0 / 0.74 / 0.77 / 0.65 / 0.65
65 / 0.9 / 1.79 / 0.62 / 3.0 / 0.82 / 0.84 / 0.68 / 0.80
66 / 0.3 / 0.50 / 0.54 / 4.0 / 0.60 / 0.64 / 0.56 / 0.56
67 / 0.9 / 0.50 / 0.58 / 0.5 / 0.89 / 0.90 / 0.86 / 0.91
68 / 0.6 / 1.79 / 0.58 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61
69 / 0.9 / 1.79 / 0.54 / 4.0 / 0.63 / 0.67 / 0.58 / 0.58
70 / 0.9 / 3.07 / 0.58 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61
71 / 0.9 / 0.50 / 0.62 / 1.0 / 0.84 / 0.86 / 0.82 / 0.85
72 / 0.9 / 0.50 / 0.54 / 4.0 / 0.60 / 0.64 / 0.56 / 0.56
73 / 0.9 / 3.07 / 0.58 / 2.0 / 0.82 / 0.86 / 0.75 / 0.76
74 / 0.6 / 3.07 / 0.62 / 2.0 / 0.83 / 0.84 / 0.72 / 0.72
75 / 0.3 / 3.07 / 0.62 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
76 / 0.6 / 0.50 / 0.54 / 1.0 / 0.82 / 0.83 / 0.73 / 0.76
77 / 0.9 / 0.50 / 0.54 / 0.5 / 0.84 / 0.86 / 0.82 / 0.85
78 / 0.9 / 0.50 / 0.58 / 3.0 / 0.74 / 0.77 / 0.66 / 0.66
79 / 0.9 / 1.79 / 0.58 / 2.0 / 0.86 / 0.86 / 0.78 / 0.79
80 / 0.6 / 1.79 / 0.62 / 3.0 / 0.78 / 0.80 / 0.65 / 0.76
81 / 0.6 / 1.79 / 0.54 / 4.0 / 0.66 / 0.70 / 0.62 / 0.61
82 / 0.3 / 1.79 / 0.62 / 1.0 / 0.82 / 0.84 / 0.73 / 0.74
83 / 0.6 / 3.07 / 0.62 / 4.0 / 0.70 / 0.74 / 0.57 / 0.69
84 / 0.3 / 0.50 / 0.62 / 4.0 / 0.69 / 0.73 / 0.56 / 0.69
85 / 0.6 / 0.50 / 0.62 / 0.5 / 0.86 / 0.87 / 0.85 / 0.84
86 / 0.9 / 1.79 / 0.62 / 0.5 / 0.90 / 0.91 / 0.85 / 0.85
87 / 0.6 / 0.50 / 0.58 / 0.5 / 0.84 / 0.86 / 0.75 / 0.80
88 / 0.6 / 0.50 / 0.54 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61
89 / 0.6 / 1.79 / 0.62 / 0.5 / 0.84 / 0.86 / 0.75 / 0.80
90 / 0.3 / 1.79 / 0.62 / 4.0 / 0.66 / 0.70 / 0.54 / 0.66
91 / 0.3 / 0.50 / 0.62 / 1.0 / 0.83 / 0.85 / 0.75 / 0.80
92 / 0.6 / 1.79 / 0.54 / 2.0 / 0.80 / 0.81 / 0.69 / 0.69
93 / 0.3 / 3.07 / 0.54 / 2.0 / 0.76 / 0.77 / 0.65 / 0.65
94 / 0.3 / 1.79 / 0.58 / 0.5 / 0.84 / 0.86 / 0.75 / 0.80
95 / 0.6 / 3.07 / 0.54 / 0.5 / 0.84 / 0.86 / 0.75 / 0.79
96 / 0.9 / 0.50 / 0.58 / 1.0 / 0.86 / 0.87 / 0.80 / 0.82
97 / 0.3 / 1.79 / 0.54 / 4.0 / 0.60 / 0.64 / 0.56 / 0.56
98 / 0.9 / 0.50 / 0.62 / 2.0 / 0.84 / 0.85 / 0.84 / 0.86
99 / 0.6 / 1.79 / 0.62 / 2.0 / 0.78 / 0.85 / 0.79 / 0.85
100 / 0.6 / 1.79 / 0.54 / 1.0 / 0.84 / 0.86 / 0.74 / 0.77
101 / 0.3 / 0.50 / 0.62 / 2.0 / 0.77 / 0.80 / 0.75 / 0.80
102 / 0.6 / 1.79 / 0.58 / 2.0 / 0.78 / 0.84 / 0.79 / 0.85
103 / 0.6 / 3.07 / 0.54 / 4.0 / 0.63 / 0.67 / 0.59 / 0.59
104 / 0.3 / 3.07 / 0.58 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61
105 / 0.9 / 3.07 / 0.62 / 1.0 / 0.86 / 0.87 / 0.78 / 0.81
106 / 0.9 / 1.79 / 0.54 / 2.0 / 0.82 / 0.87 / 0.75 / 0.75
107 / 0.3 / 3.07 / 0.58 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
108 / 0.3 / 1.79 / 0.54 / 0.5 / 0.85 / 0.80 / 0.75 / 0.80
109 / 0.6 / 1.79 / 0.62 / 4.0 / 0.73 / 0.77 / 0.59 / 0.73
110 / 0.9 / 0.50 / 0.62 / 4.0 / 0.79 / 0.84 / 0.64 / 0.79
111 / 0.3 / 1.79 / 0.62 / 2.0 / 0.71 / 0.77 / 0.72 / 0.77
112 / 0.6 / 3.07 / 0.58 / 4.0 / 0.63 / 0.67 / 0.59 / 0.59
113 / 0.3 / 1.79 / 0.54 / 3.0 / 0.68 / 0.71 / 0.60 / 0.61
114 / 0.3 / 0.50 / 0.62 / 3.0 / 0.74 / 0.76 / 0.61 / 0.72
115 / 0.3 / 3.07 / 0.54 / 4.0 / 0.63 / 0.67 / 0.59 / 0.59
116 / 0.6 / 3.07 / 0.54 / 2.0 / 0.74 / 0.77 / 0.65 / 0.65
117 / 0.9 / 1.79 / 0.62 / 4.0 / 0.76 / 0.81 / 0.61 / 0.75
118 / 0.9 / 1.79 / 0.58 / 3.0 / 0.71 / 0.74 / 0.62 / 0.62
119 / 0.9 / 1.79 / 0.62 / 1.0 / 0.85 / 0.86 / 0.79 / 0.80
120 / 0.9 / 1.79 / 0.58 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
121 / 0.6 / 3.07 / 0.62 / 0.5 / 0.86 / 0.88 / 0.80 / 0.83
122 / 0.3 / 1.79 / 0.62 / 3.0 / 0.71 / 0.73 / 0.59 / 0.69
123 / 0.6 / 1.79 / 0.58 / 0.5 / 0.91 / 0.92 / 0.82 / 0.86
124 / 0.6 / 3.07 / 0.58 / 1.0 / 0.85 / 0.85 / 0.73 / 0.80
125 / 0.3 / 1.79 / 0.58 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
126 / 0.3 / 1.79 / 0.58 / 2.0 / 0.71 / 0.77 / 0.65 / 0.65
127 / 0.9 / 3.07 / 0.58 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
128 / 0.6 / 0.50 / 0.62 / 2.0 / 0.84 / 0.85 / 0.83 / 0.88
129 / 0.9 / 3.07 / 0.54 / 0.5 / 0.86 / 0.87 / 0.80 / 0.82
130 / 0.6 / 1.79 / 0.54 / 3.0 / 0.68 / 0.71 / 0.60 / 0.61
131 / 0.3 / 0.50 / 0.54 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
132 / 0.3 / 0.50 / 0.54 / 2.0 / 0.75 / 0.77 / 0.65 / 0.65
133 / 0.9 / 1.79 / 0.54 / 1.0 / 0.81 / 0.83 / 0.71 / 0.74
134 / 0.3 / 3.07 / 0.62 / 0.5 / 0.86 / 0.87 / 0.77 / 0.82
135 / 0.6 / 3.07 / 0.58 / 3.0 / 0.69 / 0.71 / 0.60 / 0.61

Fig. S-1.Stationary phase retention Sf at flow rates of 0.5, 1, 2, 3 and 4mLmin−1 determined for head-to-tail and tail-to-head mode in dependency of the distance between the crimping in the non-polar n-hexane/acetonitrile (1:1, v/v, HAcn) and the medium polarity n-hexane/tBME/methanol/water (5:2:5:3, v/v/v/v, HTMW) solvent system.The average of the individual Sf values at the different flow rates (Sf,0.5 + Sf,1 + Sf,2 + Sf,3 + Sf,4)/5 were used to avoid fixing one parameter at a specific level.Data from Table S-2.


Fig. S-2.Stationary phase retention Sf at flow rates of 0.5, 1, 2, 3 and 4mLmin−1 determined for head-to-tail and tail-to-head mode in dependency of the crimping depth in the non-polar n-hexane/acetonitrile (1:1, v/v, HAcn) and the medium polarity n-hexane/tBME/methanol/water (5:2:5:3, v/v/v/v, HTMW) solvent system.The average of the individual Sf values at the different flow rates (Sf,0.5 + Sf,1 + Sf,2 + Sf,3 + Sf,4)/5 were used to avoid fixing one parameter at a specific level.Data from Table S-2.

Fig. S-3. Evaluationof peak widths wbof the peaks of a) methyl p-hydroxybenzoate, b)ethyl p-hydroxybenzoate, c)isopropyl p-hydroxybenzoate, d)propyl p-hydroxybenzoate, e) isobutyl p-hydroxybenzoateand f) butyl p-hydroxybenzoate obtained during the CCC separation with the medium polarity n-hexane/tBME/methanol/water (5:2:5:3, v/v/v/v, HTMW) solvent at flow rates of 0.5, 1, 2, 3 and 4 mL min-1 and at three different crimping depths.Data from Table1.


Fig. S-4.Resolution Rs obtained between the peaks of (a) methyl p-hydroxybenzoate and ethyl p-hydroxybenzoate, (b) ethyl p-hydroxybenzoate and isopropyl p-hydroxybenzoate, (c) isopropyl p-hydroxybenzoate and propyl p-hydroxybenzoate, (d) propyl p-hydroxybenzoate and isobutyl p-hydroxybenzoate and (e) isobutyl p-hydroxybenzoate and butyl p-hydroxybenzoate using the medium polarity n-hexane/tBME/methanol/water (5:2:5:3, v/v/v/v, HTMW) solvent system with three different crimping depths at five different flow rates 0.5, 1, 2, 3 and 4mLmin−1. Data from Table 1.