Agilent 1100 HPLC cystine

[bpatro]

Hi!
I came to know that cystine peak does not fluoresce and is not detected during amino acid analysis using Zorbax Eclipse-AAA column on Agilent 1100 HPLC with a fluorescence detector. The mobile phases are 40mM Na2HPO4 and ACN:MeOH:Water (45:45:10). Could anybody tell me what is the reason and is there any way cystine can be measured with this instrument and detector? Thanks very much in advance.

[bisnettrj2]

What are the excitation and emission wavelengths you are monitoring?

[bpatro]

Excitation/Emission is 340/450 nm with PMT gain 10. At 15 min, there is a wavelength switch with excitation/emission of 266/305 nm and PMT gain 9 to obtain the secondary amino acids.

Thank you.

[bisnettrj2]

I'm not sure if it helps, but I found one paper that said the following:

"The cystine molecule was predicted to absorb UV light at around 349 nm and to fluoresce at around 707 nm. The most interesting feature of our computations is the prediction of fluorescence at around 700 nm for the isolated cystine molecules. Previous experimental observations for the cystine
molecule in aqueous solution all show photodissociation for the hydrated ion. Fluorescence in the infrared region by an amino acid has not to our knowledge been observed before."

"Fluorescence of Cysteine and Cystine". Hendrik F Hameka, James O. Jensen, Kate K. Ong, Alan C. Samuels, and Constantine P. Vlahacos. J. Phys. Chem. A 1998, 102, 361-367.

[HW Mueller]

What is this ref??? Fluorescence with a single photon apparatus near 0° Kelvin? There is a reason for derivatizing amino acids for fluorescence detection.

[bisnettrj2]

Your mobile phase and column sounds like you're following this protocol:

http://www.chem.agilent.com/Library/chr ... 801193.pdf

Correct?

[danko]

I don’t think systine fluoresces at all. “Excitingâ€

[bpatro]

Yes, I am following Agilent's protocol. It mentions cystine can be detected with a diode array detector (DAD) for the same column and gradient settings but at UV 338 nm. Anyway, the samples are subjected to automated derivatization with OPA and FMOC prior to injection in both cases. We chose FLD over DAD due to higher sensitivity during the HPLC purchase.

So, any idea about cystine measurement with the same column and FLD but perhaps with other excitation/emission settings and mobile phases?

Thank you for the valuable inputs!!!

[bisnettrj2]

From the document I posted above from Agilent's site:

FLD settings:

For 75 mm column

Time Ex/Em PMT
(min) (nm) Gain
0......340/450...10
8.5*..266/305....9

For 150 mm column

Time Ex/Em PMT
(min) (nm) Gain
0......340/450...10
15*...266/305....9

*The specific time to switch fluorescence wavelengths may differ due to variations in temperature, mobile phase, etc.

Since you're using the FLD settings Agilent gave, and the column and mobile phase they specified, I have a couple questions:

1. Have you *ever* detected cystine/cysteine in this analysis using these settings?

2. Does the chromatogram you produce resemble Agilent's examples in the document posted above? Can you post you chromatogram?

[HW Mueller]

Dancho, could it be that our time is over? We do not understand that amino acid can also mean derivatized amino acid, which is a TOTALLY different chemical entity. We also don´t understand why cystine/cysteine would not derivatize, or derivatize and not fluoresce.

[bisnettrj2]

HW Mueller - perhaps it is my fault for throwing this thread off track with my goofy first response - I got tunnel-vision on the cystine-fluorescence question, Googled that, and completely missed everything else. Looking at the Agilent document that I subsequently posted, bpatro obviously meant derivitized amino acids, whether he explicitly posted it or not. I think the underlying question seems to be "Why is the Agilent setup I'm using not working for cystine/cysteine, when they say it should?"

[danko]

Hi Hans,

Dancho, could it be that our time is over?

Yes, it could be.
For instance, there are some (hopefully a few of them) guys (article writing fanatics) who have concluded that the purpose of their lives is writing articles – no matter relevance and more importantly whether or not they actually know something about the subjects they are dealing with.

I mean, how can serious scientists irradiate any given molecule with light of ca. 350 nm wavelength observe a signal at ca. 700 nm and call that signal fluorescence?

They might as well measure the signal at the same wavelength as set for excitation (would be first order light scattering) – and the bonus would be much, much stronger signal. That would be as much fluorescence as ex/em 349/707.
I think the reason for the deviation from the rule of second order light scattering will be observed at the excitation wavelength multiplied by 2, is the utilized fluorometer’s poor performance.

Best Regards

[bpatro]

I was referring to derivatized cystine.

bisnettrj2: I get almost similar chromatogram that is given in the Agilent publication

http://www.chem.agilent.com/Library/chr ... 801193.pdf

There are two tiny peaks between peaks 12 and 14, but the protocol identifies an elevation between these two tiny peaks as peak 13 (cystine) for FLD (refer to figure 8 in the protocol).

However on page 3 column 2, the protocol mentions:

"Also note in the fluorescence chromatogram, that Peak #13 (cystine) does not fluoresce under these conditions and is not detected."

So, I do not know if one should consider any of those two tiny peaks as cystine.

The protocol has the same prederivation procedure, same pump and gradient settings but different detector settings for FLD and DAD. With DAD, cystine peak can be detected. So, how come cystine fluoresce for DAD but not for FLD? Is it only the wavelength setting that is making all the difference? But again UV 338 nm is used for DAD settings.

Again from Agilent protocol:

Detector Settings
DAD:
Required Lamps:
UV lamp: yes
Vis. lamp: no
UV: 338 nm, 10 nm bandwidth
(bw), reference: 390 nm, 20 nm bw
(for OPA-amino acids)
262 nm, 16 nm bw, reference: 324
nm, 8 nm bw (for FMOC-amino
acids)
Peakwidth: >0.03 min (0.5 s)
Slit: 4 nm

You have already posted settings for FLD.

Thanks!

[HW Mueller]

Except for some very special reasons (can´t recall one) I have never understood the use of OPA and FMOC-CL simultaneously. Also, I have not heard of an amino acid which can be derivatized by FMOC-Cl to have no fluorescence.
A DAD is an absorbance apparatus, it measures absorbance not fluorescence. If the OPA derivative does not fluoresce it does not mean that it doesn´t absorb.

[bpatro]

HW Mueller: I understand OPA is used for derivatizing primary amino acids such as cystine. However, subsequent quick derivatization with FMOC-Cl helps us detecting the seconday amino acids such as hydroxyproline and proline present in a sample.
I really do not know if cystine is getting derivatized with FMOC-Cl. So in your opinion OPA derivative of cystine might be absorbing light and hence can be detected by DAD.

Is there any derivatizing agent that can make cystine fluoresce and detectable with FLD with suitable gradient settings?