• Application Note

Innovative Silica-Based RPLC Preparative Columns: Enhanced Loadability and Peak Shapes

Innovative Silica-Based RPLC Preparative Columns: Enhanced Loadability and Peak Shapes
  • Fang Xia
  • Jie Y. Cavanaugh
  • Diane M. Diehl
  • Claude R. Mallet
  • Jeffrey R. Mazzeo
  • Waters Corporation
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Abstract

In the following experiments, we demonstrate superior peak shapes (under both low and high mass loading), enhanced efficiency, direct scale-up, and the benefit of these attributes in an impurity isolation and identification application utilizing Waters SunFire analytical- and prep-scale reversed-phase HPLC columns.

Introduction

Maximizing column load is one of the goals of preparative chromatography. However, tailing peaks limit the resolution and hence the mass loading of a column. Waters SunFire family of HPLC columns was designed to provide symmetric peak shapes and high mass loading. Innovative bonding and end-capping processes were used to synthesize these silica-based reversed-phase packing materials with higher surface coverage and reduced silanol activity.

SunFire column chemistry provides higher efficiencies, more symmetric peak shapes, and extremely high mass loading for acids, bases and neutral analytes. Two chemistries (C18 and C8) were developed to meet the requirements of the chemist. Additionally, years of research led to the discovery that the packed bed density of a column is the key to manufacturing stable and efficient preparative columns. Manufactured with the unique patent-pending Optimum Bed Density (OBD) design, SunFire Prep OBD Columns have the same packed bed density as analytical columns.

Experimental Conditions and Results

1. Peak Shape - Low Mass Load

Three beta-blocker drugs were separated on both a SunFire C18 column and a Luna C18(2) column (Phenomenex Inc., Torrance, CA). The tailing factors of all the analytes are listed on the chromatograms. Less peak tailing is observed on the SunFire column at 250 μg total mass load, as shown in Figure 1.

LC Conditions

Columns:

SunFire C18 and Luna C18(2), 4.6 × 50 mm, 5 μm

Mobile phase A:

0.1% TFA in H2O

Mobile phase B:

0.1% TFA in ACN

Flow rate:

1.4 mL/min

Gradient:

5 min gradient, 5% to 70% B

Sample:

(1) nadolol (10 mg/mL)

(2) metoprolol (10 mg/mL)

(3) propranolol (5 mg/mL), all in DMSO

Injection volume:

10 μL

Mass load:

250 μg

Detection:

UV @ 260 nm

LC system:

Waters AutoPurification System
Peak shapes under low mass load conditions
Figure 1. Peak shapes under low mass load conditions. 

2. Peak Shape - High Mass Load

Three beta-blocker drugs were separated under high load on both a SunFire C18 and Luna C18(2). The tailing factors of all the analytes are listed on the chromatograms. Superior peak shapes are observed on the SunFire column under 4.5 mg total mass load, as seen in Figure 2.

LC Conditions

Columns:

SunFire C18 and Luna C18(2), 4.6 × 50 mm, 5 μm

Mobile phase A:

0.1% TFA in H2O

Mobile phase B:

0.1% TFA in ACN

Flow rate:

1.4 mL/min

Gradient:

5 min gradient, 5% to 70% B

Sample:

(1) nadolol (100 mg/mL)

(2) metoprolol (100 mg/mL)

(3) propranolol (50 mg/mL), all in DMSO

Injection volume:

18 μL

Mass load:

4.5 μg

Detection:

UV @ 260 nm

LC system:

Waters AutoPurification System
Peak shape under high mass load conditions
Figure 2. Peak shape under high mass load conditions. 

3. Mass Loading Capacity

Three beta-blocker drugs were separated on a SunFire Prep OBD C18 column. The total mass load on this column is 100 mg. As shown in Figure 3, even under such high mass load, the peak shapes for all three analytes are excellent.

Mass loading capacity
Figure 3. Mass loading capacity on a 19 x 50 mm SunFire Prep OBD C18 column. 

4. Efficiency

Separations of three antifungal drugs at 1250 μg loading on six popular commercial RPLC C18 columns are shown in Figure 4. The same mass loading and gradient conditions were applied on all the columns. Econazole (peak 2) and miconazole (peak 3) have very similar structures with only a chlorine atom difference, which challenges column efficiency.

Resolution values of the last two peaks are listed on each chromatogram. Baseline resolutions are achieved on both SunFire C18 and Waters Symmetry C18 columns. None of the other C18 columns achieve resolution higher than 1.50.

LC Conditions

Columns:

SunFire Prep OBD C18, 19 × 50 mm, 5 μm

Mobile phase A:

0.1% TFA in H2O

Mobile phase B:

0.1% TFA in ACN

Flow rate:

23.9 mL/min

Gradient:

5 min gradient, 5% to 70% B

Sample:

(1) nadolol (100 mg/mL)

(2) metoprolol (100 mg/mL)

(3) propranolol (50 mg/mL), all in DMSO

Injection volume:

400 μL

Mass load:

100 μg

Detection:

UV @ 260 nm

LC system:

Waters AutoPurification System
Comparison of column efficiencies
Figure 4. Comparison of column efficiencies among leading commercially available C18 columns. 

5. Direct Scale-up

The separation of three acidic analytes was successfully scaled-up from an analytical-scale to a preparative-scale SunFire C18 column without the need for additional method development – a significant time saver.

LC Conditions

Columns:

Six brands listed in Figure 4, 4.6 × 150 mm, 5 μm

Mobile phase A:

0.1% TFA in H2O

Mobile phase B:

0.1% TFA in ACN

Flow rate:

1.0 mL/min

Gradient:

15 min gradient, 20% to 85% B

Sample:

(1) ketoconazole (25 mg/mL)

(2) econazole (50 mg/mL)

(3) miconazole (50 mg/mL), all in DMSO

Injection volume:

10 μL

Temperature:

30 °C

Detection:

UV @ 254 nm

LC system:

Waters Alliance HT System with a Waters 2996 PDA Detector

Analytical-Scale Gradient

Time (min)

%A

%B

0.00

80

20

1.00

80

20

6.00

5

95

7.00

5

95

7.01

80

20

11.00

80

20

Prep-Scale Gradient

Time (min)

%A

%B

0.00

80

20

1.91

80

20

6.91

5

95

7.91

5

95

8.01

80

20

12.00

80

20
Direct scale-up from an analytical-scale
Figure 5. Direct scale-up from an analytical-scale to a prep-scale SunFire C18 column. 

6. Impurity Isolation and Identification

Nimodipine is a calcium channel blocker, which acts on blood and heart vessels to increase blood flow to injured tissue. Several impurities may be formed during synthesis. The existence of impurities even in small amounts may influence the efficacy and safety of the active pharmaceutical ingredient. Therefore, it is important to identify impurities.

Highly efficient isolation and direct scale-up was observed on SunFire analytical and preparative C18 columns, as shown in Figure 6. Each impurity was separated and collected on the preparative column and then analyzed by MS/MS. Daughter ion scans of nimodipine and its impurities resulted in structural identification of the impurities.

LC Conditions

Columns:

SunFire C18 (analytical-scale), 4.6 × 100 mm, 5 μm;

SunFire Prep OBD C18 (prep-scale), 19 x 100 mm, 5 μm

Mobile phase A:

0.1% Formic acid in H2O

Mobile phase B:

0.1% Formic acid in ACN

Flow rate:

1.4 mL/min (analytical-scale);

23.9 mL/min (prep-scale)

Gradient:

10min gradient, 30% to 90% B

Sample:

Crude nimodipine in DMSO (30 mg/mL)

Detection:

UV @ 290 nm

LC system:

Waters AutoPurification System

MS Conditions

Interface:

ESI+

Capillary:

3.0 kV

Source temp.:

150 °C

Cone:

30 V

Desolvation temp.:

350 °C

Extractor:

3.0 V

Cone gas:

50 L/hr

RF lens:

0.3 V

Desolvation gas:

550 L/hr

MS system:

Waters Micromass Quattro micro Mass Spectrometer
Isolation and structural identification
Figure 6. Isolation and structural identification of nimodipine and its impurities on SunFire C18 columns. 

Conclusion

Waters SunFire HPLC columns are synthesized with innovative bonding technology to achieve significantly higher surface coverage, as well as a more efficient end-capping technology to successfully reduce surface silanol activity. Therefore, for bases, acids, and neutrals, more symmetric and less tailing peak shapes can be achieved using SunFire columns, which leads to higher mass loading on preparative columns. Additionally, SunFire Prep OBD columns are created with the unique Optimum Bed Density design process, whichproduces more stable columns and ensures linear and streamlined scale-up from analytical to preparative applications. SunFire columns are also compatible with mass spectrometry detection, providing sharp peaks, good sensitivity, large peak capacities, and very low bleed – ideal for impurity isolation and identification.

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