c******m
发帖数: 884 | 1 Hi, everyone,
I have a question about HPLC separation. Hope some experts can help me.
Thanks.
Why is there a shoulder on the peak? The compound is pure through NMR. It is
acidic compound, and I diluted it to 50 uM before the injection. It is an
analytical column (C18, 250 x 4.6 mm). Flow rate is 0.8 mL/min. I tried
different solvent systems such as MeOH, water, ACN and solvent with 0.1% TFA
, as well as different gradient programs, but the shoulder keeps coming out.
What is the problem?
I greatl | h*****t
发帖数: 1226 | 2 What's your NMR detection limit?
I would say, it is not a HPLC problem, it is something in your sample.
and please make sure you did not overload the column.
is
TFA
out.
【在 c******m 的大作中提到】
: Hi, everyone,
: I have a question about HPLC separation. Hope some experts can help me.
: Thanks.
: Why is there a shoulder on the peak? The compound is pure through NMR. It is
: acidic compound, and I diluted it to 50 uM before the injection. It is an
: analytical column (C18, 250 x 4.6 mm). Flow rate is 0.8 mL/min. I tried
: different solvent systems such as MeOH, water, ACN and solvent with 0.1% TFA
: , as well as different gradient programs, but the shoulder keeps coming out.
: What is the problem?
: I greatl
| c******m
发帖数: 884 | 3 Thank you very much. I will recheck the purity of the sample.
I think the intensity of the peak with shoulder is much lower than the limit
, because I saw much larger peak intensity with other samples. I injected
20uL of 50uM sample each time (the loop is 20uL), could it cause overload? | c******m
发帖数: 884 | 4 hubtest, sorry I misunderstood u.
What did u mean of NMR detection limit? If u meant the chemical shift range,
I used -1 to 10. | b**u
发帖数: 2761 | 5 what i will do is to inject 5 or 10 micron liter instead of 20. i agree with
hubtest, the sample might be overloading the column. | t**n
发帖数: 4365 | 6 i don't think injection volume maters in this case ... how acidic is your
compound? and how big is the shoulder? | v*******y
发帖数: 1362 | 7 你跑其他的sample有shoulder吗？有可能是pre-column堵住了
is
TFA
out.
【在 c******m 的大作中提到】
: Hi, everyone,
: I have a question about HPLC separation. Hope some experts can help me.
: Thanks.
: Why is there a shoulder on the peak? The compound is pure through NMR. It is
: acidic compound, and I diluted it to 50 uM before the injection. It is an
: analytical column (C18, 250 x 4.6 mm). Flow rate is 0.8 mL/min. I tried
: different solvent systems such as MeOH, water, ACN and solvent with 0.1% TFA
: , as well as different gradient programs, but the shoulder keeps coming out.
: What is the problem?
: I greatl
| d**x
发帖数: 114 | 8 What is the pKa of your compound? It might be too acidic that both ionic
and nonionic forms exist in the mobile phase.
You can also change to a better column. I had shoulders using the old nova
pack column but after I switched to Waters new symmetry column, the peaks
just look perfect under the same mobile phase condition. | l*****a
发帖数: 375 | | c********m
发帖数: 16 | 10 Inject a standard pure sample to make sure your system/column is ok.
Generally, you will see the same shoulder for every peak in chromatography
if it's system problem, like column viod, clogged guard column or frit. You
can also collect the main peak and reinject it to see if you still see the
same shoulder.
If the system is fine, it might be due to the secondary interaction between
your acidic compound and silica stationary phase. Try to increase the salt
concentration and lower the pH. | c******m
发帖数: 884 | 11 Thank you everybody.
The pKa of my sample is aroud 8-9, the shoulder peak area is about 30% of
the major peak. It is possible there is an isomer.
Other people are using the HPLC with another column now. I will figure out
it later.
Thanks again. | W*******e
发帖数: 73 | 12 高人
You
between
【在 c********m 的大作中提到】
: Inject a standard pure sample to make sure your system/column is ok.
: Generally, you will see the same shoulder for every peak in chromatography
: if it's system problem, like column viod, clogged guard column or frit. You
: can also collect the main peak and reinject it to see if you still see the
: same shoulder.
: If the system is fine, it might be due to the secondary interaction between
: your acidic compound and silica stationary phase. Try to increase the salt
: concentration and lower the pH.
| w*********l
发帖数: 5144 | | z****l
发帖数: 97 | 14 well, if there is a shoulder with 0.1% TFA (pH ~2), I don't think that's a
pKa issue since the pKa is 8-9. |
|
