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.
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.
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.
Column efficiency, indicated as the number of theoretical plates per column, is calculated as N = 5.54 (tR / w0.5)2 where tR is the retention time of the analyte of interest and w0.5 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, often presented as As is calculated with the following equation As = 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
Resolution (Rs) 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 (tr2 and tr1) 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:
Rss = (tr2 – tr1) / ((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, Rs ≥ 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:
Rs = (tR2 – tR1) / ((1.7 * 0.5 (w0.5,1 + w0.5,2))
where w0.5,1 and w0.5,2 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.
Column Efficiency or Theoretical plate
At the beginning of a chromatographic separation the solute occupies a narrow band of finite width. As the solute passes through the column, the width of its band continually increases in a process called band broadening. Column efficiency pro- vides a quantitative measure of the extent of band broadening.
In their original theoretical model of chromatography, Martin and Synge2 treated the chromatographic column as though it consists of discrete sections at which partitioning of the solute between the stationary and mobile phases occurs. They called each section a theoretical plate and defined column efficiency in terms of the number of theoretical plates, N, or the height of a theoretical plate, H; where
A column’s efficiency improves with an increase in the number of theoretical plates or a decrease in the height of a theoretical plate.
Assuming a Gaussian profile, the extent of band broadening is measured by the variance or standard deviation of a chromatographic peak. The height of a theoreti- cal plate is defined as the variance per unit length of the column
where the variance, σ2, has units of distance squared. Because retention time and peak width are usually measured in seconds or minutes, it is more convenient to ex- press the standard deviation in units of time, τ, by dividing by the mobile phase’s average linear velocity.
When a chromatographic peak has a Gaussian shape, its width at the baseline, w, is four times its standard deviation, τ.
w = 4τ …………………12.15
The number of theoretical plates in a chromatographic column is obtained by com- bining equations 12.12 and 12.16.
Alternatively, the number of theoretical plates can be approximated as
where w1/2 is the width of the chromatographic peak at half its height.
It is important to remember that a theoretical plate is an artificial construct and that no such plates exist in a chromatographic column. In fact, the number of theo- retical plates depends on both the properties of the column and the solute. As a re- sult, the number of theoretical plates for a column is not fixed and may vary from solute to solute.
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