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Guideline for reversed-phase column choice
Taketoshi Kanda, Ph.D.
Senior Researcher
Chromatography Development Office
Materials Science Laboratory
Shiseido Research Center
Reversed-phase columns are used to at least 80% of all the HPLC applications. Packing materials for the columns basically used to be divided into silica-based type and polymer-based one, each of which has its merits and demerits. However, some companies have recently begun marketing packing materials made with hybrid silica and material with improved capping technology, allowing users to select a column from more versatile types of packing materials corresponding to their needs.
Characteristics of packing material given by its starting material
Polymer-based packing material: This type does not have silanol groups by nature, and it has superior chemical stability, although it generally tends to swell and contract by its chemical stracture. Its limited pressure resistance results in a limited separation efficiency.
Silica-based packing material: This type has a superior separation efficiency because of a availavility of mechanically strong spherical particles of small diameters. Its surface structure consisting of a lot of silanol groups, however, leads to several negative aspects, such as a limited pH range and irreversible adsorption of proteins.
Shiseido has been developing "CAPCELL PAK" series that features the merits of both polymer-based packing materials and silica-based packing materials, using a super-thin film coating technology with silicone polymer1). Recently, Shiseido developed and marketed "CAPCELL PAK C18 ACR," a high-polarity column with the strongest acid resistance in the world, and "CAPCELL PAK C18 AQ," stably usable with 100% water, by applying its unique polymer coating technology.
Conventional type Polymer coating type
【Fig. 1】 CAPCELL PAK and conventional packing material
Choice of right functional group for the target compound
Most of the packing materials for a reversed-phase column are aliphatic types (C1, C8, C18, C30), or aromatic types (Ph, etc.), obtained by silylation of silica. While cyano- or amino-types are produced in a similar chemistry, they behave as a normal phase depending on the condition used.
An appropriate column should be chosen for the target compound. Substances of small hydrophobicity, such as vitamin C or allantoin, are hardly retained in an aliphatic type column, but are retained in a normal phase mode with amino-type column. Among several aliphatic type columns, a column with a smaller carbon number is used for shortening the run time without changing the composition of the mobile phase. For an aromatic substance, using a column that has a phenyl group can provide a p-electron interaction in addition to hydrophobic one.
Analytical methods for ionic substances are basically divided into two types: those with an ion-exchange column, and ion-pair methods using a reversed-phase column with an ion-pair reagent charged oppositely to the sample. Column choice and mobile phase designs are summarized along with names of columns to be used(Fig. 2).
Fig. 2  Column selection method (selection by separation mode)
Fig. 2  Column selection method (selection by separation mode)
Fig. 2  Column selection method (selection by separation mode)
【Fig. 2】 Column selection method (selection by separation mode)
Characteristics of C18 (ODS) columns of different manufacturers
C18 columns, also called ODS columns, are the most widely available among reversed phases, and their properties differ from one company to another. The main sources of such differences are silica as a starting material (surface area, pore diameter, metal impurities, and silanol groups), and reaction conditions and types of silylation reagents.
Therefore, it is important to know the separation characteristics of various compounds which is unique to each column, before choosing a right column for a target compound. Shiseido has made every effort to clearly position Capcell Pak columns by collectively evaluating major commercially available columns under specific conditions using standard substances. The parameters evaluated are: 1) hydrophobicity; 2) hydrogen bonding; 3) surface polarity; 4) ion exchangeability; 5) coordinate bonding; and 6) planar structure recognition. An example is shown below. The parameters were obtained under the HPLC conditions listed below2), 3). Table 1 shows the values for all the columns.
ODS column evaluation method
LC conditions 6)
Table 1 Results obtained with all the columns
Column RT(N)
min
Hydro-phobicity
α(T/B)
Hydrogen bonding
α(CA/Ph)
Surface polarity
α(MB/T)
Ion exchange-ability
α(EP/B)
Coordinate bonding
α(PA/N)
Planar structure recognition
α(TP/OT)
Observed pressure
MPa
CAPCELL PAK C18
MG
36 2.09 0.33 0.43 0.35 0.65 1.32 9.1
CAPCELL PAK C18
UG80
38 2.15 0.30 0.36 0.29 0.65 1.34 8.1
CAPCELL PAK C18
UG120
29 2.09 0.31 0.39 0.31 0.64 1.27 7.9
CAPCELL PAK C18
ACR
36 2.10 0.26 0.38 0.30 0.50 1.64 8.1
A 44 2.12 0.30 0.41 0.38 0.77 1.71 17.8
B 31 2.12 0.29 0.41 0.44 0.63 1.50 9.2
C 45 2.16 0.37 0.46 0.44 0.60 1.27 10.2
D 37 2.17 0.41 0.42 0.37 0.81 1.75 10.5
E 49 2.08 0.34 0.42 0.30 0.05 1.33 7.0
F 44 2.12 0.31 0.41 0.36 0.34 1.67 8.8
G 42 2.08 0.53 0.46 * * 1.84 11.2
H 41 2.10 0.36 0.41 * * 1.66 8.6
I 36 2.12 0.30 0.43 0.37 0.41 1.19 11.1
J 35 1.98 0.29 0.40 0.34 0.54 1.42 8.6
K 32 2.13 0.31 0.41 0.50 0.65 1.48 10.7
L 36 2.06 0.49 0.48 0.60 * 1.75 9.6
M 33 2.09 0.36 0.47 0.43 0.53 1.21 12.5
N 36 2.11 0.33 0.39 0.31 0.41 1.61 10.4
O 20 1.91 0.51 0.47 0.58 0.67 2.07 11.1
P 27 2.08 0.35 0.42 0.52 0.84 1.27 11.7
Q 35 2.15 0.33 0.41 0.36 0.47 1.34 12.0
R 35 2.12 0.32 0.42 0.37 0.65 1.41 12.7
S 33 2.08 0.41 0.51 0.49 0.71 1.24 9.9
T 41 2.14 0.35 0.38 * * 1.60 10.1
U 28 2.13 0.44 0.45 0.59 0.50 1.35 8.6
V 32 2.17 0.28 0.37 0.35 0.80 1.52 8.6
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