d

br e
c th
an
ifi r
nt e
ie nd
Sc u
o old
m s

A New Phenyl-Type Stationary Phase and Its Uses

er w
Th No

Xiaodong Liu, Mark Tracy, Jinhua Chen, and Christopher Pohl

Dionex Corporation, Sunnyvale, CA, USA

Introduction Chromatographic Evaluations

Phenyl-type columns provide strong pi-pi interaction and offer selectivity High Aromatic Selectivity
complementary to other reversed-phase columns (e.g., C18). These
Triphenylene and o-terphenyl contain the same number of carbon
columns have been successfully used to separate positional isomers,
atoms, but they are arranged in different shapes—the former planar, the
tocopherols, flavonoids, polynuclear aromatics and nitroaromatic
latter propeller shaped. The retention factor ratio between triphenylene
compounds, active pharmaceutical ingredients (APIs) and related
(T) and o-terphenyl (O), α(T/O) = k’T /k’O is often used as a descriptor to
compounds. Early phenyl columns contained short alkyl phenyl ligands
measure column steric selectivity. However, with phenyl-type columns,
covalently bound to the silica surface. Later, diphenyl phases were
this descriptor measures the aromatic selectivity rather than steric
developed to enhance pi-pi interaction. Due to the short alkyl link, these
selectivity. Enhanced aromatic selectivity is beneficial for applications
columns typically lack hydrophobic retention and exhibit low hydrolytic
in drug development and testing where compounds commonly contain
stability. To improve these features, phenyl columns with longer alkyl
rings, conjugation byproducts, and ring substituents. In Figure 1, the
links were developed. By increasing the length of the alkyl spacer,
Dionex Acclaim Phenyl-1 column demonstrates the highest α(T/O) value
steric selectivity and aromatic selectivity can be enhanced. Recently, a
compared to other commercial phenyl-type phases including biphenyl,
biphenyl phase was commercialized that exhibited both hydrophobic
diphenyl, C6-phenyl, and C3-phenyl phases.
retention and pi-pi interaction compared to other commercially available
phenyl columns. Here, the authors describe a newly developed phenyl
column that features strong pi-pi interaction, high hydrophobicity, 100% Column: 4.6 × 150 mm, 3 µm
aqueous compatibility, and unique selectivity compared to C18 and other Mobile Phase: Acetonitrile/DI water
phenyl-type columns in the market. Chromatographic evaluations and v/v 80/20
Temperature: 40 °C
separations of aromatic hydrocarbons, steroids, fat-soluble vitamins, 3.5 3.30 Flow Rate: 1 mL/min
and phospholipids on this column are shown. α(T/O)=k’Triphenylene/k’o-Terphenyl Inj. Volume: 5 µL
Detection: UV at 254 nm
Sample: 0.5 mg/mL (each)
Dionex Acclaim Phenyl-1 Overview α(T/O)
Probes:
1.46
The Thermo Scientific Dionex Acclaim® Phenyl-1 column is based 0.87
1.17 1.00
on covalent modification of high-purity, spherical, porous silica
o-Terphenyl
particles with a specially designed silane ligand bearing proprietary 0
alkyl aromatic functionality. This novel column chemistry provides the Acclaim Biphenyl Diphenyl Phenyl Phenyl
Phenyl-1 Hexyl Propyl
following benefits.
Triphenylene
• High aromatic selectivity
27621
• High hydrophobic retention
Figure 1. Aromatic selectivity comparison.
• Unique and complementary selectivity
• Compatibility with highly aqueous mobile phase
• High efficiency and rugged packing
High Hydrophobic Retention Column: Acclaim Phenyl-1, 3 µm
Dimensions: 4.6 × 150 mm
Due to the incorporation of a long alkyl chain on the stationary phase, Mobile Phase: 0.1% Formic acid
the Dionex Acclaim Phenyl-1 exhibits high hydrophobicity compared Temperature: 30 °C
Flow Rate: 1 mL/min
to other commercial phenyl-type phases, including biphenyl, diphenyl, 5 Inj. Volume: 2.5 µL
C6-phenyl and C3-phenyl phases (Figure 2). This feature is desirable Detection: UV at 254 nm
4
for retaining a wide range of analytes. Sample: 0.1 mg/mL (each)
Probe: Thymine
3

Minutes
Stop-Flow Protocol:
2
1. Test the column under above conditions
Column: 4.6 × 150 mm, 3 µm
2. Stop the pump for 5 min
Mobile Phase: Acetonitrile/DI water 1
(pump pressure drops to 0)
v/v 80/20
3. Resume the flow and condition the
Temperature: 40 °C 0
0 5 10 15 20 25 column under above conditions
Flow Rate: 1 mL/min
for 5 min
Inj. Volume: 5 µL Stop-Flow Cycle Time
3 2.78 4. Inject the sample and run the test
2.67 Detection: UV at 254 nm
under the same conditions
Sample: 0.5 mg/mL (each)
5. Repeat 2 through 4
k’ of pentylbenzene

Probe: 27625
1.70
1.59
1.2 Figure 4. The Acclaim Phenyl-1 column demonstrates full compatibility with
aqueous conditions.
Pentylbenzene
0
Acclaim
Phenyl-1
Biphenyl Diphenyl Phenyl
Hexyl
Phenyl
Propyl
Applications
27622 Fullerenes
Figure 2. Hydrophobicity comparison. The increased usage of nanomaterials has triggered growing concern
regarding their environmental behavior in aquatic environments. The
ability to detect and quantify nanomaterials in complex water matrices
Column: See traces Peaks: 1. Benzo[a]pyrene (BaP) has become an important issue. C60 fullerene is of special interest
Dimensions: 4.6 × 150 mm, 3 µm because of the widespread application of nanocarbon technology.
Mobile Phase: Acetonitrile/DI water v/v 85/15
Temperature: 25 °C Figure 5 shows separations of fullerenes in fullerene soot under isocratic
Flow Rate: 1 mL/min conditions with heptane/isopropanol mobile phase using both a C18
Inj. Volume: 5 µL 2. Phenanthro
Detection: UV at 254 nm [3,4-c]phenanthrene (PhPh)
column and the Dionex Acclaim Phenyl-1. Compared to the C18
Sample: NIST SRM 869a standard mix column, the Dionex Acclaim Phenyl-1 exhibits different selectivity and
2 3 provides better resolution for minor components.
Acclaim Phenyl-1 1

3
3. Dibenzo[g,p]chrysene (TBN) Column: Acclaim Phenyl-1, 3 µm,
1 2
Pursuit® XRs, Diphenyl 3.0 × 150 mm
C60 Oxide Acclaim 120 C18, 3 µm,
AU 3 60 3.0 × 150 mm
C60 C70 Acclaim Phenyl-1 Mobile Phase: Heptane/iso-propanol
2
1 v/v 60/40 (Phenyl-1)
Pinnacle® Biphenyl C70 Oxide Heptane/iso-propanol
3 Higher than C70 fullerenes v/v 50/50 (120 C18)
Temperature: 40 °C
2
1 Flow Rate: 0.45 mL/min
Zorbax® Eclipse® Plus Hexyl Phenyl Inj. Volume: 10 µL
0 mAU C60 C70 Detection: UV at 325 nm
C60 Oxide
0 2 4 6 8 10 Acclaim 120 C18 Sample: Toluene extract of
Minutes 27624 fullerene soot
C70 Oxide
Figure 3. Polyaromatic hydrocarbon (PAH) selectivity comparison—separation Higher than C70 fullerenes
of NIST SRM 869a. 0

Fully Compatible with Highly Aqueous Mobile Phase C60 C70

0 4 8 12 16 20
Full compatibility with highly aqueous mobile phases is a desirable Minutes 28097
feature for HPLC columns. Figure 4 demonstrates that the Dionex Acclaim
Figure 5. Separations of fullerenes in fullerene soot: Acclaim Phenyl-1 vs
Phenyl-1 performs consistently well under 100% aqueous conditions.
Acclaim 120 C18.

2 A New Phenyl-Type Stationary Phase and Its Uses

Phospholipids 400
Column: Acclaim Phenyl-1, 3 µm
Lecithin is a generic term for the yellow-brown fatty substances occurring Dimension: 3.0 × 150 mm
Mobile Phase: Acetonitrile/2-propanol/ammonium
in animal and plant tissues. It is widely used for applications in human acetate (100 mM, pH 5.0) v/v/v 70/10/20
food, animal feed, pharmaceutical, paint, and other industrial applications. Temperature: 25 °C
Flow Rate: 0.45 mL/min
Depending on the source, the composition of lecithin can vary. Inj. Volume: 5 µL
Phospholipids are a class of lipids and are a major component of all cell mV Detection: Corona ultra (Gain = 100 pA; Filter = low;
Nebulizer Temp. = 25 °C)
membranes, as they can form lipid bilayers and are components in lecithin. Sample: Soybean lecithin (1 mg/mL in 2-propanol)

Figure 6 shows the profiles of lecithin from egg yolk and soybean
obtained using a Dionex Acclaim Phenyl-1 column and Thermo
Scientific ESA Corona® ultra™ Charge Aerosol Detector. While both egg
0
yolk and soybean contain phospholipids (e.g., phosphatidylcholine,
0 4 8 12 16 20
phosphatidylethanolamine, and phosphatidylinositol, shown as Minutes 27630
early-eluting peaks in the 2 to 4 min range), significant quantity of
triglycerides in egg yolk is shown as later eluting peaks from 7 to 13 Figure 7. Separation of phospholipids in soybean lecithin.
min. To obtain detailed information on phospholipid composition, a
mobile phase containing higher aqueous and less isopropanol is used to
Fat-Soluble Vitamins
resolve major components of phospholipids in soybean lecithin (Figure 7).
Fat-soluble vitamins (A, D, E, and K) are essential nutrients with small
amounts required for various roles in the human body. Fat-soluble
vitamins are stored in the liver and fatty tissues, and are eliminated at
a much slower rate. As a result, they can pose a greater risk for toxicity
than water-soluble vitamins when consumed in excess. Conversly, some
1000 O health problems may decrease the absorption of fat, and in turn, decrease
Soybean Lecithin
O
O
O O the absorption of vitamins A, D, E, and K. Analyses of fat-soluble
P N
+

O O
-
O vitamins are important and challenging assays for various products like
Column: Acclaim Phenyl-1, 3 µm pharmaceuticals, foods, and nutritional supplements. As shown in Figure
Dimension: 3.0 × 150 mm
Mobile Phase: Acetonitrile/2-propanol/ 8, the Dionex Acclaim Phenyl-1 column provides excellent selectivity for
mV ammonium acetate separating vitamins A, D2, D3, K1, K2, as well as E, E acetate and related
(100 mM, pH5.0)
Egg Lecithin v/v/v 45/45/10
substances, δ- and γ-tocopherols.
Temperature: 25 °C
Flow Rate: 0.45 mL/min
Inj. Volume: 2 µL
Detection: Corona ultra (Gain = 100 pA; Column: Acclaim Phenyl-1, 3 µm
0 60 Dimension: 3.0 × 150 mm
Filter = low;
0 4 8 12 16 20 Nebulizer Temp. = 25 °C) Mobile Phase: Methanol/water v/v 90/10
2 Temperature: 30 °C
Minutes Sample: Soybean Lecithin 3
(1 mg/mL in 2-propanol) Flow Rate: 0.5 mL/min
Egg Lecithin Inj. Volume: 2 µL
(2.5 mg/mL in 2-propanol) Detection: UV at 220 nm
Peaks: (100 ppm each)
27629 1. Retinol acetate
4 5 (vitamin A acetate)
mAU
Figure 6. Analysis of soybean and egg lecithin. 6 9 2. Vitamin D2
8 3. Vitamin D3
4. δ-Tocopherol
1 10 5. γ-Tocopherol
7 6. α-Tocopherol (vitamin E)
7. Impurity (unknown)
8. Vitamin E acetate
0 9. Vitamin K2
10. Vitamin K1
0 6 12 18 24 30
Minutes 27628

Figure 8. Separation of fat-soluble vitamins.

3
 Estrogens Column: Acclaim Phenyl-1, 3 µm
Dimension: 3.0 × 250 mm
Estrogens are a group of steroid compounds that function as the 80
Mobile Phase: Methanol/water v/v 46/54
primary female sex hormone. Three major naturally occurring estrogens Temperature: 40 °C
in women are estrone (E1), estradiol (E2), and estriol (E3). Estradiol 1 Flow Rate: 0.5 mL/min
Inj. Volume: 5 µL
(E2) is the predominant form in nonpregnant females, estrone is produced 23
4 Detection: UV at 254 nm
during menopause, and estriol is the primary estrogen of pregnancy. Peaks: (50 ppm each)
Ethynylestradiol, a derivative of estradiol, is an orally bioactive estrogen 1. Prednisone
7 2. Cortisone
used in almost all modern formulations of combined oral contraceptive mAU 56 8 3. Prednisolone
pills. While the standard C18 column fails to separate these four 4. Hydrocortisone
compounds, the Dionex Acclaim Phenyl-1 column can resolve them to 5. Dexamethasone
6. 6-Methylprednisolone
baseline isocratically (Figure 9). 7. Corticosterone
8. Deoxyhydrocortisone
0

Columns: See traces

Peaks: (100 ppm each)
Dimensions: 4.6 × 150 mm, 3 µm 0 8 16 24 32 40
1. Estriol (E3)
Mobile Phase: Acetonitrile/methanol/water OH Minutes 27626
v/v/v 15/40/45
Temperature: 40 °C H OH
Flow Rate: 1.2 mL/min Figure 10. Separation of glucocorticosteroids.
H H
Inj. Volume: 5 µL HO
Detection: UV at 220 nm
2. Estradiol (E2)
120
OH Concluding Remarks
1
H
Acclaim Phenyl-1 The Acclaim Phenyl-1 column provides unique selectivity compared to
2 3
4 HO
H H C18 and other phenyl-type reversed-phase columns. The combination
3. Estrone (E1)
of high aromatic selectivity, high hydrophobicity, 100% aqueous
O compatibility and robust column packing makes it useful for a wide
mAU
1 H range of applications, such as fullerenes, lipids, fat-soluble vitamins,
H H
steroids, and many more.
Acclaim 120 C18 HO
2 34 4. Ethynylestradiol
HO REFERENCES
0
H 1. Newton, R. Molecular Mechanisms of Glucocorticoid Action: What
0 3 6 9 12 15 H H is Important? Thorax 2000, 55 (7), 603–613.
Minutes HO
27627 2. Prohibited List, World Anti-Doping Agency. http://www.wada-ama.
Figure 9. Separation of estrogens. org/en/World-Anti-Doping-Program/Sports-and-Anti-Doping-
Organizations/International-Standards/Prohibited-List/ (accessed
Jul 5, 2011).
Glucocorticosteroids
Glucocorticosteroids are a group of naturally occurring and synthetic
hormones that moderate inflammation and other stress responses.1
All glucocorticosteroids are on the World Anti-Doping Agency’s
2005 list of substances prohibited in competition when administered
orally, rectally, intravenously, or intramuscularly.2 Separation of these
substances can be difficult. The unique chemistry of the Dionex Acclaim
Phenyl-1 provides superior selectivity to conventional C18 columns
for the separation of glucocorticosteroids. As shown in Figure 10, eight
glucocorticosteroid compounds are resolved to baseline on a 3 × 250 mm
Dionex Acclaim Phenyl column using a methanol/water mobile phase.
Acclaim and Corona are registered trademarks and ultra is a trademark of Dionex Corporation.
Zorbax and Eclipse are registered trademarks of Agilent Technologies, Inc.
Pursuit is a registered trademark of Varian, Inc.
Pinnacle is a registered trademark of Restek.

Speed • Simplicity • Solutions

Dionex Products
North America Europe Asia Pacific
1228 Titan Way U.S./Canada (847) 295-7500 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735
P.O. Box 3603 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190
South America Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Sweden (46) 8 473 3380 Taiwan (886) 2 8751 6655
Sunnyvale, CA
4
94088-3603 A New Phenyl-Type Stationary Phase and Its Uses
Brazil (55) 11 3731 5140 Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722 LPN 2871-01 06/11

(408) 737-0700 www.thermoscientific.com/dionex ©2011 Thermo Fisher Scientific, Inc.