Chromatography Forum

Hello,

I would like to ask if I reason well.

1. For DAD detector peak height decreases with the level of energy lamps - there is no reference beam, unless we often do "Set Light Exposure Time"

2. For UV-vis detector peak height is constans - we observe always a difference (sample side - reference side)

I'm right?

Hello

1. Lamp intensity decreases with lamp age. So if lamp is "old" you can see less absorbance for some wavelengths ranges.

2. What do you mean by "peak height"? If you mean detector "signal" how can it be constans? Please explain

Regards

Tomasz Kubowicz

Hello Tomasz,

I thing about the signal level for the same samples after first month, second month etc.

For Uv detector both sides are weaker and the difference is constans - like in spectrophotometr - light weakens and the result is the same.

For DAD on sensor falls less and less light and the signal should be smaler. If we make "Set light exposure time", increase the sensitivity for the current beam to the threshold of saturation and again the signal level will be like formerly (and a noise are higher).

I thing so, but I'm wating for a confirmation.

BR/Pozdrawiam
Grzegorz

Hello

You shouldn't really compare signal level (Peak height or even area) after month, two, three etc. As long as you run calibration for your method it doesn't matter. Of course it matters if signal for your lowest standards drops to level that is lower than LOQ of even LOD.
You haven't mentioned what detector you have (manufacturer) and even if theory is the same detectors have different design and can work slightly different.

Regards

Tomasz Kubowicz

Hello,

I just want to know that in case of DAD detector peaks decreasing over time, and in the case of the UVvis detector does not and that's all. The detektor is from Shimadzu:SPDM-20A and older SPDM-10Avp.

According to my experience on UV spectrophotometers ( double-beam ) ,absorbance readings ( UV range ) are little higher, with a new deuterium lamp ,against reference filters.

I think , it will apply also to HPLC UV detectors.

Hello uzman,

sorry, unfortunately, you are wrong, . The spectrophotometr must measure well regardless of energy lamps. I mean about the tests indicated the absolute values i.e grey glass filter. Is not it?

Hello Greg07,

Yes, with grey glass filters .
The transmittance values drop a little ( but within the tolerance ) , after changing the lamp.

I observed this on several occasions , also on different spectrophotometers.

I know that double beam spectrophotometers must handle the energy loss theoratically , but I think both detectors are not getting the energy at equal levels , may be because of beam splitter designs , so there may be some non-linear region below a certain energy level.

The double beam in a spectrometer will correct the %transmittance or %absorbance as the lamp changes, but the actual amount of light hitting the detectors will drop as the lamp ages. So 80% transmittance will remain 80% transmittance or absorbance depending on how you measure it, but the lumens hitting the detector will be less, which will mean at low concentrations near the limit of detection you will begin to receive a 0% absorbance with slightly higher concentrations over the age of the lamp.

Any detector using a single beam/single detector will show you the loss in sensitivity as the lamp ages for all concentrations of analyte measured, but the double beam detectors will probably only show the loss of sensitivity when analyzing the lowest standards. When you reach the point that your lowest standard and second lowest standard gives no response you know you have lost sensitivity with the ageing lamp in the double beam instrument.

Hello,

The discussion reached the limit of quantification, . I think about the normal energy levels. So again:

Detector UV
New lamp - 100% Energy level - peak height - i.e. 100mV
after 1 year - 50% Energy - peak height - still 100mV

Detector DAD
New lamp - 100% Energy level - peak height - i.e. 100mV
after 1 year - 50% Energy - peak height - 50mV - yes (I say) or no?

Regards

A DAD includes UV wavelengths in its range.The only practical difference between a "UV" detector and a "DAD" detector is that the DAD records several different wavelengths at the same time, but this does not affect the signal intensity at an given wavelength.

For a given wavelength and wavelength range (slit width) absorption by the sample is the same for a UV and a DAD. Therefore transmittance is also the same.

So why would the peak height (signal) be different between a DAD and UV detector ??

Peter

The 100mv signal is a signal that is output by the instrument after the signal is processed, not a direct reading from the detector itself. The processor in the instrument will convert absorbance or transmittance into a mv output signal. If the UV instrument is using a reference, then it will normalize the output signal to 100mv but if the DAD is not using a reference then a drop in signal will be carried through to the output and less mv will be produced.

Think of it as the difference in using a solid meter stick and one made of rubber, if you stretch the rubber one, you still get a measurement of 1m even though you are measuring a longer distance.

Is this really true? I'm not happy.
Nearly all of us have our PDA detectors set to auto-zero at the start of the run. Zero absorbance means 100% transmittance, so when the detector auto-zeroes, what it's actually doing is measuring the full intensity of the unabsorbed light-beam. It is measuring that today the lamp's current output is, say, 12429 counts, on the assumption that nothing is absorbing any light. This is declared to be a transmittance of 100% and an OD of zero.

The detector knows how bright its light is.

When, a minute later, a peak elutes and the light getting through drops to only 1245 counts, the detector knows this is 10% of its original, autozeroed value, and 10% transmittance means an OD of 1.0.

A month previously, the actual light when it autozeroed might have been 15123 counts, because the lamp was newer. In this case, the peak would have reduced this to 1512, and the detector would still have declared it an OD of 1.0 because it was still 10% of the autozeroed value.

You don't need a double beam to measure the lamp intensity in the absence of absorbance (at least, provided your lamp intensity is stable over the 15 minutes of measurement that followed the last autozero).

The consequence of deteriorating lamp output is deteriorating light-level relative to electronic noise, not a change in OD. This leads to noisier chromatograms and reduced sensitivity (worse limits of detection), but it doesn't change the absolute peak area. Autozeroing is a reference, just shifted in time rather than shifted in space, as a double-beam instrument would.