Fittings for FPLC:

How to run Pharmacia Columns on a Bio-Rad Chromatography System.

5/19/06 , A. Merz

THREAD STANDARDS
There are four incompatible thread standards that we commonly encounter:
M6 — metric threading used on many Pharmacia/Amersham/GE Healthcare fittings. The female fitting is
flat-bottom. The male fitting may be flanged or unflanged and may use a 1-piece or 2-piece ferrule/nut.
1/4–28 — SAE threading used on the Bio-Rad and many other systems. We use only flat-bottom.
NOTE that 1/4–28 and M6 fittings are about the same size and look REALLY similar, but the threads are
NOT compatible – misthreading can destroy a fitting or an entire valve port, destroying a $1000 valve. BE
CAREFUL. The M6 fittings are slightly smaller than the 1/4–28 fittings.
10–32 Fingertight — a very common conical-end SAE fitting used on many HPLC’s and on newer
Pharmacia (Tricorn, HiTrap, etc.) columns. Pharmacia calls this a 1/16” fitting. Pharmacia is the only
company that calls it this.
Luer — The fitting used on many syringes and on all BioLogic LP fittings.

CONNECTORS
The Bio-Rad system uses the Upchurch Scientific Super Flangeless fittings. To the extent possible, we will
standardize on this method of tube termination for both 1/4–28 and M6 fittings. Where possible, fittings,
adaptors, ports, junctions, etc. used downstream of the pump should be 0.020” inside diameter.

TUBING
For our medium and high-pressure applications tubing with two different outside diameters (OD) is used.
1/8” (0.125”) OD tubing, usually teflon, is used upstream of the pump. 1/16” (0.0625”) OD tubing,
generally PEEK but sometimes teflon, is used downstream of the pump. These are rigid/semirigid tubing.
Flexible tubing will not form a good seal with the connectors and should be avoided in medium and high
pressure applications. For most purposes this is 1/16” OD, 0.020” inside diameter (ID). For flow rates in
excess of 10 ml/min., 0.040” ID tubing is generally used. The pumps in our Bio-Rad unit have maximum
flow rates of 10 ml/min. so this should not be a concern.

SPECIFIC APPLICATIONS
Older Pharmacia columns typically have 1/16” tubing sticking out on both ends, terminated by a male M6
fitting. There are two ways to deal with this.
(1) Preferred alternative. Cut the 1/16” tubing on the column ends as close to the end as possible and
replace the M6 connectors with male 1/4–28 Super Flangeless connectors.
(2) Easy alternative. An M6 to 1/4–28 adaptor (Upchurch #P-621, looks like a yellow Lego brick with a
threaded hole bored down the long axis) can be used to connect a male 1/4–28 connector to a male M6
connector. Unfortunately, I have found only one adaptor that allows one to convert a male M6 connector
to a male 1/4–28 connector, and it is expensive (Jouen JR-0622, >$30).
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column inlet port, and an adaptor that converts the column’s male 10-32 outlet fitting to a female 1/4–28
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Pharmacia Superloops have two female M6 ports. To connect these columns to the Bio-Rad systems, make
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a 1/16” linker tube terminated on both ends with Super Flangeless fittings. On one end use a male 1/4–28
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nut, and on the other end use a male M6 nut (Upchurch #P-219).
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Pharmacia and Bio-Rad typically have a 100% markup over and above other vendors’ retail prices: probably IÊ Ê*UÈșUä£ÊˆÃÊ>Ê`ˆÀiVÌÊÀi«>Vi“i˜ÌÊvœÀʜÕÀÊ*UÈ{äU䣰Ê/…iʅ>À`Ê* ÊLœ`ÞʜvÊ*UÈșUä£Ê«ÀœÛˆ`iÃÊ>ÊLiÌÌiÀʓ>̈˜}Ê«œÀÌÊvœÀÊ>Ê
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130% margins. Get Upchurch, Jouen, or Valco stuff from Western Analytical – much, much less expensive.
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