HPLC for Amino Acids

[kathy]

I am a new Algilent HPLC user. I am doing a method development to analyze Amino acid. I have several questions:

1. How to set up a sequence to run automatically?
2. How to program an injector program to run every samples in the sequence?
3. How to stop the sequence after the last run?
4. How to set up a calibration table/method?

[Schmitty]

Are you using Chemstation?

[kathy]

Yeap.

[Anthony Crawshaw]

Thats quite a lot to cover!! I take it Agilent have not provided any formal training? or there is no-one else there that can use ChemStation?

The Basics...

1) On opening screen of ChemStation you should be able to see a schematic of your LC system. Injector, Pump, Column, Detector. By left clicking on each of these you can set up the appropriate parameters such as injection volume, flow rate, column temp and wavelength. Once you are happy go to Method>Save As

2) To automate a sequence goto Sequence>New Sequence and then Sequence>Sequence Table. Add the required number of lines for the number of samples. Fill in each column making sure that you select the method you have just saved. Other information will be vial number and number of injections per vial. Once you are happy with the sequence, Save As.

3) Press the big start button on the main screen.... hopefully away you go.

[kathy]

Thank you for your answer.

I have tried to follow the instruction that I received from Algilent to set up an injector program, but I don't know to set up a sequence to run all the samples in the sequence. It only runs one sample at the time.

Also, I don't know how to stop the sequence automatically.

Please help me on this.

[k66]

Also, I don't know how to stop the sequence automatically.

Try do like that :
Menu (on the top in online Instrument) Saquence -> S. parameters -> in this window in the middle on the right yuo will see "SHUTDOWN" - ther klick on the " Post-sequence comman/nacro" and select macro "shutdown"

Godspeed

[k66]

I don't know to set up a sequence to run all the samples in the sequence. It only runs one sample at the time.

You must prerare all sequence steps in "Sequence Table"
I think i know what is your problem
You must change single inject ("Run method task") to seqences ("Select sequence task") - klick to the icon with 3 vials (near to icon with 1 vial ) just below "File" and "Run control" from Menu

[Anthony Crawshaw]

Follow the instructions to set up a sequence table. Then make sure you have selected "Select Sequence Task" . This is the icon in the top left hand side of the screen that looks like 3 vials. If this is not selected then you will only do single injections as you mentioned.

If everything is ok you should see the number of vials you are going to inject under the picture of the vial tray ie 0 of 5 if you have 5 injections. Then just press start as before.

To finsh a sequence you have two options:

The 1st is to create a second method with all the parameters you want such as a low flow rate or to switch off your lamp. Just create a method as before and save it with another name such as "Shutdown". Then append an additional sample onto the end of your sequence table and point that injection to your shutdown method.

Or..

Go into the Sequence menu then Sequence Parameters and you will see an option "Post-Sequence Command/Macro" tick this on and simply select what you want the system to do from the options listed.

[kathy]

I have tried your suggestions, but it did not work.

This is my injector program:
===================
Draw 2.5ul from vial 1 (borate buffer)
Draw 0.5ul from sample(for amino acid sample)
Mix 3ul "in air", max speed, 2x
Wait 0.5min
Draw 0ul from vial 2(needle wash)
Draw 0.5ul from vial 3 (OPA)
Mix 3.5ul, max speed, 6x
Draw 0ul from vial 2(needle wash)
Draw 0.5ul from vial 4 (FMOC)
Mix 4ul, max speed, 6x
Draw 2.5ul from vial 5 (WATER)
Mix 18ul "in air", max speed, 2x
Inject
===============

I program the injector this way, but the chemstation requires me to enter amount and speed @ each step in this program "DEF" or "MAX"...

I don't know what it is, and don't know why it did not work for me.

Please help me on this.


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[Anthony Crawshaw]

Ahhh... apologies I did not realise you were trying to perform a multi-vial injection.

Unfortunately I've never had to delve into the realms of this area....

[asdfg]

Have you tired performing Amino Acid Aplication by DIONEX IC using Electrochemical detector & GOLD Electrode

[kathy]

My project is to use LC/MS to analyze Amino Acid in Bacteria.

I first program the HPLC method, but the injector program did not follow my instructions.

I have never used IC or other detectors to detect AMINO ACID.

If anyone can have any idea about this area, please help me.