v******0
发帖数: 265 | 1 大家好，上次我询问过关于HILIC column的一些问题，得到了不少帮助，但最近我又遇
到了一些问题，实在想不通，只好再来请教。
我打算分离鉴定一些phosphopeptide standard， 我先用C18做了LC-MS,大部分
phosphopeptide都可以看到明显的peak. 一些比较Hydrophilic的似乎peak很小，然后
我就用HILIC（Amide 80 HILIC) 去做,目的是检测这些Hydrophilic，浓度我调的和C18
的那个一样，不同的是C18就是water with 0.1% formic acid, HILIC用的是90%ACN，
10%water,0.1%formic acid. 我是先加了非常少的水（4ul)在sample里，然后又加了
36ul ACN。我sample量不多，所以不敢一上来加很多水去溶解。
我的结果就是用HILIC，几乎看不到什么peak, 理论上HILIC应该比C18要更有利于
phosphopeptide separation,特别是Hydrophilic的，但我却只能看到很小的几个peak,
大部分都消失了，而且也... 阅读全帖 |
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d******s
发帖数: 674 | 2 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 t... 阅读全帖 |
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a******5
发帖数: 1 | 3 各位版友好
我想请问有什么类型的HILIC column适合分离sulfamate类的化合物呢?
想分析Acesulfame potassium, saccharin, cyclamate
但已经试过用C18的column RPLC 调过gradient ,ion-pair都分不太开
想要试试看新型的HILIC COLUMN
但是stationary phase琳琅满目不知道哪个效果较好
有看到Luna Diol, ZIC-HILIC, TSK-Amide 80等等...
不知道版友们有什么推荐的HILIC COLUMN可供使用?
感激不尽 |
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v******0
发帖数: 265 | 4 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... 阅读全帖 |
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m**n
发帖数: 206 | 5 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. |
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v******0
发帖数: 265 | 6 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... 阅读全帖 |
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j*******x
发帖数: 22 | 7 知道版内挺多高手, 请教一下有关HILIC的问题.我用 ployhydroxyenthyl A pack 了一
根 75 microI.D.的column， 用UV 检测，现是用mobile phase A: 100% H2O B:100%
ACN 分typtic peptide, 在gradient里一个peak都看不到，又用了 mobile phase A:
100% H2O with 0.1% TFA B:100% ACN with 0.1%TFA.还是在gradient里一个peak都看
不到
后在Mobile pahse 里加了20mM 的盐，现在可以看到两个bubles...., 观测到column德
pressure跳来跳去的厉害，不知道问题在哪？？ （flow rate :250-300 nL/min, ACN
80-30 in 30 mins) |
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c********o
发帖数: 341 | 8 why not try a different column......
so many HILIC stationary phases |
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v******0
发帖数: 265 | 9 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 |
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v******0
发帖数: 265 | 10 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. |
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d******s
发帖数: 674 | 11 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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K*********e
发帖数: 183 | 12 如果你的化合物极性比较大, 选极性中等或教大stationary phase的就应该work. C18
对你的化合物肯定不好用.
HILIC column 其实就是正相色谱. 你的化合物极性都很大, 也许你用unbonded silica
based就可以, 你可以打电话给manufacturer, 他们回给你推荐的. |
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G*******X
发帖数: 1028 | 13 根据四个物质的性质，先选detector
1. Detetor: ELSD就是个joke。直接pass。EDTA(乙二胺四乙酸),三羧
基乙二醇，没有Uv吸收峰，所以要用质谱. Infuse injection of the four compounds
respectively to choose proper m/z for monitoring.
2. 质谱可以handle的flow rate比 diaode array小，所以column size 2.1x10cm
3.四个化合物三个是酸，极性都很强，C18、和C8留不住他们，会在void的时间流出的。
SAX是离子交换柱，一般mobile phase要加较大浓度的盐溶液，不适合质谱检测。
PS-DVB看产品介绍是只是用来将polar化合物从nonpolar化合物中分离出来的，像是纯
化用的。column本身没有官能团，对polar化合物没有分离作用。
SiOH，是normal phase column。是个选择。不过正相柱对水分很敏感，repeatability
有时候不好，不建议用。
Cyano如果是HILIC phas... 阅读全帖 |
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f*******K
发帖数: 34 | 14 Phosphopeptides是亲水性的，tag标记后的Cys peptides是相对疏水性的，可以通过
HILIC相对区分开，降低他们的相互影响。和以前的方法相比，鉴定数目明显胜出。
To dahuzi,我不知道你这个蛋白的相对丰度，不好给具体建议。不过如果你的蛋白质
里面那些包含你感兴趣的PTMs sites的peptides能够通过常规LCMSMS鉴定到,那么我的
方法应该也能。不过对单个蛋白质，你可以考虑做topdown,应该更直接。 |
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p**********m
发帖数: 472 | 15 是像在rp－HPLC一样吗， 跟柱子没啥作用？ |
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e*****r
发帖数: 379 | 16 One poss reason: MeCN too high!
try 90 or 85% MeCN
with some ammonium acetate. |
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E****F
发帖数: 10 | 18 Usually, people don't use 100% solvent as mobile phase, or it might minimize
the efficiency of the column. What we do is preparing 95/5 H2O/ACN with 0.1
% Formic Acid (or whatever you choose such as 0.1% TFA) as solvent A; 90/10
ACN/H2O with 0.1% Formic Acid as solvent B.
If you see some bubbles ocurring, that might be arised from your loading
pressure, flow rate,etc. One solution is to purge the chromatographic system
including loading and waste lines by using 2-propanol, then to increase the
f |
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L****r
发帖数: 333 | 20 DNA亲水性大，楼主使用HILIC柱就可以了。 |
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m**n
发帖数: 206 | 21 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 |
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w******g
发帖数: 2 | 22 90% Acn ia too much , Does 75% work? |
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v******0
发帖数: 265 | 23 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 |
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v******0
发帖数: 265 | 24 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. |
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m**n
发帖数: 206 | 25 did you stack your sample with high organic? Did stacking work? |
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v******0
发帖数: 265 | 26 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 |
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L****r
发帖数: 333 | 27 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. |
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v******0
发帖数: 265 | 28 I have 0.1% FA in the mobile phase, the pH between 3-4
Is that enough?
when
will |
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v******0
发帖数: 265 | 29 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 |
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m**n
发帖数: 206 | 30 try 0.5% FA. If there is no difference between 0.1% and 0.5% FA, then you
know acid is not the key. |
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d******s
发帖数: 674 | 31 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. |
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v******0
发帖数: 265 | 32 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 resu... 阅读全帖 |
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d***e
发帖数: 1215 | 33 assume你用RP-HPLC. 酸性的分子在中性和碱性条件下往往没有retention,上面提到的
那些additive对提高retention都没有太多帮助。可以加tributylamine之类的ion pair
reagent, 如果C18柱上不retain, 可以试一试带极性group的RP柱子,如果还是不行，
可以试HILIC column.
其实很多酸性不稳定的东西，在柱子上走十几分钟是没有问题的， 可以测一下不同PH
的stability. |
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m**n
发帖数: 206 | 34 peptides sure will have different solubility in different solvent. If you
prepare sample in ACN, diluted sample (10time diluted than RPLC) and
increasing injection volume (10times injection of RPLC) will probably make
those peaks visible. |
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h*****t
发帖数: 1226 | 35 氨盐可以帮助分离, 而且也可以帮助提高酸性peptide的recovery
C18
peak, |
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K*********e
发帖数: 183 | 37 All 4 are very polar or called hydrophilic, C18 or C8 are not going to
retain them.
In organic chemistry separation, we can use SiOH (use as normal phase), to
separate them. But in analytical separation,
Using HILIC column. |
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p**********g
发帖数: 378 | 38 Try -CN phase or Cation/anion hybrid C18 columns such as Imtake Sherzo SM
C18... |
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i******g
发帖数: 90 | 39 为什么choline chloride在RP上出一个峰， 而用HILIC出两个峰？ |
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