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Interesting Properties of Silica Hydride Stationary Phases
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Maria T. Matyska1, Joseph J. Pesek1, William Ciccone2 1Department
BOOTH #1974
2Microsolv
INTRODUCTION; Features & Benefits The development of stationary phases in HPLC is driven by the desire to discover capabilities for separation materials that will enhance current methodology or to solve analytical problems not readily addressed by currently available columns. One approach to retaining the mechanical properties and versatility of silica in a new stationary phase is to change the surface from one composed primarily of silanols to a new material where the predominant group on the surface of silica is a hydride. The result of this surface transformation is a number of stationary phases with fundamentally different chromatographic properties than those based on ordinary silica. Among just some of the enhanced features and benefits of silica hydride-based material, presented below, is the ability to operate in three modes of chromatography: reversed-phase, aqueous normal phase (ANP) and organic normal phase. Increasing analyte retention with increasing temperature in the ANP mode, rapid equilibrium between gradient runs and decreased water association with the hydride surface in comparison to ordinary silica are other highly beneficial properties. This material also has the ability to retain both hydrophilic and hydrophobic compounds, and a C18 that has compatibility with 100% aqueous mobile phases and excellent reproducibility.
O O O O
O Si O Si O Si O Si O
OH
O
OH
O
OH
O
OH
O
O Si OH O O Si OH O
O Si O Si O Si O Si O
O O O O
O Si O Si O Si O Si O
Hydrosilation reaction
H H
cat.
H H
HC C R
Cat=catalyst, typically hexachloroplatinic acid or free radical initiator
O Si O Si H O O O Si O Si H O O
O O Si O O Si O O Si O O Si O
O O Si O CH
CH2
O Si O O Si H O O Si O CH
CH2
16
16
CH3
CH3
O Si O Si O O O Si O Si H O O
of Chemistry, San José State University, One Washington Square, San José, CA 95192
Technology Corporation, 1 Industrial Way West, Building E, Unit D, Eatontown, NJ 07724
AQUEOUS NORMAL PHASE (ANP) RETENTION – GREAT TOOL FOR METABOLOMICS
REVERSED-PHASE (RP- HPLC) RETENTION
Cogent BDC18 BD C18
Cogent BDC8 BD C8
Cogent UDC Cogent UDC Cholesterol Cholesterol
UDC Cholesterol
O O
O
Si
Figure 5. Separation of aspirin from its major hydrolysis product, salicylic acid is shown. Peak shape is excellent. Salicylic acid can be very difficult to adequately chromatograph on columns with ordinary silica. Column: Cogent Bidentate C18™, 4µm, 100A Catalog No.40018-75P, Dimensions: 4.6 x 75 mm. Mobile Phase: 48:52 Acetonitrile: DI Water + 0.1% Phosphoric Acid. Flow rate: 1.5 mL/min. Peaks: 1. System Peak 2. Aspirin (Acetylsalicylic Acid) 3. Salicylic Acid. Injection Volume: 10 µL. Detection: UV 210 nm. Temperature: 25°C
An interesting aspect of analyzing real patient samples with ANP LCMS is illustrated in figure 1. Each chromatogram represents seven replicate samples of (1)gutamine and (2) lysine quantities taken from saliva in normal patients and patients suffering from pancreatitis and in figure 1A patients with pancreatic cancer. A cursory survey of a limited number of samples revealed that in general the lysine peak abundance was approximately equal to or greater than that for glutamine in cancer patients, while in normal patients, lysine peak abundance was significantly lower than the glutamine peak. A larger and more statistically significant number of patient samples will need to be analyzed in order to make definitive conclusions, but such correlations of metabolite patterns could prove to be a useful tool in clinical analysis for disease diagnosis.
ORGANIC NORMAL PHASE (ONP) RETENTION
Figure 1. Cogent DH column 2.1 x 150 mm. Mobile phase: A: DI Water + 10 mM ammonium acetate B: 98% acetonitrile + 2% ammonium acetate. Flow rate: 0.4 mL/min. Gradient: 0.0 min 100% B; 0.0 to 14.0 min to 60% B; 14.1 min to 5% B.
O
CH
Si O O Si H O O Si O CH O Si O O Si H O
(CH2)16
(CH 2 )16
acid and melamine were detected at 100 fg level. CH3
O O
CH3
CH3
O
Si
CH
Si O O Si H O O Si O CH O Si O O Si H
(CH2 )6
CH3
O
O
O O O
(CH2 )6
CH3
O O O
O
O
Si CH2 (CH2)9 O Si H O Si H O Si H O CH2 (CH2)9 Si O Si H
C
O
H
CH3
O
H
H
H CH3 CH3
O C
O H
H
H
H
Separation of a degraded Simvastatin tablet, on a Cogent Bidentate C18. USP Tailing Factor is 1.02 for Simvastatin. Excellent peak shape was obtained using RP-HPLC method. Mobile phase: 60:40 Acetonitrile:DI Water + 10mM Sodium Phosphate. Flow rate1.5 mL/min. Peaks: 1. Excipient 2. Hydrolysis Product 3. Simvastatin Detection, UV 238 nm,Temperature 30°C
Saliva contains a variety of hydrophilic compounds; several different classes of metabolites were detected by ANP in LCMS.
Infant formula: cyanuric
Bidentate C8
Figure 4.
Bio Markers in Saliva Detected through ANP LCMS
NOTE: Retention times are very reproducible over the concentrations ranges of the acids found in the saliva samples.
Some Examples of other moieties bonded to silica hydride
Silica Hydride columns provide very good retention when RPHPLC analysis conditions are used. Peak shape is excellent and selectivity is outstanding. These columns equilibrate in a short time even when gradient conditions are used.
The principle of ANP is simple. Retention behavior is analogous to that found in normal phase chromatography but the mobile phase has water as part of the binary solvent. In other words, normal retention with reverse phase solvents. Normal phase implies that retention is greatest for polar solutes such as acids and bases. In addition, retention must increase as the amount of the non-polar solvent in the mobile phase increases. So if the mobile phase consists of water and acetonitrile, retention will increase as the amount of acetonitrile increases. Typically the amount of the nonpolar component in the mobile phase must be 60% or greater with the exact point of increased retention depending on the solute and the organic component of the mobile phase. All Cogent TYPE-C columns exhibit ANP retention however the best separations are achieved for polar compounds when the Cogent Diamond Hydride (DH) Column is used.
Figure 2. EICs for organic acids from saliva samples on the DH column. Peak identity: (1) aconitic acid (m/z 173); (2) succinic acid (m/z 117); and (3) adipic acid (m/z 145). Flow rate 0.4 mL/min. All conditions the same as Figure 1.
Bidentate C-18
silica hydride (Microsolv Type C)
ordinary silica
PITTCON 2009
March 8th – March 13th ,2009, McCormick Place, Chicago, Illinois, USA
Ti me
% B
0.0
100
1.0
90
5.0
50
Figure 6. Detection: Atmospheric Pressure Chemical Ionization in positive mode - APCI+. Column: Bidentate C18, mobile phase: 95:5 Hexane/Ethyl Acetate. Flow rate: 1.0 mL/min.Samples: 1 – phenol with aldehyde, 2 – parent phenol, 3 – phenol with ketone, 4 – phenol with acid. Retention under above conditions follows typical organic normal phase behavior, i.e. as the amount of hexane is increased the elution times for all compounds increase. As the amount of hexane approaches 100%, the last eluting compound (phenol with an acid functional group) has for all practical purposes infinite retention. These normal phase retention characteristics provide many useful options for separation of compounds with different functionalities in the normal phase mode with a stationary phase having a hydrophobic surface.
CONCLUSIONS: Silica Hydride-based stationary phases are useful separation materials that can operate over the complete range of analytical conditions from RP-HPLC, ANP and ONP. HPLC with hydride-based columns coupled to MS is a versatile analytical tool with extensive separation capabilities and high sensitivity. Figure 3. Column: Cogent Diamond Hydride, 4um ,100A. Dimensions: 2.1 x150 mm. Solvents; A: DI water + 0.1% acetic acid B: Acetonitrile + 0.1% acetic acid. Mobile phase: Gradient, Post time 5 min,Flow rate: 0.4 mL/min. t0 = 0.8 min. Injection: 1 µL Sample Pre-fortified infant formula: 1. Cyanuric acid 1.5 microg/mL: NEG (M-H)- 128.0102 m/z analyzed in Neg mode 2. Melamine 3 microg/mL: POS (M+H)+ 127.0727 m/z analyzed in Pos mode
ACKNOWLEDGEMENT Microsolv Technology Corporation
