What is amino acid analyzer?
The instrument used to determine the amino acid composition or content of proteins, peptides, and other pharmaceutical preparations is called amino acid analyzer
.Amino acid analyser
uses cation exchange chromatography separation and post-column derivatization with ninhydrin to analyze the content of protein hydrolysate and various free amino acid components. The basic structure of the instrument is similar to that of ordinary HPLC but optimized for amino acid analysis in detail, such as nitrogen protection, inert piping, online degassing, elution gradient, and column temperature gradient control.
Amino acid analyzer working principle
Due to the differences in structure, acidity, polarity, and molecular size of the amino acid components, they can be separated from the cation exchange column. The amino acid components
can be eluted sequentially by using buffers with different pH ion concentrations, and then mixed one by one with another stream of ninhydrin reagent, after which they flow together into the spiral reaction tube. The color development reaction is carried out at a certain temperature, typically 115 to 120 degrees Celsius, resulting in the formation of a blue-violet product with maximum absorption at 570 nm. The hydroxyproline reacts with ninhydrin to produce a yellow product with maximum absorption at 440 nm.Amino acid analyzers
are usually subdivided into two systems, the protein hydrolysis analysis system (sodium salt system) and the free amino acid analysis system (lithium salt system). Among them, the free amino acid analysis system utilizes gradient elution with sodium or lithium citrate at different concentrations and pH values.
The sodium salt system analyzes up to about 25 amino acids at a time, which is faster and has better baseline straightness. The lithium salt system analyzes up to about 50 amino acids at a time, and its speed is slower and the baseline is generally not as good as that of the sodium salt system.
Classification of amino acid analyzer
Routine amino acid analysis refers to the analysis of 20 protein-hydrolyzed amino acids and more than 40 free amino acids. Since its introduction in 1958, the amino acid analyzer has been continuously updated with modern hardware and software and has now developed into automated routine testing equipment essential for amino acid analysis in modern food, feed, biotechnology, medicine and health, and life science industries.
Amino acid analyzers can be divided into two types according to their separation and detection methods.
The first type is the classical method based on cation exchange column separation and post-column ninhydrin derivative photometric determination -- ion-exchange column chromatography (IEC). This type of method was awarded the Nobel Prize in 1972 and is the international and national standard as well as arbitration and foreign-related method today.
The second category is all high-performance liquid chromatography (HPLC) methods based on reversed-phase chromatographic separation, pre-column derivatization, fluorescence or UV detection, and ion chromatography (IC) methods with direct amperometric detection of anion-exchange separation.
Comparison of the characteristics of amino acid analysis methods
|Principle||Cation exchange separation, post-column ninhydrin derivatization photometric determination||High-performance liquid chromatography, pre-column derivatization, photometric method determination
|Excess reagent||Noninterference||Need to remove||Noninterference||Need to remove||Noninterference
|Detection limit||3p to 50p mol||50p mol||1-5p mol||1p mol||3p mol
|Advantage||The method is mature and reliable, with the advantages of better stability, reproducibility, and linearity than the HPLC method, no matrix interference, long column life, and the ability to analyze 43 amino acids at one time.||The instrument is multifunctional and has a high sensitivity to some amino acids, with a short analysis time.
|Disadvantage||High-performance requirements for the whole machine, requiring dual gradients of concentration and temperature Programmed control. Instrument specialization.||The stability of derivatization reactions and products is often disturbed by matrix and excess reagents. The stability, reproducibility, linearity, and reliability of the method are not as good as those of the ninhydrin method. Short column life. Derivatization reagents are generally expensive and the method is technically demanding.
Overall, IEC is superior to HPLC, and the IEC instrument is a dedicated automated amino acid analyzer.
Application of amino acid analyzer
Automatic amino acid analyzer
A. Applications in the pharmaceutical industry
Cell physiology research cell is the basic unit of life activity. It is composed of many kinds of proteins, and the content of proteins and the composition and content of amino acids
in them vary with different physiological conditions. Therefore, in the study of cell physiology, it is necessary to analyze the protein amino acids of the cell with an amino acid analyzer.
In the biochemistry laboratory, amino acid analysis
is needed to study the composition and structure of proteins and the mechanism of enzyme action.
B. Applications in Agriculture
When cultivating good varieties of crops, it is necessary to consider high yield and adaptability to the soil, moisture, climate, etc. It is also important to focus on improving the content of protein amino acids
Adding amino acids to feed can accelerate production, so the use of amino acid analyzers
to determine the content of amino acids can help adjust the formula or add feed additives.
Due to the pollution of the environment by pesticides, the development of non-polluting pesticides has received great attention. In recent years, the use of amino acids
as raw materials for pesticide research has made great progress, and now there are herbicides made from glycine. In the development of pesticides for the development of amino acid raw materials, the use of amino acid analyzers
helps to perform many amino acid analyses
C. Applications in the food industry
In improving food processing and developing new products, developers must take into account the nutritional value and tastiness of the food. And the deliciousness of food is related to amino acids, such as glutamic acid and methylic acid are acidic, its sodium salt is sour, while glycine and alanine are sweet. The nutritional value of food is mainly determined by proteins and amino acids, so it is important to measure their amino acid content in food processing.
In process improvement, amino acid analysis needs to be compared before and after process improvement to determine whether the new process is feasible. In trial production of new products, amino acid analyzers are also needed to determine the number of amino acids in the new product to determine its nutritional value and good taste. In addition, food fortification also requires adding amino acids to the food, so amino acid analysis should also be performed.
D. Applications in the leather industry
The softness and durability of leather are related to the amount of hydroxyproline it contains, so amino acid analyzers are needed in the research of the tanning process to determine the amount of hydroxyproline and other amino acids.
E. Applications in the field of oil exploration
The content of amino acids in oil rocks is one of the important data to determine the richness of oil deposits, so amino acid analyzers are often used to determine the content of amino acids in oil rocks in petroleum development research.
Amino acid analyzer uses
Amino acid analyzer slideshare
A. Sample treatment
For the determination of various free amino acid contents in samples, the fat impurities can be removed and then directly analyzed on the column.
For the determination of the amino acid composition of proteins, the sample must be hydrolyzed by acid so that the protein is completely transformed into amino acids before being analyzed on the column.
B. Sample analysis
The treated samples are loaded onto the column for analysis. The amount of sample to be loaded on the column is determined by the sensitivity of the autoanalyzer used. Generally, it is about 0.1μ mol of each amino acid, and the dry weight of the hydrolyzed sample is about 0.3 mg. The determination must be carried out at pH 5-5.5, 100°C, and the reaction time is 10-15 min.
The resulting purple material is measured colorimetrically at 570 nm. The resulting yellow compound is measured colorimetrically at 440 nm. It takes only about 1 hour to do a full amino acid analysis. Dozens of samples can be loaded into the instrument at the same time and analyzed automatically in sequence, and the data given is calculated automatically at the end. The instrument degree is ±1 to 3%. Separation and determination of amino acids by cation exchange column.
a. The instrument has a 15-minute preparation procedure after powering on. This phase is mainly for the instrument to automatically set the working status and parameters of each component, test the communication status of the workstation and each component and set the temperature and pressure, so do not terminate this process artificially.
b. Proteolytic amino acids and physiological fluid amino acids (20 and 43) can be analyzed continuously on the autosampler, which is automatically recognized and recorded by the instrument without any additional operations by the user.
c. After a set of samples has been analyzed, the instrument will automatically start a post-program to maintain the system, so please do not terminate this program artificially.
d. The default cycle program is automatically enabled in standby mode to maintain the instrument. The reagent consumption of the cycle program is very small (1 ml/hour), so the user should not change this setting.
e. The instrument operates with a nitrogen pressure of 0.4 bar; if the nitrogen pressure is too high, the instrument will be damaged.
f. Check whether the eluent and derivative reagent flow path pressure of the automatic amino acid analyzer is normal.
g. For better maintenance of the instrument, it is recommended to add one or two cleaning procedures or manually clean the system before the post-program after each batch of samples.
h. If the instrument is temporarily set aside, it is recommended to remove the column and flush the instrument pipeline at a high flow rate, and fill the pipeline with 40% isopropyl alcohol.
i. The injection concentration range is 0.4-4 nmol. Due to the high sensitivity of the machine, if the injection concentration is too high, the analysis results will be affected and the reactor will be easily clogged.
How to buy amino acid analyzer?
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