Abstract: The author has developed a method to compare various C-18 liquid chromatography columns and C-8 liquid chromatography columns. This method is based on the retention time of various liquid chromatography columns for polar and non-polar compounds, Differences in symmetry and selectivity. This experiment for comparing various chromatographic columns reflects both the hydrophobicity of the bonded phase and the activity of the silane matrix. The author collected the retention time data of the chromatographic column and put it into a chart for selection when replacing the column and backup column.
C-18 and C-8 silane liquid chromatography columns are the most commonly used columns in high-performance liquid chromatography (HPLC), and more than 100 C-18 and C-8 columns are sold on the US market . Faced with so many columns to choose from, it is difficult for analysts to choose the appropriate column for specific use, and it is even more difficult to choose a suitable replacement column.
For non-polar samples (such as small molecular aromatics) or weakly polar samples (such as parabens), C-18 and C-8 columns are the easiest to choose. For such samples, the main difference between the columns is the retention factor (k); however, there is only a slight difference in selectivity. However, the selection of columns for polar and medium polar samples is quite difficult. For example, pharmaceutical compounds containing amino or acidic groups. Analysts will find that polar samples vary greatly in retention time, selectivity, and peak shape.
The selectivity and peak shape of the column are much more affected by the support silica than the bonded phase. In addition, studies have reported the effects of surface silanol, silicic acid, and metal impurities in reversed-phase chromatography. In special cases, the difference in selectivity can be determined by the bonding process used when preparing the filler.
Usually, the chromatographer chooses the HPLC column by comparing the specifications of the packing media provided by the column supplier. These specifications include: surface area, end capping, carbon content, particle shape, particle size, pore size, pore volume, packing density and bonding degree. The carbon content and bonding degree are only provided by the chromatography manufacturer. Without these specifications, it is impossible for the user to calculate the grams of carbon and the number of micromolecules in the bonded phase in a chromatography column. Analysts can use these two data to estimate the hydrophobic properties of a column. However, even if the manufacturer provides all the above specifications, the user cannot accurately predict the selectivity of the column to compounds containing polar functional groups.
Since the retention time of chromatography is based on many subtle interactions between the analyte and the packed matrix, we recommend using a mixture test to compare the specifications and performance of the packed matrix. Engelhardt and his colleagues reviewed the characteristics of silane reversed-phase chromatography and proposed a solute test to describe the hydrophobicity and silophilic alcohol characteristics of the stationary phase. In addition, some people also improved the test conditions and methods to interpret those chromatographic data, but they only tested a few commercial chromatographic columns, and there was no carboxylic acid in their test mixtures. In this article, we used a test mixture containing carboxylic acid to collect data from 86 C-18 and C-8 silane columns (see Table 1). We describe the test results in detail as follows. Table 1: Manufacturers of chromatographic columns used in the study (omitted).
In our comparison, we used a test mixture containing 6 substances, which are listed in Figure 1. Each substance plays a special role in the test mixture. Uracil is used to create empty volumes. Toluene is a test column for hydrophobicity. Pyridine and N, N-dimethylaniline are basic amines used to test the activity of silanol groups on basic substances. Phenol is a weak acid and is used in combination with pyridine to determine the amount of active support silicon. 4-n-Butylbenzoic acid is an active carboxylic acid used to test silanol groups against acidic substances. This aspect is often overlooked by column manufacturers when they develop alkaline deactivated chromatography columns for amine analysis.
The mobile phase we used was a mixed solution containing 65% acetonitrile and 35% potassium phosphate at a concentration of 0.05M, with a pH of 3.2. The buffer solution with pH = 3.2 can protonate 4-n-butylbenzoic acid, and at the same time can improve the reproducibility of the retention time of pyridine and N, N-dimethylaniline. We found that using a mobile phase without buffer solution, such as 65% acetonitrile and 35% water, even if we use the same bottle of mobile phase, we can not get a reproducible retention time and peak shape. High ionic strength buffer solution, such as the 0.05M buffer solution used in this test, will inhibit the activity of some silanol groups (2, 5), but for reversed-phase chromatography columns that deactivate amines from some non-basic Some elution is necessary, and some inhibition is necessary.
We have tested two other buffer solutions, but their effect is less than that of 0.05M potassium phosphate solution with pH = 3.2. When the 0.01M potassium phosphate buffer solution has a pH = 3.2, amine compounds produce forward-shifting peaks in some chromatographic columns. The 0.05M potassium phosphate buffer solution had a better peak shape at pH = 7 than at pH = 3.2. The pKa of pyridine and N, N-dimethylaniline are both approximately 5.2; therefore, these components are not protonated and neutral at pH = 7, and do not undergo ion exchange with strongly acidic silanol groups.
Experimental part A 0.05M potassium phosphate buffer solution was prepared, and 6.8 g of potassium dihydrogen phosphate was added to 1 L of HPLC-grade water. Add concentrated phosphoric acid until pH = 3.15-3.2 (controlling the pH of the buffer solution in this range is very important, because pyridine and N, N-dimethylaniline are very sensitive to pH), mix with 650ml of acetonitrile and 350ml of buffer solution To prepare the mobile phase, the mixture is filtered through a filter with a pore size of 0.45um. In order to test whether the mobile phase was prepared correctly, we injected the test mixture into a chromatographic column and compared the retention factor of this mixture in the mobile phase of another batch of in-use mobile phases.
To prepare a 100ml test mixture, first mix 65ml of acetonitrile with 4mg of uracil, 15mg of pyridine, 40mg of phenol, 15mg of N, N-dimethylaniline, 30mg of 4-n-butane benzoic acid and 400mg of toluene, ultrasonic treatment for a while, then add 35ml of HPLC grade The water is sonicated until all solids are dissolved (fill this test mixture into a 2ml bottle, number 1092, provided by Alltech Associates). At the same time prepare standards for various substances with the same concentration as in the test mixture.
The flow rate is 1.0ml / min, the syringe capacity is 10ul, the column temperature is 19-22 ° C, an ultraviolet absorption detector is used, and the wavelength is 254nm. Regarding the diameter of the chromatographic column, all the chromatograms except Alltech's alphaBond C-18 and Waters 'uBondapak C-18 column (30cm × 3.9mm) and Waters' Symmetry C-18 and C-8 columns (15cm × 3.9mm) The columns are all 15cm × 4.6mm.
Regarding the filler particle diameter, except Altech's Adsorbosphere XL ODS (3um); Waters 'Nova-Pak C-18 (4um); Synchrom's Synchropak RPP C-18, 300 (6.5um) and Waters' uBondapak C-18, Alltech's Versapack C -18, Alltech's alphaBond C-18 and Whatman's Partisil OPS and Partidil ODS-2 (all 10um), all other columns are 5um. The size of the particles is only listed as an introduction. If the manufacturer uses the same silicon-based and bonding process when preparing particles of various sizes, the size of the particles does not affect the retention time, selectivity or peak shape.
The chromatographic columns used in the comparison are all new, either just packed or recently purchased. Flush all chromatographic columns with more than 20 column volumes of mobile phase, and repeatedly inject the test mixture until the retention time of each component changes by less than 0.02 min.
We injected standard samples to determine the order of peaks of each component and used the simple chromatogram to calculate the tailing factor. We did not use a column heater to control the column temperature, but we ensured that the ambient temperature fluctuation of the experiment was less than 3 ℃, and the retention factor caused by the ambient temperature change was less than 3%, so that the temperature change was negligible in this comparison .
The retention factor is calculated by k = (tr-t0) / t0, where tr is the retention time of the peak to be detected, and t0 is the dead time of uracil. The tailing factor (T) is calculated using the formula of the United States Pharmacopoeia T = W0.05 / 2f, where W0.05 is the peak width at 5% of the peak height, and f is the leading edge of the peak at 5% of the peak to vertical The maximum half width of the peak.
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