I am following a european pharmacopoeia method for the HPLC assay of getamicins sulphate.
These are typically large molecules approx. C21H43N5O7. The method dictates to use the following conditions
Eluent 60g/L of anhydrous sodium sulphate, 1.75g/L sodium octanesulphonate, 8ml/L of tetrahydrofuran, 50ml/L of 0.2M potassium hydrogen phosphate previously adjusted to pH 3.0 with dilute phosphoric acid.
Column length 250mm Diameter 4.6mm,
stationary phase styrene-divinylbenzene copolymer (8um) with a pore size of 100nm (1000Anstoms)
temp 55 C
post column addition of 50mM NaOH
Detection is Pulse Amperiometric Detection PAD.
I am getting a very high current background. The cell was at 55 C too. So I have moved the PAD cell to a cooler environment 30 C, and background current is now much lower (approx. 300nA) But the problem is that I am getting no peaks when I inject a standard.
Any ideas what to check ?
Any advice ?
The eluent strength from the European Pharmacopoeia seems very high. Agree / disagree ?
Thanks

I haven't tried this particular system but in general it looks like there may be a problem with the concentration of your post column reagent (or at least the relative flow rate). It is important that the post column mixture be fairly alkaline and normally the post column base addition is with 0.5 molar sodium hydroxide. Check to make sure your pH for the post column effluent is at least 12.5.

Also, you might want to check on the tetrahydrofuran that you're using. I'm pretty sure you will have a problem if you're using the grade with the stabilizer in it (BHT, I think is the most common stabilizer and this is electroactive).

I have checked the post column pH. It is pH 13. Conc of NaOH 0.5M. The background is now 70nA. I got this by recycling the eluent for two days. The THF has no stablizer and the bottle used is fresh, just recently opened.
I can check the system by injecting sugar and I get peak after 5 mins. So I know the detector/eluent/post column reagent is working fine. When I inject standard for Gentamicin, I get no peak. I let it run for 80mins and still no peak.
I believe the column reccommended is either incorrect or somebody has made a mistake with the pore size. The metohod states a column length 0.25m diameter 4.6mm, a stationary phase of styrene-divinyylbenzene copolymerR (8um) with a pore size of 100nm. This has been interpreted as 1000 Anstroms.I have checked the post column pH. It is pH 13. Conc of NaOH 0.5M. The background is now 70nA. I got this by recycling the eluent for two days. The THF has no stablizer and the bottle used is fresh, just recently opened.
I can check the system by injecting sugar and I get peak after 5 mins. So I know the detector/eluent/post column reagent is working fine. When I inject standard for Gentamicin, I get no peak. I let it run for 80mins and still no peak.
I believe the column reccommended is either incorrect or somebody has made a mistake with the pore size. The metohod states a column length 0.25m diameter 4.6mm, a stationary phase of styrene-divinyylbenzene copolymerR (8um) with a pre size of 100nm. This has been interpreted as 1000Ǻ by the column suplier. Is this correct ?
I have used a Polymer Labs column PLRP-S 1000Ǻ 250mm X 4.6mm; p/n 1512-5802.
The Gentamicin standard is a certified reference standard 50mg of Gentamicin made in 100ml of eluent.
Any ideas ??

A pore size of 100 nm is a packing with a rather large pore size. Not impossible for this application. The pore size of the PLRP 1000 is about 10 nm or maybe a bit less. Look for a much larger pore size. The 10^5 A column may be about right.

Uwe,
Thanks for the reply. The method says a pore size of 100nm. Polyer labs suggest this column. Are you saying it only has a pore size of 10nm.
Then if so ploymer labs miss informed me.
The other point is. In the post above,
I used the correct Anstroms symbol. A capital A with a circle on top, for anstronms , and for some reason the software of chromatography forum has converted this A symbol to a text number &#506.
Do not get confused by the &#506 text. It is a windows thing I am sure. Replace this text with the Anstrom symbol.

Anon,

My guess is that the problem is the opposite of that suggested above. Typically, the surface area of 1000 angstrom polymeric materials is too low to be useful for reversed phase applications. I can't see why this application needs to be done on a porous polymer but my guess is that whoever wrote it accidentally wrote 100 nm when they meant 100 angstroms. This 100 angstrom pore size is typical of porous polymers used for reversed phase applications and is more likely to have sufficient capacity to accomplish the method.

By the way, in my experience the column specifications in USP classification systems are very inaccurate and this may simply be another reflection of the analogous problem in the European Pharmacopeia

I would not bet on a mistake. If it says 100 nm, I think it means 100 nm. Since you used the 10 nm column (10^3 Angstroem) column from Polymer Labs and did not see the analyte, it has too much surface area and too small a pore size.
Chris, the difficulty comes from the nomenclature that the GPC people (i.e. Polymer Labs) use for the designation of the packings. It is derived from the extended chain length of a polystyrene molecule that is just excluded from the pores. "10^3 Angstroem" means for them a real pore size of around 0.5 to 1 nm, or the equivalent of a "60 Angstroem" packing for retention chromatography. A 100 nm (or 1000 A) pore size packing translates into 100 000 or 10^5 Angstroem for the GPC people.\
To Anon: yes, indeed, I meant that the polymer people at Polymer Labs sold you their 1000 A packing, which is a packing with a true pore size of 6 nm and way too much retention for what you want.
Sianse lanquage bery bery difficold.... :-)

Uwe,

While it may be true that GPC people in use different nomenclature for designation of packings, the fact remains that if you check out the Polymer Labs website, you will see that the pore size specified for small molecules in the size range of interest is 100 angstroms. Likewise the pore size of the Hamilton equivalent generally promoted for molecules in the size range (PRP-1) is 100 angstroms. The same goes for the polymeric reversed phase column from Dionex (the MPIC column) and the polymeric reversed phase column from Supelco (SUPELCOGEL TPR-100 HPLC Column). Since in all cases the standard reported pore size for polymeric reversed phase columns is 100 angstroms for small molecule applications, it seems quite likely that the reference cited by anon above has simply transposed nm and angstroms. The failure to detect the compound could simply be because the compound is eluting near enough to the void that the pH disturbance associated with the sample injection is masking the presence of the compound. I still think anon should try a 100 angstrom pore size material.

Dear All,
The european pharmacopoeia method for the HPLC assay of getamicin sulphate was based on a paper by the group of Hoogmartens at the University of Leuven, Belgium.
Analysis of gentamycin by LC with pulsed electrochemical detection, by E. Adams, W. Roelants, R. De Paepe, E. Roets and J. Hoogmartens.
Journal of Pharmaceutical and Biomedical Analysis, 18 (1998), 689-698.
Taking a look at that paper might solve Anonymous' problems.
If not, try to contact Dr. Adams at the K.U.Leuven.
Jan

The same group (mentioned by Jan) had also published a similar paper fro a similar compound (J. Chrom. A 812 (1998) 151-157) which aslo employed a polymeric C18 (PLRP-S 1000 angstroms, 8 um). The second thing I did when I tried this method is throw out that ploymeric column and use an aqueous friendly silica based C18 column (I used Atlantis), the first was to use that PL column. The authors explain the choice of the polymer column by saying "...was chosen because of its remarkable stability and batch reproducibility". I have had much better results with the Atlantis (much better peak shape and retention stability). I did have to increase the amount of organic modifier to obtain similar retention times however (1% to 2% THF).

Just my 2 cents....

Chris - the GPC 100 Angstroem stuff does SEC of small molecules in solvents that swell the pores of the material. Under the conditions mentioned above, the 100 Angstroem stuff would be non-porous.

Uwe,

I can't know for sure but I'm fairly certain that Polymer Labs follows a practice similar ours and uses very highly cross-linked materials for the resin used in columns intended for reversed phase applications. In our case the cross-link is 55% and does not swell appreciably in common HPLC solvents. The BET measurement of pore size is approximately 100 angstroms and does not change significantly under common application conditions. Perhaps Polymer Labs uses different media for their size exclusion materials. But certainly if it were true that 100 angstrom media really had no appreciable porosity under standard usage conditions, it certainly wouldn't be the most widely used format for polymeric reversed phase media.

The current EP method for Gentamicin uses pre column sample treatment with OPA and detection with UV. It also calls out a L1 type column (silica based C18). Check it out, page 1238 EP 4th edition.

To last Anonymous:
Check out page 4016 of supplement 4.5. You'll notice the method has changed from pre-column deriv. to amperometric detection and from a silica to a polymeric type column.
Jan

Dear Jan,

Thanks,
Only had up to 4.2 sitting here !