Assay Calculation on Anhydrous basis

                   Assay Calculation on Anhydrous basis

 

What happens if my sample solvent is stronger than my mobile phase?

We do not recommend injecting in a stronger solvent because it usually results in peak distortion, broadening, poor sensitivity, and shortening of retention times.

This happens because some analytes will tend to travel too quickly through the column, instead of eluting in a symmetrical band.

If you absolutely must do this, keep the volume as small as possible and make sure the solvents are miscible.

 

 

Ideal injection volume vs column dimension

Optimal injection volumes are directly related to the cylinder volume of your  column and are, therefore, dependent on the cross sectional area (A=π r2) and length (L) of your column.

Therefore you can estimate any adjustment from an existing method for injection volume. If you are converting to a different size ID (with the same packing material and length), just multiply your current volume by the ratio of the radius squared to determine the correct volume for your new method. Injection volumes, as well as optimal flow rates, are limited by the size of the column. Ideally, your sample volumes should be, for the different column id:

Column ID	Volume (µL)
2.1 mm (30 -100 mm length)	1-3
3.0-3.2 mm (50-150 mm length)	2-12
4.6 mm (50-250 mm length)	8-40
10 mm (50-250 mm length)	40-100
21.2 mm (50-250 mm length)	150-300
30 mm (50-250 mm length)	300-700
50 mm (50-250 mm length)	1000-2000

Larger volumes may be acceptable if peaks are still symmetrical. Earlier eluting peaks will exhibit broadening first if the volume is getting too large.

If the solvent in your sample is stronger than the mobile phase (starting ratio if using a gradient), you will need to use a smaller volume.

Likewise, if your sample solvent is weaker than the mobile phase, you may be able to use a larger volume.

 

What to do when backpressure increases?

An increase in back-pressure usually suggests either a guard or analytical column problem. To find exactly where the problem lies we suggest you remove the guard column (if you are using one) and replace the old cartridge with a new one.

If the original pressure is restored, you solved the problem.

If the pressure remains high, disconnect the analytical column from the system, backflush it (do NOT connect the column to the detector while doing so) and run a few column volumes of your mobile phase through the column.

If the problem still persists you may have some strongly retained contaminants in your column coming from your previous injections.
Run the appropriate restoration procedures, as suggested by the column manufacturer, and retest the column.

If the initial pressure is not restored you may have to change the inlet frit or replace the column.

Always run your system (2 to 5 ml/min) without the guard column and the analytical column to verify that your pressure isn’t coming from another source, like a blocked in-line column prefilter, blocked/kinked tubing, particulates blocking your injector etc.
Always work your way from the detector back to the pump to isolate the problem.

 

Tailing factor ,Efficiency calculation , Resolution and Symetry

 

Column efficiency calculation

Column efficiency, indicated as the number of theoretical plates per column, is calculated as N = 5.54 (t_R / w_0.5)² where t_R is the retention time of the analyte of interest and w_0.5 the width of the peak at half height.

This half-height method enables the determination of the number of theoretical plates per column (N) even if the peak is not fully separated from a neighbouring peak (poor resolution), as long as the valley between the peaks is lower than the half-height of the peak. Half-height measurements commonly is the method of choice for automatic determination by data systems.

The larger the number of theoretical plates per column, the sharper the peak! Should you need to calculate the number of theoretical plates per meter, you must use the following equation:

Number of theoretical plates per column x 100/length of HPLC column (cm)= Number of theoretical Plates per m

 

Peak Asymmetry Factor

Peak Asymmetry Factor, often presented as A_s is calculated with the following equation A_s = b/a where b is the distance from the peak midpoint (perpendicular from the peak highest point) to the trailing edge of the peak measured at 10% of peak height and a is the distance from the leading edge of the peak to the peak midpoint (perpendicular from the peak highest point) measured at 10% of peak height. If As > 1 : tailing, et si As < 1 : fronting

 

Tailing Factor

Tailing Factor (T_f) is the USP coefficient of the peak symmetry. It is calculated using the following equation: T_f = (a+b)/2a where a is the distance from the leading edge of the peak to the peak midpoint (perpendicular from the peak highest point) measured at 5% of peak height and b is the distance from the peak midpoint (perpendicular from the peak highest point) to the trailing edge of the peak measured at 5% of peak height.

 

Resolution

Resolution (R_s) is a measure of the separation quality. In order to determine the resolution between 2 peaks we need to measure the retention times of the 2 peaks of interest (t_r2 and t_r1) and the width of the 2 peaks at baseline (w₁ and w₂) between tangents drawn to the sides of the peaks. It is normally calculated as:

R_ss = (t_r2 – t_r1) / ((0.5 * (w₁ + w₂)

 

Since nearly every peak shows some degree of tailing, so to allow for a small amount of tailing and still retain a bit of flat baseline between the peaks, R_s ≥ 2.0 generally is desired for proper resolution between 2 peaks of interest.

This equation is extremely convenient and gives good results for peak resolution calculations, but it is only useful when the peaks are resolved at baseline level. However, we are often confronted to situation where peaks are marginally separated. Peaks overlap at the bottom, and measurement of the peak width at baseline is virtually impossible.

In these cases, just like we measured the efficiency at mid peak height, the same approach can be used for the calculation of the resolution with the following equation:

R_s = (t_R2 – t_R1) / ((1.7 * 0.5 (w_0.5,1 + w_0.5,2))

where w_0.5,1 and w_0.5,2 are the peak widths measured at half the peak height. Note that the factor of 1.7 is added to the denominator to adjust for the difference in width at the half-height. The half-height technique is the way many data systems measure resolution, because it is simpler to measure than the baseline width.

The number of theoretical plates per column (performance)/symmetry factor/Tailing Factor/Resolution can and will change depending on the type of analysis and analytical conditions used.