How to use liquid chromatography column better

High performance liquid chromatography (HPLC) column is used for the rapid separation of mixed polar samples. Compared with C8 or C18 column, the hydrophobicity is smaller, and eluent with high water content can be used. It has a shorter retention time for nonpolar compounds, but has little effect on polar compounds. High performance liquid chromatography (HPLC) column is a kind of chromatographic analysis method with liquid as mobile phase. It also separates the mixture according to the difference of partition coefficient between mobile phase and stationary phase. The method for evaluating column efficiency and the formula for calculating the number of theoretical plates in gas chromatography are also suitable for high performance liquid chromatography.
Performance advantages:
1. The ideal choice for separation of biomolecules is complete termination;
2. High coverage, fully bonded, spherical silica gel carrier, completely capped, medium hydrophobic;
3. The hydrophobicity of the stationary phase is small and the separation characteristics are different from those of ODS.
The experimental conditions were as follows
1. The column 1 was 15cm long and 4.6mm in inner diameter, and packed with 10um C-18 alkyl bonded stationary phase;
2. The column 2 was 15cm in length and 4.6mm in inner diameter, packed with 5um C-18 alkyl bonded stationary phase;
3. Mobile phase: methanol: water (85:15), flow rate: 0.8ml/min;
4. The UV detection wavelength was 254nm and the sensitivity was 0.08;
5. The injection volume was 5 μ 1.
The experimental steps of HPLC column were as follows
1. According to the experimental conditions and experimental video guidance, adjust the instrument to the injection state according to the operation steps of klc321 instrument. When the instrument flow path and circuit system reach balance, and the baseline of "sampling system" of chromatographic workstation is straight, the sample can be injected;
2. Take 5 μ 1 methanol solution of benzene, naphthalene and biphenyl for injection, record the chromatographic data with chromatographic workstation, and record the file name of chromatographic data at the same time;
3. The data files are processed and recorded by the data processing system of chromatographic workstation;
4. The number of plates of column 2 was determined on the instrument, the chromatogram was printed and the column efficiency was compared.
Data recording and processing:
1. The experimental conditions were recorded.
a. Chromatographic column and stationary phase;
b. The mobile phase and its flow rate, pre column pressure;
c. Detector and its sensitivity;
d. Injection volume;
2. The retention time TR and half peak width Y12 of the corresponding peak were recorded.
3. The theoretical plate number N and theoretical plate height of benzene, naphthalene and biphenyl on the column were calculated respectively.
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