Chromatography
How a Chromatographic Method is More Than a Separation
By James Hogbin & Charis Lam.
James.Hogbin@acdlabs.com and
charis.lam@
acdlabs.com ACD/Labs, 8 King Street East, Toronto, Ontario, Canada M5C 1B5
Developing a chromatographic method is about more than separating compounds. A well-designed method complements the ultimate goals of the method- development team and helps answer research and business questions. To get to such a method, the development process must take these goals into account from the outset and follow a predefi ned plan.
Introduction
If chromatography is the science of separating mixtures into their components, then method development is the craft of turning separations to a useful purpose. Rarely do scientists separate compounds for the fun of it. Chromatography serves project or research goals, and developed methods must align with these aims.
Thus, separation of components is not the only requirement for an analytical method. Depending on the project, other needs might include long-term method stability, easy method transfer, high yield and purity for preparative scale-ups, detection of all impurities and degradation products, and more. The organised chromatographer identifi es these needs from the beginning and designs a method-development plan that achieves both separation and other method goals.
A three-tier process (Figure 1) underlies this plan [1,2]: 1. Defi ning the project purpose 2. Matching parameters to purpose 3. Validating performance This process ensures that complete methods are designed from the outset.
Unfortunately, this approach leads to continuous cycles of revision. The three impurities will be separated, only for the chromatographer to learn that there are other expected impurities, which will then be separated, only to learn that an orthogonal high performance liquid chromatography/mass spectrometry (HPLC/MS) screening approach would have been the most appropriate option from the beginning.
To tease out all the needs a method must fulfi ll from the start, begin instead with a thorough questionnaire:
1. Is the problem fully understood? 2. Have the correct questions been asked? 3. Are all the facts at hand?
These questions reinforce that the project should be understood thoroughly. Not only at the surface - which is what often emerges in the fi rst presentation of a problem - but with all the details of why the separation needs to be done, what is being separated, who is going to use the information, and how they are going to use it.
4. Have the customers asked the correct question? 5. Do they have all the facts?
A useful chromatographic method helps people make decisions. The list of people impacted can be long: developed methods may be used by entire teams, and even more people may rely on the method results for their work. It is therefore paramount to understand the entire user chain. For example, there is little purpose in developing a 90-minute ultra-high performance liquid chromatography (UHPLC) gradient if the fi nal user will be a contract house with only HPLC equipment. Each person in the chain may understand pieces of the problem, but often, no one person understands the entire picture. To get a complete view of the project, speak to multiple stakeholders.
6. What approach should be taken?
Based on what has been learned so far, design an approach from fi rst principles. Consider the expected compound structures, and use in silico tools to predict solubility, logP, logD, pKa, and degradation pathways. A logically planned approach will save time and guide the chromatographer in the right direction from the beginning.
7. How are the results reported? 8. What results are being reported? 9. What will happen to the results once reported?
Figure 1: A method-development plan should include (i) defi nition of the project purpose, (ii) use of the project purpose to select and optimise parameters, and (iii) validation of performance.
Defi ning the Project Purpose
As discussed, the ultimate purpose of an analytical method is not to separate compounds. Separation is an important proximate purpose, but it serves a higher goal.
Too often, early-career chromatographers fail to account for the multilayered purposes in a project. They start work with only a shallow understanding, jumping into the lab after a short conversation with a colleague:
Question: “I need to separate three impurities in this mixture.” Answer: “I can do that for you.”
Knowing why a method is being developed helps chromatographers to determine how the results should be reported. While many organisations use standard report templates, these should still be scrutinised for each project. What is the most important information for the project goals? Is that being reported? Is the provided information enough for downstream teams to make their decisions, or will they have to further interrogate the data?
10. Are these conditions going to be used long-term? 11. Is that the end of the work or is it a long-term project?
Methods should stand the test of time. Even when method life cycles are short, troublesome methods spawn unexpected problems, so it is always best to ensure robustness with thorough testing.
12. Is it fi t for purpose? 13. Is this GMP, GLP, development, or nice-to-know? Regardless of the reasons behind method development, the highest standards must always
INTERNATIONAL LABMATE - SEPTEMBER 2020
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84
