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Yes that's right and no I don't have a link or a book because it is simple reasoning.
Thanks for that info, so it works because the chromogenic Substrates used have a high molar absorption?
Do you have a link or book reference for that? just to be able to make my point.
What I didn't mention is the origin from the statement that the absorbance should be read between 0.200 en 1.000, this comes from the earlier photometers these were equiped with a analog logaritmic scale and tube amplifier. The ampliflier had a significant noise in the lower region and therefor the reading was less reliable in the lowerpart of the scale and a analog logaritmic scale is hard to read accurate above the 1.000
This means that the range in with you can use the law depends on the molar absorbtion from the solution, with a high molar absorbtion the maximal absorbance of a solution can be much higher then in a solution with a low molar absorbtion.
We had a simular problem and after consulting our biostatistic department we could tell if the groups where different or not.
As I remember one of the big issues was the presence of very abnormal values in 1 one of the samples and the confidence of the test depended strongly of the number of samples in the pool.
The readings from your sample looks as the didn't react, a other possibility is that the reactionproduct breaks down. I,m just guessing by lack of information.
With a tip
First pipette the blood very slowly. When taking blood in let the piston come up slowly and wait for 20-30 seconds so you're sure the blood is in.
Then slowly push the piston down and watch very carefully if all the blood from the wall of the tip is coming down, if not you're pushing to fast. It,s a very slow proces it can take minutes.
(With a glass pipet it works the same)
Much faster is using a positive displacement pipette as mentioned before.
Weight is not a solution because the specific weight of blood is not known therfor you can't calculate the volume.
I,m not sure but looking at the picture i have the feeling that the problem originates from the gel casting or the imager. I don't think the blotting or the probing caused this problem.
Bromophenol blue is an acid-base indicator its useful range lies between pH 3.0 and 4.6. It changes from yellow at pH 3.0 to purple at pH 4.6; this reaction is reversible. Bromophenol blue is structurally related to phenolphthalein-a popular indicator.
Be concentrated and let nothing distract you when you're pipetting. Try to get in a constant flow to get every thing as stable as possible as the speed of pressing the plunjer, the waiting time for the liquid to flow downwards before you press the plunjer completely down etc.
If you pick a date of wich you want to be certain it's ok then you will have to dispose a lot of perfect working solutions and make a lot of not nessecary costs.
Take for instance the NaOH 1M solution and it's used for correcting a buffer used in a test that is not influenzed by carbonate(CO2 from the air) it will be useable for indefined time.