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PRIMESEP™ (SIELC Technologies)
•
Novel silica-based mixed mode phases•
Suitable for RP, NP, ion-exchange and ion-exclusion chromatography•
Stable in 100% aqueous eluents•
LC-MS and preparative chromatography applications•
Inorganic cations and anions can be run together Primesep™ mixed-mode stationary phases have been developed by SIELC (Strong Ion Exchange Liquid Chromatography),Prospect Heights, IL. They are universal phases for separating a wide range of polar and non-polar compounds by different separation modes, based only on eluent selection. Ionizable compounds interact with the stationary phase by reversed-phase, ion-exchange or ion-exclusion mechanisms.The newer range of Obelisc columns is described on page 213. Each Primesep column has a dual chemistry stationary phase containing a hydrophobic long alkyl chain and an ionizable cationic or anionic embedded group. These columns are suit-able for analytical and preparative scale separations in iso-cratic and gradient modes, and compatible with all common detection techniques.
Primesep columns can efficiently separate organic and inor-ganic ions on the same column at the same time. This enables an organic pharmaceutical to be quantified simultane-ously with its inorganic counter ion. Also, inorganic cations and anions can be run together without a specialized ion chro-matography system. Selectivity can be altered by changing acid modifier type and concentration as well as varying organ-ic modifier concentration. Primesep Phase Particle Size (µm) Pore Size
(Å) Main Separation Mode Typical Application
A 5, 10 100 RP + cation-exchange + ion-exclusion Neutral and weak basic compounds AB 5, 10 100 RP + cation-exchange + anion-exchange Neutral, acidic and basic compounds B2 5, 10 100 RP + anion-exchange + ion-exclusion Neutral and acidic compounds C 5, 10 100 RP + cation-exchange + complex formation Amines, sulphonium, phosphonium
and metal ions
D 5, 10 100 RP + anion-exchange Small hydrophobic and acidic compounds. Low
MW plasma components and biofluids N 5, 10 100, 300 NP + cation-exchange + HILIC Basic and polar compounds SB 5, 10 100 RP + anion-exchange + ion-exclusion Neutral and acidic compounds P 5, 10 100 RP + strong cation-exchange+ π-π interaction Neutral and basic compounds.
Structural isomers of aromatic compounds 100 5, 10 100 RP + cation-exchange + ion-exclusion Neutral and basic compounds
200 5, 10 100 RP + cation-exchange + polar interaction Neutral and strong basic compounds 500 5, 10 100 RP + cation-exchange + ion-exclusion Neutral and basic compounds
Primesep A, 100, 200, 500 Primesep B, B2, D, SB Primesep C Primesep P Primesep D
Embedded acidic phases
Primesep A, 100, 200 and 500 are reversed-phase columns with different strengths of embedded acidic (anionic) ion-pair-ing groups. Primesep A is the strongest acidic column, while Primesep 500 is the weakest acidic column. Differences in functional group acidity allow selection of the most appropriate column for a particular set of basic compounds that differ in their pka value. The embedded acidic functional group can be in an ionized form, or in a non-ionized form, depending on the pH of the eluent. When these two forms are in equilibrium, the phase is half ionized and half unionized. In order to achieve component retention by ion-exchange on these Primesep columns, eluent pH should be close to or above the transition value (see Table 1).
Primesep 100 and 200 are versatile columns for separation of a broad range of compounds.
Primesep C also contains an embedded anionic group (car-boxylic acid) but with additional complex-formation properties. The latter facilitates retention of amines, sulphonium, phos-phonium and metal ions. The degree of ion-exchange and complex formation can be effectively adjusted by the alteration of eluent pH, within the range 3 - 7. The unique complex form-ing properties of Primesep C columns lead to a reversal of elu-tion order compared to ion-exchange, eg tR Li+>Na+>K+. Similarly primary amines are retained longer than secondary and tertiary amines on Primesep C columns.
Primesep Phase Transition pH A Ionized at all working pH
100 pH 1
200 pH 2
500 pH 5
C pH 3.5
PRIMESEP™ (SIELC Technologies)
Embedded acidic phases
(continued)The Primesep P phase provides three interactions with ana-lytes - reversed-phase, π-π interaction and strong cation-exchange. It contains embedded acidic ion-pairing groups combined with an aromatic moiety and is useful for the sepa-ration of structural isomers of aromatic compounds. Enhanced π-π interaction can be achieved by adding THF to CH3CN-H2O eluents.
The Primesep N phase is a normal-phase analytical column with embedded acidic groups with a pKa of about 5. In addi-tion to improving the retenaddi-tion of basic compounds by caaddi-tion- cation-exchange, this phase separates polar compounds by HILIC mechanism.
Embedded basic phases
Primesep B, B2 and SB phases are produced with embed-ded basic ion-pairing groups. In addition to improving the retention of acidic compounds by anion-exchange, the phases separate bases by an ion-exclusion mechanism. Primesep B and SB are strong basic columns for operation in the pH range 1.5 to 4 and 1.5 to 5 respectively, created by the addition of TFA, phosphoric or perchloric acids to the eluent. Primesep B2 is a weak basic column that offers an extended pH range from 1 to 7, suitable for use with appropriate buffered solutions. For development of new methods, Primesep B2, SB or D are recommended over Primesep B, due to the extended pH stability range.
Primesep B, B2, SB and D are fully ionized at all working pH values.
Primesep D comprises an anion-exchange group embedded in a long alkyl chain. It is a stronger basic column than Primesep B2. In addition, Primesep D allows direct injection of plasma and other biofluids, enabling a broad range of small molecules to be analyzed via a single column without any sample preparation. At pH 3.0, most proteins become posi-tively charged and are excluded from the stationary phase, while small hydrophobic molecules are retained.
Embedded acidic and basic phases
Primesep AB is a zwitterionic reversed-phase column with embedded cation-exchange and anion-exchange functionali-ties, combining the properties of both in a single column. With basic analytes, Primesep AB behaves as if it has a negatively charged surface. Conversely, with strong acids Primesep AB behaves as if it has a positively charged surface.
Anions and cations can be separated at the same time as neu-trals. This is useful for complex mixtures which include polar ionisable compounds both of acidic and basic nature. The col-umn is also capable of separating both the anion and cation of the same salt. This is important in analysis of pharmaceutical formulations, drug substances and other organic and inorganic salts.
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Primesep A
Primesep 100
Primesep 200
Primesep B
Primesep B2
Common C18
Columns: 150mm x 4.6mm, 5µm Flow rate: 1mL/minDetection: UV 210nm Mobile Phase:
Water/MeCN/TFA-60/40/0.1
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OBELISC™ (SIELC Technologies)
Obelisc
™
•
Multiple separation modes•
Simple eluent selection•
LC-MS, ELSD and low UV (<220nm) compatible•
Adjustable selectivityObelisc™ LC columns are based on a new generation of mixed-mode phases developed by SIELC Technologies. Two compli-mentary columns, Obelisc R and Obelisc N, based on Liquid Separation Cell (LiSC™) technology, offer a new approach to separating a variety of small polar and non-polar compounds, by multiple separation mechanisms. Like living cells, which exist in equilibrium with the outside environment, liquid separation cells exist in constant equilibrium with the eluent.
Obelisc R, with reversed-phase characteristics, and Obelisc N, with normal-phase characteristics, differ in the type and proximi-ty of their charged groups and the hydrophobiciproximi-ty of their long linking chains.
Obelisc R has cationic groups close to the 100Å silica surface separated from anionic groups by a hydrophobic chain (see Figure 1).
Obelisc N has anions close to the surface separated from cation-ic groups by a hydrophilcation-ic chain (see Figure 1).
Typical eluents used with Obelisc columns are based on ace-tonitrile, water and MS compatible buffers ammonium formate (pH 3) and ammonium acetate (pH 5), or phosphate buffer for low UV detection.
Obelisc R
Specially developed 'AQ' type columns are designed to work in low organic containing eluents, often 100% aqueous, for the analysis of polar compounds by reversed-phase. They can offer insufficient improvement in retention of highly polar molecules. Obelisc R can be used in traditional RP type applications and it offers improvement in the degree of retention of both acidic and basic polar molecules, compared with 'AQ' type phases. Obelisc R is recommended for the analysis of complex mixtures of polar and non-polar molecules, especially with MS, ELSD or CAD detection. Figure 2 shows the separation of a complex mix-ture of acids, bases, amino acids and neutral compounds on Obelisc R.
Obelisc N
Obelisc N has very polar characteristics and works well for polar and charged analytes. The positive and negative charges of the phase are well separated and independently accessible, which results in different selectivity compared to traditional HILIC and silica columns. Figure 3 shows the HILIC separation of sugars on Obelisc N. Eluent composition changes the conformation of the long hydrophilic chain, thus changing separation selectivity. Figure 4 shows the effect of buffer concentration on the resolution of isomeric aminobutyric acids.
Figure 1. Interaction of Obelisc R and Obelisc N
Figure 2. Separation of amino acids, bases, acids and neutrals on Obelisc R
Figure 3. Separation of sugars in HILIC mode on Obelisc N
ORDERING INFORMATION - (SIELC Technologies)
Primesep Phase 5µm1 Column Dimensions2(mm) Guard Column (10x2.1mm 2/pk)3 Column Dimensions2(mm) Guard Column (10x4.6mm 2/pk)3 50 x 2.1 150 x 2.1 250 x 2.1 50 x 4.6 150 x 4.6 250 x 4.6 $684 $745 $770 $275 $684 $806 $935 $275A SI5A-50AM SI5A-150AM SI5A-250AM SI5A-GC-AM SI5A-50A SI5A-150A SI5A-250A SI5A-GC-A AB SI5AB-50AM SI5AB-150AM SI5AB-250AM SI5AB-GC-AM SI5AB-50A SI5AB-150A SI5AB-250A SI5AB-GC-A B2 SI5B2-50AM SI5B2-150AM SI5B2-250AM SI5B2-GC-AM SI5B2-50A SI5B2-150A SI5B2-250A SI5B2-GC-A C SI5C-50AM SI5C-150AM SI5C-250AM SI5C-GC-AM SI5C-50A SI5C-150A SI5C-250A SI5C-GC-A D SI5D-50AM SI5D-150AM SI5D-250AM SI5D-GC-AM SI5D-50A SI5D-150A SI5D-250A SI5D-GC-A N SI5N-50AM SI5N-150AM SI5N-250AM SI5N-GC-AM SI5N-50A SI5N-150A SI5N-250A SI5N-GC-A SB SI5SB-50AM SI5SB-150AM SI5SB-250AM SI5SB-GC-AM SI5SB-50A SI5SB-150A SI5SB-250A SI5SB-GC-A P SI5P-50AM SI5P-150AM SI5P-250AM SI5P-GC-AM SI5P-50A SI5P-150A SI5P-250A SI5P-GC-A 100 SI5100-50AM SI5100-150AM SI5100-250AM SI5100-GC-AM SI5100-50A SI5100-150A SI5100-250A SI5100-GC-A 200 SI5200-50AM SI5200-150AM SI5200-250AM SI5200-GC-AM SI5200-50A SI5200-150A SI5200-250A SI5200-GC-A 500 SI5500-50AM SI5500-150AM SI5500-250AM SI5500-GC-AM SI5500-50A SI5500-150A SI5500-250A SI5500-GC-A
1
Other pore size and particle size available 2
Other dimensions available. 3
Guard column is uniquely designed for direct insert, holder not required.
Ordering Information - Primesep™ Columns (5µm, 100Å phases)
Ordering Information - Obelisc™ Columns (5µm, 100Å phases)
Obelisc Phase1 Column Dimensions2(mm) Guard Column (10x2.1mm 2/pk)3 Column Dimensions2(mm) Guard Column (10x4.6mm 2/pk)3 50 x 2.1 150 x 2.1 250 x 2.1 50 x 4.6 150 x 4.6 250 x 4.6 $684 $745 $770 $275 $684 $806 $935 $275
R SI5OR-50AM SI5OR-150AM SI5OR-250AM SI5OR-GC-AM SI5OR-50A SI5OR-150A SI5OR-250A SI5OR-GC-A N SI5ON-50AM SI5ON-150AM SI5ON-250AM SI5ON-GC-AM SI5ON-50A SI5ON-150A SI5ON-250A SI5ON-GC-A
1
Other particle sizes available 2
Other dimensions available. 3
Guard column is uniquely designed for direct insert, holder not required.
Improved peak shape of a basic compound on Primesep B2 column
Column: 150mm x 4.6mm, 5µm (P/N SI5B2-150A) Flow Rate: 1mL/min Detection: UV 250 nm Mobile Phase: MeCN/H20/TFA
20/80/0.1%
Column: 150mm x 4.6mm Flow Rate: 1mL/min Detection: UV 250 nm Mobile Phase: MeCN/H20/TFA
35/80/0.1%
Column: 150mm x 4.6mm Flow Rate: 1mL/min Detection: UV 250 nm Mobile Phase: MeCN/H20/TFA
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APPLICATIONS - PRIMESEP™ (SIELC Technologies)
Amino acids analysis in acid gradient conditionIon-exchange and hydrophobic mechanism In tertiary amines separation.
Catecholamine pathway
LC application for analysis of EDTA
Separation of organic acids Effect of both pH and organic content on a separation of sugars, amino acids, and carboxylic acids
LC analysis of active drug in a formulation
Separation of vitamin C, vitamin group B, and related impurities 10 9 8 7 6 5 4 3 2 1 0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 S uc ci na te Column : Primesep C Column size: 150 x 4.6 mm Flow rate: 1.0 ml/min Injection: 5µl
Mobile Phase: TEA 50 mM phosphate, pH 3.0/MeCN 40/60 Separation of NyQuil active ingredients
Separation of serotonin, dopamine, and related compounds
Effect of buffer on separation of buffers
Separation of paraquat and diquat
