Biochemistry kinetics

Original post by Sophlamp

I'm struggling with molar conversions and wondering if anyone can help me. I have completed a piece of work and want to check that the answers that I obtained are correct.
There are various calculations that i need to do but if i just put the basis of what i have to do down then someone can tell me if i am going in the right direction. The results are based on a practical I did.
Standard stock solution - 0.4mM
Concentration - 100uM nitrophenol and 300 uM water
I transferred 125uM to a sample plate and took readings of absorption in a plate reader.
My calculation is quite simple; concentration*volume = 125*100
But I am wondering if working with micromoles means another calculation should be used.
Any help would be much appreciated.
What I am trying to find is the amount of 4-Nitrophenol in MICROmoles. My answer was 12,500um
Thanks in advance,

You need to make sure with calculations like these that your calculations are using the same units. As you see, you have got millimolar concs of stock but micromolar concs of nitrophenol and water.

You can either express all your reagents using the same units, eg a 0.4mM conc is the same as a 400µM conc and do the calculations that way.

Alternatively, if you want to see clearly what's going on with the maths here, you can rewrite the units in index form - a 0.4mM conc = 4 x 10^-4 M, and a 100µM conc = 1 x 10^-4 M. This is your clue...!

Reply 2

OP

Original post by Reality Check

You need to make sure with calculations like these that your calculations are using the same units. As you see, you have got millimolar concs of stock but micromolar concs of nitrophenol and water.

You can either express all your reagents using the same units, eg a 0.4mM conc is the same as a 400µM conc and do the calculations that way.

Alternatively, if you want to see clearly what's going on with the maths here, you can rewrite the units in index form - a 0.4mM conc = 4 x 10^-4 M, and a 100µM conc = 1 x 10^-4 M. This is your clue...!

Thanks for the reply,

My answer must be written in micromoles which is why I have worked it out in micromoles/microlitres but I worked it out after having converted all the units into moles/litres and then converted back which gave me the answer as 0.0..... so I am doing somehting the wrong way round but I can't figure out what

Reply 3

Clearly post your working so we can see the steps and work out what went wrong. Either type it out, or take a photo of the page and post it as an image.

Reply 4

OP

Original post by Reality Check

You need to make sure with calculations like these that your calculations are using the same units. As you see, you have got millimolar concs of stock but micromolar concs of nitrophenol and water.

You can either express all your reagents using the same units, eg a 0.4mM conc is the same as a 400µM conc and do the calculations that way.

Alternatively, if you want to see clearly what's going on with the maths here, you can rewrite the units in index form - a 0.4mM conc = 4 x 10^-4 M, and a 100µM conc = 1 x 10^-4 M. This is your clue...!

Also - I am confused as to where the conversion from 0.4mM to 400uM comes into the equation because when calculating all I need is the (current) concentration which is 100 and the amount so 100*125 (this is what I thought I needed to do). Where is the 400 used?

Reply 5

Original post by Sophlamp

Also - I am confused as to where the conversion from 0.4mM to 400uM comes into the equation because when calculating all I need is the (current) concentration which is 100 and the amount so 100*125 (this is what I thought I needed to do). Where is the 400 used?

I was giving you different options of working these sort of calculations out, but this is why I'm suggesting you post up your working - otherwise I could just end up confusing you further :smile:

Reply 6

OP

Original post by Reality Check

Clearly post your working so we can see the steps and work out what went wrong. Either type it out, or take a photo of the page and post it as an image.

Sure,
So the standard stock solution has a concentration of 0.4mM, I need to calculate the amount of 4-Nitrophenol that was added in the 125 µL sample placed in the sample dish.
In the tube that I am using for this example is 100uL 4-Nitrophenol and 300uL of water.

Total volume = 400 µL

Stock = 0.4mM

Volume added = final concentration

Moles = conc*vol

Final calculation:

c = 100 µM in 125 µL

100 µM*125 µL = 12500 µM

Reply 7

OK, I need to check something here. Did the 125µL sample come from the tube containing 100µL of 4 NP and 300µL water?

Reply 8

OP

Original post by Reality Check

OK, I need to check something here. Did the 125µL sample come from the tube containing 100µL of 4 NP and 300µL water?

So basically the solution was made in a 1.5mL eppendorf tube (100uL 4-Nitrophenol and 300uL water), then 150uL of the solution was transferred to a 95 well plate.

Reply 9

I think you're out by two orders of magnitude - I calculate this as 12.5µmol of 4-NP in your aliquot. However, you've said to start with that you transferred 125µL to your plates, but your latest post says you used 150µL... :s-smilie:

Reply 10

OP