Chromatography Forum

Hi all,

I have never used a manual injector before, and was wondering if I have to injection mobile phase after I inject my sample? I injected 10ul of sample and did not get any peaks. Also when I inject and load does the method begin or do I have hit some button? Thanks.

Your manual injector has two positions. In one position it is possible to take a syringe full of your sample and fill a sample loop in the injector with it. You should thoroughly flush the sample loop with sample. Then when you manually switch the valve to the inject position mobile phase from your pump flushes the sample loop in the valve and puts the sample in the flow path to your column. Your loop will fill with mobile phase as the sample is flushed to the column.

As you switch the valve you should start your data system (if it does not start automatically) and your system pump switching (unless you are using an isocratic system) to begin your gradient in your mobile phase.

You should return the valve to the fill position and remove the syringe after the sample has been flushed from the sample loop.

When you fill the syringe with sample for the next injection be sure to take enough to thoroughly flush the sample loop.

I hope this answers your questions.

best wishes,

Rod

From what you are saying you might loading your loop when being in the inject position so everything is going to waste. You must have your manual injection valve in to load position, fill your loop with your sample by using a hamilton type syringe (make sure that the syringe is for HPLC and not for GC) and then you switch the valve to inject and direct your sample to your chromatographic column for separation. Rod has covered everything else...

So I inject in the load position and I get a drop out one of the vent capillaries. It's either waste or vent tube, not sure which one. Is this normal? The sample loop can hold 20ul, so the loop must be full of sample since I loaded 3 times with a 10ul syringe.

The method begins to run when I change to the inject position, but I don't get any peaks. Is there something else I should consider?

You should see the excess leaving the valve and going to a waste container. So that sounds correct.

It seems your chromatograph starts integrating when you manually switch the valve to the inject position. That also sounds correct (reasonable)

Is your detector lamp turned on? (are you using UV detection?)

Is it set at the right wavelength?

Do you have the correct mobile phase for the elution of the sample?

Do you have the right sample?

best wishes,

Rod

THanks,

I got it, I just needed to be a little more patient.

The sample loop can hold 20ul, so the loop must be full of sample since I loaded 3 times with a 10ul syringe.

Most valves (e.g. Rheodyne) should be overfilled 5 times (except for partial loop filling), so it's better to use a 100 ul syringe.
See http://www.rheodyne.com/pdfs/2320157C.pdf