Chromatograms-having trouble on my lab report, please help:)

Question

How do you identify the peak fraction for flowthrough (FT) on a chromatogram and identify elution peaks seen in the chromatogram. How do you identify the LDH peak and how do you know it is the LDH peak?

Also, does mAU mean absorbance or does %B mean absorbance?

Answer 1

I would love to hear more specific information about your purification process of LDH, as I could provide more specific feedback!

Your first question:

To first answer that, we have to think about what the flow through is. The flow through is simply the first liquid that leaves the column, containing items which did not readily interact with the resin. Thus, the flow through is super easy to identify as it contains anything that you do not expect to interact with the column. The elution peaks are somewhat similar, though will be found after the flow through. This type of purification is quite handy because it does a lot of the work for you, showing peaks with each fraction. But, some things can affect your elution peaks including how fast your sample is run through, what type of buffers are used, and others.

Luckily for LDH, there is already a lot of data that exists in literature and on Google. So, if you have a limited time constraint, I would try to look up a paper or two where a chromatogram was performed with LDH and look for annotations. Keep in mind that there are many fine variables in biochemistry and your data will not be the same as theirs. Although, LDH tends to elute at a similar time with similar protocols, so you could compare a study's LDH peak with your most suspicious peak.

So, without seeing your graph, it may be difficult to describe where your peak may be expected. Although, I would try to think about what time in the reaction an additional buffer was added (to create a gradient), or perhaps the salt concentration was changed. This LDH peak should be rather distinct, though may be in close proximity to other distinct peaks, depending how much you have already purified your sample.

How you would really know with certainty - refer to the literature to find the expected elution time. Then, you would run assays and protein determination on peaks with close to that expected value, until you find the one with the most specific activity.

Your second question:

Typically %B refers to the percentage of a given buffer. When using a system like a chromatogram, often times it will use two or more buffers and change automatically as time goes on. So, you may have not even recognized it changed buffers during the reaction. This can be important depending what they are, as they usually form a gradient, allowing elution to happen in steps, rather than all at once.

mAU refers to milliabsorbance units and likely is the y-axis of the chromatograph. It is simply a measure of absorbance. So, the higher the value, the more "stuff" there is. In your case, the "stuff" is probably proteins since it's likely being read at A280.