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Small peptide quantification by RP HPLC

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

12 posts Page 1 of 1
I am trying to quantify a small peptide (mw 650) by RP-HPLC. I am using a C18 column (250x4.6) and Water/ACN/TFA as mobile phase.
The solvent of peptide stock solution is DMSO. I used water for dilutions.
What we observed is a big peak at RT around 2,4 min and small peak at RT 3,4 min.
The first peak seemd for DMSO, it also appears in a blank of dmso-water (1/1000). But I am not sure about the second peak which appears also in the blank.
Can anybody suggest me any modification?
Details, details, details!

Isocratic or gradient?
If isocratic, what composition?
If gradient, what range and time?
What flow rate?
What detector (if UV, what wavelength)?

What happens if you inject just a water blank (no DMSO)?
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
sorry!!!!
Isocratic and gradient. UV detector at 215nm
Solvents: A:Water/TFA 0.1%
B:ACN/TFA 0.1%

Isocratic: A 80% / B 20%; 1mL/min

Gradient:1mL/min
time %A %B
0 100 0
60 0 100

With a water blank no peak were observed.
Wikipedia, DMSO
Varying oxidation of sulfur
Dimethyl sulfide (DMS), the corresponding sulfide, also produced by marine phytoplankton and emitted to the oceanic atmosphere where it is oxidized to DMSO, SO2 and sulfate
Methylsulfonylmethane (MSM), a related chemical often marketed as a dietary supplement
Related isomeric forms with methyl on oxygen
Dimethyl sulfite, the corresponding sulfite
Dimethyl sulfate (also DMS), the corresponding sulfate: a mutagenic alkylating compound

But "any shit" , especially in the water, may be also.
Watch should be in the process. 8)
Okay, that helps! :wink:

The fact that it doesn't show up with a pure water blank eliminates the water as a suspect (obviously!). The fact that it doesn't show up with a water blank during a gradient eliminates contamination of the "A" reservoir as well (this is a common problem). Which leaves some contaminant or degradant from the DMSO as DSP007 suggested.

As long as that small peak does not interfere with your peptide you I think you can safely ignore it.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
I am trying to quantify a small peptide (mw 650) by RP-HPLC. I am using a C18 column (250x4.6) and Water/ACN/TFA as mobile phase.
The solvent of peptide stock solution is DMSO. I used water for dilutions.
What we observed is a big peak at RT around 2,4 min and small peak at RT 3,4 min.
The first peak seemd for DMSO, it also appears in a blank of dmso-water (1/1000). But I am not sure about the second peak which appears also in the blank.
Can anybody suggest me any modification?
Do you see the peptide?
I am trying to quantify a small peptide (mw 650) by RP-HPLC. I am using a C18 column (250x4.6) and Water/ACN/TFA as mobile phase.
The solvent of peptide stock solution is DMSO. I used water for dilutions.
What we observed is a big peak at RT around 2,4 min and small peak at RT 3,4 min.
The first peak seemd for DMSO, it also appears in a blank of dmso-water (1/1000). But I am not sure about the second peak which appears also in the blank.
Can anybody suggest me any modification?
Do you see the peptide?
No, the peptide doesn´t appear.
These days I bought new peptide, i will try to use less dmso for the stock solution
I am trying to quantify a small peptide (mw 650) by RP-HPLC. I am using a C18 column (250x4.6) and Water/ACN/TFA as mobile phase.
The solvent of peptide stock solution is DMSO. I used water for dilutions.
What we observed is a big peak at RT around 2,4 min and small peak at RT 3,4 min.
The first peak seemd for DMSO, it also appears in a blank of dmso-water (1/1000). But I am not sure about the second peak which appears also in the blank.
Can anybody suggest me any modification?
Do you see the peptide?
No, the peptide doesn´t appear.
These days I bought new peptide, i will try to use less dmso for the stock solution

Hello everybody!!
I bought new peptide, dissolved it in DMSO and diluted in water 1/25000. The DMSO peak is still present at RT 3min and the peptide doesn´t appear.
Do you think I have to modify the peptide (derivatization.....) for HPLC quantification? or maybe I have to quantificate it by aa analysis??


Thank you
Is there a reason why you dissolve the peptide in DMSO? If it is a small peptide, it should be very hydrophilic. Does it even dissolve in DMSO?

I would test to dissolve some peptide in 0.1% TFA in milliQ and inject in your gradient system (starting at 0% acetonitrile). In your isocratic system (20% acetonitrile), it is very likely that the peptide elutes in the front peak.
Is it possible that you have diluted your peptide too much? A dilution of 1/25000 sounds very high but, of course, it depends on what the initial peptide concentration is.
Is it possible that you have diluted your peptide too much? A dilution of 1/25000 sounds very high but, of course, it depends on what the initial peptide concentration is.
The peptide concentration is ng/mL. This is the concentration range i found in a paper for a similar peptide.
I was talking to an experxt in peptide synthesis and purification. He told me that probably i have to modify the peptide using the Sanger method and then quantify it by hplc at 285nm.
anybody know it?
Since you have a "standard" it would seem to make sense that one first establishes some parameters like amount and wavelength necessary to detect (maybe without column), then using detectable amounts to establish retention characteristics. I would skip the DMSO initially, later check its effect.
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