Hplc

It will be necessary to calculate an approximate lag time between the flow cell and the exit point of tubing where you will be collecting peaks. This will be (60 x (internal volume of the tube coming out of the flow cell + half the volume of the flow cell) / flow rate) seconds (see Subheading 2.4.).

1. Prepare solvents (If helium sparging is used to remove dissolved gases, perform this before addition of trifluoroacetic acid. In practice, helium sparging is not generally necessary.

2. Purge pumps.

3. If sample pH is > 7, acidify sample(s) by addition of glacial acetic acid (0.05 volume). This is necessary to avoid damaging silica-based columns.

4. Equilibrate column in solvent A and run a blank chromatogram. This would also be a useful time to run approximately 10% of the original digest to determine where peaks are likely to be.

5. Load digested proteoglycan into sample loop (100-200 ^L).

6. Inject and start a gradient of 0-100% solvent B in 45-90 min.

7. Collect peaks into Eppendorf tubes by hand. Note that there is likely to be a very large void volume peak. There may occasionally be peptides here, but this is rare. Most peptides will not elute before 8 min.

When collecting peaks, start collecting after the UV absorbence has left the baseline and add the lag time that you have previously calculated. Stop collecting at the point where the UV trace is approximately halfway back to the baseline, plus the lag time. There are fraction collectors that can do some of this process automatically, but it is much more reliable to do it by hand and label the tubes based on the peak elution time (see Fig. 1).

Fig. 1. The use of lag time to ensure that peaks are collected into a single tube. To ensure that peaks from reversed-phase HPLC analyses are collected into single tubes, it is necessary to know the time between a peptide arriving in the flow cell (which translates into an absorbance change) and the peptide exiting the tube downstream from the flow cell. In a typical scenario, the tube after the flow cell will be 20-30 cm long and will have an internal diameter of 0.1 mm, resulting in a volume of 20 ^L. If the flow cell has a volume of 5 ^L, then this means that at 250 ^L / min, the lag time between the peak entering the flow cell and it appearing at the outlet will be 6 s. In practice, the point that is most important is when the apex of the peak is in the flow cell. For this, a volume of half the flow cell + the outlet tube will give a lag of 5.4 sub at 250 ^L/min and 6.75 sub at 200 ^L/ min. It is also possible to determine this empirically by injecting a colored dye into the system. Note that for maximum peptide purity, it is important to collect the peak, but not the leading and trailing edges of the peak.

Fig. 1. The use of lag time to ensure that peaks are collected into a single tube. To ensure that peaks from reversed-phase HPLC analyses are collected into single tubes, it is necessary to know the time between a peptide arriving in the flow cell (which translates into an absorbance change) and the peptide exiting the tube downstream from the flow cell. In a typical scenario, the tube after the flow cell will be 20-30 cm long and will have an internal diameter of 0.1 mm, resulting in a volume of 20 ^L. If the flow cell has a volume of 5 ^L, then this means that at 250 ^L / min, the lag time between the peak entering the flow cell and it appearing at the outlet will be 6 s. In practice, the point that is most important is when the apex of the peak is in the flow cell. For this, a volume of half the flow cell + the outlet tube will give a lag of 5.4 sub at 250 ^L/min and 6.75 sub at 200 ^L/ min. It is also possible to determine this empirically by injecting a colored dye into the system. Note that for maximum peptide purity, it is important to collect the peak, but not the leading and trailing edges of the peak.

8. Tubes can either be dried in a Savant Speedvac or frozen for storage. If peptides are being sent to a core facility, there is no need to ship them cold if they have been dried. Transferring peptides from one container to another may result in complete loss.

At the end of the run, or series of runs, rinse the injection loop with deionized water in both the load and inject positions to remove salt.

The relative size of peaks will be largely related to the amino acids that are present in the peptides. The largest peaks will likely have tryptophan as one of the constituent amino acids. The next largest peaks will likely contain tyrosine. Smaller peaks may be from contaminants or will contain no aromatic amino acids. Peaks that contain single peptides will be symetrical or may tail slightly. If the HPLC separation has been preceded by gel filtration, it is possible to estimate purity by determining how many fractions a given peak is in. Typically, a single peptide should not elute from the size-exclusion volume in a volume greater than 1 ml for a 0.5 ml sample size (two or three 0.5-ml fractions).

An estimate of the likely amount of a peptide can be obtained from the UV trace. With the UV monitor set at 220 nm and using a 2.1-mm-internal-diameter column, a "typical" peak of 15 pmol of a 12-amino acid peptide should have a peak height of 0.01-0.02 AU.

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