This is a text only instructions for HPLC operations.
Hi guys, this is a general step-by-step to do list for a batch run:
Before you even start , you should have attended these sessions Lab safety induction with Winfield Jugo (This one is a legal requirement ). Solvent , sample, and batch run preparation with Luci Mokbel.
The work here would be arranged in batches. A batch in a retention run is all %B for One column. Please check the google drive for details.
Please note, while there may be more than two people in a lab (and certainly NOT less than two. You shouldn’t be there alone. In case your partner left, grab me (Odi) from the room next door (comp chem) or someone else, and keep the door open so you can go back in), one pair would be responsible for one batch. The team of two is not only responsible for doing the pre-batch run and preparing the batch, but also doing the post batch check of the data accumulated.
Of course you would need to wear your PPE (lab coat, googles, and proper attire), and even if there’s only the two of you in the lab, maybe doing something that you think would not be dangerous, please wear them, Winfield would be checking from time to time to check on you, and he would kick you out of the lab if he catches you not wearing them more than once.
Before you go into the lab:
It is useful to know roughly how long would a run need to be for CP in your column and %B. In this case, use the runs we did last semester as an estimate, as well as the existing virtual lab. In case you’re going to use a column that have never been used before, use data from a column that’s probably going to be similar as reference. This is used to determine your max tR. However, do not worry if you can’t bring them since we would be doing a CP and blank only Pre-Batch run for obtaining them.
When you get into the lab (you will need Luci or Laurence to get into the lab, no undergrads have lab access):
Sign in to the logbook. The logbook should be on the table left of the the big screen. This is a safety as well as a legal requirement for undergraduates. So please do check in. If you’re out for lunch, check out, then check back in again. (can we make this electronic?)
Solvent and sample check.
As a rule of thumb , filling up the two litre bottle for both A and B should be sufficient for a batch. However, please do calculate the amount of solvent you actually need from the Pre-Batch run, and check if the solvent is sufficient. IF the amount of solvent needed for the whole batch exceeds 1.5 litre, It’s perhaps safer to break the batch into two sections, and then run them as two separate batches. Solvents would have a batch number and preparation date. Take a note of them.
Samples does need to be checked, and especially phenylbutazone which is known to degrade. Samples should have a preparation date, and a batch number. They are stored in the fridge. The general rule is, take a note of the batch number when you run them, and don’t use samples that are more than a week old, or two days old for phenylbutazone.
Sample Preparation ( forked from Dorothy Ko’s master branch)
Stock Solution 2.5mg/ml
Weigh out 50mg of chemical using the tin foil boats on the analytical balance. Note the amount weighed out in S4 chemical log book Transfer the tin foil boat to a labelled 20 ml standard flask Add solvent ACN:H2O (70:30) but not to the mark Place the flask in the sonicator to make sure all the chemical is dissolved ( uracil and piroxicam may take longer (5-10mins)) Let it cool down and make to the mark Store the flasks in the labelled pyrex dish and place in the fridge Labelling: give the sample a batch number , following this format: (2digits each) initials/date/month/batch number in that day. For example, Odi made a batch on 6th of August, and it’s the first batch he made that day. So the batch number will be OD060801. Take a note of the batch number that you made, and write it in the flask as well as in the google sheets for batches.
Running Solution 0.1mg/ml
Pour a small amount of solution (2-3 ml) into a small beaker Using an auto pipette withdraw 800µL of the stock solution into a labelled 20 ml standard flask. Make to the mark with ACN:H2O (70:30) Any unused solution in beaker should not be returned to the flask Store the flasks in labelled pyrex dish and place in the fridge
Filtering sample into vials
Using the 1ml disposable syringes withdraw the solution into syringe Attach the syringe filter to the syringe Push the solution in syringe through the filter into vial Cap the vial
Solvent Prep: #If using, water, obtain water from milli-Q
Use this step for ACN / Water with 0.1% formic acid. Methanol prep should skip this step.
Use 1L flask to measure 1 L of solvent. Add 1mL of formic acid (from concentrated formic acid flask) with the Eppendorf pipette to the 1 L flask. Mix well (but don’t turn flask upside down). Verify pH (should be 3) with pH paper (located near the sonicator).
For all solvents: Filter using the filtering apparatus. Use the filter in the respective fume hood, do not mix them up. Use the filter paper in the respective fume hood of the solvents, since there is a different one for eac solvent. Set the filter up, verifying that the clamp had been properly installed. The filter should consist of the filter paper (blue) sandwiched between the the funnel and filter section of the filtering apparatus, which is tightened with clamps, and then put on top the flask. Connect the hose to the vacuum pump, then turn the pump on (remember to turn on services on the fume hood first for electricity to work). Pour the solvent to the flask, filter as usual. There is no need to rinse since the filtration apparatus is only used for that solvent. Pour solvent into the bottle , and label with batch number as specified in sample prep.
Pre-Batch run:
Setup a pre-batch run just like the actual batch run, however this run only have blank and CP, as in blank, then CP, then blank again, then CP again. You do not need to do this on each %B ,but perhaps 90%, 70%, 50% and 40%. This is also the time to find if we hit 3500/4000 psi pressure at lower %B. If a pressure limit is hit , then you don’t have to do the lower %Bs, and but add some 5% fraction runs between the other %Bs to compensate.
Best strategy: When working in pairs, one should be doing the software setup while the other doing the solvent, hardware, and sample check (or prep if needed).
It would be best for the pre-batch run to be done while monitored as single runs. For this reason, set apart at least two hours for pre-batch. This would allow you to stop and re-run on the fly in case it did’t go so smoothly.
Use the time during hardware setup and pre batch run to set up the batch run, since there is a lot of data that needs to be entered. To do this, use the Offline editing tool in labsolutions. (LabSolutions > right click on machine icon > offline edit). Here you could create method files without bothering the run. It’s the same as the normal method files.
You don’t have to do the Pre-batch run the same day as the actual run, so you can arrange this with your partner.
After you finish the pre-batch run, do the tR estimation for each %B , the use Google Sheets AWESOME graph (last sem) to estimate the total time needed for the batch run. This would also give you the amount of solvent you need for your run. In case it needs more than 1,5 L, then break up the run to two equal parts, and then inform the group that another run is needed. You would most probably be doing the second part of the run(you can set it up during the post-run analysis of the first one), however the group needs to know so we can manage that.
The total time per batch that you would need to dedicate would be around 3-5 hours per batch , depending on how smoothly the batch runs.
Post-batch run:
The most important thing to check in the post batch is whether it seems that all your peaks have appeared, or had a peak “travelled” to the next run, due to the cut off time being too early. In case this happens, we might need to re-run the batch.
If they look OK, upload the data as you normally would (chromatogram, etc. ), and notify the AWESOME graph managers that you had uploaded them. If not, notify them still (and the group).
Herodion Adiwingyo Hartono