Scientists use chromatography as an effective technique in research and clinical laboratories to meticulously separate and analyze complex mixtures. Gas chromatography (GC) and high-performance liquid chromatography (HPLC) are two most commonly used techniques to do so in a Chromatography Lab.
Many consumables are needed for chromatography experiments and can be obtained from reliable chromatography consumables suppliers, such as MDM Scientific Supply. Below is a vital list of chromatography necessities.
Vials and Seals
Chromatography vials and seals are regularly employed in gas and liquid chromatography (LC) for sample analysis, collection, and preservation. Vials are typically crafted from glass but can also be made of plastic. Vial dimensions and physical specifications are vital for trouble-free operation with autosamplers and other instruments. For instance, crimp seals are used for more volatile samples, while amber glass or black plastic is employed for light-sensitive samples.
Seals consist of a cap and septum that safeguard the compound mixtures inside the vials. Similar to vials, seals must be inert and uncontaminated to avoid altering the results. Various applications necessitate specific seal types, such as push-on caps and screw caps for convenience when evaporation is not a concern and crimp caps for volatile samples and chain-of-custody obligations.
Consumables for Sample Preparation
In the analytical process, the most crucial yet critical step is sample preparation. This step involves the elimination of interferences right before analyzing the sample. As a result, the system’s lifespan is prolonged.
Syringe filters, one of the most commonly used Laboratory Consumables, are utilized to eliminate particulates from a liquid sample before analysis and are commonly employed for HPLC or ion chromatography.
To select the appropriate syringe membrane filter, the size should be based on the sample volume being filtered. Additionally, porosity is determined by the size of potential particles in the sample, while the membrane type depends on the solvent and analytes requiring filtration.
The primary selection criteria for syringe filters are solvent compatibility and porosity. The Chemical Resistance Table enumerates the most popular chromatography solvents alongside compatible membrane types.
Solid Phase Extraction (SPE)
Solid-Phase Extraction (SPE), a solvent-free sample preparation technique, involves purifying the sample before injection. This technique involves loading the sample onto the cartridge (selecting media retaining the analytes of interest) and washing out impurities. Afterward, the downstream analysis is conducted after eluting analytes of interest.
Agilent Bond Elut C18 is the most lipophilic, bonded silica sorbent available. C18 is the most sought-after SPE sorbent due to its exceptional retention of nonpolar compounds. Bond Elut C18 retains the majority of organic analytes from aqueous matrices. Examination of small to intermediate molecules can be conducted using Bond Elut C18 for desalting aqueous matrices before ion exchange, as salts pass through the sorbent unretained.
Tubing and Fittings
To utilize an HPLC system, plumbing your system with suitable tubing and connections is crucial, as it affects the equipment’s uptime. A connection refers to a system comprised of a nut and ferrule. Determining what connection system to use depends on the threads of the receiving port, the geometry of the receiving port, the size and type of tube used, the material of the port, and the amount of pressure anticipated.
The tubing used in your system can also have a significant impact on the quality of your system connections, and the tubing material can also affect your system’s performance. For instance, FEP and PFA materials offer optimal chemical resistance but have poor pressure resistance—this kind of tubing is typically used from the solvent inlet reservoir to the pump (at atmospheric pressure). PEEK and stainless-steel tubing have outstanding pressure resistance but are not as chemically inert—this kind of tubing is usually found after the pump (where pressure is typically high). Pre-cut stainless steel tubing is electrolytically cut, providing a flat-burr-free end with a clean finish. This is advised over cutting tubing in the lab.
Columns are the core of the chromatography system, where separation takes place. Different compounds pass through the column at different rates due to their polarity, molecule size, and interaction with the stationary phase, which allows the sample to be analyzed.
Columns for LC are typically made from cylinders of stainless steel or PEEK, containing bonded silica or polymer particles. Columns are available in various dimensions to fulfill the needs of different applications. Column dimensions affect sensitivity and efficiency and determine the amount of analyte that can be loaded onto the column. For instance, small ID (internal diameter) columns improve sensitivity compared to larger ID columns but with reduced loading capacity.
There are criteria for selecting columns, which can be straightforward, including column phase for application and sizes for system pressure limits. For most cases, a particular phase or column dimension may be prescribed for a pre-defined method, and your choice will mostly depend on meeting these specifications and ensuring a reliable, high-quality provider.
Both GC and LC use syringes for sample transfer from a vial to the inlet. Syringes are used for manual injection or autosamplers, and these syringes come in different sizes, needle gauges, point styles, and terminations.
A syringe needs to be matched perfectly in the injection port to tolerate repeated injecting cycle without bending or breaking. Syringes must have the capability to resist chemical forces within a specific range, should be inert, which means they should not introduce interfering compounds into sample and deliver accurate volumes with no dead volume or carryover between injections.
Solvent and Reagents
Solvents serve as liquids utilized for the extraction and/or dissolution of substances, functioning as the mobile phase in LC. It is always recommended to opt for HPLC grade or superior solvents when selecting mobile-phase solvents for reverse-phase chromatography. Likewise, for Ultra-High-Performance Liquid Chromatography (UHPLC) and mass spectrometry, LC/MS grade solvents or higher are advisable.
Variations in selectivity and sample retention are observed among different mobile phases. The solubility of samples is also subject to variation, thereby influencing the choice of specific solvent(s).
With ionic compounds, retention experiences significant alterations with changes in pH, underscoring the importance of pH control to stabilize retention and selectivity. Method development with most samples, including basic compounds and typical weak acids, typically recommends maintaining a pH between two and four.
To ensure reproducibility, the pH utilized should ideally be within ± 1 pH unit above or below the pKa (acid dissociation constant) of the solutes being separated. In cases where the pKas of analytes are unknown, testing multiple mobile phase pH values may yield optimal results. Reversed-phase columns typically tolerate pH ranges from 2 through 8 or higher, offering versatility to identify the optimum mobile phase pH for separation. When determining the mobile phase pH, it is essential to measure and adjust it in the aqueous component before blending with organic modifiers to attain the most accurate and reproducible outcomes.
Reagents are used to enhance the selectivity and specificity of compounds. In GC, derivatization is used to change the functional groups of an analyte so that separation or detection can be done. Some of the benefits derivatization provides are higher volatility in samples, better selectivity, and chromatographic efficiency with increased detectability.
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