The establishment of liquid chromatographic detection methods requires the selection of different separation modes and detectors tailored to specific samples and analytical objectives, fully leveraging instrument performance to efficiently achieve analytical goals. Typically, a single detector is employed for direct detection to achieve the desired results, and if necessary, derivatization can be performed prior to detection or multiple detectors can be combined for analysis. The principles guiding the selection of separation modes and detectors are essentially unified, involving preliminary assessments based on the structural properties of the analyte, separation mode, sample handling methods, and mobile phase selection. The compatibility of the detector is determined based on the characteristics of the analyte, separation mode, sample treatment, and mobile phase. The selection of separation modes and detectors represents an inseparable and mutually unified essential aspect of chromatographic method development.

Regarding the selection of separation modes and detectors,

The following examples may illustrate the principles:

01 Analysis of Aliphatic Biogenic Amines

Sample Characteristics of Aliphatic Biogenic Amines: These substances exhibit extremely weak absorption in the ultraviolet-visible range, and their derivatization process is intricate, inefficient, and prone to error. They are highly polar and water-soluble compounds.

Method Development Approach: Considering these fundamental conditions, we have determined that the Hydrophilic Interaction Liquid Chromatography (HILIC) mode with an Evaporative Light Scattering Detector (ELSD) is a suitable choice for analysis.

Detection Results are shown in the following figure:
Welch Ultisil® HILIC Amphion II column for the separation of four aliphatic biogenic amines.

02 Glycerides Analysis by Non-Aqueous Reversed-Phase Chromatography

Sample Characteristics of Glycerides: Glycerides are lipophilic substances that are sparingly soluble in water and insoluble in methanol, exhibiting non-polar properties.

Method Development Approach: For such substances, a non-aqueous reversed-phase chromatography method is deemed suitable.

03 Amino Acid Analysis

Sample Characteristics of Amino Acids: Amino acids exhibit weak ultraviolet (UV) absorbance, high polarity, and water solubility.

Method Development Approach: Two feasible approaches for analysis are derivatization method and non-derivatization method.

The separation effects of the two methods are shown in the figure below:

Welch Ultisil® Amino Acid Plus is a non-derivatization method used for the separation of 23 amino acids.
Ultisil® Amino Acid for Derivatization-based Separation of 19 Amino Acids.

04 Monosaccharide and Oligosaccharide Analysis

Sample Characteristics of Monosaccharides and Oligosaccharides: These samples are water-soluble and polar substances with weak UV-Vis absorbance.

Method Development Approach: For this type of substances, a commonly employed detection method is the differential refractive index (RI) detector.

Welch Ultisil® XB-NH2 for Fructose Analysis

05 Protein, RNA, Virus, and other Sample Analysis

Sample Characteristics of Protein, RNA, Virus, etc.: These samples are characterized by large molecular sizes.

Method Development Approach: Size exclusion chromatography (SEC) is a suitable choice for analysis.

Welch Xtimate® SEC-120 for RNA Analysis
Welch Xtimate® SEC-2000 for Analyzing Attenuated Genes of Vaccinia Virus.
Welch Xtimate® SEC-300 for Analysis of IgG Proteins.
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