Chromatography Forum

Hai,

I am facing a pecular problem . I have an intermediate inhouse reference material. Now When I get it analysed, sometimes it is showing peak splitting and most of the time it is a single peak (actually we qualified this with three different analysts and on two different systems). Now though the column is new, a freshly prepared MP and sample with different analysts it is not matching .

Can anaybody suggest a solution

raman

Peak Splitting can have a number of causes. The most likely is a column void or channel. Replace the column and this should go away.

A second likely candidate is that the sample diluent is stronger than the MP. This usually a problem for early eluting peaks. Replace the sample diluent with a weaker solvent, and this should fix it.

Dear Raman,
usually, a doctor will ask how the detail about your headache like the exact location and the strength and pattern and so on after you tell him you got a headache.
So if you can provide us the detail: sample preparation, concentration, dilent, injection volume, mobile phase, even molecule structure, and so on, experts, not me, here will be able to help you much better.
how many different LC and columns you tried is good information though it does not matter how many chemist have tested on this unlss they followed different route. Thanks for sharing and good luck

Also, is it just one analyte peak that is splitting, or are all your analyte peaks splitting? If you only have one peak in your chromatogram then add an appropriate internal standard and see if that splits as well. If both peaks split then the split is probably happening before the column rather than on column (i.e. some kind of misinjection).

I was having a similar problem with an HPLC-ICPMS method. It turned out that the positioning of a trigger command in the method file was causing my injection valve to turn twice with each one injection, resulting in a sample delivered in two "slugs" to the column. Weird, but fixed with a method rewrite.

Dear YM3132

The mobile phase is SLS and acetonitrile at pH 4.0 and the diluent is acetonitrile. The problem is we are using the same method for 4 intermediates. Out of these only one intermediate is behaving like this.

The column new, fresh mobile phase, freshly prepared sample

One day it give one peak. The other day it gives splitting

Raman

Dear YM3132

The mobile phase is SLS and acetonitrile at pH 4.0 and the diluent is acetonitrile. The problem is we are using the same method for 4 intermediates. Out of these only one intermediate is behaving like this.

The column new, fresh mobile phase, freshly prepared sample. One day it give one peak. The other day it gives splitting

Raman

I would suggest dissolving your sample in the mobile phase, or at least the same water/acetonitrile ratio. Also, I would try injecting different volumes and/or concentrations to ascertain if you have a solubility issue.

Why regard to your other question in your signature, check PubMed for more details of the following article.

J Pharm Biomed Anal. 2006 Jun 16;41(4):1152-6.
Validated chiral liquid chromatographic method for the enantiomeric separation of Pramipexole dihydrochloride monohydrate.
Pathare DB, Jadhav AS, Shingare MS.

The enantiomers of Pramipexole dihydrochloride monohydrate were resolved on a Chiralpak AD (250 mm x 4.6 mm, 10 microm) column using a mobile phase system containing n-hexane:ethanol:diethylamine (70:30:0.1, v/v/v). ... The presence of diethylamine in the mobile phase has played an important role in enhancing chromatographic efficiency and resolution between the enantiomers. ....The proposed method was found to be suitable and accurate for the quantitative determination of (R)-enantiomer in bulk drugs.

I remember the chiralpak AD only allow the ratio of alkane/ethanol to be 100/0 - 85/15 or 40/60 - 0/100. The ratio outside of this range might have some adverse effect against the column. Is it safe to use at 70:30? Is there any other list of separation enhancer we could use such as Diethylamine?
Thanks,

According to the manufacturer, the baseline stability may be adversely affected, presumably not the column. In my limited experience with a Chiralpak OD-H column, ethanol can provide improved resolution, but the lower ethanol content can cause standard pump seala on an Agilent 1100 to leak after a few hours. Polyethylene normal phase seals have to be used.

Typically, the potential use of TFA or DEA depends on the functional groups on the enantiomers you're trying to separate.

http://www.daicel.co.jp/chiral/e/produc ... on/ad.html
" Some of the most popular eluents for HPLC (such as acetone, acetonitrile, chloroform, DMF, DMSO, ethyl acetate, water, methanol, methylene chloride, and THF) remaining in your HPLC system may destroy this column....Suitable Mobile Phases include:
- Hexane/2-Propanol (100/0 to 0/100 v/v) Typical Mobile Phase used is Hexane/2-Propanol (90/10 v/v).
- Hexane/Ethanol (100/0 to 85/15 v/v), (40/60 to 0/100 v/v)
(NOTE: The range 85/15 to 40/60 (v/v) may adversely affect baseline stability.)
- Suitable Mobile Phase Modifiers include N,N-Diethylamine for a basic sample, and Trifluoroacetic acid for an acidic sample.
(NOTE: Minimize use of the modifier. Typical use is 0.1%; maximum 0.5%).

Bruce Hamilton

Have you considered that by using SLS in your MP that you essentially have an Ion Pair reagent.

Couple this with a brand new column, and it sounds to me like your going to have problems.

If the column has not been bedded in enough, the SLS will essntially retain on the column, this will then in turn retain a certain amount of the analyte in question.

...hence probably why you saw split peaks when using a new column.

Does the effect lessen with an older column?

One question is..why have you SLS in the mobile phase?

Anthony