HPLC quantitation formula doubt

[vellaikumar]

Dear Members,
I have one doubt on HPLC quantitation formula,
Amount in micro g / g (ppm) = As/Astd X M/M1 X V/V1 X F
where,
As= sample area
Astd= std.area
M= std injected (micro lit )
M1= wt.of the sample (g)
V= vol of final extract (ml)
V1= sample injected (micro lit)

As per the calculation, the final result accounts for ml/g and
not for micro g/g.

Can any one explain this.

[tom jupille]

Where did you get that formula?

You have M and M1 flipped, but it's very confusing to use the same primary symbol (M) for both a mass and a volume (M1).

Look at it this way (I'll refer to your standard as "1" and sample as "2"):

for equal injection volumes, the concentration of sample is equal to the area ratio times the concentration of standard:

M2/V2 = (A2/A1)*(M1/V1)
where
M1 is mass of standard
V1 is volume of standard solution
M2 is mass of sample
V2 is volume of sample solution

If you inject different volumes of sample and standard, you have to correct that by the inverse of the injection volume ratio (we'll call injection volume "I"). That makes the final formula:

M2/V2 = (A2/A1)*(I1/I2)*(M1/V1)
where
M1 is mass of standard
V1 is volume of standard solution
M2 is mass of sample
V2 is volume of sample solution
I1 is injection volume of standard
I2 is injection volume of sample

the final result (M2/V2) will have the same units you used for M1 and V1 (e.g., g/mL, g/L, μg/mL, . . . , whatever). If you want ppm, you will also need to use either μg/mL or mg/L and then factor in the specific gravity of the solvent).

[vellaikumar]

Dear Tom,

Thanks for your reply. Can u please explain me in practical.

Thanks

[vellaikumar]

Dear Tom,

Thanks for your reply. Can u please explain me in practical.

Thanks

[AdrianF]

If you want to understand how to work it out I suggest you forget about the formula and work it out from first principles.

The principle is that the area is proportional to the amount injected.

1. From a knowledge of the concentration work out the amount you injected in the standard

eg 100ug gives an area of 1000

therefore 1ug will give an area of 1000/100 = 10

2. From the area of the sample work out the amount in the sample

Say your sample has an area of 700 the amount in the sample is 700/10 = 70ug.

3. Calculate the amount in your original sample
Lets say the volume of the injection is 20ul and the sample of 1 tablet was dissolved in was 100ml

Amount in the flask is 100x1000(to convert to ul)/20 = number of times 20ul can be fitted into one flask. = 5000

amount in flask is 5000 x 70ug = 350,000ug

= 350,000/1000 mg = 350mg

I think it is important to think through your calculations from scratch. By doing so I have found a number of errors in methods I have been given.

Sorry if this seems very basic - hope its helpful