hplc area variation

[jiangds06]

hi everyone,

is it normal for you guys to see 3 out 14 injections that are outliers. How could I improve the robustness of analytical methods?

[Consumer Products Guy]

hi everyone,

is it normal for you guys to see 3 out 14 injections that are outliers. How could I improve the robustness of analytical methods?

Is that 14 injections of the same sample/vial? Then that's terrible. Are you doing autosampler injection, or manual.

Normal for us would be all 14 injections well within 2%RSD.

[DJ]

Make sure your pump is putting out the correct flow rate.

[jiangds06]

how do you evaluate consistency of flow rate?

Make sure your pump is putting out the correct flow rate.

[uzman]

Check retention times , if your pump is good you can get consistent retention times .
Of course there are other variables effecting retention times ; such as temperature change , mobile phase composition change etc. , but normaly you must get good retention time repeatibility.

You may also check the pump flow rate by collecting waste from the detector outlet against time.

[jiangds06]

retention time is very consistent. see below.
3.81
3.81
3.81
3.81
3.81
3.81
3.81
3.81
3.81
3.81
3.81
3.82
3.81

Looks like pump and temperature control is very good.

Check retention times , if your pump is good you can get consistent retention times .
Of course there are other variables effecting retention times ; such as temperature change , mobile phase composition change etc. , but normaly you must get good retention time repeatibility.

You may also check the pump flow rate by collecting waste from the detector outlet against time.

[KM-USA]

Are you doing autosampler injection, or manual?

Are you doing autosampler injection, or manual?

I'd suspect/check out the autosampler. Maybe set up to do a simple standard/check-out standard. What brand/model of autosampler are you using?

If you're doing manual injections, anything can happen if your technique is not reproducible; time to get into the 1980s!

[DJ]

Are you doing autosampler injection, or manual?

time to get into the 1980s!

My Ph.D. adviser *still* uses strip chart recorders. "cm" was our unit for retention time and integration was done gravemetrically.



Are you doing partial or full loop injections? What size is your sample loop?

What is the column size, flow rate?

[M Farooq]

My Ph.D. adviser *still* uses strip chart recorders. "cm" was our unit for retention time and integration was done gravimetrically. Are you doing partial or full loop injections? What size is your sample loop?
What is the column size, flow rate?

Glad to know that old school still exists when the retention times increased from right to left! Manual injections can be irreproducible if the waste line is incorrectly configured i.e. at times it can drain the whole sample loop by siphoning off. Another common mistake is to inject the "exact volume" as the nominal loop volume. The loop should be filled by several loop volumes to make a reliable manual injection.

[jiangds06]

Partial loop is used.
my column size is 2.1mm and the size of sample loop is 50ul.

Are you doing autosampler injection, or manual?

time to get into the 1980s!

My Ph.D. adviser *still* uses strip chart recorders. "cm" was our unit for retention time and integration was done gravemetrically.



Are you doing partial or full loop injections? What size is your sample loop?

What is the column size, flow rate?

[skunked_once]

Partial loop is used.
my column size is 2.1mm and the size of sample loop is 50ul.

How much are you loading onto your 50 uL sample loop? The correct technique is to load at least 3X the loop volume if you wish to inject 50 uL. For lesser volumes, the amount loaded should be 50% or less of the loop volume. Also be sure to not remove the syringe from the injection port until after the injection has been made.

[jiangds06]

is this the problem why peak area varies?
or is this because S/N is too small?

Partial loop is used.
my column size is 2.1mm and the size of sample loop is 50ul.

How much are you loading onto your 50 uL sample loop? The correct technique is to load at least 3X the loop volume if you wish to inject 50 uL. For lesser volumes, the amount loaded should be 50% or less of the loop volume. Also be sure to not remove the syringe from the injection port until after the injection has been made.