Chromatography Forum

Hello, I'm new to HPLCs and I was wondering if you could help me with what most labs do during a typical autosampler run. For a non-regulated run and you wanted to analyze 5 samples; what would the order of the sequence look like. Do you run blanks first- how many, run samples - all in a row, run standard - how many? Also for system suitability - minimum accetable parameters to look at and values. My last question is minimum levels for a calibration curve- how many calibration points would be considered acceptable ?All these questions are for a non-regulated sample, and I just wanted to get a feel as to what most labs would do for setting up a sequence given good lab practices. Thanks for your help.

Hi Student,

I think I may be able to help. I'm new to the forum, and I have never submitted a reply. But your request seems simple enough, and I'm a little surprised that no one has answered it yet.
In a GLP/GMP environment, your run might be as follow: Blank, Blank, Std 1-1, Std 1-2, Std 1-3, Std 2-1, Std 2-2, Std 2-3, Smp 1-1, Smp 1-2, Smp 2-1, Smp 2-2, Smp 3-1, Smp 3-2, Std 1-4, Std 1-5, Smp 4-1, Smp 4-2, Smp 5-1, Smp 5-2, Std 1-6, Std 1-7
Typically, for a GLP/GMP HPLC Analysis, system suitability needs to be completed prior to every sequence of samples for a particular method. In the example above, two standards have been prepared (of two different weights) and bracketed around the five sample injections. All sample injections and intervening standard injections have been made in duplicate (samples not prepped in duplicate, just injected in duplicate). The primary system suitability assessment will take place within the first several injections. In this case, the six initial standard injections could be used to measure the precision of the autosampler (I'll address the autosampler because your question was based on that particular component, but keep in mind these initial injections would likely be used to assess other components of the HPLC system; i.e. theoretical plates and peak shape might be used to assess the health of the column). Precision is a measure of the consistency of your injections, which is directly related to the performance of your autosampler and measured in terms of peak area. So, for those initial six standard injections, you’ll want to determine the % RSD amongst the area of those injections. Keep in mind, if you have two standard preps as shown above, you will need to correct for the weight discrepancies; use the response factors (area/weight). The bracketed injections are used to monitor the system’s performance throughout the run. You may include the areas from those injections in your overall precision calculation, but the SOP’s of some labs require that you determine the suitability of the system prior to injecting your samples.
A typical spec for precision (as defined by USP) is less than or equal to 2.0 %. However, for standard injections from the same standard prep (same weighing) you should expect to see less than 0.5 %RSD.
As far as your calibration curve, you should try to use at least two points (two separate standard weights as shown above). In this case, the final concentration of the chosen standard weights should bracket the final concentration of the sample preparations. Other methods might employ a standard curve of more than two standard weights, but I’m not sure if that is necessary.
I hope this puts you in the right direction, let me know if I can help you with anything else.

Ryan