Agilent Technologies GPC/SEC Bedienungsanleitung

GPC/SEC

Column User Guide

カラムユーザーガイド

色谱柱用户指南

Manual do usuário

de colunas

Installation

Tubing and connectors

Stainless steel tubing of 1/16 in od and

0.12 mm id or 0.17 mm id is recommended

for column connections of analytical columns,

and 0.5 mm id for 25 mm preparative columns.

Connecting tubing lengths between columns,

detectors and injection volumes should be

minimized to avoid excessive dead volume

which will diminish system performance.

Column connections should be made using

Parker compatible 1/16 in nuts and ferrules. The

compatibility of column connectors is illustrated

in Figure 1.

Figure 1. Compatible connectors

The distance “X” for the standard column end

tting is 0.090 in and a minimum male nut

length of 0.210 in is required. Some ttings from

other manufacturers may not be compatible, for

example Waters and Rheodyne. If unsure, please

contact Agilent Technologies.

Column connection

Connect the GPC column in the eluent ow

direction indicated and tighten the 1/16 in nut

and ferrule using wrenches on the 1/16 in nut

and the actual end tting.

It is recommended that several drops of eluent

have been pumped before the column outlet

is connected to another column or detector to

clean out the end tting of any particulate matter

which may be present.

To avoid loosening the end ttings and causing

leaks, wrenches must be used on the end tting

adjacent to the connecting nut and NOT on the

column barrel or the opposite end tting,

see Figure 2.

Figure 2. Do not use wrenches on the ats

English

Eluent Flow Rate

For conventional GPC columns using 7.5 and 7.8

mm id columns, 1.0 mL/min is an optimum ow

rate for most separations. When column id is

increased or decreased, the volumetric ow rate

should be adjusted accordingly in order to give

an equivalent linear velocity through the column.

The recommended ow rates are given in

Table 1, however, in order to avoid excessively

high pressures, higher viscosity eluents should

be used at reduced ow rates or elevated

temperature. Flow rates should be changed

progressively and pressure pulses limited. At no

time should the maximum operating pressure of

the column be exceeded (see Table 4).

Table 1. Recommended ow rate

Column Typical ow rates

(mL/min)

Recommended ow

rate (mL/min)

2.1 mm id 0.04 – 0.1 0.06

4.6 mm id 0.2 – 0.5 0.3

7.5 & 7.8

mm id

0.5 – 1.5 1.0

10 mm id 2.0 – 5.0 2.0

25 mm id 5.0 – 20.0 10.0

Sample Preparation and Injection

If maximum resolution and expected column

lifetime are to be achieved, care must be taken in

sample preparation.

To avoid blockage of the column frits, sample

ltration is recommended (0.5 – 2.0 μm porosity

depending on MW). A guard column will further

protect the columns with little detrimental effect

on performance.

Optimum sample volumes and concentrations

are best determined for each type of analysis

and are dependent on sample MW. Broad

distribution polymer can generally be injected at

higher concentrations than lower polydispersity

samples. Overloading will not damage the

column, but distorted peaks and therefore

spurious results will be obtained.

Excessive injector loop volume can contribute

to band broadening and reduce system

performance, particularly with high efciency or

narrow bore columns. Agilent’s injection volume

recommendations are shown in Table 2.

Table 2. Injection volume recommendations

Column Recommended

concentration (%)

Recommended

injection (µL) per

column

2.1 mm id 0.01 – 0.2 0.2 – 2

4.6 mm id 0.01 – 0.2 1 – 20*

7.5 & 7.8 mm id 0.05 – 0.5 20 – 50**

10 mm id 0.05 – 0.5 20

25 mm id 0.5 – 5.0 500 – 2,000

* PL Multisolvent, MesoPore and ResiPore are 1 – 10 µL.

** PL Multisolvent, MesoPore and ResiPore are 10 – 20 µL,

PL Rapide/PL Rapide Aqua are 20 µL

English

Eluents

All eluents should be of high purity and should be

ltered and degassed prior to use.

GPC columns can be transferred to other

eluents. When transferring to another eluent,

miscibility and viscosity of the new eluent are of

primary consideration. See Figure 3 for 7.5 mm

id columns. For other id columns, apply the ow

rate shown in Table 1.

When heating or cooling columns in high

viscosity eluents (for example, TCB, NMP,

DMF) a low solvent ow rate must always be

maintained. Typically, 0.2 mL/min for 7.5 mm id

and 0.1 mL/min for 4.6 mm id should be used

prior to raising the temperature, see Figure 3.

Organic

Organic GPC columns are compatible with

an extensive range of organic solvents

ranging in polarity from peruoroalkanes to

dimethylformamide. Mixed organic solvent

systems can also be used, but water should not

be used except at concentrations less than 10%

by volume in a miscible organic eluent. Columns

are normally supplied in ethylbenzene unless

otherwise stated, and can be ushed directly

from ethylbenzene to THF at 0.5 mL/min.

Unstabilized THF (for example, HPLC grade) is

not recommended as an eluent due to the attack

of peroxide on the gels.

Aqueous and polar

Aqueous/polar GPC columns are normally

supplied in water containing 0.02% sodium azide.

Buffered eluent systems within the pH range

2 – 10 (of high and low ionic strength) may be

used, with no detrimental effect on the column.

When transferring from one eluent to another

the compatibility and solubility of any salts or

additives must be checked to prevent on-column

precipitation which would irreversibly damage

the column. PolarGel columns are compatible

with an extensive range of organic solvents and

with aqueous based eluents. Mixed organic

solvent systems can be used, assuming full

miscibility of the components. The only organic

solvent which is recommended for use with the

PL aquagel-OH and PL Rapide Aqua columns is

methanol (up to 50% by volume).

PL Multisolvent

PL Multisolvent columns are supplied in

stabilized THF, and may be transferred directly

to organic solvents, or to pure water and then

to buffer. Unstabilized THF (for example, HPLC

grade) is not recommended as an eluent due

to the attack of peroxide on the gels. Buffered

eluents within the pH range 2 – 8.5 (of high

and low ionic strength) may be used. When

transferring from one eluent to another the

compatibility and solubility of any salts or

additives must be checked to prevent on-column

precipitation which would irreversibly damage

the column. Polar organic solvents, such as

DMF, DMSO, or DMAc are not recommended as

eluents.

English

Organic columns (PLgel; PlusPore; PL Rapide)

Column supplied in ethylbenzene

Transfer to

low viscosity

solvents e.g.

THF, Chloroform,

Dichloromethane

Transfer

to medium

viscosity

solvents e.g.

Toluene, DMF,

DMSO, HFIP

Transfer to

high viscosity

solvents e.g.

TCB, m-Cresol,

NMP

Flush column

with acetone at

0.5 mL/min for 2

column volumes

Flush column

with acetone at

0.5 mL/min for 2

column volumes

Set column oven

to 50 °C, ow at

0.1 mL/min

Flush with new

eluent at

0.5 mL/min for 2

column volumes

Flush with new

eluent at

0.2 mL/min for 2

column volumes

Flush column

direct with new

eluent** at 50 °C

at 0.1 mL/min for

2 column volumes

Increase column

temperature to

30 - 40 °C* as

required for

analysis at

1 °C/min

Increase column

temperature to

50 - 80 °C* as

required for

analysis at

1 °C/min

Increase column

temperature to

100 - 220 °C*

as required for

analysis at

1 °C/min

Operate column

in new eluent at

required ow rate

Operate column

in new eluent at

required ow rate

Operate column

in new eluent at

required ow rate

*Always ensure operating temperature is at least 10 °C below

boiling point of solvent. **Always ensure miscibility. If unsure,

use acetone at room temperature.

Figure 3. Eluent transfer guide

English

Aqueous & Polar columns

(PolarGel, PL aquagel-OH, PL Rapide Aqua)

Column supplied in water containing 0.02% NaN3

Transfer to

aqueous e.g.

Water, Buffer

Transfer to polar

organic** e.g.

DMSO, DMF

Transfer to

mixed solvent

systems e.g.

Water/THF*,

Water/methanol*

Flush column with

pure water at

1.0 mL/min for 2

column volumes

Flush column

with pure water at

1.0 mL/min for 2

column volumes

Flush column

with pure water at

1.0 mL/min for 2

column volumes

Flush with new

buffer at

1.0 mL/min for 2

column volumes

Flush with

acetone at 0.5

mL/min for 2

column volumes

Flush with new,

premixed

eluent at

0.2 mL/min for 2

column volumes

Increase column

temperature*

as required for

analysis at

1 °C/min

Flush with new

eluent at

0.2 mL/min for 2

column volumes

Increase column

temperature*

as required for

analysis at

1 °C/min

Operate column

in new eluent at

required ow rate

Increase column

temperature to

50 - 80 °C* as

required for

analysis at

1 °C/min

Operate column

in new eluent at

required ow rate

Operate column

in new eluent at

required ow rate

*Always ensure operating temperature is at least 10 °C below

boiling point of solvent. ** Polargel only

Multisolvent columns (PL Multisolvent)

Column supplied in THF containing 0.015% BHT

Transfer to aqueous e.g.

Water, Buffer

Transfer to organic e.g.

Chloroform*

Dichloromethane**

Flush column with pure water

at 1.0 mL/min for 2 column

volumes

Flush with new eluent at

0.5 mL/min for 2 column

volumes

Flush with new buffer at

1.0 mL/min for 2 column

volumes

Increase column

temperature as required for

analysis at 1 °C/min

Increase column temperature

as required for analysis at

1 °C/min

Operate column in new

eluent at required ow rate

Operate column in new eluent

at required ow rate

*Always ensure operating temperature is at least 10 °C below

boiling point of solvent

Column Testing and Specications

Every column is supplied with a test certicate

indicating the test conditions and the column

performance. Measurements of column

performance are described below:

N = 5.54 []

W1/2

N = 25 t

W5δ

Efciency (1/2 ht)

(Plates/m)

Efciency (5δ)

(Plates/m)

Symmetry a/b

Where “t” is the peak elution time, “W1/2“ is the

peak width at half peak height, “W5σ” is the peak

width at 4.4% of peak height, “L” is the column

length in meters and “a” and “b” are the peak

widths either side of the perpendicular measured

at 10% of peak height.

Column efciency is dependent on many

experimental factors (system dead volume,

eluent, ow rate, test probe, temperature, and

so forth) and test results may differ from those

quoted on the column certicate due to variability

in these parameters. Band broadening effects

are more severe when using high efciency and/

or narrow bore GPC/SEC columns. It is vital to

ensure that the system dispersion is minimized

in order to obtain the full potential of Agilent

columns.

We recommend measuring performance when a

column set is rst used and at regular intervals after

that. Consider replacing columns when efciency

falls below 80% of the starting value.

Table 3. Column specications

Column Typical

operating

pressure

psi (bar)

Maximum

operating

pressure psi

(bar)

Maximum

operating

temp. °C2

PLgel 3 µm 750 (50) 2,700 (180) 110

PLgel 5 µm 450 (30) 2,200 (150) 150

PLgel 9 & 10 µm 150 (10) 2,200 (150) 220

PLgel 20 µm 50 (3) 2,200 (150) 220

PLgel Olexis 90 (6) 2,250 (150) 220

PLgel Prep 10 µm 350 (25) 2,200 (150) 150

PolyPore 450 (30) 2,200 (150) 160

ResiPore 750 (50) 2,700 (180) 120

MesoPore 750 (50) 2,700 (180) 120

OligoPore 450 (30) 1,500 (100) 120

PL Rapide-F 350 (25) 1,500 (100) 150

PL Rapide-L 225 (15) 1,100 (75) 150

PL Rapide-M 75 (5) 1,100 (75) 220

PL Rapide-H 75 (5) 1,100 (75) 220

PolarGel 450 (30) 1,600 (112) 80

PL aquagel-OH 5 µm 900 (60) 1,600 (112) 90

PL aquagel-OH 8 µm 450 (30) 1,600 (112) 90

PL aquagel-OH 15 µm 150 (10) 1,600 (112) 90

PL Rapide Aqua 225 (15) 1,600 (112) 90

PL Multisolvent

(Water)

1450 (100) 1,600 (112) 80

PL Multisolvent

(Organic)

750 (50) 1,600 (112) 80

1Based on THF (organic columns) or H20 (aqueous columns) at

20 °C, 4.6 mm id at 0.3 mL/min, 7.5 mm id at 1.0 mL/min, 10 mm

id at 1.8 mL/min (2 mL/min for PL Rapide 10 x 100 mm) and

25 x 300 mm at 10 mL/min.

2At very high temperatures, column lifetimes are likely to be

reduced. OligoPore and PL Rapide-F may show deterioration to

lifetimes at >80°C, while PL Multisolvent may degrade at > 50 °C.

Amend ow rates and temperatures accordingly.

[]

English

Storage

On removing the column from the system,

the end plugs must be replaced to prevent the

column from drying out by evaporation, since

subsequent shrinkage of the gel and disruption

of the packing will occur. The end plugs

need only be applied nger-tight. All eluents

mentioned previously are suitable for storage,

but unstabilized THF and halogenated solvents

should not be used.

For long term storage of aqueous columns, ush

with water and store in water containing 0.02%

sodium azide.

Warranty

The columns are covered by warranty for 90

days following delivery. For columns used

at or above 170 °C, the warranty period

is reduced to 30 days. Agilent Technologies

cannot accept liability from improper handling

and use of columns above their maximum

operation temperature and pressure (see Table

3) and void the warranty. For a full warranty

statement, please request Agilent’s General

Conditions of Sale.

Maintenance

Deterioration in column performance may occur

as a result of damage to the packed bed or as

a result of blockage in the column frits. In the

case of frit blockage, the column can be reverse

ushed for 1 minute at the recommended ow

rate to remove loosely retained material. If a

column blockage exists despite back ushing,

then some column inlet frits can be replaced by

following the procedure outlined below.

English

Frit replacement procedure

1. Seal the end tting at the outlet end of the

column. Clamp the column vertically with the

inlet end uppermost. Undo the end tting on the

inlet end of the column and gently remove it. (For

aqueous columns, ush eluent into water prior to

this procedure.)

2. After removing any gel from the end tting,

remove the frit, seal and spreader with the frit

removal tool, and wash all traces of gel from

the end tting. Then place a new frit, seal and

spreader in the bottom of the end tting and

ensure that they are properly seated.

3. Some gel (up to 2 mm) may have expanded

out of the column. Using a spatula, place a dome

of repair gel onto the top of the column and

add sufcient solvent to bind the gel particles

together.

4. Hold the column at an angle of 45° pointing

downwards and replace the end tting complete

with frit, seal and spreader onto the column.

Tighten the end tting one at past nger-tight.

5. Back ush the column with eluent at 0.5

mL/min to remove trapped air. Check for gel

particles in the eluent in case the frit has not

seated correctly. Return the column to normal

ow conditions and check the pressure drop and

column efciency.

Agilent recommends that preparative columns

are returned to Agilent for tment of replacement

frits or repair, however, a similar procedure to the

above can be applied if necessary. For further

technical assistance, or to use the Column Repair

Service, please contact Agilent Technologies.

English

日本語版

取り付け

チューブとコネクタ

分析カラムのカラム接続には外径 1/16 インチ、

内径 0.12 mm または 0.17 mm のステンレスス

チールチューブを、25 mm 分取カラムには内径

0.5 mm のステンレススチールチューブを推奨

します。システムパフォーマンスの低下を招く

デッドボリュームの増加を防ぐには、カラム、

検出器、インジェクター間の接続チューブの長

さをできるだけ短くする必要があります。カラ

ム接続には、Parker 互換 1/16 インチナットと

フェラルを使用します。図１に、カラムコネク

タの互換性を示します。

図１. 互換コネクタ

標準カラムエンドフィッティングの距離 “X” は

0.090 インチであるため、長さ 0.210 インチ以上

のオスナットが必要です。Waters、Rheodyne

など、他の製造元の一部のフィッティングは、

使用できない可能性があります。ご不明の場合

は、アジレント・テクノロジーにお問い合わせ

ください。

カラム接続

溶離液が示された向きに流れるように GPC カラ

ムを接続し、1/16 インチナットと実際のエンド

フィッティング上でスパナを使って、1/16 イン

チナットとフェラルを締めます。

カラム出口を別のカラムまたは検出器に接続す

る前に、溶離液を数滴ポンプで送り、存在する

可能性のある粒子状物質をエンドフィッティン

グから追い出すことを推奨します。

エンドフィッティングが緩んで漏れが生じるの

を防ぐため、スパナは、カラムバレルや反対側

のエンドフィッティング上ではなく、接続ナッ

トに隣接するエンドフィッティング上で使用し

てください ( 図２を参照してください )。

図 2. スパナをフラット上で使用しない

フラット

溶離液の流速

内径 7.5 mm または 7.8 mm のカラムを使用す

る従来の GPC カラムの場合、流速 1.0 mL/min

がほとんどの分離に適しています。カラムの内

径がそれより大きいか、小さいときには、カラ

ムを通過する際に同等の線速度が得られるよう

に、内径に合わせて流量を調整する必要があり

ます。

表 1 に、推奨流速を示します。ただし、圧力が

過剰に高くなるのを防ぐために、粘度の高い溶

離液を使用する場合は、流速を遅くするか、昇

温してください。流速を徐々に変化させ、脈流

を抑えます。カラムの最大稼働圧を超えてはな

りません ( 表４を参照してください )。

表１. 推奨流速

カラム代表流速

（mL/min）

推奨流速（mL/min）

内径 2.1 mm 0.04 ～ 0.1 0.06

内径 4.6 mm 0.2 ～ 0.5 0.3

内径 7.5/7.8

0.5 ～ 1.5 1.0

内径 10 mm 2.0 ～ 5.0 2.0

内径 25 mm 5.0 ～ 20.0 10.0

サンプル前処理と注入

最高の分離能と期待されるカラム寿命を達成す

るには、サンプルの前処理に注意を払う必要が

あります。

カラムフリットの目詰まりを防ぐため、サンプ

ルのろ過を推奨します (MW に応じた 0.5 ～ 2.0

μ m の多孔質フィルタ )。ガードカラムは、パ

フォーマンスへの悪影響がほとんどないため、

カラムの保護に有効です。

分析のタイプごとに最適なサンプル量と濃度が

決定されており、値はサンプルの MW に依存し

ます。通常、分子量分布の広いポリマーは、多

分散性の低いサンプルより高濃度で注入できま

す。過剰添加によってカラムが損傷を受けるこ

とはありませんが、ピークが歪むため、偽の結

果が現れます。

インジェクターのループ容量が大きすぎると、

特に高速高分離カラムやナローボアカラムで、

バンドが広がり、システムパフォーマンスが低

下する可能性があります。表２に、アジレント

の推奨する注入量を示します。

表 2. 注入量の推奨値

カラム推奨濃度（%）カラムあたりの

推奨注入（µL）

内径 2.1 mm 0.01 ～ 0.2 0.2 ～ 2

内径 4.6 mm 0.01 ～ 0.2 1 ～ 20*

内径 7.5/7.8 mm 0.05 ～ 0.5 20 ～ 50**

内径 10 mm 0.05 ～ 0.5 20

内径 25 mm 0.5 ～ 5.0 500 ～ 2,000

* PL Multisolvent、MesoPore と ResiPore は 1 ～ 10 µL.

** PL Multisolvent、MesoPore と ResiPore は 10 ～ 20 μ L、

PL Rapide/PL Rapide Aqua は 20 μ L

溶離液

すべての溶離液は、高純度であり、使用前に

フィルタでろ過し、脱気する必要があります。

GPC カラムを別の溶離液に置き換えることがで

きます。別の溶離液に置き換える際には、まず

新しい溶離液の混和性と粘性を検討します。内

径 7.5 mm のカラムの場合、図 3 を参照してく

ださい。その他の内径のカラムには、表１の流

速を適用してください。

日本語版

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