Chromatography Forum

Hi All

I've been given a BP/EP method for analysis of Polysorbate 80 raw material which has a Fatty Acid Composition test for assessment, and would like some advise please.

The method conditions are:
Column: 30m x 0.32mm Macrogol 2000 (o.5µm film) - (DBWax?)
Injector and detector (FID): 250°C
Temperature ramp: 0-14 minutes: 80 - 220°C
14 -54 minutes: 220°C
Carrier: He @ 50cm/sec
Split: not specified

Specification:
Myristic Acid: NMT 5.0%
Palmitic Acid: NMT 16.0%
Palmitoleic Acid: NMT 8.0%
Stearic Acid: NMT 6.0%
Oleic Acid: NLT 58.0%
Linoleic Acid: NMT 18.0%
Linolenic Acid: NMT 4.0%

Sample prep:
Methylate 0.5g using BF3/MeOH complex and extract into heptane.

Reference Solution A:
Accurately weigh 0.5g of the following mixture of calibrating substances into a 50mL vol flask. Dissolve and dilute to vol in heptane:
Substance Eq. Chain length Composition(%m/m)
Isothermal Lin Temp Gradient
Methyl Myristate 14.0 5 15
Methyl palmitate 16.0 10 15
Methyl Stearate 18.0 15 20
Methyl Arachidate 20.0 20 15
Methyl Oleate 18.3 20 15
Methyl Eicosenoate 20.2 10 10
Methyl Behenate 22.0 10 5
Methyl Lignocerate 24.0 10 5


Ref Soln B:
Dilute Ref Soln A 1:10 in Heptane (for S/N on methyl myristate peak)

Ref soln C:
Accurately weigh 0.5g of the following mixture of fatty acid methyl esters into a 50mL vol flask. Dissolve and dilute to vol in heptane: (Commercially available mixtures of fatty acid methyl esters may be used)

** List given as per specification above **
(Note: listed as acids NOT methyl esters, with no instruction to methylate)

For system suitability
inject ref soln A for resolution between methyl oleate and methyl stearate (NLT 1. and NLT 30000 plates calculated on Methyl Stearate peak.

Ref Soln B for S/N on methyl myristate peak (NLT 5)

Procedure:
Inject the Test Soln and Ref Soln C and identify the peaks; the peaks may also be identified by drawing calibration curves using the c'gram obtained with ref soln A and the info given in the table above.

a)
Using isothermal conditions giving the logarithms of reduced retention times as a function of the No. of carbon atoms of the fatty acid; identify the peaks by means of the straight line thus obtained and the "equivalent chain lengths" of the different peaks. The calibration curve of the saturated acids is a straight line. The logarithms of reduced retention times of unsaturated acids are situated on this line at points corresponding to non-integer values of carbon atoms known as "equivalent chain lengths";

b)
Using linear temperature programming giving the retention times according to the No. of carbon atoms of the fatty acid; identify by reference to the calibration curve.

Quantify by normalisation disregarding peaks with area less than 0.05%

OK finally to my questions:
If the solution of fatty acids (ref soln C) is injected will we see any peaks?
Note that the method says fatty acid methyl esters, but then specifies the ffa's and does not methylate.

Procedure a)
Using isothermal conditions...
In your opinion, what temperature should be used for this?

The table given in ref soln A gives Composition (%m/m) for isothemal and temp gradient;
Does the % composition change depending on the temperature program?

And, finally, can anyone explain the section in Procedure A) regarding 'logarithms of reduced retention times' and 'equivalent chain lengths'?


My apologies for such a long post.
I hope you all didn't lose interest half way through.
Any insights would be much appreciated before we start playing with this to see if we can get it to work.
And, yes I did a google search, but haven't had a chance to get to the library.
My main issues are regarding the logarithms and equivalent chain lengths to identify the peaks. (should we inject individual ID solutions?)

Regards
Gary

Quick Answers, possibly not correct, quick peek at my 5th Edition EP without supplements - haven't checked for a revision.

The monograph says to use Table 2.4.22-3 with the linear temperature programme composition ( because the monograph specifies oven conditions ) to prepare Ref Soln A, so isothermal is irrelevant. However, there are some additional foornotes which may affect you.

The table uses FAME, so you should see peaks. The FFAs composition is only in the monograph for when you calculate the sample composition.

Calculation uses the ECLs which are determined by reference to the retention of saturated FAs. ECLs are very column dependent, and you should calculate using the retention times for the reference mixture. Indications of ECL for the specificed column are in Table 2.

I'm sure ECL calculations and explanations will be available on WWW.

Hope this helps, please keep having fun,

Bruce Hamilton