ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

[ CARE AND USE MANUAL ]

1

CONTENTS

I. INTRODUCTION

II. 

GETTING STARTED

a.  Column Connectors

b.  Column Installation

c.  Column Equilibration

d.  Procedure for Using New, 

  Out-of-Box Columns

e.  eCord Installation

f.  Column QR Code

g.  Initial Column Efficiency Determination

h.  VanGuard Pre-Columns

i.  Installation Instructions

II. 

COLUMN USE

a.  Sample Preparation 

b.  pH Range 

c. Solvents 

d. Pressure 

e. Temperature

III. 

COLUMN CLEANING, REGENERATING, 

AND STORAGE

a.  Cleaning and Regeneration 

b. Storage

IV. eCORD INTELLIGENT CHIP TECHNOLOGY
 

a. Introduction 

b. Installation 

c.  Manufacturing Information 

d.  Column Use Information

V. 

ADDITIONAL INFORMATION

a.  Tips for Maximizing ACQUITY UPLC

  HSS T3 Column Lifetimes

VI. 

REPRESENTATIVE TEST CHROMATOGRAM

VII. 

CAUTIONARY NOTE

I. INTRODUCTION
Thank you for choosing a Waters™ ACQUITY™ UPLC™ and/or 
ACQUITY PREMIER Peptide HSS T3, 100 Å Column that 
contains our High Strength Silica particle technology to deliver 
increased peptide retentivity and different selectivity compared 
to results generated on the Peptide CSH™ C

18, 130 Å or Peptide 

BEH C

18, 130 Å and 300 Å column offerings. The manufacture of 

our Peptide HSS T3 particles begins with ultrapure reagents to 
control the chemical composition and purity of the final product. 
Peptide HSS T3 columns are manufactured in a cGMP, ISO 
9001:2000 certified plant with each step being conducted within 
narrow tolerances. Each batch of ACQUITY UPLC and ACQUITY 
PREMIER HSS T3, 100 Å material is chromatographically tested 
with acidic, basic, and neutral analytes, as well part of Waters 
standard batch qualification procedure. In addition, each batch 
of HSS T3, 100 Å material is QC tested with a gradient separation 
of a tryptic digest of cytochrome c and must pass stringent 
performance criteria before acceptance for use in an ACQUITY 
UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Column. 
A final QC test for packed bed efficiency is also performed on 
each column with this as well as the batch test results provided 
on the column’s attached eCord™ Intelligent Chip.

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 
100 Å Columns were designed and tested specifically for 
use on ACQUITY UPLC Systems and will deliver maximum 
chromatographic performance when used on the holistically-
designed ACQUITY Systems. 

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

2

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

[ CARE AND USE MANUAL ]

II. GETTING STARTED
Each ACQUITY UPLC and ACQUITY PREMIER Peptide 
HSS T3 Column comes with a Certificate of Analysis and a 
Performance Test Chromatogram embedded within the eCord 
intelligent chip. The Certificate of Analysis is specific to 
each batch of packing material contained in the ACQUITY 
UPLC and ACQUITY PREMIER Peptide HSS T3 Column and 
includes the gel batch number, analysis of unbonded particles, 
analysis of bonded particles, and chromatographic results and 
conditions. The Performance Test Chromatogram is specific to 
each individual column and contains such information as: gel 
batch number, column serial number, USP plate count, USP 
tailing factor, capacity factor, and chromatographic conditions. 
These data should be stored for future reference. 

a. Column Connectors
The ACQUITY UPLC System utilizes tubing and gold 
plated compression screws which have been designed 
to meet stringent tolerance levels and to minimize extra 
column volumes.

Optimized column inlet tubing (p/n: 430001084) is supplied 
with the ACQUITY UPLC System. The inject valve end of 
the tubing is clearly marked with a blue shrink tube marker. 
Insert the opposite end of the tubing into the ACQUITY 
UPLC Column and tighten the compression fitting using 
two 5/16-inch wrenches.

For information on the correct column outlet tubing, please 
refer to the relevant detector section in the ACQUITY UPLC 
System Operator’s Guide (p/n: 71500082502).

b. Column Installation
Note: The flow rates given in the procedure below are for a 
typical 2.1 mm I.D. by 50 mm length 1.8 µm column. Scale the 
flow rate up or down accordingly based upon the flow rate and 
pressure guide provided in Section V (Additional Information).

1.  Purge the pumping system of any buffer-containing mobile 

phases and connect the inlet end of the column to the  
injector outlet.

2.  Flush column with 100% organic mobile phase (methanol 

or acetonitrile) by setting the pump flow rate to 0.1 mL/min 
and increase the flow rate to 0.5 mL/min over 5 minutes.

3.  When the mobile phase is flowing freely from the column 

outlet, stop the flow and attach the column outlet to the 
detector. This prevents entry of air into the detection 
system and gives more rapid baseline equilibration.

4.  Gradually increase the flow rate as described in Step 2.

5.  Once a steady backpressure and baseline have been 

achieved, proceed to the next section.

Note: If mobile phase additives are present in low concentrations 
(e.g., ion-pairing reagents), 100 to 200 column volumes may be 
required for complete equilibration. In addition, mobile phases 
that contain formate (e.g., ammonium formate, formic acid, etc.) 
may also require longer initial column equilibration times.

C. Column Equilibration
ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3 
Columns are shipped in 100% acetonitrile. It is important 
to ensure mobile-phase compatibility before changing to a 
different mobile-phase system. Equilibrate the column with 
a minimum of 10-column volumes of the mobile phase to be 
used (refer to Table 1 for a list of column volumes). The column 
may be considered thermally equilibrated once a constant 
backpressure is achieved.

Table 1: Empty column volumes in mL 
(multiply by 10 for flush solvent volumes)

Column length (mm)

Column internal diameter (mm)

1.0 mL

2.1

50

0.04 mL

0.2 mL

100

0.08 mL

0.4 mL

150

0.12 mL

0.5 mL

 
To avoid precipitating mobile-phase buffers on your column or 
in your system, flush the column with five column volumes of a 
water/organic solvent mixture, using the same or lower solvent 
content as in the desired buffered mobile phase. (For example, 
flush the column and system with 60% methanol in water prior 
to introducing 60% methanol/40% buffer mobile phase). 

d. Procedure for Using New, Out-of-Box Columns
Prior to using a new column, it is important to confirm that 
it produces reproducible chromatography and the desired 
level of chromatographic resolution. To this end, it is useful 
to benchmark column performance with a sample that is 
representative of the intended application. The number of 
injections necessary to achieve reproducible performance 
may be dependent on sample characteristics and system 
type. Method variables like pH, mass load, ionic strength, and 
ion pairing could also have impact. The ACQUITY PREMIER 
Columns have MaxPeak™ High Performance Surfaces that can 
reduce the number of injections necessary to achieve desired 
performance due to the improved hardware inertness.

3

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

[ CARE AND USE MANUAL ]

e. eCord Installation
The eCord button should be attached to the side of the column 
heater module. The eCord button is magnetized and does not 
require specific orientation.

f. Column QR Code
The quick reference (QR) code that is located on the column 
label provides column-specific information (i.e., the part and 
serial numbers that are unique identifiers for the column), and 
its encoding follows a widely adopted industry-standard. 

1.  Scan QR code using any device that is capable of 

scanning QR codes (i.e., for smart phones and tablets, 
use the built-in camera app).

2.  Be directed to the column’s information hub 

on waters.com.

3.  Access technical and scientific information for the column 

(i.e., certificate of analysis, application notes).

g. Initial Column Efficiency Determination
1.  Perform an efficiency test on the column before using it. 

Waters recommends using a suitable solute mixture, as 
found in the “Performance Test Chromatogram”, to analyze 
the column upon receipt. 

2.  Determine the number of theoretical plates (N) and use 

this value for periodic comparisons. 

3.  Repeat the test at predetermined intervals to track column 

per formance over time. Slight variations may be obtained 
on two different UPLC systems due to the quality of the 
connections, operating environment, system electronics, 
reagent quality, column condition, and operator technique.

h. VanGuard Pre-Columns
VanGuard™ Pre-Columns are 2.1 mm I.D. x 5 mm length guard 
column devices designed specifically for use in the ACQUITY 
UPLC System. VanGuard Pre-Columns are packed with the 
same UPLC Chemistries and frits as our 2.1 mm I.D. UPLC 
Columns. VanGuard Pre-Columns are designed to be attached 
directly to the inlet side of an ACQUITY UPLC and ACQUITY 
PREMIER Column.

Note: In order to ensure void-free and leak-free connections, 
the VanGuard Pre-Column is shipped with the collet and ferrule 
NOT permanently attached. Care must be taken when removing 
the O-ring that holds these two pieces on the pre-column tubing.

i. Installation Instructions
1.  Remove the VanGuard Pre-Column from its box and 

shipping tube and remove plastic plug.

2.  Orient the pre-column so that male end is facing up and 

carefully remove rubber O-ring that holds collet and ferrule 
in place during shipping (collet and ferrule are not yet 
permanently attached).

3.  Orient the ACQUITY UPLC and ACQUITY PREMIER Peptide 

HSS T3 Column perpendicular to the work surface so that 
column inlet is on the bottom (column outlet on top).

4.  From below, insert the VanGuard Pre-column into the 

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3 
Column inlet and hand-tighten (collet and ferrule are not yet 
permanently attached).

5.  While pushing the VanGuard Pre-Column into the column 

inlet, turn assembled column and pre-column 180˚ so that 
the pre-column is now on top.

6.  Tighten with two 5/16" wrenches placed onto the ACQUITY 

UPLC and ACQUITY PREMIER Column flats and the 
VanGuard Pre-column hex nut (male end) as shown.

7.  Tighten 1/4 turn to set collet and ferrule.

8.  Check that the ferrule is set by loosening the connection 

and inspecting the ferrule depth. A properly set ferrule 
depth will resemble other connections in the ACQUITY 
UPLC and ACQUITY PREMIER System.

9.  Reattach pre-column, apply mobile-phase flow, and 

inspect for leaks.

Figure 1. VanGuard Pre-Column installation diagram.

ACQUITY UPLC Column

VanGuard Pre-Column

Place wrench here

Ferrule

Collet

Place wrench here

Flow

4

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

[ CARE AND USE MANUAL ]

II. COLUMN USE
To ensure the continued high performance of ACQUITY 
UPLC and ACQUITY PREMIER Peptide HSS T3 Columns, 
follow these guidelines:

a. Sample Preparation
1.  Sample must be dissolved in a diluent compatible 

with initial strength of mobile phase. 

2.  Sample must be completely in solution and free 

of particulates. 

3.  To remove particulates, the sample may be filtered 

with a 0.2 µm membrane. 

If the sample is dissolved in a solvent that contains an organic 
modifier (e.g., acetonitrile, methanol, etc.) ensure that the 
membrane material does not dissolve in the solvent. Contact 
the membrane manufacturer with solvent compatibility 
questions. Alternatively, centrifugation for 20 minutes at 
15,000 rpm, followed by the transfer of the supernatant 
liquid to an appropriate vial, could be considered.

b. pH Range
The recommended operating pH range for ACQUITY UPLC 
and ACQUITY PREMIER Peptide HSS T3 Columns is pH 2 
to 8. A listing of commonly used buffers and additives is 
given in Table 2. Additionally, the column lifetime will vary 
depending upon the operating temperature, and the type 
and concentration of buffer used. Note: Working at the 
extremes of pH, temperature and/or pressure will result in 
shorter column lifetimes.

c. Solvents
To maintain maximum column performance, use high quality 
chromatography grade solvents. Filter all aqueous buffers prior 
to use through a 0.2 µm filter. Solvents containing suspended 
particulate materials will generally clog the outside surface of 
the inlet distribution frit of the column. This will result in higher 
operating pressure and poorer performance. 

d. Pressure
ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3 
Columns can tolerate operating pressures up to 18,000 psi 
(1241 bar or 124 MPa). Note: Working at the extremes of pressure, 
pH and/or temperature will result in shorter column lifetimes.

e. Temperature
The maximum recommended temperature for ACQUITY UPLC 
and ACQUITY PREMIER Peptide HSS T3 Columns is 60 °C. 
Higher temperatures can be used, but may result in shorter 
column lifetimes. When operating with mobile phases that 
are close to the lower pH limit, using temperatures above 60 
°C may result in shorter column lifetimes due to hydrolysis 
of the bonded phase. High temperatures should also be 
avoided when operating close to the upper pH limit, where 
the dissolution of silica particles starts to occur. To maximize 
column lifetime near the upper pH limit, temperatures lower 
than 45 °C are recommended. The rate of silica dissolution 
also varies with the type and concentration of the buffer, with 
carbonate and phosphate buffers giving some of the highest 
rates. As a result, when using these buffers near the upper pH 
limit, lower temperatures and lower concentrations should 
be considered. Note: Working at the extremes of temperature, 
pressure and/or pH will result in shorter column lifetimes.

Table 2. Buffer Recommendations for Using ACQUITY UPLC and 
ACQUITY PREMIER Peptide HSS T3, 100 Å Columns from pH 2 to 8 

Additive/Buffer

pK

a

Buffer 

range  

Volatility 

(±1 pH unit)

Used for 

Mass 

Spec

Comments

TFA

0.3

–

Volatile

Yes

Ion pair additive, can suppress MS signal, 
used in the  0.02–0.1% range.

Acetic acid

4.76

–

Volatile

Yes

Maximum buffering obtained when used with 
ammonium acetate salt. Used in 0.1–1.0% range.

Formic acid

3.75

–

Volatile

Yes

Maximum buffering obtained when used with 
ammonium formate salt. Used in 0.1–1.0% range.

Acetate 
(NH

4CH2COOH)

4.76

3.76–5.76

Volatile

Yes

Used in the 1–10 mM range. 
Note that sodium or potassium salts are not volatile.

Formate (NH

4COOH)

3.75

2.75–4.75

Volatile

Yes

Used in the 1–10 mM range. 
Note that sodium or potassium salts are not volatile.

Phosphate 1

2.15

1.15–3.15 Non–volatile

No

Traditional low pH buffer, good UV transparency.

5

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

[ CARE AND USE MANUAL ]

III. COLUMN CLEANING, REGENERATING, 

AND STORAGE

a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the 
peak, shifts in retention, change in resolution or increasing 
backpressure may indicate contamination of the column. 
Flushing with a neat organic solvent, taking care not to 
precipitate buffers, is usually sufficient to remove the 
contaminant. If the flushing procedure does not solve the 
problem, purge the column using the following cleaning 
and regeneration procedures.

Use the cleaning routine that matches the properties of the 
samples and/or what you believe is contaminating the column 
(see Table 3). Flush columns with 20-column volumes of solvent. 
Increasing column temperature increases cleaning efficiency. 
If the column performance is poor after regenerating and 
cleaning, call your local Waters office for additional support.

b. Storage
For periods longer than four days at room temperature, 
store reversed-phase ACQUITY UPLC and ACQUITY 
PREMIER HSS Columns in 100% acetonitrile. For elevated 
temperature applications, store immediately after use in 
100% acetonitrile for the best column lifetime. Do not store 
columns in buffered eluents. If the mobile phase contained a 
buffer salt, flush the column with 10-column volumes of HPLC-
grade water (see Table 1 for common column volumes) and 
replace with 100% acetonitrile for storage. Failure to perform 
this intermediate step could result in precipitation of the buffer 
salt in the column when 100% acetonitrile is introduced. 
Completely seal column to avoid evaporation 
and drying out of the bed.

Note: If a column has been run with a mobile phase that contains 
formate (e.g., ammonium formate, formic acid, etc.) and is then 
flushed with 100% acetonitrile, slightly longer equilibration times 
may be necessary when the column is re-installed and run again 
with a formate-containing mobile-phase.

Table 3. Reversed-Phase Column Cleaning Sequence 

Proteinaceous Samples

Water

Option 1: Inject repeated 100 µL aliquots of dimethyl sulfoxide (DMSO) 
using a reduced flow rate delivering 50% Eluent A and 50% Eluent B  

Methanol

Option 2: Gradient of 10% to 90% B where: A = 0.1% trifluoroacetic acid (TFA) 
in water, B = 0.1% trifluoroacetic acid (TFA) in acetonitrile (CH3CN)  

Isopropanol

Option 3: Flush column with 7 M guanidine hydrochloride, or 7 M urea

Note: To avoid potentially damaging precipitation within your column (e.g., if your separation eluent contains phosphate buffer), be 
certain to flush column with 5 to 10 column volumes of water BEFORE using suggested organic eluent column wash procedures.

6

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

[ CARE AND USE MANUAL ]

IV. eCORD INTELLIGENT CHIP TECHNOLOGY

a. Introduction
The eCord Intelligent Chip Technology provides the history 
of a column’s performance throughout its lifetime. The eCord 
will be permanently attached to the column to assure that the 
column’s performance history is maintained in the event that 
the column is moved from one instrument to another.

c. Manufacturing Information

d. Column Use Information
The eCord will automatically capture column use data. The 
top of the screen identifies the column including chemistry 
type, column dimensions, and serial number. The overall 
column usage information includes the total number of 
samples, total number of injections, total sample sets, date 
of first injection, date of last injection, maximum pressure, 
and temperature. The information also details the column 
history by sample set including date started, sample set 
name, user name, system name, number of injections in the 
sample set, number of samples in the sample set, maximum 
pressure, and temperature in the sample set and if the 
column met basic system suitability requirements.

Figure 2. eCord Intelligent Chip.

eCord Intelligent Chip

Figure 3. eCord inserted into side of column heater. 

eCord inserted into 

side of column heater

At the time of manufacture, tracking and quality control 
information will be downloaded to the eCord. Storing this 
information on the chip will eliminate the need for a paper 
Certificate of Analysis. Once the user installs the column, 
the software will automatically download key parameters 
into a column history file stored on the chip. In this manual, 
we explain how the eCord will provide a solution for easily 
tracking the history of the columns, reduce the frustration of 
paperwork trails, and give customers the reassurance that a 
well-performing column is installed onto their instruments.

b. Installation
Install the column into the column heater. Plug the eCord into 
the side of the column heater. Once the eCord is inserted into 
the column heater the identification and overall column usage 
information will be available allowing the user to access column 
information on their desktop.

Figure 5. The eCord chip provides 

the user with QC test conditions 

and results on the column run by 

the manufacturer. The information 

includes mobile phases, running 

conditions, and analytes used 

to test the columns. In addition, 

the QC results and acceptance 

is placed onto the column.

Figure 4. The eCord chip provides the user with 

an overview of the bulk material QC test results.

Figure 6. An example of column use information provided by the eCord chip.

7

ACQUITY UPLC and ACQUITY PREMIER Peptide HSS T3, 100 Å Columns

[ CARE AND USE MANUAL ]

V. ADDITIONAL INFORMATION

a. Tips for Maximizing ACQUITY UPLC and ACQUITY 
PREMIER Peptide HSS T3 Column Lifetimes
1.  To maximize ACQUITY UPLC and ACQUITY PREMIER 

Peptide HSS T3 Column lifetime, pay close attention to:

■

Water quality (including water purification system)

■

Solvent quality

■

Mobile-phase preparation, storage, and age

■

Sample, buffer, and mobile-phase solubilities

■

Sample quality and preparation

2.  When problems arise, often only one improper practice 

must be changed.

3.  Always remember to:

■

Use in-line filter unit or, preferably, a VanGuard  

 Pre-column.

■

Discourage bacterial growth by minimizing the use 

  of 100% aqueous mobile phases where possible.

■

Change aqueous mobile phase every 24–48 hours  

  (if 100% aqueous mobile phase use is required).

■

Discard old 100% aqueous mobile phases every  

  24–48 hours to discourage bacterial growth.

■

Add 5–10% organic modifier to mobile  

  phase A and adjust gradient profile.

■

Filter aqueous portions of mobile phase through 

  0.2 µm filter.

■

Maintain your water purification system so that 

  it is in good working order.

■

Only use ultra pure water (18 megohm-cm) water 

  and highest quality solvents possible. HPLC-grade 
  water is not UPLC grade water.

4.  Avoid (where possible):

■

100% aqueous mobile phases (if possible)

■

HPLC-grade bottled water

■

“Topping off” your mobile phases

■

Old aqueous mobile phases. Remember to 

  rinse bottles thoroughly and prepare fresh 
  every 24 to 48 hrs

■

Using phosphate salt buffer in combination with high  

  ACN concentrations (e.g., >70%) due to precipitation

5.  Don’t: assume a “bad” column is the culprit when  

high backpressure or split peaks are observed:

■

Investigate cause of column failure

■

Backpressure

■

Mobile phase(s), bacteria, precipitation, 

  and/or samples

■

Peak splitting

■

Sample quality

■

Injection solvent strength

6.  Remember: the diameter of UPLC columns (1.0, 2.1, and  

3.0 mm I.D.) are often lower than that of a conventional 
HPLC column and therefore, mobile phases last 
much longer. To reduce the chances of mobile-phase 
contamination or degradation, only prepare what you 
need for analysis or store excess bulk quantities in a 
refrigerated environment.

7.  Mobile-phase-related questions to ask:

■

Am I using 100% aqueous mobile phases?  

  Am I able to add a small amount of organic  
  modifier to my mobile phase A?

■

Do I filter my aqueous mobile phases  

  through 0.2 µm filters?

■

How old is my mobile phase? Do I label the 

  bottle with preparation date?

■

Do I “top off” or do I prepare fresh mobile phases 

  every 24–48 hrs?

■

What is the quality of my water? Has the quality  

  recently changed? How is my water purification  
  system working? When was it last serviced?

■

Am I working with pH 7 phosphate buffer (which is  

  VERY susceptible to bacterial growth)?

8.  Sample-related questions to ask:

■

If I inject neat standards prepared in mobile phase 

  do I observe these problems?

■

If I prepare my standards in water and prepare  

  them like samples (e.g., SPE, filtration, etc.) do I  
  still observe these problems?

■

Has the quality of my samples changed over time?

[ CARE AND USE MANUAL ]

Waters Corporation 
34 Maple Street 
Milford, MA 01757 U.S.A. 
T: 1 508 478 2000 
F: 1 508 872 1990 
www.waters.com

[ CARE AND USE MANUAL ]

Waters, The Science of What’s Possible, ACQUITY, UPLC, CSH, eCord, MaxPeak, and VanGuard are 
trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2020 Waters Corporation  October 2020  720005940EN  Rev C  IH-PDF

Figure 4. Separation of tryptic digest of cytochrome c on an ACQUITY UPLC HSS T3, 1.8 µm Column.

VI. REPRESENTATIVE TEST CHROMATOGRAM

IX. CAUTIONARY NOTE
Some products may be hazardous during and after use and 
are to be used by professional laboratory personnel trained in 
the competent handling of such materials. The responsibility 
for the safe use of products rests entirely with the purchaser 
and user. The safety data sheets (SDS) for these products are 
available at www.waters.com/sds.

T1

T13T14

T1

4 

T4

T9T1

0 

T1

0 

T19C

T8

T1

5 

T1

9 

T5

T12T13

T1

2 

AU

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

  40 min

Chromatographic Conditions: 
Column: 

ACQUITY UPLC Peptide HSS T3, 
100 Å, 1.8 µm, 2.1 × 150 mm (p/n: 186008756)

Sample: 

Cytochrome c Digestion Standard 
(p/n: 186006371)

Inject: 

7.5 µL

Mobile phase A: 

0.045% TFA in water

Mobile phase B: 

0.045% TFA in acetonitrile

Temp.: 

35 °C

Wavelength: 

214 nm

  
Gradient: Time  Flow 

%A 

%B1 

Curve 

0  0.2 100 0  – 
9.0 0.2  85  15  6 
39.0 0.2  64 

36  6 

43.0 0.2  40 

60  11 

58.0 0.2  100  0 

11 

67.0 0 

100  0 

11