Chromatography Forum

Hello,

I meet in my work with DAD and spectrophotometric detectors. Recently, a situation occurred that on the chromatogram from DAD the peak from substance A for lambda max (280nm) is higher than for 240nm - area 140k:70k and that is OK. The same substance, the same phase, the same column, the same injection and the spectrophotometric detector for 280nm/240nm is 135k/160k. Why? Tested on 3 HPLC sets with SPD and 3 with DAD.

Regards
Greg

Hello,

I meet in my work with DAD and spectrophotometric detectors. Recently, a situation occurred that on the chromatogram from DAD the peak from substance A for lambda max (280nm) is higher than for 240nm - area 140k:70k and that is OK. The same substance, the same phase, the same column, the same injection and the spectrophotometric detector for 280nm/240nm is 135k/160k. Why? Tested on 3 HPLC sets with SPD and 3 with DAD.

Regards
Greg

Most detectors do not measure the signal at exactly the wavelength selected so the wavelength range unique to each detector setup can make a difference.

I know many DAD you have to set a wavelength window, if it is +/- 20 lambda it will accept all signal from 260-300 and from 220-260 but the SPD may use a slit which gives narrower or wider lambda cutoff. If one substance has a sharp peak near lambda max and another has a broad peak, then the difference in the wavelength range actually recorded by the detector can give different peak areas/heights. For two substances that have the same absorbance at exactly their lambda max, the one with the narrow absorbance band will give a smaller peak response than the substance with a wide absorbance band.

Some years ago I developed a method where I determined relative response factors for different impurities (and these factors were put in the method). It turned out later that the relative reponse factors were not the same on DAD and single wavelength detectors. Not sure if this is commonly known, since you see relative response factors in many methods, including compendial.

I have no explaination why though. Could be a matter of different slit witdh as described above

I do not understand the terminology being used here as it does not relate to HPLC. "DAD and spectrophotometric detectors". ?
All HPLC DAD (or PDA) detectors function as, and are basically spectrophotometric detectors. So are single or multi-wavelength detectors too. So what are you comparing? Are you comparing a bench-top spectrophotometer with cuvette holder (or a cuvette with sipper tube) to some type of HPLC detector with conventional flow cell? Please explain.

Next, any comparison made between different HPLC systems, different detectors or for that matter a bench-top spectrophotometer would likely yield different data for the same samples. Specifically in their output. Besides tubing, plumbing, column and setup differences, the detectors are different too. Hardware and Software differences account for most of the changes in output.

Hardware:
The detector optical blocks are different and optimized to transmit light in different ways to the detectors (different designed used). The parts and electronics are different and setup for arbitrary outputs. the flow cells used (or cuvettes) may be made of different quartz material, usually have different path lengths and volumes. The detector's bandwidth is often different. On some detectors bandwidth is fixed, on others it is adjustable. Lamp slits are sometime used to reduce or increase the amount of lamp energy focused through the system to the detector. Again, all of these things can cause well documented output differences.
Software: There is a long list of detector software functions which can manipulate the raw or processed data output from the detector. A great example of one feature that can completely change the output (add signal, delete signal, change the entire signal obtained) is the software feature known as the "Reference Wavelength" (not to be confused with the auto-zero that takes place at the start of a run). We see this mess up a lot of data for users, and they are often unaware that it is happening because they do not understand what the feature does.

A few links for more info:
https://hplctips.blogspot.com/2011/09/u ... width.html

https://hplctips.blogspot.com/2011/03/r ... -hplc.html

Maybe some of this answers your question, but maybe you could explain what you are comparing in more detail so everyone can understand the question better.

Some years ago I developed a method where I determined relative response factors for different impurities (and these factors were put in the method). It turned out later that the relative reponse factors were not the same on DAD and single wavelength detectors. Not sure if this is commonly known, since you see relative response factors in many methods, including compendial.

I have no explaination why though. Could be a matter of different slit witdh as described above

I have seen relative response factors a lot in industrial applications(QA/QC labs) but for environmental where I work they have to be determined and verified each day. I have seen shifts in the RRF that can be over 20% from one day to the next. A relative response factor is only valid if the exact same conditions exist as when it was determined, any contamination of the cell, change in lamp intensity, calibration of the wavelengths, ect can cause them to change, but often that is not taken into account and the method writers assume they are always constant. If they are always constant, then instruments could run for decades and never need recalibration, which would be wonderful

I do not understand the terminology being used here as it does not relate to HPLC. "DAD and spectrophotometric detectors". ?
All HPLC DAD (or PDA) detectors function as, and are basically spectrophotometric detectors. So are single or multi-wavelength detectors too. So what are you comparing? Are you comparing a bench-top spectrophotometer with cuvette holder (or a cuvette with sipper tube) to some type of HPLC detector with conventional flow cell? Please explain.

Next, any comparison made between different HPLC systems, different detectors or for that matter a bench-top spectrophotometer would likely yield different data for the same samples. Specifically in their output. Besides tubing, plumbing, column and setup differences, the detectors are different too. Hardware and Software differences account for most of the changes in output.

Hardware:
The detector optical blocks are different and optimized to transmit light in different ways to the detectors (different designed used). The parts and electronics are different and setup for arbitrary outputs. the flow cells used (or cuvettes) may be made of different quartz material, usually have different path lengths and volumes. The detector's bandwidth is often different. On some detectors bandwidth is fixed, on others it is adjustable. Lamp slits are sometime used to reduce or increase the amount of lamp energy focused through the system to the detector. Again, all of these things can cause well documented output differences.
Software: There is a long list of detector software functions which can manipulate the raw or processed data output from the detector. A great example of one feature that can completely change the output (add signal, delete signal, change the entire signal obtained) is the software feature known as the "Reference Wavelength" (not to be confused with the auto-zero that takes place at the start of a run). We see this mess up a lot of data for users, and they are often unaware that it is happening because they do not understand what the feature does.

A few links for more info:
https://hplctips.blogspot.com/2011/09/u ... width.html

https://hplctips.blogspot.com/2011/03/r ... -hplc.html

Maybe some of this answers your question, but maybe you could explain what you are comparing in more detail so everyone can understand the question better.

Just one of the reasons we call them instruments instead of machines, each one is unique just like musical instruments and each, while very accurate, will behave a little differently to the next.