How do you calculate injection volume HPLC?

For example, if you are currently injecting 20 µL on a 150 x 4.6 mm column and then switch to a 150 x 3.0 mm column, you could estimate the adjusted volume by multiplying 20 x (1.52)/(2.32). Your new volume should be about 8.5 µL.

How do you calculate HPLC?

Should you need to calculate the number of theoretical plates per meter, you must use the following equation:

  1. Number of theoretical plates per column x 100/length of HPLC column (cm)= Number of theoretical Plates per m.
  2. Rss = (tr2 – tr1) / ((0.5 * (w1 + w2)
  3. Rs = (tR2 – tR1) / ((1.7 * 0.5 (w0.5,1 + w0.5,2))

How do you calculate retention factor in HPLC?

The retention factor is calculated by multiplying the distribution constant by the volume of stationary phase in the column and dividing by the volume of mobile phase in the column.

How is resolution calculated in HPLC?

Resolution is an important HPLC performance indicator usually assessed by how quickly and how completely target components in a sample separate as they pass through a column. Resolution is measured by dividing the difference in peak retention times by the average peak width.

How do you inject in HPLC?

In the load position a sample loop is filled with sample while the system is equillibrating. When turning to the inject position, the sample loop is switched to the high pressure part of the HPLC system. The flow delivered by the pump flows through the loop and feeds the sample onto the column.

How does injection volume effect HPLC?

The effect of the injection volume on the performance of RP-amide fused-core HPLC chromatography has been established and demonstrated to be more pronounced than on conventional columns. Varying the injection volume within the 2–100 μL range results in altered performance characteristics.

What is K value in HPLC?

The retention (or capacity) factor (k) is a means of measuring the retention of an analyte on the chromatographic column. Determination of Retention Factor (k) A high k value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase.

What is the tailing factor limit in HPLC?

Acceptable Tailing In practical terms, an As value below 1.5 is usually OK to work with, and up to As = 2.0 may be acceptable depending on the separation and resolution of the peaks. If the As value is greater than 2.0, then there is a problem that needs to be identified and fixed.

What is injection in HPLC?

In HPLC, whether the injection is manual or automatic, the injection is performed with an injection loop that must be filled and then injected into the system. The volume of the injection loop plays a major role in the volume of sample injected. On a general basis, injection loops vary from 1 to 100 µL.