what factors affect injection repeatability?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

12 posts Page 1 of 1
Greetings.

We currently have an injection repeatability issue. Here are chromatographic conditions:

Injection size:5uL
Diluent:methanol
gradient
flow: 1mL/min
Column: C18 15cm*4.6mm*5um
mobile phases: MeOH with 0.05% TFA;H2O with 0.05% TFA

RSD of 5 injections of std can be upto 1.6%. And RSD for 5 injections of std varies from 0.1 to 1.6% for injection repeatability. This is tough for us to pass the spec of 2% RSD for sample analysis repeatability.

Please recommend typical contibuting factors to injection RSD.

Thanks

I suppose the first question should be are your standard injections coming from one vial or multiple vials? Meaning, n=5 from one vial or do you perform a single injection from each of five vials?

Assuming you are doing "partial fill" injection (i.e., using the autosampler's syringe to meter out a volume of sample that is less than the loop volume), you can run into repeatability problems if the injection volume is a small fraction of the syringe volume. The solution is to mount a smaller syringe.

Very viscous samples can have problems if the draw rate of the syringe is too fast.

Poor lubrication and general wear in the mechanicals can also cause problems.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374

Tom, I don't see where moon says he/she is even using an autosamper, needs to post that. Also, what brand/model HPLC and/or autosampler, e.g. Agilent model #1050 or whatever.

We use 5ul injections or less for our assays (autosamplers), and get way better repeatability than that. Manual injection will not produce as accurate results, but should meet your criteria if done correctly with a 5ul loop.

If the peaks look good, like good shape and consistent retention times, but only the areas are off, then concentrate on the injections (assuming all from same vial, as noted above).

Agilent 1200. Autosampler G1329A with injection loop size of 100uL.
Out of one vial instead of multiple vials.

And RSD varies. I did four 6-injections on two LCs. The RSD was (1.6, 0.1) and (0.8, 0.1.)

Draw: 200uL/min
Eject: 200 uL/min

diluent is methanol

injection loop size of 100uL
What size is the syringe?
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374

syringe volume 100uL

1.6% really isn't that bad. How tall/wide are the peaks? Manual or auto integration. Have you looked at the baselines of the integration to see if anything funny is causing poor integration decisions?

syringe volume 100uL
I suspect that's a big part of your problem. A 2% error in injecting 5 microliters translates to an error of only 0.1% of the syringe volume. I believe the syringes are driven by stepper motors which probably have a "resolution" of 1024 steps.

Go back and look at the sequences that have high RSD. If the high RSD is caused by one "outlier", then I'd be willing to bet that you could improve things by fitting a smaller syringe.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374

We get better %RSD with Agilent 1050 and 1100 autosamplers than that, with 5ul injections (even 3ul injections) and same loop, draw conditions.

Have you ever changed the metering seal (same part as the piston seal for pump) ? Is the connection to your needle tight? You don't have a tiny leak in your loop at the end weld, do you?

I always try to keep it as simple as possible. If the areas are consistently growing then it is probably a problem with the methanol evaporating in the vial between injections. Either switch caps or inject from new vials for each standard.

Re:

Blazer wrote:
I suppose the first question should be are your standard injections coming from one vial or multiple vials? Meaning, n=5 from one vial or do you perform a single injection from each of five vials?

What is the case with separate vials? We have RSD from separate way higher than from the same. Solution is the same. Instrument Shimadzu, autosampler moves to each vial. Inj. 5 uL. Injections don't depend on particular position or vial. Thanks.
12 posts Page 1 of 1

Who is online

In total there are 17 users online :: 0 registered, 0 hidden and 17 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: No registered users and 17 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry