Logbook_Entry-of-the-Day

HPLC (High Performance Liquid Chromatography) - Ion Exchange

Sample Pretreatment

Perform sample concentration of fractions with the highest enzymatic activity via centrifugation with amicon tube.

HPLC

A pH gradient is used to elute the enzymes via cation exchange. Lactate Dehydrogenase (LDH) should elute in the later fractions since it should remain bound until a pH equivalent to its pI is attained.

Only a single peak was obtained and this peak coincided with fractions 1 and 2. Therefore, LDH eluted first.

Problems

1. A pH gradient should not be used.
2. Tris buffer is not suitable.

Rectification

The fractions were concentrated via amicon tubes to salvage LDH in sample. Protocol must be re-planned. Next run will proceed on Wednesday, 25th October.

Conclusion

The fractions with the highest enzyme activity were concentrated after gel filtration. HPLC did not proceed as planned since LDH eluted immediately. The fractions from HPLC had to be concentrated again after being diluted from the run.

Therefore, the sample was concentrated, diluted and re-concentrated.

Nett work done = 0.

Ultimate result:

Purification Experiment FAILED.

--End of Log--

After my lab session, I had to eat. Just eat. Ate steamboat with me, myself and I at SRC. After that, we had NUSIS (NUS Invitational Shoot) meeting.