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Method development: an alternative method for API HPLC analyses.

Since their introduction in the late 1970s, Bio-Rad's Aminex HPLC (high-performance liquid Chromatography) columns have become a standard in a number of different industries as evidenced by the several hundred scientific publications using these columns. For instance, they have been used to detect the ethanol and glycerol content of wine, which is an excellent indicator of both fermentation and the quality of the final product-the amount of glycerol correlating well with the smoothness of wine.

Aminex polystyrene-divinylbenzene (PS-DVB) ion exchange resins are complementary to reversed phase columns and best suited for water-soluble and partially water-soluble small organic compounds, including sugar alcohols and short-chain organic acids.

However, there is a large group of compounds in the middle range of solubility that are amenable to analysis on both the PS-DVB and reversed phase columns. For these compounds, Aminex resins offer excellent if not superior performance over reversed phase. They exhibit high pressure as well as pH stability over a wide range. Moreover, these resins exhibit long life times and maintain reproducibility over hundreds of injections. In a study of the PS-DVB column performance, retention times varied only up to 0.3% while efficiencies decreased by only up to 6.3% after 200 injections.

Separation mechanism

Aminex HPLC columns offer many advantages for the analysis of carbohydrates, alcohols and organic acids using simple isocratic normal phase eluents like water or dilute acid, which are greener than reversed phase solvents, typically acetonitrile. The resin packing allows a variety of partition type separations without the disadvantage of the limited lifetime associated with bonded phase silica materials.

The fundamental partition process responsible for separation is moderated by the ionic group bound to the resin and by the choice of the counter ion. Termed ion-moderated partitioning, this unique process enables these resins to separate compounds via multiple modes of interaction, involving a combination of ion exclusion, ion exchange, ligand exchange, size exclusion, reversed phase and normal phase partitioning.

Column selection

Selectivity for a particular separation can be optimized by choosing the appropriate column type based on three characteristics:

1. Resin cross-linkage: The lower the cross-linkage the more open the structure and the more permeable it is to higher molecular weight substances. A 4% cross-linked resin (Aminex HPX-42A and HPX-42C) can resolve higher oligosaccharides, whereas an 8% cross-linked resin (Aminex HPX-87C and HPX-87H) is ideal for smaller molecular weight oligosaccharides.

2. Resin ionic form: Aminex columns are fixed-ion resin columns-the resin is converted to a specific ionic form before packing. The ionic form of the resin contributes to selectivity by virtue of its charge co-ordination stability, steric hindrance with the pores of the resin, or through a combination of these factors.

3. Column configuration: Column length and diameter affect resolution and analysis time. For compounds of interest that are strongly retained by the resin, a short column can significantly decrease analysis time.

Separation of APIs

While Aminex columns are widely employed, their use in the separation of small APIs (active pharmaceutical ingredients) has been limited. Analysis of the purity of an API as well as measurement of its concentration relative to side products and precursor building blocks (for example, organic acids, aldehydes, ketones, alcohols and carbohydrates) is important in monitoring the progress and efficiency of the reaction.

Aminex HPLC methods not only increase the efficiency with which a sample component can be determined but also allow determination of compounds that were previously undetectable. Several of these columns are available for small acidic and polar molecule analysis.

When many compounds in a reaction mixture must be analyzed or when high-resolution separations are required, the 300- x 7.8-mm ID HPX-87H columns are recommended, although the HPX-87C columns are also suited for analyzing organic acids in combination with carbohydrates. The smaller Aminex Fast Acid Analysis (FAA) monitoring colunms provide fast separations of specific organic acids in samples where only certain components are of interest. With these columns analyses can be completed in 3 to 5 min.

The HPX-87H columns separate organic acids using primarily the ion exclusion and reversed phase mechanisms. When dilute sulfuric acid is used as the eluent, organic acids elute from the column in order of increasing pKa. Anions are eluted near the void volume and acids, which have been ionized in the acidic eluent, elute according to the fraction of the acid ionized. Column selectivity is controlled by changing the column temperature, the pH of the eluent, or by adding organic modifiers such as acetonitrile to the eluent to reduce resin/compound interactions and to cause strongly bound compounds such as aromatic acids to elute more rapidly.

[FIGURE 1 OMITTED]

The HPX-87H columns separate neutral species such as carbohydrates and alcohols by reversed phase partitioning. The eluent is polar while the resin matrix is nonpolar, so aliphatic nonpolar alcohols are adsorbed by the resin and are eluted after charged molecules. This ability to separate a variety of compounds makes the columns ideal for separating mixtures that contain several classes of components.

HPX-87H columns perform most analyses in about 20 min with sensitivity to the nanogram level. A simple isocratic elution with slightly acidified water is all it takes to analyze organic acids in most samples with a 300- x 7.8-ram column. Filtration is the only sample preparation required before injection. Separations can be performed at ambient temperatures up to 60 C at flow rates between 0.4 to 1.0 mL/min.

FAAs separating APIs

FAA columns are optimized for the analysis of alcohols, glycols and hydrophobic organic acids. Typical analyses can be shortened four-fold over the time required with research length (300- x 7.8-mm) colunms. Because the FAA columns are shorter, components elute as taller, narrower peaks. Detection limits are improved and smaller sample loads can be used.

The authors recently investigated conditions to explore the effectiveness of FAA columns (100 x 7.8 mm) in separating common building blocks (uracil, 5-bromo-3-cyanopyridine, HATU, 2-hydroxycinnamic acid and phenyl acetic acid) used in the synthesis of APIs and in separating a mixture of water-soluble vitamins (nicotinamide, thiamine and nicotinic acid).

[FIGURE 2 OMITTED]

Previous work using these columns was done using acetonitrile and water as the mobile phase. Due to the shortage of acetonitrile, methods were developed employing a combination of water and methanol. Alcohols had not been previously recommended with Aminex columns, as they were known to shrink the polymer backbone of resins, potentially resulting in high backpressures under HPLC conditions.

When a 1:1 mixture of uracil and 5-bromo3-cyanopyridine was separated under isocratic conditions using 90%, 80% and 70% methanol in water (flow rate: 1 mL/min; injection volume: 10 mL; detection: 254 nm) the retention time was found to increase with increasing concentrations of methanol. The nonpolar 5-bromo-3-cyanopyridine was retained more strongly than uracil, which shows that reversed phase partitioning is involved in the separation. The uracil and 5-bromo-3-cyanopyridine molecules distribute between the methanol and water mobile phase and the aromatic resin backbone.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Another set of common building blocks--phenyl acetic acid, trans-2-hydroxycinnamic acid, uracil, HATU and 5-bromo-3-cyanopyridine-were separated using gradient conditions 1% to 20% B over 15 min (A: MeOH:H20:H2SO4 (95:5: 0.05); B: MeOH:H2O:H2SO4 (5:95:0.05)). In the absence of acid, the mobile phase showed poor peak shape and long retention for HATU and 5-bromo-3-cyanopyridine.

The presence of 0.05-M H2SO4 in the mobile phase helped decrease the retention time of HATU and 5-bromo-3-cyanopyridine by 4 min. Significant peak tailing was observed in the absence of acid.

Uracil, which under reversed phase chromatography is poorly retained and is used as a standard void volume marker, was retained by the FAA columns and eluted at 3.6 min under these conditions. This method can be used to measure the concentration of uracil in the crude product, allowing determination of the progress and efficiency of a reaction where uracil is used as the starting material.

The FAA columns were also used to separate a mixture of water soluble vitamins, nicotinamide, thiamine and nicotinic acid under gradient conditions 2% to 15% B over 15 min (A: MeOH:H2O:HOAc (95:5:0.1); B: MeOH:H2O:HOAc (5:95:0.1)). Nicotinic acid had the longest retention time (6 min) followed by thiamine (3 min) and nicotinamide (2.3 min).

In conclusion, mixtures of APIs were separated in a short time (10 to 15 min) with excellent efficiency on FAA columns using a combination of methanol and water. Methanol did not appear to shrink the polymer or result in high backpressures. These findings indicate that Aminex HPLC columns offer an attractive if not superior alternative to reversed phase HPLC for the determination of polar analytes in APIs.

Industrial Applications of Aminex HPLC Columns

* Food and beverage industry

* Dairy products

* Fruits and vegetables

* Wine and beer

* Clinical applications

* Metabolite analysis

* Bacteria and yeast studies

* Oligosaccharides analysis

* Glycoprotein and glycoconjugate analysis

* Nucleic acid analysis

* Cellulose and wood analysis

* Plant biochemistry

* Fermentation monitoring

Shahnaz Ghassemi, SynPure International, an Farah Mavandadi, Bio-Rad Laboratories

Shalnaz Ghassemi is the president of SynPure Intl. Farah Mavandadi is the product manager in the Separations Div. of Bio-Rad Laboratories.
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Title Annotation:Chromatography Techniques
Author:Ghassemi, Shahnaz; Mavandadi, Farah
Publication:Laboratory Equipment
Article Type:Cover story
Date:Jun 1, 2009
Words:1519
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