Выходные данные
Статус student, 5th year of Specialist Degree program
Организация Novosibirsk state University, Institute of solid state chemistry and mechanochemistry SB RAS, Nivisibirsk, Russia
Подсекция Аналитическая химия
Метки студент
Chromatographic determination of amino acids
Dome Karina Viktorovna
Dear Karina, according to this data, you have adopted the existing procedure (i.e. both procedures: derivatization and gradient HPLC separation from ref. [5]) to your type of samples. What is your personal scientific impact in these parts of the work. Thanks in advance.
Dear Andrey Stavrianidi,
Indeed, the methodology [5] was taken as the basis for this work. However, it could not be used immediately as written. The sorbent used in my column has a different composition. Because of this, the order of release of amino acid derivatives has changed. I injected one amino acid at a time to determine the exact retention time under our conditions. Under the gradient elution conditions of Method 5, amino acid separation with satisfactory peak resolution was not achieved. Thus, several derivatives of amino acids (isoleucine and leucine, phenylalanine and tryptophan, proline and a certain impurity from the reagents) had a resolution criterion of less than 0.5. This is absolutely insufficient to talk about peak resolution. I managed to find the necessary conditions to achieve greater resolution of the peaks without increasing the time of registration of the chromatogram (Table 1). Better resolution can be achieved with increasing time. Also, from the point of view of the resolution of the peaks, I checked the optimal elution rate and column temperature, their optimal value was the same as in methodology [5].
For the derivatization of amino acids, experiments were carried out to determine the effect of the purity of triethylamine. Was used «chemically pure» (99%) and «for HPLC» (99,5%). It was shown that the effect of impurities does not interfere with the determination of analytes. We also selected the optimal drying option in terms of time, availability and convenience. Drying on a rotary evaporator was chosen instead of drying with an inert gas or in a vacuum oven.
All the work I have done will be further used to determine free amino acids by HPLC in the products of processing plant materials, for example, acid and enzymatic hydrolysates, which were discussed in the introduction.
Could you explain how trueness and precision of measurement were carried out.
Dear Ivan V. Mikheev,
I determined the trueness according to the «entered-found» method.
Precision was calculated under repeatability conditions. The standard deviation was used as a measure of the scatter of the measurement results under repeatability conditions. Unfortunately, it was not possible to determine the reproducibility (use other equipment, another method, invite another specialist).
The statistical processing of experiments data was perfomed based on the results of 5 parallel experiments and represented rhe mean with a confidence interval (n=5, P=0,95).
I want to note that the instability of aqueous solutions of amino acids was observed. So fresh solutions were prepared an every time.