Agilent Technologies GPC/SEC User manual

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

2
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

3
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

4
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

5
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

6
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 []
t
W1/2
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.
[]
/L
2
/L
English

7
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

8
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

9
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

10
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
mm
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