Chromatography Forum

Hello, I am operating a HP 1050 hplc and running a older version of chemstation.

I am unsure how to have my calculation set based upon my method and injection to receive correct results.
(sample amount, multiplier, dilution)

I take my sample liquid medicinal tincture. I do a 10x dilution 100ul sample into 900ul meoh. my injection is 5ml. my standards from restek are 1,000 μg/mL

For dry plant samples I am receiving correct results extracting .1g into 20ml.
5ul injection. sample amount set at 5000. dilution:1 multiplier:1

Thank you. honestly I am testing medical marijuana tinctures for potency and activation. This medicine is mostly for epileptic children and is being brought to me by their moms. I would like to know how to get correct results so we can standardize dosing for these kids and stop their seizures.

Hello, I am operating a HP 1050 hplc and running a older version of chemstation.

I am unsure how to have my calculation set based upon my method and injection to receive correct results.
(sample amount, multiplier, dilution)

I take my sample liquid medicinal tincture. I do a 10x dilution 100ul sample into 900ul meoh. my injection is 5ml. my standards from restek are 1,000 μg/mL

For dry plant samples I am receiving correct results extracting .1g into 20ml.
5ul injection. sample amount set at 5000. dilution:1 multiplier:1

I would like to know how to get correct results so we can standardize dosing.

This is for versions like A.06 and later. If you're using A.03, will be a little different.

Inject the Calibration Standard, must have this to make a new Calibration Table. We put concentration of calibration standard in g/ml into the Calibration Table (make a new Cal Table). So 1e-6 goes into the amount on the Cal Table, will calculate the Area/concentration for you.
Under Specify Report we choose ESTD%. And Save the Method.

In Sequence, we always discard the first injection.
So first row is Calibration Standard, Level 1, Replace RF, No Update on RT, 2 injections (just uses last one, replaces whatever was stored in computer's memory, important.
Second row is Calibration Standard, Level 1, Average RF, No Update on RT, 2 injections or more (averages results for each injection on this line with the last injection of the previous row). This automated calibration will be used to assay subsequent samples in lower rows unless a new calibration sample is injected.
Third row: Sample for dry plant samples. Put weight 0.1 in Sample Amount, put 20.0 in the Dilution column. Multiplier can be blank or "1".
Fourth row: liquid medicinal tincture. We'd put 0.100 into sample amount (as it's 0.100 ml) and 1.000 into the Dilution column (as that's the total amount). For these liquid samples we'd put the density of the methanol into the Multiplier column so results will be corrected to weight percent.

Of course 3rd and 4th rows can be in either order, any number of injections, etc. And you can add in more rows as necessary into your sequence.

Now: you get the results as a Sequence Summary Report, part of the Sequence. Each day under Sequence Parameters, change Subdirectory to a different name, such as the date, so you don't over-write older files.

Under Sequence Output, click on "Print Sequence Summary Report". The to the right click Setup, and click number 9 and change to "Compound Summary" instead of "Sample Summary", click OK, OK, and Save the Sequence.

So either automatically or if you re-process (click to use Sample Table entries if you re-process), use Sequence Summary Report to get the results. For sample injections, results will be in Percent (nomatter what the header might say, there is a software glitch there). For calibration injections, results will be in g/ml. That's just what it does, you just need to know that.

Remember: always try some results with manual calcualtions, make sure they agree.

And remember to purge all for lines (5 minutes at 5 ml/min) even if only two channels being used. If changing solvent in a reservoir and 1050 degasser is used, remember their hold-up volume is 40 ml per channel, and adjust for that.

Ok I am starting to understand... however. why would I enter 1e-6 in the amount for the calibration standard. restek standard is at 1,000 μg/mL

Ok I am starting to understand. I am receiving correct results for dry herb samples using the calculations supplied to me above

I am not getting reasonable results on the tincture samples when done as recommended above. Should the dilution be 10 to represent the dilution. not 1 to represent the total? If I do not enter the .79 density of meoh into the multiplier are my results percent by quantity (1ml) not percent by weight?

I am considering extracting the liquid tincture sample as I would the dry herb sample. by weight. .1g of liquid into 20ml. would this give me correct percent? having sample at .1g of liquid. dilution at 20 and multiplier at 1?

Ok I am starting to understand. I am receiving correct results for dry herb samples using the calculations supplied to me above

Good

I am considering extracting the liquid tincture sample as I would the dry herb sample. by weight. .1g of liquid into 20ml. would this give me correct percent? having sample at .1g of liquid. dilution at 20 and multiplier at 1?

Yes, I think it does make sense to make up the liquid tincture sample as weight to volume (like you did with the dry material). Sure, you could weigh 0.1g liquid tincture sample and take to volume of 10.0 ml or 25ml using a volumetric flask. Enter the weight in the Sample Amount column and the volumetric flask size/volume into the Dilution column, and leave the Multiplier column empty. Then both calculations will be in the weight percent mode. I'd really go with volumetric flask for the liquid sample; for the dry sample you can pipet in 20.0 ml solvent and the liquid volume will stay at about 20.0 ml. With the liquid sample if you mix 0.1 gram sample with 20 ml methanol, you theoretically have slightly different volume than 20.00 ml. But maybe that "error" is so slight as to be negligible.

Your calibration standard is already weight/volume (1,000 μg/mL ) so that way everything matches up.

Would I then weigh my tincture sample in order to determine the concentration by volume? lets say 1gram is 1.1ml. At lets say 5% that would mean 50mg/gram of tincture. or 50mg/1.1ml. yes? and use that To calculate the amount of medicine in 1ml?


100ul of tincture is .085grams. I am using a measuring pump not a volumetric flask so I cannot adjust as so. would a extra 100ul volume significantly screw results? Should I add that to the dilution total?

Does the 5ul vs a 10ul injection effect any of this?

Its making sense. Thank you so much.

Does the 5ul vs a 10ul injection effect any of this?

Injection size can be important depends on your column diameter, flow, organic content and likely other factors that others can point out.

If you are using reverse phase, and injecting methanol (as opposed to aqueous or mixed solvents), I'd go with the smaller injection size if your peak sizes are OK. If one injects too much organic, can "behave" as a solvent, even from a few microliters. Typically one injects less volume and if the peak shape is way better, go with that. Here, typically 5µl injection is the largest we use.

Of course: in a Sequence, keep the injection volume constant throughout, part of the saved Method, don't use that column in the Sequence Table.

Hello Claysulak, i am on the same boat too, im trying to figure out how to check the potency on the flower, i have an agilent 1100 with chemstation for LC rev. A 10.02 {1757}.

I Also have the standard from Restek. i try to calibrate the LC with 3 point linear calibration/ 5x, 25x, and 125x dilution. But so far is not working properly.
Any suggestion?

feel free to write me an email, im located in California. duaoposs@hotmail.com

Best Regards.

claysulak@gmail