Problems with FLR (no autozero possible)

UPLC_User

<p>Hello,</p><p></p><p>I created an instrument method for the use with FLR.</p><p></p><p>Excitation: 276 nm</p><p>Emission: 308 nm</p><p>Gain: 50</p><p>AutoZero after Injection</p><p></p><p>When I monitor the method, the signal goes down to -10'000 immediately and there are messages and beeps due to signal saturation.</p><p></p><p>Thus, I stop monitoring and try to make Autozero. But this does not work. Only a Reset of the FLR brings the signal to zero.</p><p></p><p>After starting the monitoring again, the problem remains the same as described.</p><p></p><p>My mobile phase is methanol/water 52:48</p><p></p><p>Could anybody help me?</p><p></p><p>Florian</p>

KWB

Hi Florian,

Were you monitoring Emission Units or Energy Units? Emission Units are meant to be independent of Gain settings, although you can still have too high a gain when using either unit. This is specified in the instrument method. I believe the service engineer would be monitoring Energy Units for the Gain/Linearity test. Using Emission Units allows multiple FLR detectors to have their response normalized against Raman spectrum of water..

Regards,

Ken

RD

Lower the Gain to 1 and see what happens.

Regards,

Rich

UPLC_User

Dear Rich,

thank you for you reply.

Gain 1 works.

But: why do we have the possibility to increase the gain up to 1000, when the simple monitoring of a methanol/water baseline already leads to a signal saturation when gain 50 is used?

Regards

Florian

KWB

Hi Florian,

Try a monitoring with the Gain set to 1. The Photomultiplier Tube protects itself when overloaded. In this state, the signal will go below zero and the detector starts beeping. If this resolves the problem, make an injection with a Gain of 1 and see if you can see your peaks. The next step is to optimize the Gain setting so that your highest standard does not oversaturate the PMT. You want to use the highest gain possible to have the most sensitivty without overloading the PMT. The Acquity FLR getting started guide describes how to do this.

Sometimes, even with the PMT gain set correctly for the peaks of interest, you may still have an overload condition when the unretained portion of your sample passes through the detector, but this is only a temporary condition. If it does not interfere with the separation, there is no need to change the gain. You can also program the gain as a timed event.

I believe that the FLR will autozero itself by default at the beginning of an injection (this may be an option you select). You can program autozeroing during the run, but don't try to autozero when the signal is being driven high or low.

Regards,

Ken Blakeslee

UPLC_User

Hello Ken,

thank you so much for your sophisticated answer; I really appreciate it.

During some gain trials, I noticed that the area of my peak is the same for gain 10 as well as for gain 20.

But the Waters service man performed a gain linearity test acc. to Waters standard procedure (using an anthracene solution).

Shouldn't a comparable test also work for my analyte (estriol)?

Additional note: we have installed the large volume cell.

Regards

Florian

UPLC_User

Dear Ken,

I just tried it out with the monitoring of the energy. Now, the peak area is dependent on the gain setting.

I performed four runs:

RunNo	Gain	Area	Area/Gain	S/N ratio
1	1	2012727	2012727	97
2	5	11088737	2217747	89
3	10	22805036	2280504	101
4	20	42794218	2139711	109

Up to now, I thought the gain might be a tool for better sensitivity of small peaks. But now, I am disappointed due to the fact that the S/N ratio is more or less independent on the gain. The increasing peak areas are worthless.

I also wanted to try additional experiments with gains higher than 20 (up to 1000 is possible). But when I monitor the baseline, the instrument beeped and indicated a signal saturation, which shall arise from a methanol/water mobile phase. I am really puzzled about that.

So, please help me:

- am I too stupid to see the advantages of higher gains?

- am I too stupid to see the reason for the beeps when monitoring MeOH/H2O with a gain of 30? Non-answering this question is not really a problem due to the fact that higher gains do not improve my sensitivity.

Thank you very much for helping me.

Regards

Florian

KWB

Hi Florian,

Its logical to think or hope that a higher Gain setting will create a corresponding increase in detector sensitivity, but increasing the Gain only increases the amplification of the photomultiplier tube of the light coming out of the flow cell. Everything is amplified, including background noise originating in the cell. What is noise in the detector? Some of it originates in the electronics, some of it originates in the flow cell. Mobile phase can have fluoresence, depending on the mobile phase composition and the excitation and emission settings. It is well known that dissolved oxygen can contribute to a noisy background fluorescence signal.

As you found, increasing gain does not lead to great increases in sensitivity. For any given method however, there is a usable range which must be determined. It is possible to have the Gain set too low to detect your lowest standard and samples. Its like turning the volume down on the TV. The softest spoken voices will not be heard. You need to establish the minimum Gain needed for your method's detection range. You want to find the highest Gain setting which is good for the high end of the method. This will give you the highest Gain to apply to the low end of the curve and still not oversaturate at the high end.

If you have more than one peak and they respond differently, you can program Gain changes within the run - if there is enough separation between the peaks.

To maximize signal to noise, don't forget to optimize the data detection rate (around 20 - 25 points per narrowest peak) and select an appropriate Filter setting (usually 'Normal'). Too many data points will generate unneccesary noise. Too few points will lose peak response.

Regards,

Ken