When many analysts operate liquid chromatogram, columns work at room temperature. If the temperature in the laboratory can be kept constant, problem barely occurs. However, most working places’ temperature constantly changes. If the liquid chromatography method developed by one laboratory is applied to other laboratories, the temperature difference will cause more complicated problems. So today, we would like to introduce how temperature affects chromatographic separation.
Change in Selectivity
Fig.1 Column temperature : 30℃
The blank ingredients appear before the principal component and are not separated
Fig. 2 Column temperature: 25℃, other conditions kept consistent
As can be seen from the superposition diagram, when the column temperature decreases, the separation degree becomes worse.
Fig. 3 Column temperature : 40℃, other conditions kept consistent
As can be seen from the overlapping diagram, when the temperature of the column is raised to 40 degrees, the peak of the blank ingredient, the peak of the principal component and the peak of the impurity I can be completely separated.
Comparing with figure 1, 2 and 3, it is obvious that temperature change can cause significant change in selectivity. In Figure 1, the blank ingredient and the main peak are very close at 30℃, but when the temperature decreases, the blank ingredient and the main peak are closer. When the temperature rises to 40℃, the two peaks are separated. From the three figures, the optimal separation condition for the two peaks is 40℃. It is interesting to see that the variation in selectivity is related to the composition. For instance, the relative positions of the first two peaks in the figure are almost unchanged when the temperature changes.
Changes in Retention Time
Most analysts know that the retention time will be affected by the temperature at isocratic elution and gradient elution. When the temperature rises, all chromatographic peaks will move forward. In the case of isocratic elution, the retention time will be shortened by 1-3% for every 1℃ increase of temperature. Laboratories with temperature control systems usually have all-day temperature control setting, but this setting can cause significant changes in room temperature, which will influence chromatographic systems that operate overnight.
In different seasons, the laboratory’s nighttime temperature may be lower than the temperature of normal working day, but this change in temperature will lead to “retention time window” of chromatographic peak, resulting in a series of invalid data in the continuous injection. Another possible factor influences temperature is the location of the chromatographic instrument in the laboratory. When the column is directly opposite the air supply outlet of the air conditioner, the temperature of the chromatographic system will constantly change although that of the whole laboratory is very stable. That’s why we use column oven.
We must control the column temperature. The best way is to use the column oven. Commercial column ovens come in two forms: direct heating of columns and heat transfer through air. Each design has its advantages and disadvantages, and LC systems designed by various manufacturers have different limitations.
In the direct heating type column oven, the column is sandwiched in the metal heater or wrapped by the heater. But air heat transfer type of column oven and column oven for GC column are similar, the column is suspended in the static or flow air, by heating the air to maintain the column temperature.
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