Determination of selectivity of HPLC systems

by correspondence factor analysis

Yuan Wang, Jun Yang, Xin Lu, Guo Wang Xu *

National Chromatographic Research & Analysis Center, Dalian Institute of Chemical Physics,

Chinese Academy of Sciences, Dalian 116023, China

Received 31 December 2006

Abstract

Correspondence factor analysis (CFA) was employed to study the selectivity of 14 HPLC systems. The tested LC systems were

classified as reversed-phase (RP), ion-exchange (IE) and hydrophilic interaction chromatography (HILIC) modes. It was found that

the retentions of the hydrophilic solutes on HILIC column were significantly influenced by the second-order effects besides their

hydrophilic properties. Organic modifiers and residue silanol groups on silica surface both participated in retention. HypersilTM

amino column performed separation in the HILIC mode at appropriate conditions, and its retention mechanism was more similar to

that of HILIC silica column than that of HILIC column coating poly(aspartamide) groups.

# 2007 Guo Wang Xu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: CFA; HILIC; Liquid chromatography

Correspondence factor analysis (CFA) [1] and hierarchical ascending classification (HAC) [1–3] have been proved

feasible in chromatography. CFA is feasible for investigating complex chromatographic behaviors and affinity of

solutes to specific separation mode, since it focuses on the relative aspects of the data and highlights second-order

effects which often play key roles in chromatographic retention. HAC characterizes objects by revealing their

relationships. In chromatography, it has been used to classify selectivity of chromatographic separation systems

according to their similarity.

The aims of this study are to evaluate the similarity of selectivity of several LC systems using water-soluble

solvents by CFA, and understand retention mechanisms in specific separation mode.

Distilled water was purified with a Milli-Q water purification system (Millipore Co., USA). The modifiers in LC

separation included acetonitrile (ACN), methanol (MeOH), formic acid (FA) (HPLC grade, TEDIA, USA) and

ammonium formate (A.R., Sinopharm Chemical Reagent Co. Ltd., Shanghai). Thirty-four standard solutes (Sigma–

Aldrich Inc., USA or Tianjin Chemical Reagent Co. Inc., Tianjin) were diluted or dissolved in water or ethanol before

injection. An Ultra-Plus II capillary liquid chromatograph (Micro-Tech Scientific Inc., USA) coupled with a UVIS 200

detector (Linear Instruments, USA) was employed for all the capillary columns packed in lab. An Agilent 1100 liquid

chromatograph series (Agilent Technologies, USA) was employed for the common columns. The detection wavelength

was set at 254 nm. The details of column dimensions and chromatographic conditions were summarized in Table 1.

www.elsevier.com/locate/cclet

Chinese Chemical Letters 18 (2007) 565–568

* Corresponding author.

E-mail address: xugw@dicp.ac.cn (G.W. Xu).

1001-8417/$ – see front matter # 2007 Guo Wang Xu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2007.03.026

Y. Wang et al. / Chinese Chemical Letters 18 (2007) 565–568566

Table 1

Column dimensions and chromatographic conditions

Code Packing material Column dimensions Mobile phase Flow rate

(mL/min)

C18-A ODS2 (MicroTech Scientific) 300 mm i.d. � 250 mm 86% ACN in water 5.0

C18-M ODS2 (MicroTech Scientific) 300 mm i.d. � 250 mm 90% MeOH in water 5.0

C8-A C8W (MicroTech Scientific) 300 mm i.d. � 150 mm 60% ACN in water 5.0

C8-M C8W (MicroTech Scientific) 300 mm i.d. � 150 mm 70% MeOH in water 5.0

Ph-A Phenyl (KromasilTM) 300 mm i.d. � 200 mm 50% ACN in water 5.0

Ph-M Phenyl (KromasilTM) 300 mm i.d. � 200 mm 60% MeOH in water 5.0

CN-A Cyano (KromasilTM) 300 mm i.d. � 200 mm 50% ACN in water 5.0

CN-M Cyano (KromasilTM) 300 mm i.d. � 200 mm 60% MeOH in water 5.0

NH2(K)-A Amino (KromasilTM) 250 mm i.d. � 200 mm 90% ACN, 0.005 mol/L

NH4COOH with 0.1% FA

3.5

NH2(K)-M Amino (KromasilTM) 250 mm i.d. � 200 mm 90% MeOH, 0.005 mol/L

NH4COOH with 0.1% FA

3.5

NH2(H)-A Amino (HypersilTM) 4.6 mm i.d. � 250 mm 90% ACN, 0.005 mol/L

NH4COOH with 0.1% FA

1000

NH2(H)-M Amino (HypersilTM) 4.6 mm i.d. � 250 mm 90% MeOH, 0.005 mol/L

NH4COOH with 0.1% FA

1000

HILIC (Poly) Poly(aspartamide)

(PolyHYDROXY-ETHYL ATM, PolyLC Inc.)

300 mm i.d. � 200 mm 90% ACN, 0.002 mol/L

NH4COOH with 0.1% FA

5.0

HILIC (Si) Silica (AtlantisTM HILIC Silica, Waters Co.) 2.1 mm i.d. � 150 mm 90% ACN, 0.01 mol/L

NH4COOH with 0.1% FA

250

Fig. 1. CFA map for retention parameters (Xi) of 34 solutes on 14 LC systems in the plane of the principal factorial axes 1 and 2. Solutes: (1) phenol;

(2) 2,6-dimethyl phenol; (3) benzene; (4) toluene; (5) 2-chlorotoluene; (6) 4-chlorotoluene; (7) 1,2,4-trimethyl benzene; (8) biphenyl; (9) 4-chloro

biphenyl; (10) naphthalene; (11) 2,3-dimethyl naphthalene; (12) 2,7-dimethyl naphthalene; (13) acenaphthene; (14) pyrene; (15) fluoranthene; (16)

4-methylbenzyl alcohol; (17) ethyl benzoate; (18) diethyl phthalate; (19) uracil; (20) uridine; (21) pseudouridine; (22) palmatine; (23) atenolol; (24)

cinchonine; (25) nicotine; (26) caffeine; (27) alprenolol; (28) propanolol; (29) hippuric acid; (30) 3,5-dinitrobenzoic acid; (31) 4-chlorobenzoic acid;

(32) 4-hydroxybenzoic acid; (33) 4-nitrobenzoic acid; (34) 4-bromobenzoic acid.

The average retention time of triplicate injections of each solute on each separation system was measured and

normalized to a relative retention value named as ‘‘retention parameters’’ (Xi) [4]:

Xi ¼tRi � t0

tR f � t0

(1)

where t0 was the dead time on a separation system; tRi the retention time of solute i; tRf was the retention time of the

solute with the longest retention time on this system. Thus, each retention time was scaled ranging from 0 to 1 in spite

of its absolute magnitude.

HAC were achieved with the Statistical Package for the Social Science (SPSS) software (V13.0 for windows). CFA

was achieved with a plug-in unit for SPSS software; its validation had been certified by processing an example in Ref. [5].

By CFA, 14 tested LC systems were classified into three separation modes, reversed-phase (RP), ion-exchange (IE)

and hydrophilic interaction chromatography (HILIC), based on similarity of their selectivity. Resemblance or the

affinity of the solutes with specific separation modes was also plotted in CFA maps (Fig. 1).

When ACN was used as organic modifier, non-endcapping HypersilTM amino column was projected in the HILIC

section, and located near the HILIC Si column instead of PolyHYDROXYETHYL ATM column coating with

Y. Wang et al. / Chinese Chemical Letters 18 (2007) 565–568 567

Fig. 2. Group linkage dendrograms of 14 tested separation systems.

Fig. 3. Effect of ACN concentrations on retentions of four solutes on HypersilTM amino column.

poly(aspartamide) groups. It was regarded that the organic modifier and residue silanol groups on the surface of silica

particles both participated in retention of polar solutes on the amino column.

The classification of the LC systems by CFA was verified by widely used HAC technique (Fig. 2). Meanwhile, as

shown in Fig. 3, retentions of some tested solutes increased along with the increase of ACN concentration. The

phenomenon certified HILIC separation character of HypersilTM amino column in our study.

In conclusion, CFA was proved to be a powerful technique not only for the classification of the separation systems

and relevant solutes based on similarity of their selectivity, but also for exploitation of the key factors in retention

mechanisms. Hydrophobic solutes were separated on non-polar RP columns mainly based on their hydrophobicity.

Acidic solutes were strongly retained on amino columns under tested chromatographic conditions in IE mode. In

HILIC mode, retentions of hydrophilic solutes on polar stationary phases were multi-influenced by the second-order

interactions. Organic modifiers and functional groups on silica particle surface played significant roles for retention. It

was verified that HypersilTM amino column could perform separation in HILIC mode. The retention behavior of the

non-endcapping amino column was very similar to that of HILIC silica column instead of that of

PolyHYDROXYETHYL ATM column.

Acknowledgments

This study has been supported by the National Natural Science Foundation of China (No. 20675082), the

Foundation for Distinguished Young Scholars from National Natural Science Foundation of China (No. 20425516)

and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-SW-329, KGCX2-SW-213).

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Y. Wang et al. / Chinese Chemical Letters 18 (2007) 565–568568