There are no references but you can check www.chromacademy.com  As I recall about 60% (depends on packing and particle size) of the volume of a column is available for the mobile phase.  Since 1 mL is 1 cubic cm (1 cm3) for water at STP, means there are 10,000 cubic cm in a cubic m (m3).  Similarly, 1 mL is 1,000 uL.  The rest is just basic Math (don't have my calculator with me).

Thank you for the calculation advice. I did not find the agilent guide to backup the less than 2% after my lecturer told me this and she did not have it. 

That is true.  But there is requirement to have a maximum 2.0 %RSD for 5 injections (common for an assay) in USP section .  While this requirement applies to a chromatographic method, you can't achieve this requirement if the instrument (HPLC, GC, CE...) is not running at its best.

The chromatographic instrument (like an HPLC) is calibrated (injector, pump, detector...) per SOP without the column.  Usually, this company requirement is 'way above' the supplier's specifications but ensures the 'new' analytical method can be easily validated per ICH Q2(r1).

Why do you want to perform these calculations if they have little effect on the method you will develop and validate?

Because it was potential to earn more marks in the thesis and thank you for the view point it allows me to make sure that I do that optimisation step injection volume correctly.    I don't have lots of experience in hplc method development and comparing hplc calibration specs to the column specifications is genius. Plus I was not familiar with the standards and documents. Compare and contrast at it's best.

So you are saying instrument variation can throw off the 2% guideline and specs between column pump injector, detector are mismatched compared to column volume required RSD percentage. So the injector can handle more but the columns cannot because of overload. Hence it depends on the calibration of the instrument performed by the company and not the user?. 

My supervisor does not calibrate the equipment I think it does that automatically when it is switched on.

5.1ml injection volume is what the pump can do as the 2% relative std deviation.

Not necessarily.  In our SOP (my past life) we had it written that if you dial in a flow of 5.0 mL/min you received that volume in a 25 mL calibrated volumetric in 4.9 to 5.1 minutes (that is 2.0%).

The injector vial was weighed before and after making several injections at 10, 50, and 100 uL.  The variation was NMT 2.0%.

I think Waters has calibration procedures online.

I noticed 25-100ul can be injected. Yet I found my setup and plate count is good for 3ul to keep as much sample as possible. Should I try past 7ul?. That will only increase the mAu and not the separation. Also I am embarrased because I do not know what is NMT abbreviation?. I am not super intelligent. I break things down into apples and oranges and take away facts to make problems smaller. Its the attitude that counts.

NMT is 'not more than'.  Most HPLC methods require 2.0% RSD, but you cannot achieve this goal if you inject < 10 uL.  Usually 10 uL is injected for a highly concentrated molecule around 100 ug/mL.  The exception is UPLC or UHPLC which rivals injection volumes with a GC (10 uL or less and still have a good %RSD).

You may want to change the system suitability requirements to 'resolution between peaks of baseline separation (R>1.5)' rather than plate count (HETP) since most modern columns can 'blow' this requirement away.

For chromatographic equations see www.chromacademy.com

my software obtains the half the peak width. Does that mean its HETP is wrong. When I compare my calculations of N to the plate count of the software there is a variation of 50 only. So I expect the resolution to be reasonably correct aswell as the retention times used to obtain resolution. I will check the option on Monday. What do you explain by that modern columns blow away the requirement. They have a USP tailing factor and specification of N plate count calculated by their specific method.

If I have peak tailing then that baseline width would be wrong and hence my calculated resolution and that of the software?. Plus my supervisor does not know how to calibrate the instrument when the slit width and wavelengths are changed the software demands calibration for the test runs. The software should not know when to stop calculating across the baseline. 

Your methods must meet the validation requirements of ICH Q2(r1) and review system suitability requirements of the USP section .  This assumes you make or use pharmaceuticals or biopharmaceuticals.  The FDA (a regulator) has an office for animals (VICH?).

I dont know that. I will look up the validation requirements because I am at that stage.

Nothing related to veterinary product so VICH animal products is not crucial right now. 

Evolution is key for my success.

I will be using this method for bacterial cell metabolites in the future if it works or it may be postponed depending on validation outcome. It is an academia project. Sufficient for me to gain experience. 

Most 'modern' HPLC columns have a small particle size (3 um, microns or less).  Since particle size influences HETP modern columns, they easily achieve >5000 HETP also N.  Thus modern columns are usually 5 cm long and achieve the same resolving power of a 25 cm and 10 um particle size common 30 years ago.  Tailing tells me residual silanol groups of your column are reacting with your molecule (a very bad thing).  Again modern columns are more non-polar (have a higher %Carbon) and are endcapped so residual silanols cannot react.

Lots of misinformation in this thread. Let's address some basic fundamentals of chromatography to get you back on track.

• We use ~ 3% of packed column volume as a "Max" injection load per column, BUT you must determine it and any optimized load volume using a proper load study first.
• Your formula for "column volume" is incorrect as it does not take into account the the actual volume of the packing material inside. Depending on the type of material used, it can vary, but below is a link to a table with typical values as well as the actual formula to use for porous HPLC silica based packing material ["Determination of HPLC Column Dead Volume / Dead Time (T zero)"].
• The best injection volume to use with your method should not determined by guessing. First, it must be based on the range and specifications of your Autoinjector (A/I) or syringe (most show poor RSD at the low and high ranges). For example, most A/I's used in analytical HPLC have a range of 0.1 to 100 ul. Most of those units will perform poorly below 5 ul and above 95 ul, so try and use volumes that are between those values and are far below the ~3% max volume (or whatever value it turns out to be). Don't inject volumes that are at or near the limit of the injector used. Second, the sample concentration needs to be adjusted to get the actual injection volume used within this ideal range. These steps contribute to accuracy, precision and good RSD's.
• Read up on the basic of HPLC. Many great books on the topic with the earliest ones by Dolan and Snyder still the best. The basic concepts (which few ever seem to learn) are still 100% applicable and needed to perform method development at any scale.

BTW: Mel. Great performance in "Braveheart" (1995).

https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html

Had to be done for Scotland and her pride. That is excellent information and the article was really simple and packs a lot of quality.