injection volume

[sudhis]

hi every one ,
in general and maximum times we analyse drug with 10 micro litres as injection volumes
ofcourse sometimes as 20 and 50 micro litres. but on what basis we will give as 10microlitres. what is specific reason for 10microlitres.
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[amaryl]

hi every one ,
in general and maximum times we analyse drug with 10 micro litres as injection volumes
ofcourse sometimes as 20 and 50 micro litres. but on what basis we will give as 10microlitres. what is specific reason for 10microlitres.
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I think injection volume has no relation with method so developed. Its just that how much analyzing solution is available with you and what your HPLC system is equipped with, provided its well calibrated.

May be someone can come up with a reason

Regards,

Amaryl.

[james little]

I can't address your specific methods, but injection volume is important in ours.

Normally we are doing protein precipitation (100 ul's plasma ppt with 250 ul's of acetonitrile). I normally use between 5-10 ul's. If I use more, I loose peak shape due to the presence of primarily acetonitrile in my supernatant after centrifuging the sample.

If I need more sensitivity, I remove the supernatant from the 96-well (Normally just offset the syringe and inject from above the protein pellet in the well). After removing, concentrate by blowing down with nitrogen at 40 C. THen reconstitute in mobile phase A used for the gradient.

THen you can inject much more because the analyte stays focused on the column. Only limited on my autosampler by the miniminum volume I can inject out of the 96-well how much solvent A I add to the well.

I use the Waters UPLC sample manager and need to have the needle properly calibrated for distance to get to the bottom of the well and know how much to offset the needle when injecting directly over the pellet.

[Srinivas SK]

Hello:

The most commonly used LC column is 25 mm x 4.6 mm ID, 5 - 10 micron particle size. For these parameters, the usual optimum sample load is around 10 to 50 micrograms. For a sample concentration of 1 mg/ml, this would work out to an injection volume of about 10 to 50 microliters.

Since a larger injection volume would increase band spreading and contribute to lower resolution and distorted peaks, 10 to 20 ul is the preferred injection volume.

Since a higher sample concentration would, in principle, overload the column and contribute to peak fronting, many LC methods that use 25x4.6 columns are optimised using 1 mg/ml and 10 ul volume. ( More or less).

Of course, with reduction in particle size and column dia, and with micro/nano HPLC becoming more popular these days, we will see a gradual shift towards 1 ul injection volumes or lower.

But for now, it's still 25x4.6, so it's still 10 ul injection volume.

Hope this helps.

[Consumer Products Guy]

Because of narrow bore 2 mm columns, and short columns, and high-organic concentration sample solvents we use, we oftenmost use 5 ul or 3 ul injections. In the days of mostly manual injections, one achieved best accuracy by completely filling a loop, rather than partially filling it, reason why full-loop volumes like 10 ul or 20 ul were oftentimes used. With today's autosamplers one can obtain great precision with smaller injection volumes.

[amaryl]

Hello:

The most commonly used LC column is 25 mm x 4.6 mm ID, 5 - 10 micron particle size.

25 cm or 250 mm.

Amaryl.

[Victor]

There is some confusion in the answers which have been given to the original question.

Volume overload and mass overload are separate issues. Of course they can be connected, in that a larger volume will result in a larger injected mass, however, they are not necessarily connected. For example it is possible to obtain bad peak shape by injecting 100ul of a very dilute solution due to the volume overload, not the mass overload. It is also possible to see mass overload by injecting 1ul of a highly concentrated solution.

The volume injected should be small compared with the volume of mobile in which the peak elutes. 10ul is a reasonable limit for a standard size column (say 15cm x 0.46cm). Volume overload will get worse for peaks of low retention time, or as the diameter of the column is reduced.

These remarks assume you are injecting the sample made up in the mobile phase. The problem may be worse if your injected sample contains excess organic modifier (Mr Little's example). The problem of volume overload is reduced if you are doing gradient elution, where the sample can be concentrated on the column first.

[DR]

The default injection volume for most instruments (probably for the reasons specified by Srinivas SK) is 10µL. I suspect that even when switching column dimensions, most analysts would just as soon tweak their concentrations for different column jackets than have to enter a number other than 10µL on their HPLCs.

This is probably less of an issue if a CDS with instrument control is in place, but yes


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I'm attributing it to laziness.