avatar: chicho
chicho
3 posts
Replied to chicho's post on November 14th, 2011

I just wanted to know what is the highest OD/Absorbance the machine can read reliably.

Or, in other words, at what OD reading does the machine "saturate"?

Replied to chicho's post on November 14th, 2011

The absorbance value is calculated by the

equation

A = -log(I/Io),

where Io is the light intensity without any

sample and I is the intensity after an

absorbing or reflecting medium.

So, 4.

avatar: chicho
chicho
3 posts
Replied to I wish to remain anonymous post on November 14th, 2011

Quote I wish to remain ano

The absorbance value is calculated by the

equation

A = -log(I/Io),

where Io is the light intensity without any

sample and I is the intensity after an

absorbing or reflecting medium.

So, 4.

Can you please explain how do you come up with the Absorbance of 4 being where the instrument saturates?  That just tells me that I/Io = 0.0001 or 1/10,000

Morevoer, I thought the saturation was instrument-specific.

avatar: Bob Kafato
Bob Kafato
40 posts
Replied to chicho's post on November 14th, 2011

The machine spec is Accuracy @ 2 OD < 2 % Precision @ 2 OD < 0.1%

Saturation is instrument specific, but once you are over 2 OD beers law starts to fail and you get a non-linear response.  I think it is best practice to keep your OD under 2, and preferrably under 1.0.  Dilute your sample if necessary.  I would not count on any OD over 2 as being a reliable result - most certainly accuracy and precision get considerably worse.

avatar: chicho
chicho
3 posts
Replied to Bob Kafato's post on November 14th, 2011

Quote Bob Kafato

The machine spec is Accuracy @ 2 OD < 2 % Precision @ 2 OD < 0.1%

Saturation is instrument specific, but once you are over 2 OD beers law starts to fail and you get a non-linear response.  I think it is best practice to keep your OD under 2, and preferrably under 1.0.  Dilute your sample if necessary.  I would not count on any OD over 2 as being a reliable result - most certainly accuracy and precision get considerably worse.

I agree with the information you provided.  In my previous work, I used to keep all my Absorbance values (pertaining to ELISAs) under OD = 1.0.  Currently where I am now, I see results reported with Absorbances much higher; in the rage of 2.0 - 5.0.  The machine currently used is the Perkin Elmer Envision and I was wondering if this machine was special in some way that it allowed reliable results at such high ODs.

avatar: Bob Kafato
Bob Kafato
40 posts
Replied to chicho's post on November 15th, 2011

I'm old school, I don't know this machine specifically.  Calibration was always  linear and unless your machine allows some sort of non-linear calibration curve to be applied to it then the results will be off...by what factor is anyone's guess.  It depends on the kind of accuracy you are looking for, but if the error at 2 OD is around 2%, then at 3 OD your error is likely going to be 5% and at 4 OD probably in the neighborhood of 10 to 20 percent or maybe more.  Run some standards and find out or contact Perkin Elmer and get the scoop. Old school says keep it under 2 and you'll be fine.