v******0
发帖数: 265 | 1 我最近在使用HILIC Column，是bare silica的那种。因为在测试摸条件阶段，所以就
拿一些tryptic digested peptide做sample,但我分出的峰基本上都很难看，很宽，而
且拖尾严重，重复性也不好，有时同一个sample,两天后用同一个column,本来peak
shape还凑或的峰变得又不好了。
我用的是HILIC-MS,mobile phase也试过加ammonium formate, pH基本在3-4，gradient
也试了不少，sample浓度从100fmol injection到10pmol injection都试过，sample一
般都resuspend在90% ACN中 （先用水溶解，再稀释到90% ACN),可结果大多不理想。不
过同样的system, 换成C18 column就非常好。
我在网上看到的一些example似乎都还不错，但大多数用的是UV detector,也有mass
spec的，不过column基本都是Amide 80或者zic-HILIC之类的，好像是有modified,不是
bared silica.
目前手头只有这个column,想请教专家们，我的问题主要可能在哪里？
1）新手，经验不足，条件没有优化好? 如果是，请问有什么"trick"
2）column本身的问题? | m**n
发帖数: 206 | 2 加酸在溶剂中试试，应该可以减少silanol的吸附，比如0.1% tfa or 0.5%formic acid
gradient
【在 v******0 的大作中提到】
: 我最近在使用HILIC Column，是bare silica的那种。因为在测试摸条件阶段，所以就
: 拿一些tryptic digested peptide做sample,但我分出的峰基本上都很难看，很宽，而
: 且拖尾严重，重复性也不好，有时同一个sample,两天后用同一个column,本来peak
: shape还凑或的峰变得又不好了。
: 我用的是HILIC-MS,mobile phase也试过加ammonium formate, pH基本在3-4，gradient
: 也试了不少，sample浓度从100fmol injection到10pmol injection都试过，sample一
: 般都resuspend在90% ACN中 （先用水溶解，再稀释到90% ACN),可结果大多不理想。不
: 过同样的system, 换成C18 column就非常好。
: 我在网上看到的一些example似乎都还不错，但大多数用的是UV detector,也有mass
: spec的，不过column基本都是Amide 80或者zic-HILIC之类的，好像是有modified,不是
| w******g
发帖数: 2 | 3 90% Acn ia too much , Does 75% work? | v******0
发帖数: 265 | 4 I didn't try TFA since it's not good for MS signal, but I did try higher
percentage of formic acid, it seems not helping a lot.
acid
【在 m**n 的大作中提到】
: 加酸在溶剂中试试，应该可以减少silanol的吸附，比如0.1% tfa or 0.5%formic acid
:
: gradient
| v******0
发帖数: 265 | 5 I read many materials, they all say you have to resuspend the sample in high
organic solvent, so most of them use 90%, but I did try one of my peptide
mixture sample in 75%, the peak shape is not improved either.
【在 w******g 的大作中提到】
: 90% Acn ia too much , Does 75% work?
| m**n
发帖数: 206 | 6 did you stack your sample with high organic? Did stacking work? | v******0
发帖数: 265 | 7 So you mean start from high organic and let it run some time before the
gradient applies? If yes, I do have a 5 min 90% ACN in the begining
【在 m**n 的大作中提到】
: did you stack your sample with high organic? Did stacking work?
| m**n
发帖数: 206 | 8 I suggest using acid in the mobile phase. I saw noticeable improvements when
i used acidic mobile phase for amide HILIC column. Not sure whether it will
do the same thing to bare silica. But it is normal that HILIC is less
efficient than RPLC. |
| L****r
发帖数: 333 | 9 1. If you analyze peptides, you can add 0.1-1% acids like acetic acid.
2. Thoroughly equilibration, for example, the start condition: 100% ACN or
95% ACN, you should let it equilibrate for a while. | v******0
发帖数: 265 | 10 I have 0.1% FA in the mobile phase, the pH between 3-4
Is that enough?
when
will
【在 m**n 的大作中提到】
: I suggest using acid in the mobile phase. I saw noticeable improvements when
: i used acidic mobile phase for amide HILIC column. Not sure whether it will
: do the same thing to bare silica. But it is normal that HILIC is less
: efficient than RPLC.
| | | v******0
发帖数: 265 | 11 I have 0.1% FA inside
So how long the equilibration time should be, I usually let it run 5 min at
250min/ul, I can try to increase to see whether it got improved
【在 L****r 的大作中提到】
: 1. If you analyze peptides, you can add 0.1-1% acids like acetic acid.
: 2. Thoroughly equilibration, for example, the start condition: 100% ACN or
: 95% ACN, you should let it equilibrate for a while.
| m**n
发帖数: 206 | 12 try 0.5% FA. If there is no difference between 0.1% and 0.5% FA, then you
know acid is not the key.
【在 v******0 的大作中提到】
: I have 0.1% FA inside
: So how long the equilibration time should be, I usually let it run 5 min at
: 250min/ul, I can try to increase to see whether it got improved
| c********o
发帖数: 341 | 13 why not try a different column......
so many HILIC stationary phases | d******s
发帖数: 674 | 14 First of all, wrong approach: bare silica column in HILIC mode uses silanol
group for retention, which is also the main cause of peak tailing. Adding
TFA which is the common practise in RPC to suppress silanol interference
will not work here.
Why use this column in HILIC mode? Please don't say that you only have this
column.
Reverse phase for peptide/protein digest is the golden standard, and you
will have much better luck to start here. Or, if you are trying to do something
different, you can try ion exchange (either anion or cation exchange), much easier
to work with than HILIC.
Or if you have to use HILIC becaues neither of the above works, try use
other columns such as the ones you mentioned. Those columns have stationary
phase built on silica beads but have silanol groups protected/endcapped so
interference is minimized, and/or you can also add up to 0.05% TFA to
improve peak shape.
Good luck.
gradient
【在 v******0 的大作中提到】
: 我最近在使用HILIC Column，是bare silica的那种。因为在测试摸条件阶段，所以就
: 拿一些tryptic digested peptide做sample,但我分出的峰基本上都很难看，很宽，而
: 且拖尾严重，重复性也不好，有时同一个sample,两天后用同一个column,本来peak
: shape还凑或的峰变得又不好了。
: 我用的是HILIC-MS,mobile phase也试过加ammonium formate, pH基本在3-4，gradient
: 也试了不少，sample浓度从100fmol injection到10pmol injection都试过，sample一
: 般都resuspend在90% ACN中 （先用水溶解，再稀释到90% ACN),可结果大多不理想。不
: 过同样的system, 换成C18 column就非常好。
: 我在网上看到的一些example似乎都还不错，但大多数用的是UV detector,也有mass
: spec的，不过column基本都是Amide 80或者zic-HILIC之类的，好像是有modified,不是
| v******0
发帖数: 265 | 15 Thanks a lot.
Currently, I am only having this bare silica HILIC column, and the reason I
am still doing that is because I need test whether they are working properly
. My purpose is not to find a way separating some peptides, since I already
got good one with C18 column.
I might get some Amide 80 one, but it will take a while. I have little
experience on HILIC, so based on your opinion, I won't have good results (I
mean nice peak) using bare silica, no matter how I optimize the condition,
right?
silanol
this
something
much easier
【在 d******s 的大作中提到】
: First of all, wrong approach: bare silica column in HILIC mode uses silanol
: group for retention, which is also the main cause of peak tailing. Adding
: TFA which is the common practise in RPC to suppress silanol interference
: will not work here.
: Why use this column in HILIC mode? Please don't say that you only have this
: column.
: Reverse phase for peptide/protein digest is the golden standard, and you
: will have much better luck to start here. Or, if you are trying to do something
: different, you can try ion exchange (either anion or cation exchange), much easier
: to work with than HILIC.
| v******0
发帖数: 265 | 16 If I am not using peptide standards, do you know whether there are some
other standards could give nice peak in bare Silica HILIC using nanoflow?
silanol
this
something
much easier
【在 d******s 的大作中提到】
: First of all, wrong approach: bare silica column in HILIC mode uses silanol
: group for retention, which is also the main cause of peak tailing. Adding
: TFA which is the common practise in RPC to suppress silanol interference
: will not work here.
: Why use this column in HILIC mode? Please don't say that you only have this
: column.
: Reverse phase for peptide/protein digest is the golden standard, and you
: will have much better luck to start here. Or, if you are trying to do something
: different, you can try ion exchange (either anion or cation exchange), much easier
: to work with than HILIC.
| d******s
发帖数: 674 | 17 I know you can achieve good peak shapes for small molecules on bare silica,
e.g. nicotine. But won't be appropriate using nanoflow, there is nothing
wrong with nanoflow for small molecules just the throughput will be too low.
【在 v******0 的大作中提到】
: If I am not using peptide standards, do you know whether there are some
: other standards could give nice peak in bare Silica HILIC using nanoflow?
:
: silanol
: this
: something
: much easier
| v******0
发帖数: 265 | 18 Thanks again. I am still a little confused, the low throughput means ...?
Why the low throughput can affect my purpose? (using nanoflow to achieve
nice peak on HILIC)
,
low.
【在 d******s 的大作中提到】
: I know you can achieve good peak shapes for small molecules on bare silica,
: e.g. nicotine. But won't be appropriate using nanoflow, there is nothing
: wrong with nanoflow for small molecules just the throughput will be too low.
| d******s
发帖数: 674 | 19 low throughput means it takes longer (than desirable) to complete the
analysis
If you just trying to demonstrate that you can achieve nice peak shape on
HILIC, you can do whatever you want
【在 v******0 的大作中提到】
: Thanks again. I am still a little confused, the low throughput means ...?
: Why the low throughput can affect my purpose? (using nanoflow to achieve
: nice peak on HILIC)
:
: ,
: low.
| v******0
发帖数: 265 | 20 Ok, but the peaks I got from bare silica on small molecules are also ugly.
But the molecules I tested until now are something people use 80%ACN as
isocratic to elute, such as uracil, cytosine, etc.
Is that because these are not retained well with my column, so I don't have
good peaks. If I goes for some other more hydrophilic small molecules, could
it be better?
The other question is since Silanol makes the peptide peak tailing, if I use
UV as detector, which doesn't require acid, could the result be better at
higher pH? No positive charge interactiing with silanol group. Not sure
whether I am right or not
【在 d******s 的大作中提到】
: low throughput means it takes longer (than desirable) to complete the
: analysis
: If you just trying to demonstrate that you can achieve nice peak shape on
: HILIC, you can do whatever you want
|
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