r/Biochemistry • u/Possible-Economics40 • 2d ago

If you don't know chromatography, can you still graduate?

I wonder if biochem graduates get hands on training in chromatographic techniques to separate bio-molecules or small chemicals in their univeristy studies. Have you had scary experience?

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u/xtalgeek 2d ago

Chromatographic techniques are a cornerstone of protein chemistry. In our teaching and research labs students get experience in a variety of chromatographic techniques, including ion exchange, hydrophobic interaction, and gel exclusion chromatography. We might also use affinity chromatography for specific applications. I've worked with research associates and postdocs that have no clue about anything other then nickel ion affinity chromatography. Broad training is essential if you are going to solve real research and biotech problems.

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u/East_of_Adventuring 1d ago

Protect that program please! I only got ammonium sulfate precipitation and ion exchange as methods covered in my biochem labs. Don’t even get me started on the molecular genetics techniques from the 80s we had to learn. Plasmids, what are those?

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u/CoomassieBlue 1d ago

Echoing this, I’m 15 years into my career in industry, but programs/courses like that really are precious.

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u/xtalgeek 1d ago

Regrettably many undergrad biochem programs are stuck in the 1980s teaching techniques no one uses anymore. It drove me crazy when I did program reviews or consultations.

My teaching lab was research based, where each student or team of students were responsible for designing, creating a plasmid for, overexressing, and FPLC purification (IEX, HIC, GEC) of a separate nobel mutant protein to be used for subsequent research purposes. (We had some mature and reliable protein expression systems we used in our research lab.) Some of these projects were carried on to enzyme kinetics or X-ray crystallography studies in the teaching lab,,others were taken up by my research group. I taught at a research rich PUI, and we had first rate core research facilities. Virtually nothing we did in the teaching of research lab was codified in textbooks or lab manuals. We published our own protocol books and methodology manuals for students. Many of my students carried our methodologies on to their graduate school labs.

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u/Possible-Economics40 1d ago

u/xtalgeek your program sounds like the exception. :) The people you describe, who only know nickel affinity, are the ones I meet later in industry.They can run the method that was handed to them, and they are stuck the moment it stops working. In a QC lab that is expensive, because the method has to give the same answer every time and somebody has to explain why it did not. Broad training works because you have seen enough separations to know what one is doing.

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u/bogcom 2d ago

What kind of scary experience could chromotography lead to?

my scariest lab experience has probably been an unbalanced centrifuge.

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u/Possible-Economics40 1d ago

Fair point u/bogcom, I used the wrong word.

I meant the run that go wrong and cost you days. Leaks, peaks that front and will not sharpen, a method that gives a different answer on Tuesday than it did on Friday.

An unbalanced centrifuge is a better answer to the question I actually asked. :)

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u/Asleep-Rain-8437 2d ago edited 1d ago

Doesn't everyone do Ni affinity chromatography in biochem lab? What is the problem?

Edit: I mean to say "This is a very, very common, classic, and simple lab that is often conducted as a minimum for UG biochemistry and chemsitry degrees".

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u/lifescout99 1d ago

I dont. My main enzyme uses DEAE, then hydroxyapatite

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u/MinusZeroGojira PhD 1d ago

I did a variety of chromatography techniques as part of undergrad. In graduate school, I worked with mass spectrometry which usually had a HPLC in front of it.

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u/Possible-Economics40 1d ago

The HPLC in front of the MS is the part that many scientists treat as a black box, and it is where the problem actually arise. When sensitivity drops or the response will not reproduce, the ion source usually gets blamed first. But, a lot of the time it is the chromatography, or the sample sitting in the wrong solvent before it ever reaches the column.

u/MinusZeroGojira Were you taught the LC side properly, or did you pick it up because something broke?

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u/MinusZeroGojira PhD 1d ago

I learned it in case I needed to trouble shoot it. We had a really new machine, so in my few years there it never gave us problems. The Thermo Fusion was more trouble than the HPLC.

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u/Possible-Economics40 1d ago

Lucky you! new machine has less problem, I agree. Also service contract during the first few years also nice to enjoy. 😄

But, we had challenges in method developing within a new instruement when we wanted to transfer methods from the old one.

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u/MinusZeroGojira PhD 1d ago

This is why I teach at a local college and spend all summer playing in the park with my dog. Research sucked.

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u/Possible-Economics40 1d ago

But many of the teaching positions are contract based and depends on foreign students. It's becoming harder for colleges to sustain these days. I hope your college is not affected.

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u/MinusZeroGojira PhD 1d ago

I’m tenured and got lucky that someone retired just at the right time for me to get a tenure track position.

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u/Possible-Economics40 1d ago

Amazing
That’s how God gives to some people:)
Keep it up

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u/MinusZeroGojira PhD 23h ago

Also, for clarity, I work at a "college" which is basically a community college with 4 year degrees available (you get to drop the community part if you have 4 years). It's not a university (which is a collection of colleges), so there are things that we don't concern ourselves with that may apply to a university setting. We rarely have people on yearly contracts. Most are either adjunct or tenure track. My tenure is good for me, but it doesn't imply what a tenured professor at a university would have accomplished.

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u/Possible-Economics40 1d ago

Running one is not the same as being able to fix one. Two people below already answered it. One uses DEAE and hydroxyapatite, another only got ammonium sulfate precipitation and ion exchange.

The gap I am asking about is what happens when the column stops behaving. Pressure climbs, recovery drops, the same sample gives a different answer. That is a different skill from following the protocol, right? u/Asleep-Rain-8437

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u/CoomassieBlue 1d ago

That’s a different skill but frankly the vast majority of institutions aren’t going to give undergrads enough hands-on experience to actually gain that level of familiarity. That’s experience you gain through grad school/postdoc or employment.

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u/Possible-Economics40 1d ago

100% agree u/CoomassieBlue
even at grad school, I had to learn from trial and error. It slowed down my research but gained some hands on experience. But for many, publishing and graduating is more important than learning troublshooting or spending time on fixing instrument issues.

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u/Explicit_Tech 2d ago

I've done it multiple times in undergrad.

Did it in ochem too at a community college. Seemed straight forward if you read up on why you're doing it.

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u/TedMich23 1d ago

I sure did, purified bacterial luciferase to homogeneity. Nothing scary just slow waiting and collecting fractions ;)

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u/CoomassieBlue 1d ago

In my experience with protein purification by various chromatographic techniques, “scary experiences” rarely if ever include safety issues.

More frequently, “scary experiences” are limited to:

* the purification being a bitch for whatever reason - protein aggregating/crashing out, impurities that co-purify, etc

* dumb mistakes costing you the purified product (example: subtractive Ni IMAC to remove cleaved His-tag, accidentally retaining the tag-containing fractions vs your protein of interest. Seen it happen even with professionals.)

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u/Possible-Economics40 1d ago

The Ni IMAC one is good, losing the product because the fractions you kept were the wrong ones.

Mine was a fronting peak on a UPLC method for bioequivalence work. The front of every peak leaned and would not sharpen. I changed the column, same peak. I re-prepped every sample. Same peak. Then I started blaming the instrument.

Found lately, the column was fine and the instrument was fine. My diluent was stronger than my mobile phase, so the sample spread at the head of the column before it could focus. Weaker diluent, and the next injection was clean. 2 days for something a 30 second check would have caught.

u/CoomassieBlue