Chromatography Forum

Hello all,

I have a LC system as below.

Buffer Solution:
Adjust the pH of the 0.05 M KH2PO4 solution with 5 N KOH to 6.80

Mobile Phase:
Mobile Phase(a) : Buffer
Mobile Phase(b) : The mobile phase is prepared by mixing Buffer, acetonitrile and methanol in 70: 25: 5 ratio (v/v/v) and degas the solution by sonication

Chromatographic Conditions:
Column : Atlantis T3, 150 * 4.6 mm, 3 µm
Column temperature : 25 C
Flow rate : 1.0 mL/min
Injection volume : 10 µL
Detector : UV 300 nm
Gradient Table
Time (min) Mobile phase A(%) Mobile phase B(%)
0 100 0
37 0 100
45 100 0
50 100 0

When we perform this chromatogram system and inject a blank injection (mobile phase B), we always get a ghost peak at about retention time 24 min. Because we have an analyte (drug impurity) at the same retention time, this ghost peak interferes this impurity significantly.

We have tried many ways to solve this problem, such as

1. Replace buffer solution salt (KH2PO4), pH modifier (KOH), different water source
2. Different column lot
3. Different instrument brand (Agilent & Waters)
4. Different lab

This ghost peak still exist. Please assist me to solve this issue.

Have you tried running a different sample? If YES, is the ghost peak still there? Sounds like it's in your sample.
Tony Vella www.hplcworks.net

Hello all,

I have a LC system as below.

Buffer Solution:
Adjust the pH of the 0.05 M KH2PO4 solution with 5 N KOH to 6.80

Mobile Phase:
Mobile Phase(a) : Buffer
Mobile Phase(b) : The mobile phase is prepared by mixing Buffer, acetonitrile and methanol in 70: 25: 5 ratio (v/v/v) and degas the solution by sonication

Chromatographic Conditions:
Column : Atlantis T3, 150 * 4.6 mm, 3 µm
Column temperature : 25 C
Flow rate : 1.0 mL/min
Injection volume : 10 µL
Detector : UV 300 nm
Gradient Table
Time (min) Mobile phase A(%) Mobile phase B(%)
0 100 0
37 0 100
45 100 0
50 100 0

When we perform this chromatogram system and inject a blank injection (mobile phase B), we always get a ghost peak at about retention time 24 min. Because we have an analyte (drug impurity) at the same retention time, this ghost peak interferes this impurity significantly.

We have tried many ways to solve this problem, such as

1. Replace buffer solution salt (KH2PO4), pH modifier (KOH), different water source
2. Different column lot
3. Different instrument brand (Agilent & Waters)
4. Different lab

This ghost peak still exist. Please assist me to solve this issue.

Garbage peaks in gradient blanks are common. Run the "3 blank gradients" test described here:
http://www.lcresources.com/resources/TSWiz/hs400.htm

If the garbage peak gets bigger with longer equilibration, then you have contamination in your "A" reservoir. If it does not, then you have carryover or contamination in your autosampler.