Chromatography Forum

this is the most confusing topic I have ever seen.
please point out if I am wrong:
I cut the Van Deemter plot to three sections with incresing flow I) plate height gets lower, 2) not much change 3) higher. correspoding to this:
1) for the peak height/plate no./column efficiency. a) increasing b) not much change( for the bottom limited part of the plot) and c) decreasing with increasing flow rate
2) UV detecting time is proportional to the peak width seen on a C-gram;
3) then UV detecting time x)decreased y) not much changed; z) increased ;----this is really interesting that the faster the mobile phase runs the longer the analyte stays in the UV cell-----(?)
4) for UV area, there are three possibilities too, o) not clear p) Not much changed; q) getting less . the reason is that the column efficiency effect was less than the flow rate effect for the uV detecting time with the bonus of lower peak heigth.
thanks

sorry I have to try again from my last post;
4) the signal or peak area is determined by the peak width times peak height; there is no way we can tell from the Van Deemter how the result of the width times the height will change.

II ran some experiment at different flow rates recently and was wondering about the same thing. Here’s the data based on the main peak (from Agilent 1200 HPLC, UV at 220 nm). Uwe was right, the peak area multiplied by the flow rate remained constant.


Flow rate (mL/min): 1.0; 1.5; 2.0; 2.5
Data rate (Hz): 10; 20; 20; 20
Peak area (mAU*s): 2597; 1734; 1302; 1037
Peak height (mAU): 947; 933; 909; 883
Width H ½ (min): 0.0422: 0.0287: 0.0217: 0.0181
Plates: 108506: 107212; 108635; 101699
Area x flow rate: 2597; 2601; 2604; 2593

I couldn't get the table to appear correctly, so I had to list it this way. could someone tell me how to post a table? Thanks.

Peak area times flow rate is constant.

Peak height decreases with flow rate.

This is all normal and exactly as it should be. What is the question?

Uwe, why "Peak area times flow rate is constant. " not something else such as peak width, peak height, plate number, plate height?
Thanks

to YM3142:

The first question was about peak area. If you multiply peak area with flow rate, it makes no difference what you do, what the flow rate is, or how long your molecules are in the detector cell (unless they get destroyed by UV).

Peak area times flow rate does not care about van Deemter... UV detection time is about one million times faster than chromatography, so no concern there...

Peak height will change with the column efficiency, which is a function of the flow rate, and in most practical cases, peak height will increase with decreasing flow. But nobody cares, and the effect can be very small...

to YM3142:

The peak height is proportional to concentration. When you integrate, you get the peak area in the form of concentration times time. This is per se not very useful, but if you multiply with flow rate, you get mass:

area = C*t = m/V *t

area * F = m / (V/t) *F = m / ((V/t) * (V/t) = m

(F is flow rate = volume / time)

(Concentration C = mass / Volume)

(m = mass)

Thanks, Uwe. this is a wonderful lesson.

One implication of peak area varying inversely iwth flow is that flow rate repeatability and reproducibility become extra terms in the uncertainty budget. For routine work I would guess that all the HPLC pumps are more than repeatable enough, but what about for highly accurate and precise analyses such as value assignments for reference mterials and certified reference materials.

How repeatable and reproducible are HPLC flow rates, and how accurate are the instrument read-outs of actual flow.

Since it is flow at the detector that matters, how much different is this from flow at the pump due to liquid phase compressibility and temperature changes (the column will usually be thermostatted but the pump will not be) ?

Thanks

Peter

Peter, remember that flow rate changes immediatly result in retention time changes, provided other things are kept constant. Also, I have always advocated measuring the flow rate separatly (not relying on pump rotation rate....). The flow rates given in publications should be from flow meters (behind detector), not pump settings/readings.

Thanks Hans,

Retention time does give an easily accessible measure of average flow (or at least if retention is constant average flow must also be constant). I like the idea of monitoring actual flow just downstream of the detector - is there hardware available to do this ?

Peter

Yes- a measuring cylinder and a stopwatch. Maybe you might want use some sort of stopper to limit solvent evaporation.

OK Victor

Doubtless that would work but what I had in mind was something that would monitor flow continuously in real time, during automated sequences of analyses and output its data to statistics software to be used to correct peak areas for flow.

Actually, what I would really like is for the experts on pumps to tell me that their hardware maintains flows at the detectors that do not vary by more than 0.1 %.

Peter

There are all kinds of electronic flow meters on the market: Bronkhorst, Analyt-MTC.....
google flowmeter, if you are not swamped let me know, I´ll check my catalogs etc.

Peter,

Flow rate accuracy is a subject that has confused many people. What counts (from my standpoint) is that the flow rate at the detector is accurate. This means what goes into the pump at atmospheric pressure should come out of the column at atmospheric pressure. Consequently, compressibility compensation is nothing but strange voodoo. Heating of the mobile phase make things more complex, but the old high-quality high-temperature GPC instruments were constructed such that mobile phase, pump, columns, and detectors were all at the same elevated temperature, and thus no concern about flow accuracy. With column heaters you add a constant expansion factor to the accurate flow coming from the pump. This adds some complication, but in practice, the thermal expansion is a constant factor.