Chromatography Forum

We have analysing some compound for some days.Unfortunately our column is not giving good performance after just 20 to 25 injections.

Mobile phase A: Ph 7.0 buffer ( 3.0ml of ohosphoric aci in 2000ml Milli Q water and adjusted Ph with tryethyamine)

Mobile phase B:Above buffer and methanol in the ratio of 10:90

Diluent : Above Ph 7.0 buffer and methanol in the ratio of 20:80
Gradient programme.
TIME %A %B

0 100 0
15 100 0
25 85 15
35 75 25
45 60 40
55 60 40
65 30 70
70 30 70
72 100 0
80 100 0

Chromotographic conditions:a) Uv-detector ,289nm
b)250*4.6 mm Xterra RP-8 ,5 micron column

c) column temperature:Ambient
d)Flow rate:0.8 ml/min
e)Injection volume :10 microliters.

The method was validated and given for QC.But we are getting just fewer number of injections not more than 25.
I request members of forum to guide me in resolving this proble.

What is the nature of the failure?

UWE NEUE, the problem is not consistant .Some times peak spilitting , some times improper peak shapes i.e more taling and some time poor resolution etc.Different analysts getting different problem after 20 to 25 injections.

Is this a method that has been used sucessfully for a long time and then sudden problems?
Comments Chromforum folks please:
The "buffer" seems a little strange to me.
Do you measure the pH correctly and consistently?
You could try adding the phosphoric acid and triethyl amine to the water by weight each time for more consistency.

Is it reasonably consistent, i.e. EVERYBODY is getting about 20 to 25 injections?

What was the column lifetime during development and validation of the method?

What is the sample? Is there a difference between the sample used for validation and the samples currently used (e.g. drug product vs. standards etc.)?

You seem to imply that the short life happens with multiple operators and multiple columns. How many?

The buffer is OK. All things considered, my suspicion is that bugs are growing in the instrument.

Others have made the obvious comments, and without knowing more about your samples, it's hard to comment, but, in my experience, C8 columns are less robust than C18 for yucky samples, such as biological extracts and fermentation media.

Your sample diluent has a relatively high concentration of MeOH, is there a reason for that choice, such as it's not fully soluble in aqueous?.

Please keep having fun,

Bruce Hamilton

Is it reasonably consistent, i.e. EVERYBODY is getting about 20 to 25 injections?

What was the column lifetime during development and validation of the method?

What is the sample? Is there a difference between the sample used for validation and the samples currently used (e.g. drug product vs. standards etc.)?

You seem to imply that the short life happens with multiple operators and multiple columns. How many?

I asked R&D guys ,they are telling there is no change difference between my samples and samples used for validation study.

During validation ,they get this problem but once only.

Others have made the obvious comments, and without knowing more about your samples, it's hard to comment, but, in my experience, C8 columns are less robust than C18 for yucky samples, such as biological extracts and fermentation media.

Your sample diluent has a relatively high concentration of MeOH, is there a reason for that choice, such as it's not fully soluble in aqueous?.

Please keep having fun,

Bruce Hamilton

What they are saying is that Me OH concentration used for better extraction puposes.

"Better extraction" from what?.

You still need to provide information on some of the other questions asked earlier about what your samples are and what they are extracted from.

My first concern is that you are loading a sample in 80% methanol with pH 7 control, but your gradient is only going to 63% methanol at same pH and the column remaining at ambient temperature

Bruce Hamilton

Bruce has a good point. It is possible that something accumulates on the column that is not washed out. An intermediate wash would solve this. Also, if this is the case, columns that have deteriorated could be recovered by a suitable wash.

The other possibility still remains. You have not commented on the possibility of bacterial growth in the instrument. These bugs love water with a pH 7 buffer and some stuff to eat (such as the triethylamine).