Home | HPLC Calculator

113

Questions Asked

Answers Given

518

Community Members

Questions Today

Questions

Ask Your Question

slucas

10 m ago

Peak area variability in DMSO analysis

Hello,
I am running an impurity analysis method for DMSO with the following parameters:

FID Temperature: 250 °C
Injector Temperature: 250 °C
Column: Agilent DB-624
Flow: 5 mL/min (Hydrogen)
Initial temp: 100 °C, hold for 2 min
Ramp from 100 to 200 °C at 10 °C/min
Final temp: 200 °C, hold for 3 min
Injection volume: 0.5 microliters
Injection syringe volume: 1 microliter (auto-injector)
Split: 1:10
Liner: Ultrainert 5190-2295
FID flows:
• Hydrogen: 35 mL/min
• Air: 400 mL/min
• Nitrogen: 35 mL/min

I’m having serious issues with area repeatability when injecting the same vial 6 times in a row. I’m getting %RSD values around 9%, and my requirement is ≤ 5.0%
I’ve tried all kinds of adjustments—changing injection speeds, adding a 3-second viscosity delay… but nothing seems to solve the issue.

Can you help me?

Thank you very much.

0 Like 3 answers 326 views

Alan Cervantes 10 m ago

distribute your STD on single injections vials, not the same vial.

👍 1 | 👎 0

youhana Hosny 10 m ago

you have to exclude first that the problem is not related to the instrument itself.
is the instrument calibrated?
have you tried another method using the same instrument and the results are good ?

👍 0 | 👎 0

slucas 10 m ago

Yes, the instrument is fully calibrated and other methods report good results. The only remarkable thing is that this method is the only one I have that use hydrogen as carrier gas.

Pathan Sohil 10 m ago

I think first you should lower your Column flow according to your Column ID, and need to check the glass wool in your linear.

👍 0 | 👎 0

slucas 10 m ago

I can’t lower the column flow, this is a requirement of USP… The liner is new and the glass wool is in perfect conditions.

Frequently Asked Questions: HPLC Analysis & Chromatography

High-Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify each component in a mixture. It relies on a pump to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

Column efficiency is typically measured by the number of Theoretical Plates ($N$). The most common formula is $N = 16 \times (t_r / W)^2$, where $t_r$ is the retention time and $W$ is the peak width at the base. A higher number of theoretical plates indicates a sharper peak and better analytical separation. You can calculate this instantly using our Theoretical Plates Calculator.

The ICH (International Council for Harmonisation) Q2(R1) guidelines mandate specific validation parameters for HPLC methods. These include assessing Accuracy, Precision (Repeatability and Intermediate Precision), Specificity, Detection Limit (LOD), Quantitation Limit (LOQ), Linearity, and Range. Our calculators are designed specifically to help analysts easily compute these linearity, LOD/LOQ, and %RSD parameters in compliance with ICH standards.

Popular Search Trends & Keywords

HPLC Calculator Agilent HPLC metrics HPLC detector (DAD/UV) HPLC MS analysis HPLC pump pressure RP-HPLC parameters Chromatography principles UPLC method scaling Hb HPLC method Liquid chromatography tools

Download the HPLC Calculator App: The Ultimate HPLC Analysis Tool

Questions

Peak area variability in DMSO analysis

Mobile App

Frequently Asked Questions: HPLC Analysis & Chromatography

What is HPLC (High-Performance Liquid Chromatography)?

How do I calculate HPLC column efficiency and Theoretical Plates?

What are the ICH guidelines for HPLC method development?

Popular Search Trends & Keywords