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Guideline for reversed-phase column choice
Hydrophobic interaction
Hydrophobic interaction is the basis of ODS silica. It is affected by the type and reaction conditions of silylation reagents used for introducing C18 groups. A separation factor (value) of toluene and benzene was defined as a hydrophobic interaction value. In general, a packing material with a larger number of carbon atoms tends to have a larger value. Retention of neutral compounds increases with increasing hydrophobic interaction.
CAPCELL PAK shows an average value among commercially available columns, and can be viewed as a "standard" C18. (UG80 uses silica with a large surface area as a starting material. Its hydrophobicity is large in comparison with those of other CAPCELL PAK columns.)
Hydrogen bonding
Hydrogen bonding depends on residual silanol groups on silica or hydroxyl groups derived from a reagent (di- or tri-functional silylation agents). Packing materials with their OH groups end-capped well tend to show a smaller value, and the value varies according to the surface polarity. Three types of residual silanol groups exist: isolated, vicinal and geminal (refer to Fig. 5). Here, hydrogen bonding is defined as a separation factor of caffeine and phenol. CAPCELL PAK shows relatively small values among the commercially available columns. This is because of its unique polymer coating technology produced by controlling the amount of the three types of silanols. CAPCELL PAK C18 ACR has the smallest value, and is hardly affected by the residual silanol groups.
Surface polarity
Surface polarity was defined as a separation factor of methyl benzoate and toluene. Packing materials with a small surface polarity tend to let polar substances elute quickly and sharply. CAPCELL PAK is formed by coating silica with silicone polymer, resulting in a relatively small surface polarity. However, CAPCELL PAK C18 MG, produced under a special process, has a relatively large value. CAPCELL PAK C18 AQ shows the largest value.
Ion exchangeability
Ion exchangeability was defined as a separation factor of 2-ethylpyridine and benzene. Packing materials having large ion exchangeability values strongly adsorb the basic substance, causing delayed retention or a tailing phenomenon. The sources of ion exchangeability are residual silanol groups and metallic impurities in silica. CAPCELL PAK columns show small values, being free from residual silanol groups and metallic impurities.
Coordinate bonding
Adsorption of coordinating compounds is caused by the metal impurities on silica. The parameter was defined as a peak area ratio of phenylacetyl acetone and naphthalene. CAPCELL PAK C18 MG had the smallest value.
Planar structure recognition
Planar structure recognition was defined as a separation factor of o-terphenyl (non-planar) and triphenylene (planar). Polymeric ODS phases have hydrophobic chains extending three-dimensionally, and are known to show a high value. Empirically, a value exceeding 1.6 corresponds to a polymeric ODS.
【Fig. 3 】 Hydrophobicity
 【Fig. 3】 Hydrophobicity
【Fig. 4】  Hydrogen bonding
 【Fig. 4】 Hydrogen bonding
【Fig. 5】  Three types of residual silanol groups
 【Fig. 5】 Three types of residual silanol groups
【Fig. 6 】 Surface polarity
 【Fig. 6】 Surface polarity
【Fig. 7】  Ion exchangeability
 【Fig. 7】 Ion exchangeability
【Fig. 8 】 Coordinate bonding
 【Fig. 8】 Coordinate bonding
【Fig. 9】  Plane recognition capacity
 【Fig. 9】 Planar recognition capacity
Acidic and basic resistances were defined as relative retention values over the initial ones, obtained as follows.
Acidic resistance is evaluated by a step gradient method composed of a mobile phase (pH = 1) containing 2 vol% TFA. The column temperature is set to 60
°C, making the evaluation method quite severe. Our CAPCELL PAK C18 ACR has the highest acidic resistance among commercial columns.
Basic resistance is also obtained through continuous operation in a mobile phase of pH = 10 containing 10% acetonitrile with the column temperature set at 50°C. CAPCELL PAK C18 MG has the highest basic resistance, 95%, among Shiseido columns.
Long-term acidic resistance of ACR
LC conditions for acidic resistance
Column: 4.6 mm i.d. 150 mm
Mobile phase: (A) 2 vol% TFA, H2O, pH 1
(B) 2 vol% TFA, CH3CN
B 65% (20 min)
→ 0% (60 min) → 98% (5 min) → 65% (5 min)
Flow rate: 1.0 mL/min
Temperature: 60
Detection: UV 254 nm
Samples: Uracil, Amylbenzene
*Residual ratio of retention factor for amylbenzene after 14 cycles
Superior basic resistance
LC conditions for basic resistance
Column: 4.6 mm i.d. 150 mm
Mobile phase: 4 mmol/L Na2B4O7/CH3OH = 90/10, pH 10.0 Flow rate: 1.0 mL/min
Temperature: 50°C
Detection: UV 254 nm
Samples: Uracil, Amylbenzene
*Residual ratio (%) of retention factor (k) for benzyl alcohol after 20 hours of liquid flow
1) Y. Ohtsu, H. Fukui, T. Kanda, K. Nakamura, M. Nakana, O. Nakata and Y. Fuziyama, Chromatographia, 24, 351 (1987).
2) O. Shirota, Y. Ohtsu and O. Nakata, J. Chromatogr., Sci., 28, 553 (1990).
3) S. Kobayashi, I. Tanaka, O. Shirota, T. Kanda and Y. Ohtsu, J. Chromatogr. A, 828, 75 (1998).
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