Please advise if the following statements are correct:

1. In running reversed-phase chromatography (HPLC), (using C18 columns), we have to make the pH of the mobile phase to be lower than the pKa of the analyte in order to suppress ionization of the compound -- so that the compound can retain in the column and thus we could get a good peak shape. Also, we have use a buffer that has a pKa close to the mobile phase pH, is that right? But how do we know what is the concentration of the buffer? (2mM or 10mM). How do we determine the optimal buffer concentration as I read from the book that a mobile phase with margial buffer capacity will affect the peak shape.

2. However, is it that, if we are using a LC/MS, then the mobile phase pH should be above the pKa of the analyte because we want the analyte to be ionized in the source? (It is because I am trying to separate an analyte with pKa around 8.0 and the internal standard with pKa 6.6, I am using a mobile phase of 97% acetonitrible, with 2mM ammonium bicarbonate, pH 8.75 (for isocratic separation)). I was previously using ammonium acetate as the buffer and pH around 8.0 (unadjusted), but there is a peak tailing problem. Changing the pH and buffer help the situation a bit, but not much. I just do not know how to determine the optimum pH in the mobile phase relative to the analytes' pKa (or pKb) either in LC/MS or HPLC.

3. During sample preparation (for reversed-phase), is it that we want the sample to be non-ionized as well (using solvents below the pKa of the analytes) in order to extract the analyte out?

4. How would this change if we are using ion-pair or normal phase chromatography? (relationship of pKa versus mobile phase pH and sample preparation procedure).

5. Is there a standard procedure (or theory) for liquid-liquid or solid-phase extraction?

Thank you for your help.

April,

I'll try to comment point-by-point to your message...I'm sorry this is lengthy...

1. In running reverse phase HPLC on C18 columns, you are correct in that you generally want to suppress the ionization of your analyte(s) in order to get good peak shape and retention. You want to choose your buffering system based on the pKa of the species. For example, phoshphate has pKa's at 2.1, 7.2 and 12.3. You want to operate in a range of +/- 1 pH unit from the pKa. So if you decide you need a mobile phase pH of 4.5, phosphate is not a good choice (outside the window for buffering capacity). Use acetate instead (pKa of acetate is 4.8). Buffer concentration is also important in that if your buffer is too dilute, it may not have sufficient buffering capacity to actually act as a buffer. Unless I am running LC/MS, I prefer to operate in the 10 - 50 mM range. Less than 10 mM can be problematic depending on your analyte, and >50-100 mM has the potential to precipitate depending on your organic component.

2. If you are running LC/MS electrospray, you do need to form your ions in solution. You can do this by buffering you mobile phase pH very high (assuming your column is stable at these pHs and you can get good chromatography) or, you may want to set up a post-column reagent addition. The post-column addition can be as simple as a second pump, a tee and a couple of check valves. You can then add acid/base as you need to maximize your response. Remember, though, to keep your system as simple as possible (less problems down the road).

3, 5. Depends on your sample preparation techniques and what you are trying to accomplish. This is a tough question to answer without knowing specifically what you are trying to do.

Hope this helps. Good luck.

You got some good answers from Joe, but let me add a few comments.
With today's columns, I am not as worried any more about the ionization of the sample as with older columns. You can get very good results even if the sample is completely or partially ionized. If you are in a state with only partial ionization, you need good pH control to control retention.
The MS ionization can be interesting. We found good ionization under conditions where the analyte should not have been ionized at all. My MS colleagues shrugged their shoulders and mumbled something about "gas-phase ionization is not the same as liquid-phase ionization".
During sample prep, you can use the ionization of the analyte or the matrix to manipulate sample clean-up. If you are interested in this I can tell you more, but I would need to understand what the sample matrix is and what you want to clean.

FIRST PART :

I'd like to add the following items
You can divide the problem from a chemical point of view, in ACID, BASIC AND NEUTRAL compounds. The general following rules can be useful:
A. Reverse phase are "organic" so that "neutral organic compounds" have more affinity for RP than polar compounds. The lesser polar the more retention in RP (like C18).
B. Buffers in mobile phases (MP) are highly polar so that polar compounds prefer MP. An ionized form of a given compound is more polar than the non-ionized one so that the last one wil be more retained that the first one (rule A)
C. An ACID has its ionized species at pH > pKa in other words, if the buffer pH < pKa then the acid will be more retained.
D. A BASE is ionized (protonated) if buffer pH < pKa. That means a basic compound will be more retained in RP as pH increase above pKb. But for most aliphatic amines pKa = 8-10 then for regular buffer in RP-HPLC (pH < 8) most of them will be ionized and therefore are not easily retained by RP (C18).
E. The more polar is a functional group the more is the affinity for the mobile phase, the lesser is the retention.
F. The longer and linear is a H-C chain, the more is the affinity for the stationary reverse-phase, the higher is the retention in the RP column.
G. Check the ratio of polar groups to hydrocarbon chains

SECOND PART:
For instance, let's take a look to the catecholamines norepinephrine (NE), epinephrine (EP) and dopamine (DA). (OH)2-Ar-CHR-CH2-NHR'


2 x -OH (catechol) All of them
1 x aliph. N group All of them
CH3 bonded to N (R') EP
1 x -OH aliphatic (R) EP + NE

¿Can you suggest a mobile phase? ¿elution order in RP?

NE y EP has 1 additional OH (polar group) ==> DA more retained than NE and EP
EP has 1 CH3 bonded to the N (secondary amine) more C atoms ==> less polar compound ==> more retained than NE
Order NE, EP, DA. Mobile Phase 2 or 3 OH + protonated amino is VERY POLAR ==> NE and EP are practically unretained with buffer pH 2.5 - 7 with 2 - 5% Methanol or ACN.
How can we do?
In order to obtain a less polar compound we can add an ion-pairing agent which has two main requeriments and two mains efects:
Requeriments:
1. A counter ion group (for amines is useful suphonic acids)
2. An important non polar group (aliphatic alquil mostly)
Effects
1. To build a ion-pair, which neutralized the ionic group wraping it in a non-ionic shell made by the C carbons of the agent
2. To cover the column RP surface with the counter-ion group of the agent enhancing retention by ionic-exchange mechanisms
As a result, you can increase retention for ionic compouds.
A good basic book of organic chemistry could be also very useful.
Hope this helps and sorry for my English and the lenght of this message.

Regards.

How to decide weighing factor during interpreting the goodnes of fitting of calibraton curve ?
It will be appriciated if some body given web link for down load the theory behind of the above question .

SIVA PRASAD
RESEARCH ASSOICATE

How to decide weighing factor during interpreting the goodnes of fitting of calibraton curve ?
It will be appriciated if some body given web link for down load the theory behind of the above question .

SIVA PRASAD
RESEARCH ASSOICATE