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14 July 2010

John Dolans

HPLC Solutions
John Dolan is best known as one of the worlds foremost troubleshooting authorities. Separation Science and John Dolan have collaborated to
offer this weekly digital learning platform providing valuable advice on everyday issues, problems and challenges faced by LC practitioners.
Importantly, you will also have the opportunity to interact with John through our online questions submission system.

Tech Tip
End-capping

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To understand end-capping, we need to step back and look at the bonded
phase on the HPLC column. Reversed-phase HPLC columns usually
comprise a silica particle with a stationary phase, such as a C18
hydrocarbon bonded onto the surface. Click here to read more..
www.sepscience.com

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problems with carryover, ghost peaks or any other
HPLC issues then click here to contact John.

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End-capping
Recently a reader sent an Ask the Doctor email to us asking what was meant when a column is referred to as end-capped, and what
the function of the end-capping was. To understand end-capping, we need to step back and look at the bonded phase on the HPLC column. Reversed-phase HPLC columns usually comprise a silica particle with a stationary phase, such as a C18 hydrocarbon bonded onto
the surface.
Silica is an amorphous polymer of silicon and
oxygen. This polymer terminates at the surface
of the particle as Si-OH groups, commonly
called silanols. These silanol groups serve as
attachment sites for the bonded phase. A silane
reagent, such as Cl(CH3)2SiC18H37 is reacted with
the silanol to form a silyl ether (Si-O-Si-). The
bulk of the C18 group prevents bonding to all
of the exposed silanols. This results in a surface
that looks much like that on the left side of
Figure 1, where there is a fairly high population
of unbounded silanols, often termed residual
silanols. The residual silanols are somewhat
acidic and can be overly reactive with sample
components, especially basic analytes, so it is
preferable to reduce the population of residual
silanols.
To further deactivate the surface of the
particle, a smaller reagent is used in the endcapping reaction. For example, Cl(CH3)2SiCH3

Figure 1

Figure 1: The end-capping process. Left, silica surface following bonding with the primary stationary phase. Right, reduced surface silanol population after
end-capping.

is one common end-capping reagent. You

can see that substituting a methyl group for
the large C18 group used above makes this
reagent much smaller, allowing it to have access
to some of the residual silanol groups on the
surface. After this second reaction is completed,
we say that the column is end-capped (right
side of Figure 1).
It is interesting to note that even when endcapping is done as thoroughly as possible,
such as repeating the reaction (double endcapping), approximately half of the silanols
are still unbonded. You might think that this is
a problem, but on the contrary, I suspect that
if we were to eliminate the silanols completely,
we might be disappointed at the performance
of the column. For example, if silica is replaced
by a polymer, such as in the polymer reversedphase materials, we often find that these
columns lack adequate selectivity to get
satisfactory separations. More important is to
have the silanols well shielded so that their
interactions with the sample and mobile phase
are controlled.
If only the end-capping reagent is used
(no C18), the surface is not very stable, with
rapid loss of end-capping at low pH (e.g.,
pH<2). However, column stability under acidic
conditions increases with the chain length
of the bonded phase, so C8 and C18 phases
are quite stable under conditions that would
destroy an end-capped-only phase. When C8
or C18 phases are end-capped, the bulk phase
serves to protect the end-capping group from
hydrolysis and the end-capping reduces the
reactivity of the unbounded surface a win-win
situation. As a result, most reversed-phase HPLC
columns today are end-capped.
In addition to protecting and deactivating the
silica surface, sometimes end-capping is used

for other purposes. For example, if the endcapping reagent contains >
a polar moiety, such
as a diol function, the reagents imparts some
polar characteristics to the surface. This can be
used to create an AQ or polar-embedded-phase
column that can be used with 100% aqueous
mobile phases to avoid phase dewetting (see
HPLC Solutions, Issue #6 for more on dewetting).

John Dolan is best known as one of

the worlds foremost HPLC
troubleshooting authorities. He is
also known for his ongoing research
with Lloyd Snyder, resulting in more
than 100 technical publications
and three books. Contact John at
TechTips@sepscience.com

LIVE
ON THE
WEB

Featured Course: HPLC Basics, Equipment, and Troubleshooting

September 14-30, 2010
Times are 08:00 - 10:30 (pacific time). Sessions are recorded and available for later viewing.
If you use chromatography on the job and would like a better understanding of how it works, HPLC Basics, Equipment, and
Troubleshooting is the course for you. Its designed for chemists and biochemists who use HPLC as a regular part of their
jobs, but technicians with some HPLC experience will also find the course valuable. No previous HPLC training is assumed;
however, much of the course will appeal to the experienced chemists who want a firm grounding in the basics of HPLC.

What does it cover?

You will acquire a solid understanding of the fundamentals of chromatography and learn simple tips and guidelines to make
your chromatography work easier and more efficient. What you learn will demystify your instrument. Youll find that all of the
perplexing and frustrating problems your experience have simple and logical solutions. And, better yet, youll learn that most
of these problems are preventable. If youve never taken a formal course or if you need a refresher, its time to learn HPLC the
right way. Students tell us that this extremely practical course is a must for everyone who uses HPLC.

http://www.lcresources.com/training/trfund.html

Next weeks

John Dolan, Tom Jupille and their team

at LC Resources are available to answer
your specific method development and
troubleshooting HPLC questions.
Submitted Q & As will also form the
basis of future Tech Tips.
NOTE! Help! I need a method to separate ___
Unfortunately, this is a question that John cant
help you with. However, here are a few hints: (1)
do a literature search using Pub Med or one of
the free search engines; (2) a good source of
methods are Journal of Chromatography A and
B issues; (3) consult the applications literature of
various manufacturers an extensive database
of searchable methods can be found at
www.sepscience.com; (4) visit Chrom Forum at
www.chromforum.org

Click here to submit your question

Tech Tip

Recommend a Colleague

Next week, John Dolan returns to his Back to

Basics series with a look at peak tailing.

If you have a work colleague, collaborator or staff

member who would benefit from this weekly
publication then send us their details below.

Dont miss the next instalment of John Dolans HPLC

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Ask the Doctor

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